Potential Therapeutic Effects of Underground Parts of...

11
Research Article Potential Therapeutic Effects of Underground Parts of Kalanchoe gastonis-bonnieri on Benign Prostatic Hyperplasia Antonio Palumbo, 1 Livia Marques Casanova, 2 Maria Fernanda Paresqui Corrêa, 2 Nathalia Meireles Da Costa, 3 Luiz Eurico Nasciutti , 1 and Sônia Soares Costa 2 1 Instituto de Ciˆ encias Biom´ edicas, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil 2 Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil 3 Programa de Carcinogˆ enese Molecular, Centro de Pesquisas, Instituto Nacional do Cˆ ancer, 20231-050 Rio de Janeiro, RJ, Brazil Correspondence should be addressed to Luiz Eurico Nasciutti; [email protected] and onia Soares Costa; [email protected] Received 28 July 2018; Accepted 9 December 2018; Published 2 January 2019 Academic Editor: Ester Pagano Copyright © 2019 Antonio Palumbo et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Benign Prostatic Hyperplasia (BPH) affects mainly older men. It is estimated to affect 50% of 51-60-year-old men and 70% of 61- 70-year-old men. BPH is a nonmalignant proliferation of epithelial and stromal cells of the prostate gland regions. Despite the use of conventional pharmacological therapy, herbal medicines are used in BPH therapy, and several mechanisms of action have been suggested based on their complex chemical composition. Considering the ethnomedicinal uses of Kalanchoe gastonis-bonnieri (KGB), we evaluated the inhibitory effects on the proliferation of stromal cells from primary benign prostatic hyperplasia (BPH) of four different aqueous extracts from this plant: underground parts from specimens in flower (T1 treatment), leaves from specimens in flower (T2 treatment), and flowers (T3 treatment) and leaves from specimens not in flower (T4 treatment). T1, T2, T3, and T4 treatments at 250 g/ml for 72 hours inhibited BPH cells by 56.7%, 29.2%, 39.4%, and 13.5%, respectively, showing that the KGB underground parts extract (T1 treatment) was the most active. Our findings show that the extract of the KGB underground parts (150 and 250 g/ml) stimulates important changes in the BPH cells, modulating crucial processes such as proliferation, viability, and apoptosis. HPLC-DAD-MS/MS analysis provided a tentative identification of glycosylated syringic acid derivatives, glycosylated forms of volatile compounds, and lignans in this extract. Finally, these results suggest that there is a potential therapeutic use for KGB in BPH, which could improve the clinical management of the disease. 1. Introduction Benign Prostatic Hyperplasia (BPH) is a nonmalignant pro- liferation of epithelial and stromal cells of the prostate gland, causing an enlargement of the gland that may or may not be associated with lower urinary tract symptoms (LUTS) which affect the quality of life [1–4]. BPH affects mainly older men; and the prevalence increases with age. BPH is estimated to affect 50% of 51-60- year-old men, and this number reaches 80 to 90% for men over 80 years old [2, 5, 6]. Two antagonistic phenomena are involved in maintaining the normal size of the prostate: the rate of cell proliferation and apoptosis (cell death). In normal tissue, these ratios are similar for both the epithelial and stromal cells. However, in BPH there is an imbalance where the cell proliferation rate increases considerably more than the rate of the apoptosis process [4, 7, 8]. ere are evidences that androgens, estrogen, growth factors, and neurotransmitters may play an important role in the etiology of BPH [3]. Additionally, scientific and clinical studies have shown that an inflammatory process may also influence the onset of this disease [2, 9–11]. Currently, six categories of drugs are used in the treat- ment of BPH: herbal agents, selective -adrenergic block- ers, inhibitors of the enzyme 5 -reductase, antimuscarinic agents, 3-adrenergic agonists, and, more recently, inhibitors Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2019, Article ID 6340757, 10 pages https://doi.org/10.1155/2019/6340757

Transcript of Potential Therapeutic Effects of Underground Parts of...

Research ArticlePotential Therapeutic Effects of UndergroundParts of Kalanchoe gastonis-bonnieri on BenignProstatic Hyperplasia

Antonio Palumbo1 Livia Marques Casanova2 Maria Fernanda Paresqui Correcirca2

Nathalia Meireles Da Costa3 Luiz Eurico Nasciutti 1 and Socircnia Soares Costa 2

1 Instituto de Ciencias Biomedicas Universidade Federal do Rio de Janeiro 21941-902 Rio de Janeiro RJ Brazil2Instituto de Pesquisas de Produtos Naturais Universidade Federal do Rio de Janeiro 21941-902 Rio de Janeiro RJ Brazil3Programa de Carcinogenese Molecular Centro de Pesquisas Instituto Nacional do Cancer 20231-050 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Luiz Eurico Nasciutti luiznasciuttihistoufrjbr andSonia Soares Costa sscostabhgmailcom

Received 28 July 2018 Accepted 9 December 2018 Published 2 January 2019

Academic Editor Ester Pagano

Copyright copy 2019 Antonio Palumbo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Benign Prostatic Hyperplasia (BPH) affects mainly older men It is estimated to affect 50 of 51-60-year-old men and 70 of 61-70-year-old men BPH is a nonmalignant proliferation of epithelial and stromal cells of the prostate gland regions Despite theuse of conventional pharmacological therapy herbal medicines are used in BPH therapy and several mechanisms of action havebeen suggested based on their complex chemical composition Considering the ethnomedicinal uses ofKalanchoe gastonis-bonnieri(KGB) we evaluated the inhibitory effects on the proliferation of stromal cells from primary benign prostatic hyperplasia (BPH) offour different aqueous extracts from this plant underground parts from specimens in flower (T1 treatment) leaves from specimensin flower (T2 treatment) and flowers (T3 treatment) and leaves from specimens not in flower (T4 treatment) T1 T2 T3 and T4treatments at 250 120583gml for 72 hours inhibited BPH cells by 567 292 394 and 135 respectively showing that the KGBunderground parts extract (T1 treatment) was the most active Our findings show that the extract of the KGB underground parts(150 and 250120583gml) stimulates important changes in the BPHcells modulating crucial processes such as proliferation viability andapoptosis HPLC-DAD-MSMS analysis provided a tentative identification of glycosylated syringic acid derivatives glycosylatedforms of volatile compounds and lignans in this extract Finally these results suggest that there is a potential therapeutic use forKGB in BPH which could improve the clinical management of the disease

1 Introduction

Benign Prostatic Hyperplasia (BPH) is a nonmalignant pro-liferation of epithelial and stromal cells of the prostate glandcausing an enlargement of the gland that may or may not beassociated with lower urinary tract symptoms (LUTS) whichaffect the quality of life [1ndash4]

BPH affects mainly older men and the prevalenceincreases with age BPH is estimated to affect 50 of 51-60-year-old men and this number reaches 80 to 90 for menover 80 years old [2 5 6]

Two antagonistic phenomena are involved inmaintainingthe normal size of the prostate the rate of cell proliferation

and apoptosis (cell death) In normal tissue these ratios aresimilar for both the epithelial and stromal cells However inBPH there is an imbalance where the cell proliferation rateincreases considerably more than the rate of the apoptosisprocess [4 7 8]There are evidences that androgens estrogengrowth factors and neurotransmitters may play an importantrole in the etiology of BPH [3] Additionally scientific andclinical studies have shown that an inflammatory processmayalso influence the onset of this disease [2 9ndash11]

Currently six categories of drugs are used in the treat-ment of BPH herbal agents selective 120572-adrenergic block-ers inhibitors of the enzyme 5 120572-reductase antimuscarinicagents 1205733-adrenergic agonists and more recently inhibitors

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 6340757 10 pageshttpsdoiorg10115520196340757

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Extraction and yield of different parts of K gastonis-bonnieri

Fresh plant materialmass (g) LyophilizedExtract (g)

Yield

Leaves of plants not in flower (11392) 238 21Leaves of plants in flower (1495) 29 19Underground parts of plants in flower (829) 08 12Flowers (873) 34 39

of the enzyme phosphodiesterase type 5 [12ndash14] Addi-tionally there are current evidences that nonsteroidal anti-inflammatory drugs (NSAID) can improve LUTS [15]

Herbal medicines are used in BPH therapy and severalmechanisms of action have been put forward based onthe complex chemical composition present in plants Thepresence of different substances acting on specific targetsmakes herbal medicines a relevant therapeutic strategy in thetreatment of prostatic hyperplasiaThemain herbal medicineused in the treatment of BPH is an extract of Serenoa repensfruit (Arecaceae) popularly known as ldquosaw palmettordquo forwhich there is strong evidence of clinical efficacy [16ndash18]

Many natural products are also used for the improvementof physiological functions as well as the symptoms of BPHAmong them the pollen extract Cernitin (Secale cerealeCernilton) Pygeum africanum (Tadenan) Urtica dioicaScutellaria baicalensis Cucurbita pepo lycopene and 120573-sitosterol are known for their beneficial effects on BPH [16ndash18] Besides many medicinal plant species have been testedin vitro and in vivo after they have shown potential for BHPtreatment [19] Some secondary metabolites from plants havealso shown promising results in vitro and in vivo amongthem phenolic substances such as isoflavones lignans andthe stilbene resveratrol [20ndash24]

Kalanchoe gastonis-bonnieri Raym-Hamet amp H Perrier(synKalanchoe adolphi-engleriRaym-Hamet) is a medicinalherb from the family Crassulaceae It is used in LatinAmerican medicine as a vaginal contraceptive as well asin the treatment of genital-urinary and vaginal infections[25] In a previous study we reported the isolation of thenew flavonoid quercetin 3-O-120572-rhamnopyranoside-7-O-120573-D-glucopyranosyl-(1997888rarr3)-120572-L-rhamnopyranoside as well asvicenin-2 a C-glycosyl flavone from the leaf extract of KGB[26] We also demonstrated that aqueous extracts from Kgastonis-bonnieri (KGB) are effective in controling dentalbacterial plaque and calculus in dogs [27]

Extracts from KGB have been shown to immobilize toclump together and to promote structural changes in ratsperm [28] The production and storage of seminal fluidand other components of semen are intrinsically linkedto the functions of the prostate [29 30] Considering theethnomedicinal use of KGB this study aimed to determinethe efficacy of this herb in the treatment of BPH by usingan in vitro model of BPH primary cell culture particularlyfocusing on the main hallmarks related to the developmentof the disease

2 Materials and Methods

21 Plant Material In this study leaves were collected fromspecimens in flower and from specimens not in flower ofKalanchoe gastonis-bonnieri cultivated in a residential gardenin the city of Rio de Janeiro Underground parts and flowerswere also obtained from specimens growing in the samegarden A sample of a flowering specimen was identifiedand its voucher specimen (RGA 31592) is deposited in theHerbarium of the Botany Department at the Institute ofBiology of the Federal University of Rio de Janeiro

22 Extraction Fresh leaves fromK gastonis-bonnieri (KGB)(average length of leaves 15 cm) were rinsed with distilledwater cut into small pieces and crushed in a blender Theextract obtained from the leaves of specimens not in flowerwas filtered and resulted in a clear yellow liquid The color ofthe leaf extract of the specimens in flower was a salmon pinkThe flowers were extracted by infusion with distilled water(20 ww) The same procedure was applied for extractingthe underground parts harvested from the other specimensin flower Table 1 shows the mass of the different parts ofthe K gastonis-bonnieri specimens and the yield obtainedfrom the extraction of each part All the extracts were frozenlyophilized and kept in a freezer at -20∘C

23 HPLC-DADMSMS High-Performance Liquid Chro-matography analyses with a Diode Array Detector coupledto a Tandem Mass Spectrometry (HPLC-DAD MSMS)were carried out at the Center for Mass Spectrometry ofBiomolecules-CEMBIO (IBCCF UFRJ) The ProminenceShimadzu Liquid Chromatography system used was com-posed of an LC-20AD pump a degasser system DGU-20A and a DAD detector SPD-M20A coupled to a MaxisImpact Q-TOF mass spectrometer (Bruker) equipped withan electrospray ionization (ESI) interface An ODS-Hypersilreverse phaseC-18 column (ThermoScientific 3120583m 150mm21mm) protected by a precolumnof the samematerial (3120583m1 mm and 21 mm) was used The columns were maintainedat 40∘C

A sample of the extract (4 mgml) from the undergroundparts of KGB was diluted in a mixture of water acidified withformic acid 01 and acetonitrile (191)The injection volumewas 20 120583l

Evidence-Based Complementary and Alternative Medicine 3

The mobile phase consisted of eluent A water containing01 formic acid (Sigma-Aldrich) and eluent B acetoni-trile (Merck) containing 01 formic acid (Sigma-Aldrich)The samples were run for 40 min at 03 mlmin and theabsorbance was monitored between 210 and 400 nm Thegradient used was as follows 0minus5 min (5minus20 B) 5minus11 min(20minus22 B) 11minus12 min (22minus100 B) 12minus26 min (100 B)and 26minus40 min (100minus5 B)

The Q-TOF mass spectrometer was operated in thenegative ion mode using the following parameters capillaryvoltage 5000 V endplate offset -500 V pressure of nebulizer4 Bar drying gas temperature 200∘C Nitrogen was usedas both the sheath and drying gas at a flow rate of 80lmin The mass range analyzed was set at mz 50-1200 andcollision energy at -5 eV An external calibration solution(sodium formate 100 mM in waterisopropanol 11) wasinjected in the columnanddetected in the dead time ensuringmass accuracy throughout the chromatographic analysisThe elemental composition of the detected compounds wasdetermined considering mass errors below 5 ppm The datawas processed using the Bruker Compass Data Analysissoftware

24 BPH Cell The stromal cell cultures were obtained frompatients undergoing a clinical and histological diagnosis forBPH BPH stromal cells were isolated according to previouslydescribed methods [31] Briefly prostate tissue was washedwith phosphate-buffered saline (PBS) before being diced intoapproximately 1 mm3 pieces The fragments were transferredto 10 ml dissociation flasks containing a solution of DMEMsupplemented with 10 FBS and 1mgml of type I collagenase(Sigma St Louis MO) Tissue specimens were dissociated byconstant stirring with a magnetic stir bar for 2-4 h at 37∘CThe supernatant was frozen at 4∘C and the remaining tissueswere submitted to a new cycle of dissociation as describedabove After that the supernatants from the first and thesecond cycles were centrifuged and washed with balancedsaline solution without calciumandmagnesiumat 1200RPMthree times The resulting cells were seeded in 25 mm3 flasksand left to allow attachment in a defined medium composedof supplemented DMEM (10 FBS antibioticantimycoticmixture (Gibco) Penicillin 100 Uml Streptomycin 100120583gml and Fungizone 25120583gml) and placed in a tissue cultureincubator at 37∘C in humidified air containing 5 CO

2 Cells

were fed 3 times a week At subconfluence (approximately90 occupancy in each bottle) they were harvested using005 trypsinEDTA (both from Sigma) and replated

25 BPH Cell Proliferation Assay The cell proliferation assaywas performed using 1x104 BPH stromal cells per well in96-well plates using Dulbeccorsquos Modified Eagles Medium(DMEM) containing 05 ethanol and 1 Fetal BovineSerum (FBS) Cells were treated with the extract of theunderground parts extract of leaves from the specimens inflower and not in flower and extract of flowers of KGB for72 hours The treated BPH stromal cells and controls werewashed once with PBS fixed in a solution of 100 ethanol for10 minutes and then stained with 005 solution of crystalviolet (Vetec) for 10 minutes After staining the cells were

washed with distilled water and incubated in methanol for 5minutes on a plate shaker and the supernatant was collectedThe absorbance was measured on an ELISA reader (iMARKBIO-RAD) at 570 nm

In this assay we evaluated the inhibitory effects of thefour treatments (T1ndashT4) on cell proliferation of BPH stromalcellsThe four treatmentswere the extract of the undergroundparts (T1) leaf extract of the flowering specimens (T2)extract of the flowers (T3) and leaf extract of the specimensnot in flower (T4) at 250 120583gml for 72 hours

26 BPH Cell Viability Assay The cytotoxic potential of KGBunderground parts extract (T1) against BPH stromal cellswas assessed by a quantitative MTT colorimetric assay Thisassay is based on the reduction of MTT by the mitochondrialenzyme NADH dehydrogenase tetrazolium dye in violetcrystals known as formazan to detect and determine cell pro-liferation and viability [32] The supernatants were removedfromeachwell and replaced by the sampleT1 in quadruplicatewells except for the zero time where after removing thesupernatant 100 120583l MTT (3-[45-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide 05 mgml Sigma) was addedAfter addition of MTT the culture plate was kept at 5 CO

2

and temperature at 37∘C for three hours After this timeMTTwas removed and 100 120583l of DMSO (Sigma) was added Theabsorbance was read in an ELISA reader (BIO-RAD iMARK)at 570 nm The same procedure was repeated 72 hours afterthe addition of the treatment and control

The absorbance (optical density) of the treatments wascalculated and their values were subtracted from the valuesfor the wells incubated only with DMEMThen the percent-age of cell viability was expressed using the formula samplevalue (DMEMDMEM+ FCS or T1)mean value at time zero(T = 0) x 100

27 BPH Cell Apoptosis Assay After trypsinization and cen-trifugation 1x105 cells were resuspended in 200 120583l of propid-ium iodide solution (PBS Triton X-100 01 and propidiumiodide 50 120583gml Sigma) and incubated on ice for 5 minutesAfter the incubation period cell death was measured by flowcytometry (FACScalibur Becton Dickison) after acquiring20000 events The excitation of the fluorochrome was mea-sured using an argon laser with a wavelength of 488 nm andthe emission was collected through a filter 63022 nm

28 Statistical Methods All data represent the mean plusmnstandard deviation values of three independent experimentsDifferences between groups were analyzed using one-wayANOVA followed by the multiple comparison Newman-Keuls test The value p lt005 (lowast) was considered statisticallysignificant

3 Results and Discussion

Four aqueous extracts were prepared from leaves flowersand underground parts of K gastonis-bonnieri (KGB) spec-imens The yield of the extract from leaves collected from thespecimens not in flower (21 ) was similar to that observedfor leaves from flowering specimens (19 ) while the yields

4 Evidence-Based Complementary and Alternative Medicine

lowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

Pro

lifer

atio

nCo

ntro

l

(a)

5673

2923

3934

1351

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

P

rolif

erat

ion

Cont

rol

(b)

Figure 1 Proliferation of BPH stromal cells treated with different extracts of Kalanchoe gastonis-bonnieri (KGB) (a) Graph representing theproliferation of cells with the different extracts (T1-T4) of KGB at 250 120583g ml after 72 h lowast plt005 lowastlowast plt0001 Data represent the mean plusmnstandard deviation values of three independent experiments (b) Percentage of BPH stromal cells after treatment with four different extracts(T1-T4) of KGB Data represent the mean plusmn standard deviation values of three independent experiments

from the underground parts from flowering specimens andflowers were 12 and 39 respectively (Table 1)

We evaluated the inhibitory effects of the four KGBpreparations on the proliferation of stromal cells fromprimary benign prostatic hyperplasia (PBH) Undergroundparts extract from the flowering specimens of KGB (T1treatment) leaf extract of KGB fromflowering specimens (T2treatment) flowers extract from KGB (T3 treatment) andleaf extract of KGB not in flower (T4 treatment) were testedat 250 120583gml for 72 hours T1 T2 T3 and T4 treatmentsinhibited the cells proliferation by 567 292 394 and135 respectively (Figure 1)

The best results were observed for the underground partsextract (T1 treatment) which encouraged the continuation ofour experiments with this sample in order to corroborate thepreliminary detection of its activity in BPH

31 BPH Cell Viability Assay The extract of KGB under-ground parts (T1 treatment) significantly reduced the viabil-ity of BPH stromal cells treated with 250 120583gml promotingdecay of more than 50 cell viability (Figure 2)

32 BPH Cell Apoptosis Assay In order to clarify whetherthe reduction in the number of BPH stromal cells observedafter the treatment with KGB underground parts was due toa blockage in the proliferation activity of these cells or due toan induction of cell death we performed an apoptosis assayto address this question

Thepercentage of dead cells detected in the subG0 regionof the cell cycle after 72 hours was significantly higher whenthe cells were seeded in the presence of T1 treatment in theconcentrations of 150 120583gml and 250 120583gml (Figure 3(a))

However the activity was not dependent on the concen-tration used We observed that both concentrations of the

extract were able to suppress the progression of BPH cellsalong the cell cycle with no significant difference betweenthem Additionally Figure 3(b) shows that there was inthe control group a distribution throughout the differentcell cycle phases including the S and G2M phases thusindicating a proliferating profile of the BPH stromal cells inthe absence of the KGB treatment On the other hand thetreatment with T1 showed that BPH stromal cells were almostexclusively at the subG0 phase of the cell cycle

Despite numerous reports on the use of plants or deriva-tives of natural products of plant origin for the treatment ofbenign prostatic hyperplasia these activities have not alwaysbeen proven in pharmacological studies

The inhibitory activity observed for KGB in BPH cellproliferation was very effective since the KGB undergroundparts (T1) at the concentration of 150 120583gmL was able todrastically reduce the proliferation activity and the viability ofBPH stromal cells in 72 hours Moreover the same treatmentalso induced a strong increase in the apoptosis rates ofBPH stromal cells since a large percent of these cells wererestricted in the subGo phase of the cell cycle as revealed bythe flow cytometry analysis In addition the cell cycle profilepresented by BPH stromal cells after the treatment with theKGB underground parts corroborated the proliferation dataas the normal transition throughout the cell cyclewas blockedby the KGB treatment Therefore these results that show aconcomitant decrease in the cell viability and proliferationcombined with an induction of cell death by apoptosis mayreveal a beneficial role of KGB in combating the process ofprostate growth that culminates in the development of BPH

Our results with KGB underground parts are comparablewith those observed for extracts from two plants clini-cally used to treat BHP The first one Pygeum africanum(Tadenan) inhibits the proliferation of cultured human

Evidence-Based Complementary and Alternative Medicine 5

72 hours

lowastlowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1

DMEM 1 + T 1 250 gml

T = 00

50

100

150

200

250

Cel

l Via

bilit

y

(a)72 hours

5284

12388

18659

DMEM 10

DMEM 1

DMEM 15 + T 1 250 gml

T = 00

50

100

150

200

250

C

ell V

iabi

lity

(b)

Figure 2 Cell viability assessed by MTT (a) Graph representing the viability of BPH stromal cells with the underground parts extract ofKGB (T1 treatment) at the concentration of 250 120583gml lowast plt005 lowastlowast plt0001 Data represent the mean plusmn standard deviation values of threeindependent experiments (b) Percentage of BPH stromal cells after treatment with extract of KGB underground parts (T1 treatment) Datarepresent the mean plusmn standard deviation values of three independent experiments

prostatic myofibroblasts and fibroblasts as well as enhancesapoptosis at concentrations from 25 to 100 120583gml [33 34]Serenoa repens (Permixon) the second one revealed atissue-selective action resulting in morphological changesand augmented apoptosis rates in addition to the inhibition ofnuclear membrane bound 5120572-reductase isoenzymes catalyticactivity in prostate cells at the concentration of 10 120583gml[35] Additionally an increment of Bax-to-Bcl-2 expressionand caspase 3 activity molecules involved in the apoptoticpathway has already been documented in prostatic tissuesamples from BPH symptomatic patients under treatmentwith Permixon for at least 3 months [36] Also an invitro study showed that treatment with S repens leads tothe lightening of BPH symptoms due to antiproliferativeand proapoptotic effects exerted on prostate epithelia andtriggered by the downregulation of IGF-1 signaling pathwayand induction of JNK [37] Finally BPH treatment withfinasteride the main therapeutic approach employed forthis disease management also demonstrated a mechanismexclusively observed in epithelial cells caspase-dependentapoptosis initiation through activation of caspases 3 and 6[38] In fact the success of the main treatment approaches(phytotherapic or conventional pharmacologic drugs) thatare routinely used in the treatment of BPH are related tothe control of BPH growth by reducing proliferation andinducing apoptosis [33 34 36] Thus the effects of KGBon BPH seem very interesting since this disease is largelycharacterized by an imbalance between the proliferation andapoptosis [4 7 8 11] Furthermore it was recently shown thatKGB underground parts were able to abrogate the androgen

signaling in prostate malignant cell lineages besides inducingthe apoptosis via caspase 8 activation thus reinforcing thetherapeutic potential of KGB in prostatic diseases [39]

Although the greater activity is in the subterraneanparts from K gastonis-bonnieri and therefore could be adisadvantage for a phytomedicinal preparation due to thenonrenewable characteristics of this part of the plant thissucculent herb is a fast growing-species that propagates easilyby asexual reproduction [40]

33 Chemical Composition of KGB Underground Parts Theextract from the KGB underground parts had its chemicalcomposition assessed byHPLC-DADMSMS in the negativeion mode As the TOF analyzer enables high-resolutionmass measurements with mass errors below 5 ppm itwas possible to infer the molecular formula of the majorconstituents detected in the KGB underground parts Theresulting chromatogram is shown in Figure 4 while data onthe major compounds detected are summarized in Table 2

Peak 1 (Rt 45 min 120582max 261 nm) presented the [M-H]minus ion at mz 3590994 (C

15H19O10) as base peak MSMS

spectrum showed a fragment at mz 1970458 (C9H9O5)

suggesting the loss of a hexose unity This substance couldpossibly correspond to a glycosylated form of syringic acidsuch as syringate 4-O-120573-glucopyranoside [41] Howeverisomers of syringic acid cannot be ruled out Peak 3 (Rt 52min 120582max 282 nm) also showed a [M-H]minus ion for which themolecular formula C

15H19O10

was proposed and a similarfragment at mz 1970457 We hypothesize that this substancecould correspond to a glycosyl ester of syringic acid Syringic

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Extraction and yield of different parts of K gastonis-bonnieri

Fresh plant materialmass (g) LyophilizedExtract (g)

Yield

Leaves of plants not in flower (11392) 238 21Leaves of plants in flower (1495) 29 19Underground parts of plants in flower (829) 08 12Flowers (873) 34 39

of the enzyme phosphodiesterase type 5 [12ndash14] Addi-tionally there are current evidences that nonsteroidal anti-inflammatory drugs (NSAID) can improve LUTS [15]

Herbal medicines are used in BPH therapy and severalmechanisms of action have been put forward based onthe complex chemical composition present in plants Thepresence of different substances acting on specific targetsmakes herbal medicines a relevant therapeutic strategy in thetreatment of prostatic hyperplasiaThemain herbal medicineused in the treatment of BPH is an extract of Serenoa repensfruit (Arecaceae) popularly known as ldquosaw palmettordquo forwhich there is strong evidence of clinical efficacy [16ndash18]

Many natural products are also used for the improvementof physiological functions as well as the symptoms of BPHAmong them the pollen extract Cernitin (Secale cerealeCernilton) Pygeum africanum (Tadenan) Urtica dioicaScutellaria baicalensis Cucurbita pepo lycopene and 120573-sitosterol are known for their beneficial effects on BPH [16ndash18] Besides many medicinal plant species have been testedin vitro and in vivo after they have shown potential for BHPtreatment [19] Some secondary metabolites from plants havealso shown promising results in vitro and in vivo amongthem phenolic substances such as isoflavones lignans andthe stilbene resveratrol [20ndash24]

Kalanchoe gastonis-bonnieri Raym-Hamet amp H Perrier(synKalanchoe adolphi-engleriRaym-Hamet) is a medicinalherb from the family Crassulaceae It is used in LatinAmerican medicine as a vaginal contraceptive as well asin the treatment of genital-urinary and vaginal infections[25] In a previous study we reported the isolation of thenew flavonoid quercetin 3-O-120572-rhamnopyranoside-7-O-120573-D-glucopyranosyl-(1997888rarr3)-120572-L-rhamnopyranoside as well asvicenin-2 a C-glycosyl flavone from the leaf extract of KGB[26] We also demonstrated that aqueous extracts from Kgastonis-bonnieri (KGB) are effective in controling dentalbacterial plaque and calculus in dogs [27]

Extracts from KGB have been shown to immobilize toclump together and to promote structural changes in ratsperm [28] The production and storage of seminal fluidand other components of semen are intrinsically linkedto the functions of the prostate [29 30] Considering theethnomedicinal use of KGB this study aimed to determinethe efficacy of this herb in the treatment of BPH by usingan in vitro model of BPH primary cell culture particularlyfocusing on the main hallmarks related to the developmentof the disease

2 Materials and Methods

21 Plant Material In this study leaves were collected fromspecimens in flower and from specimens not in flower ofKalanchoe gastonis-bonnieri cultivated in a residential gardenin the city of Rio de Janeiro Underground parts and flowerswere also obtained from specimens growing in the samegarden A sample of a flowering specimen was identifiedand its voucher specimen (RGA 31592) is deposited in theHerbarium of the Botany Department at the Institute ofBiology of the Federal University of Rio de Janeiro

22 Extraction Fresh leaves fromK gastonis-bonnieri (KGB)(average length of leaves 15 cm) were rinsed with distilledwater cut into small pieces and crushed in a blender Theextract obtained from the leaves of specimens not in flowerwas filtered and resulted in a clear yellow liquid The color ofthe leaf extract of the specimens in flower was a salmon pinkThe flowers were extracted by infusion with distilled water(20 ww) The same procedure was applied for extractingthe underground parts harvested from the other specimensin flower Table 1 shows the mass of the different parts ofthe K gastonis-bonnieri specimens and the yield obtainedfrom the extraction of each part All the extracts were frozenlyophilized and kept in a freezer at -20∘C

23 HPLC-DADMSMS High-Performance Liquid Chro-matography analyses with a Diode Array Detector coupledto a Tandem Mass Spectrometry (HPLC-DAD MSMS)were carried out at the Center for Mass Spectrometry ofBiomolecules-CEMBIO (IBCCF UFRJ) The ProminenceShimadzu Liquid Chromatography system used was com-posed of an LC-20AD pump a degasser system DGU-20A and a DAD detector SPD-M20A coupled to a MaxisImpact Q-TOF mass spectrometer (Bruker) equipped withan electrospray ionization (ESI) interface An ODS-Hypersilreverse phaseC-18 column (ThermoScientific 3120583m 150mm21mm) protected by a precolumnof the samematerial (3120583m1 mm and 21 mm) was used The columns were maintainedat 40∘C

A sample of the extract (4 mgml) from the undergroundparts of KGB was diluted in a mixture of water acidified withformic acid 01 and acetonitrile (191)The injection volumewas 20 120583l

Evidence-Based Complementary and Alternative Medicine 3

The mobile phase consisted of eluent A water containing01 formic acid (Sigma-Aldrich) and eluent B acetoni-trile (Merck) containing 01 formic acid (Sigma-Aldrich)The samples were run for 40 min at 03 mlmin and theabsorbance was monitored between 210 and 400 nm Thegradient used was as follows 0minus5 min (5minus20 B) 5minus11 min(20minus22 B) 11minus12 min (22minus100 B) 12minus26 min (100 B)and 26minus40 min (100minus5 B)

The Q-TOF mass spectrometer was operated in thenegative ion mode using the following parameters capillaryvoltage 5000 V endplate offset -500 V pressure of nebulizer4 Bar drying gas temperature 200∘C Nitrogen was usedas both the sheath and drying gas at a flow rate of 80lmin The mass range analyzed was set at mz 50-1200 andcollision energy at -5 eV An external calibration solution(sodium formate 100 mM in waterisopropanol 11) wasinjected in the columnanddetected in the dead time ensuringmass accuracy throughout the chromatographic analysisThe elemental composition of the detected compounds wasdetermined considering mass errors below 5 ppm The datawas processed using the Bruker Compass Data Analysissoftware

24 BPH Cell The stromal cell cultures were obtained frompatients undergoing a clinical and histological diagnosis forBPH BPH stromal cells were isolated according to previouslydescribed methods [31] Briefly prostate tissue was washedwith phosphate-buffered saline (PBS) before being diced intoapproximately 1 mm3 pieces The fragments were transferredto 10 ml dissociation flasks containing a solution of DMEMsupplemented with 10 FBS and 1mgml of type I collagenase(Sigma St Louis MO) Tissue specimens were dissociated byconstant stirring with a magnetic stir bar for 2-4 h at 37∘CThe supernatant was frozen at 4∘C and the remaining tissueswere submitted to a new cycle of dissociation as describedabove After that the supernatants from the first and thesecond cycles were centrifuged and washed with balancedsaline solution without calciumandmagnesiumat 1200RPMthree times The resulting cells were seeded in 25 mm3 flasksand left to allow attachment in a defined medium composedof supplemented DMEM (10 FBS antibioticantimycoticmixture (Gibco) Penicillin 100 Uml Streptomycin 100120583gml and Fungizone 25120583gml) and placed in a tissue cultureincubator at 37∘C in humidified air containing 5 CO

2 Cells

were fed 3 times a week At subconfluence (approximately90 occupancy in each bottle) they were harvested using005 trypsinEDTA (both from Sigma) and replated

25 BPH Cell Proliferation Assay The cell proliferation assaywas performed using 1x104 BPH stromal cells per well in96-well plates using Dulbeccorsquos Modified Eagles Medium(DMEM) containing 05 ethanol and 1 Fetal BovineSerum (FBS) Cells were treated with the extract of theunderground parts extract of leaves from the specimens inflower and not in flower and extract of flowers of KGB for72 hours The treated BPH stromal cells and controls werewashed once with PBS fixed in a solution of 100 ethanol for10 minutes and then stained with 005 solution of crystalviolet (Vetec) for 10 minutes After staining the cells were

washed with distilled water and incubated in methanol for 5minutes on a plate shaker and the supernatant was collectedThe absorbance was measured on an ELISA reader (iMARKBIO-RAD) at 570 nm

In this assay we evaluated the inhibitory effects of thefour treatments (T1ndashT4) on cell proliferation of BPH stromalcellsThe four treatmentswere the extract of the undergroundparts (T1) leaf extract of the flowering specimens (T2)extract of the flowers (T3) and leaf extract of the specimensnot in flower (T4) at 250 120583gml for 72 hours

26 BPH Cell Viability Assay The cytotoxic potential of KGBunderground parts extract (T1) against BPH stromal cellswas assessed by a quantitative MTT colorimetric assay Thisassay is based on the reduction of MTT by the mitochondrialenzyme NADH dehydrogenase tetrazolium dye in violetcrystals known as formazan to detect and determine cell pro-liferation and viability [32] The supernatants were removedfromeachwell and replaced by the sampleT1 in quadruplicatewells except for the zero time where after removing thesupernatant 100 120583l MTT (3-[45-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide 05 mgml Sigma) was addedAfter addition of MTT the culture plate was kept at 5 CO

2

and temperature at 37∘C for three hours After this timeMTTwas removed and 100 120583l of DMSO (Sigma) was added Theabsorbance was read in an ELISA reader (BIO-RAD iMARK)at 570 nm The same procedure was repeated 72 hours afterthe addition of the treatment and control

The absorbance (optical density) of the treatments wascalculated and their values were subtracted from the valuesfor the wells incubated only with DMEMThen the percent-age of cell viability was expressed using the formula samplevalue (DMEMDMEM+ FCS or T1)mean value at time zero(T = 0) x 100

27 BPH Cell Apoptosis Assay After trypsinization and cen-trifugation 1x105 cells were resuspended in 200 120583l of propid-ium iodide solution (PBS Triton X-100 01 and propidiumiodide 50 120583gml Sigma) and incubated on ice for 5 minutesAfter the incubation period cell death was measured by flowcytometry (FACScalibur Becton Dickison) after acquiring20000 events The excitation of the fluorochrome was mea-sured using an argon laser with a wavelength of 488 nm andthe emission was collected through a filter 63022 nm

28 Statistical Methods All data represent the mean plusmnstandard deviation values of three independent experimentsDifferences between groups were analyzed using one-wayANOVA followed by the multiple comparison Newman-Keuls test The value p lt005 (lowast) was considered statisticallysignificant

3 Results and Discussion

Four aqueous extracts were prepared from leaves flowersand underground parts of K gastonis-bonnieri (KGB) spec-imens The yield of the extract from leaves collected from thespecimens not in flower (21 ) was similar to that observedfor leaves from flowering specimens (19 ) while the yields

4 Evidence-Based Complementary and Alternative Medicine

lowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

Pro

lifer

atio

nCo

ntro

l

(a)

5673

2923

3934

1351

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

P

rolif

erat

ion

Cont

rol

(b)

Figure 1 Proliferation of BPH stromal cells treated with different extracts of Kalanchoe gastonis-bonnieri (KGB) (a) Graph representing theproliferation of cells with the different extracts (T1-T4) of KGB at 250 120583g ml after 72 h lowast plt005 lowastlowast plt0001 Data represent the mean plusmnstandard deviation values of three independent experiments (b) Percentage of BPH stromal cells after treatment with four different extracts(T1-T4) of KGB Data represent the mean plusmn standard deviation values of three independent experiments

from the underground parts from flowering specimens andflowers were 12 and 39 respectively (Table 1)

We evaluated the inhibitory effects of the four KGBpreparations on the proliferation of stromal cells fromprimary benign prostatic hyperplasia (PBH) Undergroundparts extract from the flowering specimens of KGB (T1treatment) leaf extract of KGB fromflowering specimens (T2treatment) flowers extract from KGB (T3 treatment) andleaf extract of KGB not in flower (T4 treatment) were testedat 250 120583gml for 72 hours T1 T2 T3 and T4 treatmentsinhibited the cells proliferation by 567 292 394 and135 respectively (Figure 1)

The best results were observed for the underground partsextract (T1 treatment) which encouraged the continuation ofour experiments with this sample in order to corroborate thepreliminary detection of its activity in BPH

31 BPH Cell Viability Assay The extract of KGB under-ground parts (T1 treatment) significantly reduced the viabil-ity of BPH stromal cells treated with 250 120583gml promotingdecay of more than 50 cell viability (Figure 2)

32 BPH Cell Apoptosis Assay In order to clarify whetherthe reduction in the number of BPH stromal cells observedafter the treatment with KGB underground parts was due toa blockage in the proliferation activity of these cells or due toan induction of cell death we performed an apoptosis assayto address this question

Thepercentage of dead cells detected in the subG0 regionof the cell cycle after 72 hours was significantly higher whenthe cells were seeded in the presence of T1 treatment in theconcentrations of 150 120583gml and 250 120583gml (Figure 3(a))

However the activity was not dependent on the concen-tration used We observed that both concentrations of the

extract were able to suppress the progression of BPH cellsalong the cell cycle with no significant difference betweenthem Additionally Figure 3(b) shows that there was inthe control group a distribution throughout the differentcell cycle phases including the S and G2M phases thusindicating a proliferating profile of the BPH stromal cells inthe absence of the KGB treatment On the other hand thetreatment with T1 showed that BPH stromal cells were almostexclusively at the subG0 phase of the cell cycle

Despite numerous reports on the use of plants or deriva-tives of natural products of plant origin for the treatment ofbenign prostatic hyperplasia these activities have not alwaysbeen proven in pharmacological studies

The inhibitory activity observed for KGB in BPH cellproliferation was very effective since the KGB undergroundparts (T1) at the concentration of 150 120583gmL was able todrastically reduce the proliferation activity and the viability ofBPH stromal cells in 72 hours Moreover the same treatmentalso induced a strong increase in the apoptosis rates ofBPH stromal cells since a large percent of these cells wererestricted in the subGo phase of the cell cycle as revealed bythe flow cytometry analysis In addition the cell cycle profilepresented by BPH stromal cells after the treatment with theKGB underground parts corroborated the proliferation dataas the normal transition throughout the cell cyclewas blockedby the KGB treatment Therefore these results that show aconcomitant decrease in the cell viability and proliferationcombined with an induction of cell death by apoptosis mayreveal a beneficial role of KGB in combating the process ofprostate growth that culminates in the development of BPH

Our results with KGB underground parts are comparablewith those observed for extracts from two plants clini-cally used to treat BHP The first one Pygeum africanum(Tadenan) inhibits the proliferation of cultured human

Evidence-Based Complementary and Alternative Medicine 5

72 hours

lowastlowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1

DMEM 1 + T 1 250 gml

T = 00

50

100

150

200

250

Cel

l Via

bilit

y

(a)72 hours

5284

12388

18659

DMEM 10

DMEM 1

DMEM 15 + T 1 250 gml

T = 00

50

100

150

200

250

C

ell V

iabi

lity

(b)

Figure 2 Cell viability assessed by MTT (a) Graph representing the viability of BPH stromal cells with the underground parts extract ofKGB (T1 treatment) at the concentration of 250 120583gml lowast plt005 lowastlowast plt0001 Data represent the mean plusmn standard deviation values of threeindependent experiments (b) Percentage of BPH stromal cells after treatment with extract of KGB underground parts (T1 treatment) Datarepresent the mean plusmn standard deviation values of three independent experiments

prostatic myofibroblasts and fibroblasts as well as enhancesapoptosis at concentrations from 25 to 100 120583gml [33 34]Serenoa repens (Permixon) the second one revealed atissue-selective action resulting in morphological changesand augmented apoptosis rates in addition to the inhibition ofnuclear membrane bound 5120572-reductase isoenzymes catalyticactivity in prostate cells at the concentration of 10 120583gml[35] Additionally an increment of Bax-to-Bcl-2 expressionand caspase 3 activity molecules involved in the apoptoticpathway has already been documented in prostatic tissuesamples from BPH symptomatic patients under treatmentwith Permixon for at least 3 months [36] Also an invitro study showed that treatment with S repens leads tothe lightening of BPH symptoms due to antiproliferativeand proapoptotic effects exerted on prostate epithelia andtriggered by the downregulation of IGF-1 signaling pathwayand induction of JNK [37] Finally BPH treatment withfinasteride the main therapeutic approach employed forthis disease management also demonstrated a mechanismexclusively observed in epithelial cells caspase-dependentapoptosis initiation through activation of caspases 3 and 6[38] In fact the success of the main treatment approaches(phytotherapic or conventional pharmacologic drugs) thatare routinely used in the treatment of BPH are related tothe control of BPH growth by reducing proliferation andinducing apoptosis [33 34 36] Thus the effects of KGBon BPH seem very interesting since this disease is largelycharacterized by an imbalance between the proliferation andapoptosis [4 7 8 11] Furthermore it was recently shown thatKGB underground parts were able to abrogate the androgen

signaling in prostate malignant cell lineages besides inducingthe apoptosis via caspase 8 activation thus reinforcing thetherapeutic potential of KGB in prostatic diseases [39]

Although the greater activity is in the subterraneanparts from K gastonis-bonnieri and therefore could be adisadvantage for a phytomedicinal preparation due to thenonrenewable characteristics of this part of the plant thissucculent herb is a fast growing-species that propagates easilyby asexual reproduction [40]

33 Chemical Composition of KGB Underground Parts Theextract from the KGB underground parts had its chemicalcomposition assessed byHPLC-DADMSMS in the negativeion mode As the TOF analyzer enables high-resolutionmass measurements with mass errors below 5 ppm itwas possible to infer the molecular formula of the majorconstituents detected in the KGB underground parts Theresulting chromatogram is shown in Figure 4 while data onthe major compounds detected are summarized in Table 2

Peak 1 (Rt 45 min 120582max 261 nm) presented the [M-H]minus ion at mz 3590994 (C

15H19O10) as base peak MSMS

spectrum showed a fragment at mz 1970458 (C9H9O5)

suggesting the loss of a hexose unity This substance couldpossibly correspond to a glycosylated form of syringic acidsuch as syringate 4-O-120573-glucopyranoside [41] Howeverisomers of syringic acid cannot be ruled out Peak 3 (Rt 52min 120582max 282 nm) also showed a [M-H]minus ion for which themolecular formula C

15H19O10

was proposed and a similarfragment at mz 1970457 We hypothesize that this substancecould correspond to a glycosyl ester of syringic acid Syringic

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

The mobile phase consisted of eluent A water containing01 formic acid (Sigma-Aldrich) and eluent B acetoni-trile (Merck) containing 01 formic acid (Sigma-Aldrich)The samples were run for 40 min at 03 mlmin and theabsorbance was monitored between 210 and 400 nm Thegradient used was as follows 0minus5 min (5minus20 B) 5minus11 min(20minus22 B) 11minus12 min (22minus100 B) 12minus26 min (100 B)and 26minus40 min (100minus5 B)

The Q-TOF mass spectrometer was operated in thenegative ion mode using the following parameters capillaryvoltage 5000 V endplate offset -500 V pressure of nebulizer4 Bar drying gas temperature 200∘C Nitrogen was usedas both the sheath and drying gas at a flow rate of 80lmin The mass range analyzed was set at mz 50-1200 andcollision energy at -5 eV An external calibration solution(sodium formate 100 mM in waterisopropanol 11) wasinjected in the columnanddetected in the dead time ensuringmass accuracy throughout the chromatographic analysisThe elemental composition of the detected compounds wasdetermined considering mass errors below 5 ppm The datawas processed using the Bruker Compass Data Analysissoftware

24 BPH Cell The stromal cell cultures were obtained frompatients undergoing a clinical and histological diagnosis forBPH BPH stromal cells were isolated according to previouslydescribed methods [31] Briefly prostate tissue was washedwith phosphate-buffered saline (PBS) before being diced intoapproximately 1 mm3 pieces The fragments were transferredto 10 ml dissociation flasks containing a solution of DMEMsupplemented with 10 FBS and 1mgml of type I collagenase(Sigma St Louis MO) Tissue specimens were dissociated byconstant stirring with a magnetic stir bar for 2-4 h at 37∘CThe supernatant was frozen at 4∘C and the remaining tissueswere submitted to a new cycle of dissociation as describedabove After that the supernatants from the first and thesecond cycles were centrifuged and washed with balancedsaline solution without calciumandmagnesiumat 1200RPMthree times The resulting cells were seeded in 25 mm3 flasksand left to allow attachment in a defined medium composedof supplemented DMEM (10 FBS antibioticantimycoticmixture (Gibco) Penicillin 100 Uml Streptomycin 100120583gml and Fungizone 25120583gml) and placed in a tissue cultureincubator at 37∘C in humidified air containing 5 CO

2 Cells

were fed 3 times a week At subconfluence (approximately90 occupancy in each bottle) they were harvested using005 trypsinEDTA (both from Sigma) and replated

25 BPH Cell Proliferation Assay The cell proliferation assaywas performed using 1x104 BPH stromal cells per well in96-well plates using Dulbeccorsquos Modified Eagles Medium(DMEM) containing 05 ethanol and 1 Fetal BovineSerum (FBS) Cells were treated with the extract of theunderground parts extract of leaves from the specimens inflower and not in flower and extract of flowers of KGB for72 hours The treated BPH stromal cells and controls werewashed once with PBS fixed in a solution of 100 ethanol for10 minutes and then stained with 005 solution of crystalviolet (Vetec) for 10 minutes After staining the cells were

washed with distilled water and incubated in methanol for 5minutes on a plate shaker and the supernatant was collectedThe absorbance was measured on an ELISA reader (iMARKBIO-RAD) at 570 nm

In this assay we evaluated the inhibitory effects of thefour treatments (T1ndashT4) on cell proliferation of BPH stromalcellsThe four treatmentswere the extract of the undergroundparts (T1) leaf extract of the flowering specimens (T2)extract of the flowers (T3) and leaf extract of the specimensnot in flower (T4) at 250 120583gml for 72 hours

26 BPH Cell Viability Assay The cytotoxic potential of KGBunderground parts extract (T1) against BPH stromal cellswas assessed by a quantitative MTT colorimetric assay Thisassay is based on the reduction of MTT by the mitochondrialenzyme NADH dehydrogenase tetrazolium dye in violetcrystals known as formazan to detect and determine cell pro-liferation and viability [32] The supernatants were removedfromeachwell and replaced by the sampleT1 in quadruplicatewells except for the zero time where after removing thesupernatant 100 120583l MTT (3-[45-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide 05 mgml Sigma) was addedAfter addition of MTT the culture plate was kept at 5 CO

2

and temperature at 37∘C for three hours After this timeMTTwas removed and 100 120583l of DMSO (Sigma) was added Theabsorbance was read in an ELISA reader (BIO-RAD iMARK)at 570 nm The same procedure was repeated 72 hours afterthe addition of the treatment and control

The absorbance (optical density) of the treatments wascalculated and their values were subtracted from the valuesfor the wells incubated only with DMEMThen the percent-age of cell viability was expressed using the formula samplevalue (DMEMDMEM+ FCS or T1)mean value at time zero(T = 0) x 100

27 BPH Cell Apoptosis Assay After trypsinization and cen-trifugation 1x105 cells were resuspended in 200 120583l of propid-ium iodide solution (PBS Triton X-100 01 and propidiumiodide 50 120583gml Sigma) and incubated on ice for 5 minutesAfter the incubation period cell death was measured by flowcytometry (FACScalibur Becton Dickison) after acquiring20000 events The excitation of the fluorochrome was mea-sured using an argon laser with a wavelength of 488 nm andthe emission was collected through a filter 63022 nm

28 Statistical Methods All data represent the mean plusmnstandard deviation values of three independent experimentsDifferences between groups were analyzed using one-wayANOVA followed by the multiple comparison Newman-Keuls test The value p lt005 (lowast) was considered statisticallysignificant

3 Results and Discussion

Four aqueous extracts were prepared from leaves flowersand underground parts of K gastonis-bonnieri (KGB) spec-imens The yield of the extract from leaves collected from thespecimens not in flower (21 ) was similar to that observedfor leaves from flowering specimens (19 ) while the yields

4 Evidence-Based Complementary and Alternative Medicine

lowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

Pro

lifer

atio

nCo

ntro

l

(a)

5673

2923

3934

1351

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

P

rolif

erat

ion

Cont

rol

(b)

Figure 1 Proliferation of BPH stromal cells treated with different extracts of Kalanchoe gastonis-bonnieri (KGB) (a) Graph representing theproliferation of cells with the different extracts (T1-T4) of KGB at 250 120583g ml after 72 h lowast plt005 lowastlowast plt0001 Data represent the mean plusmnstandard deviation values of three independent experiments (b) Percentage of BPH stromal cells after treatment with four different extracts(T1-T4) of KGB Data represent the mean plusmn standard deviation values of three independent experiments

from the underground parts from flowering specimens andflowers were 12 and 39 respectively (Table 1)

We evaluated the inhibitory effects of the four KGBpreparations on the proliferation of stromal cells fromprimary benign prostatic hyperplasia (PBH) Undergroundparts extract from the flowering specimens of KGB (T1treatment) leaf extract of KGB fromflowering specimens (T2treatment) flowers extract from KGB (T3 treatment) andleaf extract of KGB not in flower (T4 treatment) were testedat 250 120583gml for 72 hours T1 T2 T3 and T4 treatmentsinhibited the cells proliferation by 567 292 394 and135 respectively (Figure 1)

The best results were observed for the underground partsextract (T1 treatment) which encouraged the continuation ofour experiments with this sample in order to corroborate thepreliminary detection of its activity in BPH

31 BPH Cell Viability Assay The extract of KGB under-ground parts (T1 treatment) significantly reduced the viabil-ity of BPH stromal cells treated with 250 120583gml promotingdecay of more than 50 cell viability (Figure 2)

32 BPH Cell Apoptosis Assay In order to clarify whetherthe reduction in the number of BPH stromal cells observedafter the treatment with KGB underground parts was due toa blockage in the proliferation activity of these cells or due toan induction of cell death we performed an apoptosis assayto address this question

Thepercentage of dead cells detected in the subG0 regionof the cell cycle after 72 hours was significantly higher whenthe cells were seeded in the presence of T1 treatment in theconcentrations of 150 120583gml and 250 120583gml (Figure 3(a))

However the activity was not dependent on the concen-tration used We observed that both concentrations of the

extract were able to suppress the progression of BPH cellsalong the cell cycle with no significant difference betweenthem Additionally Figure 3(b) shows that there was inthe control group a distribution throughout the differentcell cycle phases including the S and G2M phases thusindicating a proliferating profile of the BPH stromal cells inthe absence of the KGB treatment On the other hand thetreatment with T1 showed that BPH stromal cells were almostexclusively at the subG0 phase of the cell cycle

Despite numerous reports on the use of plants or deriva-tives of natural products of plant origin for the treatment ofbenign prostatic hyperplasia these activities have not alwaysbeen proven in pharmacological studies

The inhibitory activity observed for KGB in BPH cellproliferation was very effective since the KGB undergroundparts (T1) at the concentration of 150 120583gmL was able todrastically reduce the proliferation activity and the viability ofBPH stromal cells in 72 hours Moreover the same treatmentalso induced a strong increase in the apoptosis rates ofBPH stromal cells since a large percent of these cells wererestricted in the subGo phase of the cell cycle as revealed bythe flow cytometry analysis In addition the cell cycle profilepresented by BPH stromal cells after the treatment with theKGB underground parts corroborated the proliferation dataas the normal transition throughout the cell cyclewas blockedby the KGB treatment Therefore these results that show aconcomitant decrease in the cell viability and proliferationcombined with an induction of cell death by apoptosis mayreveal a beneficial role of KGB in combating the process ofprostate growth that culminates in the development of BPH

Our results with KGB underground parts are comparablewith those observed for extracts from two plants clini-cally used to treat BHP The first one Pygeum africanum(Tadenan) inhibits the proliferation of cultured human

Evidence-Based Complementary and Alternative Medicine 5

72 hours

lowastlowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1

DMEM 1 + T 1 250 gml

T = 00

50

100

150

200

250

Cel

l Via

bilit

y

(a)72 hours

5284

12388

18659

DMEM 10

DMEM 1

DMEM 15 + T 1 250 gml

T = 00

50

100

150

200

250

C

ell V

iabi

lity

(b)

Figure 2 Cell viability assessed by MTT (a) Graph representing the viability of BPH stromal cells with the underground parts extract ofKGB (T1 treatment) at the concentration of 250 120583gml lowast plt005 lowastlowast plt0001 Data represent the mean plusmn standard deviation values of threeindependent experiments (b) Percentage of BPH stromal cells after treatment with extract of KGB underground parts (T1 treatment) Datarepresent the mean plusmn standard deviation values of three independent experiments

prostatic myofibroblasts and fibroblasts as well as enhancesapoptosis at concentrations from 25 to 100 120583gml [33 34]Serenoa repens (Permixon) the second one revealed atissue-selective action resulting in morphological changesand augmented apoptosis rates in addition to the inhibition ofnuclear membrane bound 5120572-reductase isoenzymes catalyticactivity in prostate cells at the concentration of 10 120583gml[35] Additionally an increment of Bax-to-Bcl-2 expressionand caspase 3 activity molecules involved in the apoptoticpathway has already been documented in prostatic tissuesamples from BPH symptomatic patients under treatmentwith Permixon for at least 3 months [36] Also an invitro study showed that treatment with S repens leads tothe lightening of BPH symptoms due to antiproliferativeand proapoptotic effects exerted on prostate epithelia andtriggered by the downregulation of IGF-1 signaling pathwayand induction of JNK [37] Finally BPH treatment withfinasteride the main therapeutic approach employed forthis disease management also demonstrated a mechanismexclusively observed in epithelial cells caspase-dependentapoptosis initiation through activation of caspases 3 and 6[38] In fact the success of the main treatment approaches(phytotherapic or conventional pharmacologic drugs) thatare routinely used in the treatment of BPH are related tothe control of BPH growth by reducing proliferation andinducing apoptosis [33 34 36] Thus the effects of KGBon BPH seem very interesting since this disease is largelycharacterized by an imbalance between the proliferation andapoptosis [4 7 8 11] Furthermore it was recently shown thatKGB underground parts were able to abrogate the androgen

signaling in prostate malignant cell lineages besides inducingthe apoptosis via caspase 8 activation thus reinforcing thetherapeutic potential of KGB in prostatic diseases [39]

Although the greater activity is in the subterraneanparts from K gastonis-bonnieri and therefore could be adisadvantage for a phytomedicinal preparation due to thenonrenewable characteristics of this part of the plant thissucculent herb is a fast growing-species that propagates easilyby asexual reproduction [40]

33 Chemical Composition of KGB Underground Parts Theextract from the KGB underground parts had its chemicalcomposition assessed byHPLC-DADMSMS in the negativeion mode As the TOF analyzer enables high-resolutionmass measurements with mass errors below 5 ppm itwas possible to infer the molecular formula of the majorconstituents detected in the KGB underground parts Theresulting chromatogram is shown in Figure 4 while data onthe major compounds detected are summarized in Table 2

Peak 1 (Rt 45 min 120582max 261 nm) presented the [M-H]minus ion at mz 3590994 (C

15H19O10) as base peak MSMS

spectrum showed a fragment at mz 1970458 (C9H9O5)

suggesting the loss of a hexose unity This substance couldpossibly correspond to a glycosylated form of syringic acidsuch as syringate 4-O-120573-glucopyranoside [41] Howeverisomers of syringic acid cannot be ruled out Peak 3 (Rt 52min 120582max 282 nm) also showed a [M-H]minus ion for which themolecular formula C

15H19O10

was proposed and a similarfragment at mz 1970457 We hypothesize that this substancecould correspond to a glycosyl ester of syringic acid Syringic

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

lowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

Pro

lifer

atio

nCo

ntro

l

(a)

5673

2923

3934

1351

DMEM 10

DMEM 1 + T4

DMEM 1 + T3

DMEM 1 + T2

DMEM 1 + T1

DMEM 1T = 0

0

45

90

135

180

P

rolif

erat

ion

Cont

rol

(b)

Figure 1 Proliferation of BPH stromal cells treated with different extracts of Kalanchoe gastonis-bonnieri (KGB) (a) Graph representing theproliferation of cells with the different extracts (T1-T4) of KGB at 250 120583g ml after 72 h lowast plt005 lowastlowast plt0001 Data represent the mean plusmnstandard deviation values of three independent experiments (b) Percentage of BPH stromal cells after treatment with four different extracts(T1-T4) of KGB Data represent the mean plusmn standard deviation values of three independent experiments

from the underground parts from flowering specimens andflowers were 12 and 39 respectively (Table 1)

We evaluated the inhibitory effects of the four KGBpreparations on the proliferation of stromal cells fromprimary benign prostatic hyperplasia (PBH) Undergroundparts extract from the flowering specimens of KGB (T1treatment) leaf extract of KGB fromflowering specimens (T2treatment) flowers extract from KGB (T3 treatment) andleaf extract of KGB not in flower (T4 treatment) were testedat 250 120583gml for 72 hours T1 T2 T3 and T4 treatmentsinhibited the cells proliferation by 567 292 394 and135 respectively (Figure 1)

The best results were observed for the underground partsextract (T1 treatment) which encouraged the continuation ofour experiments with this sample in order to corroborate thepreliminary detection of its activity in BPH

31 BPH Cell Viability Assay The extract of KGB under-ground parts (T1 treatment) significantly reduced the viabil-ity of BPH stromal cells treated with 250 120583gml promotingdecay of more than 50 cell viability (Figure 2)

32 BPH Cell Apoptosis Assay In order to clarify whetherthe reduction in the number of BPH stromal cells observedafter the treatment with KGB underground parts was due toa blockage in the proliferation activity of these cells or due toan induction of cell death we performed an apoptosis assayto address this question

Thepercentage of dead cells detected in the subG0 regionof the cell cycle after 72 hours was significantly higher whenthe cells were seeded in the presence of T1 treatment in theconcentrations of 150 120583gml and 250 120583gml (Figure 3(a))

However the activity was not dependent on the concen-tration used We observed that both concentrations of the

extract were able to suppress the progression of BPH cellsalong the cell cycle with no significant difference betweenthem Additionally Figure 3(b) shows that there was inthe control group a distribution throughout the differentcell cycle phases including the S and G2M phases thusindicating a proliferating profile of the BPH stromal cells inthe absence of the KGB treatment On the other hand thetreatment with T1 showed that BPH stromal cells were almostexclusively at the subG0 phase of the cell cycle

Despite numerous reports on the use of plants or deriva-tives of natural products of plant origin for the treatment ofbenign prostatic hyperplasia these activities have not alwaysbeen proven in pharmacological studies

The inhibitory activity observed for KGB in BPH cellproliferation was very effective since the KGB undergroundparts (T1) at the concentration of 150 120583gmL was able todrastically reduce the proliferation activity and the viability ofBPH stromal cells in 72 hours Moreover the same treatmentalso induced a strong increase in the apoptosis rates ofBPH stromal cells since a large percent of these cells wererestricted in the subGo phase of the cell cycle as revealed bythe flow cytometry analysis In addition the cell cycle profilepresented by BPH stromal cells after the treatment with theKGB underground parts corroborated the proliferation dataas the normal transition throughout the cell cyclewas blockedby the KGB treatment Therefore these results that show aconcomitant decrease in the cell viability and proliferationcombined with an induction of cell death by apoptosis mayreveal a beneficial role of KGB in combating the process ofprostate growth that culminates in the development of BPH

Our results with KGB underground parts are comparablewith those observed for extracts from two plants clini-cally used to treat BHP The first one Pygeum africanum(Tadenan) inhibits the proliferation of cultured human

Evidence-Based Complementary and Alternative Medicine 5

72 hours

lowastlowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1

DMEM 1 + T 1 250 gml

T = 00

50

100

150

200

250

Cel

l Via

bilit

y

(a)72 hours

5284

12388

18659

DMEM 10

DMEM 1

DMEM 15 + T 1 250 gml

T = 00

50

100

150

200

250

C

ell V

iabi

lity

(b)

Figure 2 Cell viability assessed by MTT (a) Graph representing the viability of BPH stromal cells with the underground parts extract ofKGB (T1 treatment) at the concentration of 250 120583gml lowast plt005 lowastlowast plt0001 Data represent the mean plusmn standard deviation values of threeindependent experiments (b) Percentage of BPH stromal cells after treatment with extract of KGB underground parts (T1 treatment) Datarepresent the mean plusmn standard deviation values of three independent experiments

prostatic myofibroblasts and fibroblasts as well as enhancesapoptosis at concentrations from 25 to 100 120583gml [33 34]Serenoa repens (Permixon) the second one revealed atissue-selective action resulting in morphological changesand augmented apoptosis rates in addition to the inhibition ofnuclear membrane bound 5120572-reductase isoenzymes catalyticactivity in prostate cells at the concentration of 10 120583gml[35] Additionally an increment of Bax-to-Bcl-2 expressionand caspase 3 activity molecules involved in the apoptoticpathway has already been documented in prostatic tissuesamples from BPH symptomatic patients under treatmentwith Permixon for at least 3 months [36] Also an invitro study showed that treatment with S repens leads tothe lightening of BPH symptoms due to antiproliferativeand proapoptotic effects exerted on prostate epithelia andtriggered by the downregulation of IGF-1 signaling pathwayand induction of JNK [37] Finally BPH treatment withfinasteride the main therapeutic approach employed forthis disease management also demonstrated a mechanismexclusively observed in epithelial cells caspase-dependentapoptosis initiation through activation of caspases 3 and 6[38] In fact the success of the main treatment approaches(phytotherapic or conventional pharmacologic drugs) thatare routinely used in the treatment of BPH are related tothe control of BPH growth by reducing proliferation andinducing apoptosis [33 34 36] Thus the effects of KGBon BPH seem very interesting since this disease is largelycharacterized by an imbalance between the proliferation andapoptosis [4 7 8 11] Furthermore it was recently shown thatKGB underground parts were able to abrogate the androgen

signaling in prostate malignant cell lineages besides inducingthe apoptosis via caspase 8 activation thus reinforcing thetherapeutic potential of KGB in prostatic diseases [39]

Although the greater activity is in the subterraneanparts from K gastonis-bonnieri and therefore could be adisadvantage for a phytomedicinal preparation due to thenonrenewable characteristics of this part of the plant thissucculent herb is a fast growing-species that propagates easilyby asexual reproduction [40]

33 Chemical Composition of KGB Underground Parts Theextract from the KGB underground parts had its chemicalcomposition assessed byHPLC-DADMSMS in the negativeion mode As the TOF analyzer enables high-resolutionmass measurements with mass errors below 5 ppm itwas possible to infer the molecular formula of the majorconstituents detected in the KGB underground parts Theresulting chromatogram is shown in Figure 4 while data onthe major compounds detected are summarized in Table 2

Peak 1 (Rt 45 min 120582max 261 nm) presented the [M-H]minus ion at mz 3590994 (C

15H19O10) as base peak MSMS

spectrum showed a fragment at mz 1970458 (C9H9O5)

suggesting the loss of a hexose unity This substance couldpossibly correspond to a glycosylated form of syringic acidsuch as syringate 4-O-120573-glucopyranoside [41] Howeverisomers of syringic acid cannot be ruled out Peak 3 (Rt 52min 120582max 282 nm) also showed a [M-H]minus ion for which themolecular formula C

15H19O10

was proposed and a similarfragment at mz 1970457 We hypothesize that this substancecould correspond to a glycosyl ester of syringic acid Syringic

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

72 hours

lowastlowast

lowastlowast

lowastlowast

DMEM 10

DMEM 1

DMEM 1 + T 1 250 gml

T = 00

50

100

150

200

250

Cel

l Via

bilit

y

(a)72 hours

5284

12388

18659

DMEM 10

DMEM 1

DMEM 15 + T 1 250 gml

T = 00

50

100

150

200

250

C

ell V

iabi

lity

(b)

Figure 2 Cell viability assessed by MTT (a) Graph representing the viability of BPH stromal cells with the underground parts extract ofKGB (T1 treatment) at the concentration of 250 120583gml lowast plt005 lowastlowast plt0001 Data represent the mean plusmn standard deviation values of threeindependent experiments (b) Percentage of BPH stromal cells after treatment with extract of KGB underground parts (T1 treatment) Datarepresent the mean plusmn standard deviation values of three independent experiments

prostatic myofibroblasts and fibroblasts as well as enhancesapoptosis at concentrations from 25 to 100 120583gml [33 34]Serenoa repens (Permixon) the second one revealed atissue-selective action resulting in morphological changesand augmented apoptosis rates in addition to the inhibition ofnuclear membrane bound 5120572-reductase isoenzymes catalyticactivity in prostate cells at the concentration of 10 120583gml[35] Additionally an increment of Bax-to-Bcl-2 expressionand caspase 3 activity molecules involved in the apoptoticpathway has already been documented in prostatic tissuesamples from BPH symptomatic patients under treatmentwith Permixon for at least 3 months [36] Also an invitro study showed that treatment with S repens leads tothe lightening of BPH symptoms due to antiproliferativeand proapoptotic effects exerted on prostate epithelia andtriggered by the downregulation of IGF-1 signaling pathwayand induction of JNK [37] Finally BPH treatment withfinasteride the main therapeutic approach employed forthis disease management also demonstrated a mechanismexclusively observed in epithelial cells caspase-dependentapoptosis initiation through activation of caspases 3 and 6[38] In fact the success of the main treatment approaches(phytotherapic or conventional pharmacologic drugs) thatare routinely used in the treatment of BPH are related tothe control of BPH growth by reducing proliferation andinducing apoptosis [33 34 36] Thus the effects of KGBon BPH seem very interesting since this disease is largelycharacterized by an imbalance between the proliferation andapoptosis [4 7 8 11] Furthermore it was recently shown thatKGB underground parts were able to abrogate the androgen

signaling in prostate malignant cell lineages besides inducingthe apoptosis via caspase 8 activation thus reinforcing thetherapeutic potential of KGB in prostatic diseases [39]

Although the greater activity is in the subterraneanparts from K gastonis-bonnieri and therefore could be adisadvantage for a phytomedicinal preparation due to thenonrenewable characteristics of this part of the plant thissucculent herb is a fast growing-species that propagates easilyby asexual reproduction [40]

33 Chemical Composition of KGB Underground Parts Theextract from the KGB underground parts had its chemicalcomposition assessed byHPLC-DADMSMS in the negativeion mode As the TOF analyzer enables high-resolutionmass measurements with mass errors below 5 ppm itwas possible to infer the molecular formula of the majorconstituents detected in the KGB underground parts Theresulting chromatogram is shown in Figure 4 while data onthe major compounds detected are summarized in Table 2

Peak 1 (Rt 45 min 120582max 261 nm) presented the [M-H]minus ion at mz 3590994 (C

15H19O10) as base peak MSMS

spectrum showed a fragment at mz 1970458 (C9H9O5)

suggesting the loss of a hexose unity This substance couldpossibly correspond to a glycosylated form of syringic acidsuch as syringate 4-O-120573-glucopyranoside [41] Howeverisomers of syringic acid cannot be ruled out Peak 3 (Rt 52min 120582max 282 nm) also showed a [M-H]minus ion for which themolecular formula C

15H19O10

was proposed and a similarfragment at mz 1970457 We hypothesize that this substancecould correspond to a glycosyl ester of syringic acid Syringic

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table2Major

chem

icalcompo

unds

inthee

xtractof

undergroun

dpartsfrom

Kgasto

nis-b

onnieribyHPL

C-DADM

SMS

Peak

No

Rt(m

in)

Molecular

form

ula[M

-H]-

Measuredmz[M

-H]-

Calculated[M

-H]-

Error(

ppm)

UV120582max

(nm)

MSMSfragmention

sProp

osed

compo

und

145

C 15H19O10

3590

986

3590

984

-06

261

1970

457

Syrin

gica

cidhexosid

e2

49

C 12H20NO8

306119

4306119

40

nd

20503801610455

Unk

nown

352

C 15H19O10

3590

996

3590

984

-36

282

1970

4582390

572

Syrin

gica

cidhexosid

e4

55

C 18H24N5O6

4061732

4061721

01

255

307104

0Unk

nown

560

C 16H21O9

3571196

3571191

-13

273

1770

556

Unk

nown

662

C 16H29O10

3811780

3811766

-35

nd

235119

61610

458

Alkyldiglycoside

773

C 19H27O10

4151618

4151610

-19

nd

2691

0371610

456

Benzyldiglycoside

880

C 17H31O10

3951935

3951923

-3nd

2491

3521610

461

Alkyldiglycoside

982

C 26H33O11

5212

035

5212

028

-12

nd

3591

507

Glycosylatedlignan

1085

C 26H33O11

5212

022

5212

028

13283

3591

508

Glycosylatedlignan

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

G2M

SG1

SubG0

Cel

lula

r Eve

nts

G2M

S

G1SubG0

G2M

S

G1SubG0

T1 (150 gml)CONTROL T1 (250 gml)

Propidium Iodide

0 1023 0 1023 0 1023

(a)

SubG0 G0G1 S G2

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

T1 (250

gml)

T1 (150

gml)

Control

0

20

40

60

80

100

120

C

ells

0

10

20

30

40

0

5

10

15

20

0

15

30

45

60

(b)

Figure 3 BPH cell death evaluation by flow cytometry (a) Histogram representing the distribution of BPH stromal cells in different phasesof the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri (T1) at concentrations of 150 120583gml and250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of three independent experiments (b) Percentageof BPH stromal cells in different phases of the cell cycle after treatment with the underground parts extract of Kalanchoe gastonis-bonnieri(T1) at concentrations of 150 120583gml and 250 120583gml after 72 h lowast (p lt0001) Data represent the mean plusmn standard deviation values of threeindependent experiments

2 4 6 8 10 Time [min]000

025

050

075

100

125

150

Intens

1 2

3

4

5

6

7

8

9

10

x105

Figure 4 Base peak chromatogram (LC-MS) of aqueous extract from KGB underground parts (4 mgml) in the negative ion mode

acid 120573-D-glucopyranosyl ester has already been reported forleaves of Kalanchoe pinnata and a derivative of this substancewas recently reported in the underground parts of the samespecies [42 43] Thus peak 1 could correspond to a syringicacid glycosylated at the phenolic hydroxyl having a freecarboxylmoiety and peak 3 to the same aglycone glycosylated

at the carboxyl moiety This is corroborated by their UVspectra which correspond to those of the aforementionedsubstances and the order of elution since an ester is less polarthan a carboxylic acid

Peak 6 (Rt 62min) showed the [M-H]minus ion atmz 3811780(C16H21O9) with fragments at mz 2351196 (C

10H19O6) and

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

1610458 (C6H9O5) at the MSMS spectrum corresponding

to the loss of a deoxyhexose (eg rhamnose) and a C4H10O

(butanol) unity respectively Peak 7 (Rt 73 min) in itsturn presented the [M-H]minus ion at mz 4151618 (C

19H27O10)

and fragments at mz 2691037 (C13H17O6) and 1610456

(C6H9O5) corresponding to the loss of a deoxyhexosemoiety

and a C7H8O (benzyl alcohol) unity Peak 8 (Rt 80 min)

gave a [M-H]minus ion at mz 3951935 and fragments at mz2491352 (C11H21O6) and 1610461 (C6H9O5) correspondingagain to a loss of a deoxyhexose and a C

5H12O (pentanol

or methylbutanol) unity Thus peaks 6 7 and 8 were tenta-tively attributed to glycosidically bound volatile substanceswhich are commonly found in plants [44ndash46] Alcohol andmonoterpene glycosides are found in the roots of plantsfrom Rhodiola species which also belong to the Crassulaceaefamily [47]

Peaks 9 (Rt 82 min) and 10 (Rt 85 min) presentedthe [M-H]minus ions at mz 5212022 and 5212035 respectivelyBoth presented C

26H33O11

as molecular formula [M-H]minusand a MSMS fragment at mz 3591508 indicative of theloss of a hexose unity As we did not observe any furtherfragmentation of the aglycones many structural possibilitieswere found for these substances All the possible substances ofnatural origin with this molecular formula found in the Sci-Finder database belonged to the class of lignans with severalpossible skeletons Thus we postulate that peaks 9 and 10correspond to glycosylated lignans In the underground partsof Kalanchoe pinnata a glycosylated aryltetralin lignan wasrecently reported [43] Lignans are also present in roots ofRhodiola species [48]

There are several reports on the activity of lignans in BPHand prostate cancer For instance a lignan-enriched extractfrom flaxseed (Beneflax) was capable of improving LUTSin patients with BPH in a double-blind placebo-controlledclinical trial [23] A similar extract from flax hulls preventedthe development of testosterone propionate- (TP-) inducedBPH in rats [49] Also secoisolariciresinol diglucoside themajor lignan in flaxseed was able to inhibit BPH in TP-induced BPH in rats Enterolactone a metabolite of thissubstance was shown to block the proliferation of a humanprostatic stromal cell line by a mechanism involving the Gprotein-coupled estrogen receptor 1 [50] Furthermore thelignans from the medicinal species Campylotropis hirtella(Fabaceae) were shown to inhibit prostate specific antigenand to decrease the androgen receptor expression in aprostate cancer cell linage The most potent of those lignans(dehydrodiconiferyl alcohol) was further investigated andexhibited proapoptotic effects in these cells [24]

It was not possible to identify peaks 2 (Rt 49 min) 4(Rt 55 min) and 5 (Rt 60 min) We reported the molecularformulas considered most likely here with the smallestpossible errors However we do not discard other structuralpossibilities for these peaks

4 Conclusion

Thepresent results seem very encouraging since they reveal apotential use of the underground parts ofKalanchoe gastonis-bonnieri in the treatment of benign prostatic hyperplasia a

condition that causes significant chronic morbidity for menFurthermore the increment in the ldquophytotherapic productsportfoliordquo currently available could improve themanagementof this disease since a large number of natural compoundshas been described as reliable safe and cost effective in thetreatment of several diseases

In addition the main mechanisms related to KGB treat-ment seem to be the inhibition of the proliferation activityalong with the induction of apoptosis

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

Maria Fernanda Paresqui Correarsquos Current address is Insti-tuto Nacional de Propriedade Industrial (INPI) 20090-910Rio de Janeiro RJ Brazil

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Antonio Palumbo carried out the BPH assays contributedwith the data analysis and discussion andwith themanuscriptpreparation Livia Marques Casanova performed the HPLC-ESI-MSMS analysis and data interpretation and contributedto the literature search and background information as well aswith the manuscript preparation Maria Fernanda ParesquiCorrea carried out the KGB extraction and provided thesamples for BPH assays Nathalia Meireles Da Costa con-tributed with the flow cytometry experiments Luiz EuricoNasciutti supervised the BPH assays and contributed to thediscussion of the data Sonia Soares Costa supervised thephytochemical study contributing to the discussion of theresults and themanuscript preparation Luiz Eurico Nasciuttiand Sonia Soares Costa were responsible for the study designAll the authors read the final manuscript

Acknowledgments

Special thanks are due to Mariana Neubarth Coelho forhelp with HPLC-DAD-MSMS analysis and Eduardo Matosfor skillful technical support with HPLC-DAD-MSMSMaria Fernanda Paresqui Correa (process 1415702007-5)Antonio Palumbo (process 1423182011-6) and Livia Mar-ques Casanova (process 1402772013-7) were granted withPhD fellowships from CNPq Antonio Palumbo receiveda postdoctoral fellowship from Fundacao do Cancer AryFrauzino Brazil (Oncobiology ProgramIBqMUFRJBrazil)Livia Marques Casanova currently receives a postdoctoralfellowship from CAPES (PNPDprocess 1723490)

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

References

[1] G Corona L Vignozzi G Rastrelli et al ldquoBenign ProstaticHyperplasia A New Metabolic Disease of the Aging Male andIts Correlationwith Sexual Dysfunctionsrdquo International Journalof Endocrinology vol 2014 Article ID 329456 14 pages 2014

[2] B Fibbi G Penna A Morelli L Adorini and M MaggildquoChronic inflammation in the pathogenesis of benign prostatichyperplasiardquo International Journal of Andrology vol 33 no 3pp 475ndash488 2010

[3] C G Roehrborn ldquoPathology of benign prostatic hyperplasiardquoInternational Journal of Impotence Research vol 20 no 3 ppS11ndashS18 2008

[4] K T Foo ldquoPathophysiology of clinical benign prostatic hyper-plasiardquo Asian Journal of Urology vol 4 no 3 pp 152ndash157 2017

[5] C Vuichoud and K R Loughlin ldquoBenign prostatic hyperplasiaEpidemiology economics and evaluationrdquoThe Canadian Jour-nal of Urology vol 22 pp 1ndash6 2015

[6] K B Lim ldquoEpidemiology of clinical benign prostatic hyperpla-siardquo Asian Journal of Urology vol 4 no 3 pp 148ndash151 2017

[7] A Prajapati S Gupta and B Mistry ldquoProstate stem cells inthe development of benign prostate hyperplasia and prostatecancer Emerging role and conceptsrdquo Biomed Res Int vol 201310 pages 2013

[8] L Minutoli M Rinaldi H Marini et al ldquoApoptotic PathwaysLinked to Endocrine System as Potential Therapeutic Targetsfor Benign Prostatic Hyperplasiardquo International Journal ofMolecular Sciences vol 17 no 8 p 1311 2016

[9] B Chughtai R Lee A Te and S Kaplan ldquoInflammation andbenign prostatic hyperplasia Clinical implicationsrdquo CurrentUrology Reports vol 12 no 4 pp 274ndash277 2011

[10] G Robert A Descazeaud Y Allory F Vacherot and A dela Taille ldquoShould We Investigate Prostatic Inflammation forthe Management of Benign Prostatic Hyperplasiardquo EuropeanUrology Supplements vol 8 no 13 pp 879ndash886 2009

[11] B Kruslin D Tomas T Dzombeta M Milkovic-Perisaand M Ulamec ldquoInflammation in Prostatic Hyperplasia andCarcinomamdashBasic Scientific Approachrdquo Frontiers in Oncologyvol 7 2017

[12] R V Nunes J Manzano J C Truzzi A Nardi A Silvinato andW M Bernardo ldquoTreatment of benign prostatic hyperplasiardquoRevista da Associacao Medica Brasileira vol 63 no 2 pp 95ndash99 2017

[13] K Gupta M Yezdani T Sotelo and J B Aragon-Ching ldquoAsynopsis of drugs currently in preclinical and early clinicaldevelopment for the treatment of benign prostatic hyperplasiardquoExpert Opinion on Investigational Drugs vol 24 no 8 pp 1059ndash1073 2015

[14] C De Nunzio and A Tubaro ldquoInnovations in medical andsurgical treatmentrdquo Nature Reviews Urology vol 12 no 2 pp76ndash78 2015

[15] A Kahokehr R Vather ANixon andAGHill ldquoNon-steroidalanti-inflammatory drugs for lower urinary tract symptomsin benign prostatic hyperplasia Systematic review and meta-analysis of randomized controlled trialsrdquo BJU International vol111 no 2 pp 304ndash311 2013

[16] E Pagano M Laudato M Griffo and R Capasso ldquoPhytother-apy of benign prostatic hyperplasia A minireviewrdquo Phytother-apy Research vol 28 no 7 pp 949ndash955 2014

[17] O Allkanjari and A Vitalone ldquoWhat do we know aboutphytotherapy of benign prostatic hyperplasiardquo Life Sciencesvol 126 pp 42ndash56 2015

[18] A Keehn J Taylor and F C Lowe ldquoPhytotherapy for BenignProstatic Hyperplasiardquo Current Urology Reports vol 17 no 72016

[19] H Azimi A-A Khakshur I Aghdasi M Fallah-Tafti and MAbdollahi ldquoA review of animal and human studies for man-agement of benign prostatic hyperplasia with natural productsperspective of new pharmacological agentsrdquo Inflammation ampAllergymdashDrug Targets vol 11 no 3 pp 207ndash221 2012

[20] K-S Chung S-Y Cheon and H-J An ldquoEffects of resveratrolon benign prostatic hyperplasia by the regulation of inflamma-tory and apoptotic proteinsrdquo Journal of Natural Products vol78 no 4 pp 689ndash694 2015

[21] C Eleazu K Eleazu andW Kalu ldquoManagement of benign pro-static hyperplasia Could dietary polyphenols be an alternativeto existing therapiesrdquo Frontiers in Pharmacology vol 8 2017

[22] A E Katz ldquoFlavonoid and botanical approaches to prostatehealthrdquo The Journal of Alternative and ComplementaryMedicine vol 8 no 6 pp 813ndash821 2002

[23] W Zhang X Wang Y Liu et al ldquoEffects of dietary flaxseedlignan extract on symptoms of benign prostatic hyperplasiardquoJournal of Medicinal Food vol 11 no 2 pp 207ndash214 2008

[24] H-Y Han X-H Wang N-L I Wang M-T Ling Y-CWong and X-S Yao ldquoLignans isolated from Campylotropishirtella (Franch) Schindl decreased prostate specific antigenand androgen receptor expression in LNCaP cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 16 pp 6928ndash69352008

[25] A L Ososki P Lohr M Reiff et al ldquoEthnobotanical literaturesurvey of medicinal plants in the Dominican Republic used forwomenrsquos health conditionsrdquo Journal of Ethnopharmacology vol79 no 3 pp 285ndash298 2002

[26] S S Costa M F P Correa and L M Casanova ldquoA newtriglycosyl flavonoid isolated from leaf juice of kalanchoegastonis-bonnieri (crassulaceae)rdquoNatural ProductCommunica-tions (NPC) vol 10 no 3 pp 433ndash436 2015

[27] S L Abdalla S S Costa M A Gioso et al ldquoEfficacy of a Kalan-choe gastonis-bonnieri extract to control bacterial biofilms anddental calculus in dogsrdquo Pesquisa Veterinaria Brasileira vol 37no 8 pp 859ndash865 2017

[28] M M De la Luz Miranda-Beltran A M Puebla-Perez AGuzman-Sanchez and L Huacuja Ruiz ldquoMale rat infertilityinductionspermatozoa and epididymal plasma abnormalitiesafter oral administration ofKalanchoe gastonis bonnieri naturaljuicerdquo Phytotherapy Research vol 17 no 4 pp 315ndash319 2003

[29] S Nieto-Ceron H Vargas-Lopez M Perez-Albacete et alldquoAnalysis of cholinesterases in human prostate and spermrdquoChemico-Biological Interactions vol 187 no 1-3 pp 432ndash4352010

[30] Y Gat M Gornish M Heiblum and S Joshua ldquoReversal ofbenign prostate hyperplasia by selective occlusion of impairedvenous drainage in the male reproductive system Novel mech-anism new treatmentrdquo Andrologia vol 40 no 5 pp 273ndash2812008

[31] A Palumbo L B Ferreira P A V Reis de Souza et alldquoExtracellular matrix secreted by reactive stroma is a maininducer of pro-tumorigenic features on LNCaP prostate cancercellsrdquo Cancer Letters vol 321 no 1 pp 55ndash64 2012

[32] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

[33] D Boulbes L Soustelle P Costa et al ldquoPygeum africanumextract inhibits proliferation of human cultured prostaticfibroblasts and myofibroblastsrdquo BJU International vol 98 no5 pp 1106ndash1113 2006

[34] M TQuilesMA Arbos A Fraga I MDe Torres J Reventosand J Morote ldquoAntiproliferative and apoptotic effects of theherbal agent Pygeum africanum on cultured prostate stromalcells from patients with Benign Prostatic Hyperplasia (BPH)rdquoThe Prostate vol 70 no 10 pp 1044ndash1053 2010

[35] C W Bayne M Ross F Donnelly and F K Habib ldquoTheselectivity and specificity of the actions of the lipido-sterolicextract of serenoa repens (Permixon) on the prostaterdquo TheJournal of Urology vol 164 no 3 part 1 pp 876ndash881 2000

[36] R Vela-Navarrete M Escribano-Burgos A Lopez Farre JGarcıa-Cardoso F Manzarbeitia and C Carrasco ldquoSerenoarepens treatment modifies BaxBcl-2 index expression andCaspase-3 activity in prostatic tissue from patients with benignprostatic hyperplasiardquoThe Journal of Urology vol 173 no 2 pp507ndash510 2005

[37] T LWadsworth JMCarroll R AMallinson C T Roberts Jrand C E Roselli ldquoSaw palmetto extract suppresses insulin-likegrowth factor-I signaling and induces stress-activated proteinkinasec-Jun N-terminal kinase phosphorylation in humanprostate epithelial cellsrdquo Endocrinology vol 145 no 7 pp 3205ndash3214 2004

[38] A Bozec A Ruffion M Decaussin et al ldquoActivation ofcaspases-3 -6 and -9 during finasteride treatment of benignprostatic hyperplasiardquo The Journal of Clinical Endocrinology ampMetabolism vol 90 no 1 pp 17ndash25 2005

[39] N Shamaladevi S Araki D A Lyn et al ldquoThe andean anti-cancer herbal product BIRM causes destabilization of androgenreceptor and induces caspase-8 mediated-apoptosis in prostatecancerrdquo Oncotarget vol 7 no 51 pp 84201ndash84213 2016

[40] HMGarces C E Champagne B T Townsley et al ldquoEvolutionof asexual reproduction in leaves of the genus KalanchoerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 104 no 39 pp 15578ndash15583 2007

[41] K Wolfram J Schmidt VWray C Milkowski W SchliemannandD Strack ldquoProfiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)rdquo Phytochemistry vol 71no 10 pp 1076ndash1084 2010

[42] K Furer M Raith R Brenneisen et al ldquoTwo new flavonolglycosides and a metabolite profile of bryophyllum pinnatuma phytotherapeutic used in obstetrics and gynaecologyrdquo PlantaMedica vol 79 no 16 pp 1565ndash1571 2013

[43] M Cryer K Lane M Greer et al ldquoIsolation and identificationof compounds from Kalanchoe pinnata having human alpha-herpesvirus and vaccinia virus antiviral activityrdquo Pharmaceuti-cal Biology vol 55 no 1 pp 1586ndash1591 2017

[44] A Kilic H Kollmannsberger and S Nitz ldquoGlycosidicallybound volatiles and flavor precursors in Laurus nobilis LrdquoJournal of Agricultural and Food Chemistry vol 53 no 6 pp2231ndash2235 2005

[45] C-C Chyau P-T Ko C-H Chang and J-L Mau ldquoFreeand glycosidically bound aroma compounds in lychee (Litchichinensis Sonn)rdquo Food Chemistry vol 80 no 3 pp 387ndash3922003

[46] P Wu M-C Kuo and C-T Ho ldquoGlycosidically Bound AromaCompounds in Ginger (Zingiber officinale Roscoe)rdquo Journal ofAgricultural and Food Chemistry vol 38 no 7 pp 1553ndash15551990

[47] Y-C Ma X-Q Wang F Hou et al ldquoSimultaneous quantifica-tion of polyherbal formulations containing Rhodiola rosea Land Eleutherococcus senticosus Maxim using rapid resolutionliquid chromatography (RRLC)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 55 no 5 pp 908ndash915 2011

[48] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[49] J-F Bisson SHidalgo R Simons andMVerbruggen ldquoPreven-tive effects of lignan extract from flax hulls on experimentallyinduced benign prostate hyperplasiardquo Journal of MedicinalFood vol 17 no 6 pp 650ndash656 2014

[50] G Ren C Chen W Chen Y Huang L Qin and L ChenldquoThe treatment effects of flaxseed-derived secoisolariciresinoldiglycoside and itsmetabolite enterolactone on benign prostatichyperplasia involve the G protein-coupled estrogen receptor 1rdquoApplied Physiology Nutrition andMetabolism vol 41 no 12 pp1303ndash1310 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom