UNIVERSIDADE FEDERAL DO CEARÁ CENTRO DE … · “Os sonhos são como uma bússola, indicando os...

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UNIVERSIDADE FEDERAL DO CEARÁ

CENTRO DE TECNOLOGIA

DEPARTAMENTO DE ENGENHARIA QUÍMICA

PROGRAMA DE PÓS-GRADUAÇÃO EM ENGENHARIA QUÍMICA

ADRIANA DUTRA SOUSA

EFEITO DO MÉTODO DE EXTRAÇÃO E DA SECAGEM SOBRE O

CONTEÚDO FENÓLICO E A COMPOSIÇÃO QUÍMICA DE QUEBRA-PEDRA

(PHYLLANTHUS AMARUS E PHYLLANTHUS NIRURI)

FORTALEZA

2017

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ADRIANA DUTRA SOUSA

EFEITO DO MÉTODO DE EXTRAÇÃO E DA SECAGEM SOBRE O CONTEÚDO

FENÓLICO E A COMPOSIÇÃO QUÍMICA DE QUEBRA-PEDRA (PHYLLANTHUS

AMARUS E PHYLLANTHUS NIRURI)

Tese apresentada ao Programa de Pós-

Graduação em Engenharia Química da

Universidade Federal do Ceará, como

requisito parcial à obtenção do Título de

Doutor em Engenharia Química. Área de

Concentração: Processos Químicos e

Bioquímicos

Orientador: Prof. Dr. Edy Sousa de Brito

FORTALEZA

2017

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Dados Internacionais de Catalogação na Publicação Universidade Federal do Ceará

Biblioteca Universitária Gerada automaticamente pelo módulo Catalog, mediante os dados fornecidos pelo(a) autor(a)

S696e Sousa, Adriana Dutra.

Efeito do método de extração e da secagem sobre o conteúdo fenólico e a composição química de quebra- pedra (Phyllanthus amarus e Phyllanthus niruri) / Adriana Dutra Sousa. – 2017.

119 f. : il. color.

Tese (doutorado) – Universidade Federal do Ceará, Centro de Tecnologia, Programa de Pós-Graduação em Engenharia Química, Fortaleza, 2017.

Orientação: Prof. Dr. Edy Sousa de Brito.

1. Fenólicos. 2. Ultrassom. 3. Líquido pressurizado. 4. Quimiometria. 5. Secagem convectiva. I. Título. CDD 660

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ADRIANA DUTRA SOUSA

EFEITO DO MÉTODO DE EXTRAÇÃO E DA SECAGEM SOBRE O CONTEÚDO

FENÓLICO E A COMPOSIÇÃO QUÍMICA DE QUEBRA-PEDRA (PHYLLANTHUS

AMARUS E PHYLLANTHUS NIRURI)

Tese apresentada ao Programa de Pós-

Graduação em Engenharia Química da

Universidade Federal do Ceará, como

requisito parcial à obtenção do Título de

Doutor em Engenharia Química. Área de

Concentração: Processos Químicos e

Bioquímicos

Aprovada em: _22_/_02_/_2017.

BANCA EXAMINADORA

________________________________________

Dr. Edy Sousa de Brito (Orientador)

Embrapa Agroindústria Tropical

_______________________________________

Prof. Dr. Fabiano André Narciso Fernandes

Universidade Federal do Ceará (UFC)

_________________________________________

Dr. Guilherme Julião Zocolo


_________________________________________

Drª. Henriette Monteiro Cordeiro de Azeredo


_________________________________________

Dr. Kirley Marques Canuto


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AGRADECIMENTOS

A Deus, por sempre ter iluminado meus caminhos e por ter me proporcionado

força e coragem durante toda a minha jornada de trabalho.

Aos meus pais, Helena e Flávio, pelo grande amor, carinho, estímulo,

ensinamentos e dedicação em todas as etapas da minha vida.

Ao meu esposo Franzé Júnior, por sempre acreditar em minha capacidade e por

todo apoio, amor, carinho e compreensão.

Ao meu orientador, Dr. Edy Sousa de Brito, pela paciência, amizade, confiança

em meu trabalho e conhecimentos compartilhados, de grande importância para minha

vida acadêmica.

Ao Dr. Kirley Marques Canuto, ao Dr. Guilherme Julião Zocolo, ao Prof. Dr.

Fabiano André Narciso Fernandes, e à Dra. Henriette Monteiro Cordeiro de Azeredo,

pelas orientações para o enriquecimento deste trabalho.

Ao Programa de Pós-graduação em Engenharia Química e a todos os seus

professores, pela oportunidade de realização do doutorado e pelos ensinamentos

transmitidos.

À Embrapa Agroindústria Tropical pelas instalações concedidas durante a

realização da parte experimental da minha tese. Em especial aos amigos Dra. Isabel

Maia, Caroline Gondim, Karine Nojosa, Dra. Tigressa Rodrigues, Dr. Paulo Riceli, Dra.

Lorena Silva, Marcelo Victor, Dr. Jéfferson Malveira, Aline Cavalcante, Luiz Bruno,

Paloma Lira, Náyra de Oliveira e Francilene Silva do Laboratório Multiusuário de

Química de Produtos Naturais pela ajuda e bons momentos.

À turma de doutorado, em especial aos amigos, Maria de Fátima, Valéria Melo,

Valéria Santos e Ana Cristina, pelo companheirismo.

À FUNCAP, pelo apoio financeiro.

A todos que direta ou indiretamente tornaram possível o cumprimento de mais

esta etapa.

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“Os sonhos são como uma bússola,

indicando os caminhos que seguiremos e

as metas que queremos alcançar. São

eles que nos impulsionam, nos

fortalecem e nos permitem crescer.”

(Augusto Cury)

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RESUMO

O gênero Phyllanthus, conhecido popularmente no Brasil como quebra-pedra, é

composto de plantas ricas em compostos bioativos, principalmente fenólicos. Na

obtenção desses compostos de interesse, a secagem da matéria-prima e o processo de

extração são fundamentais. Atualmente, tem se buscado a utilização de técnicas de

extração “verde” que reduzam o impacto ao meio ambiente. Dentre estas técnicas

destacam-se a extração assistida por ultrassom (EAU) e a extração com líquido

pressurizado (ELP). Neste estudo, a extração aquosa das partes aéreas de P. amarus e

P. niruri foi realizada por EAU, ELP e extração convencional. Foi avaliado o efeito do

tempo, intensidade ultrassônica e razão líquido/sólido na EAU e do tempo e

temperatura na ELP na extração de fenólicos totais e ácido gálico. A composição

química dos extratos foi determinada por UPLC-QTOF-MS/MS em conjunto com

técnicas quimiométricas (PCA e OPLS-DA). Também foram investigados parâmetros

de secagem das plantas. Partes aéreas das duas espécies foram secas em estufa de

circulação de ar e dados de cinética de secagem foram obtidos. O efeito da temperatura

do ar de secagem (50, 60 e 70°C) sobre o conteúdo fenólico e a composição química

também foi estudado. O maior conteúdo de fenólicos totais foi observado nos extratos

obtidos por ELP em 192°C/15 min para as duas espécies, mas esta temperatura

elevada levou à degradação de alguns compostos. Os extratos obtidos por ELP na

temperatura de 120°C apresentaram um alto conteúdo fenólico e sem degradação

química. As outras técnicas de extração promoveram menor rendimento de compostos

fenólicos e maior consumo de solvente. Portanto, a ELP na temperatura de 120°C e

pressão de 110 bar mostrou-se um método adequado para extrair compostos fenólicos,

incluindo os compostos com importância medicinal. A composição química dos

extratos apresentou principalmente taninos hidrolisáveis e flavonóides. Com relação à

secagem, o aumento da temperatura do ar de secagem reduziu o tempo de secagem e

aumentou a difusividade efetiva de umidade. A melhor temperatura testada para se

obter um maior conteúdo fenólico para ambas as espécies foi de 60°C. Os resultados

indicam a importância do controle da temperatura de secagem para manter a qualidade

da matéria-prima e do processo de extração na obtenção dos compostos de interesse.

Palavras-chave: Fenólicos. Ultrassom. Líquido pressurizado. Quimiometria. Secagem

convectiva.

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ABSTRACT

The genus Phyllanthus, popularly known as quebra-pedra in Brazil, is composed of

plants rich in bioactive compounds, mainly phenolics. Drying of raw material and the

extraction process are essential to achieve those compounds. Nowadays, "green"

extraction techniques are required to reduce the environmental impacts. Among these

techniques, ultrasound-assisted extraction (UAE) and pressurized liquid extraction

(PLE) stand out. In this study, aqueous extraction from aerial parts of P. amarus and P.

niruri was performed using UAE, PLE and conventional extraction. It was evaluated the

effect of time, ultrasonic intensity, and liquid/solid (L/S) ratio in UAE and of time and

temperature in PLE on total phenolics and gallic acid extraction. The chemical

composition of the extracts was determined by UPLC-QTOF-MS/MS in conjunction

with chemometric techniques (PCA and OPLS-DA). Also plants drying parameters

were investigated. Aerial parts of the two species were dried in a circulating air-drying

oven and drying kinetics data were obtained. The effect of air-drying temperature (50,

60 and 70°C) on phenolic content and on chemical composition was also studied. The

highest total phenolics content was observed in the extracts obtained by PLE at

192°C/15 min for the two species, but this high temperature led to degradation of some

compounds. The extracts obtained by the PLE at 120°C presented a high phenolic

content without chemical degradation. The other extraction techniques produced a lower

yield of phenolic compounds and higher solvent consumption. Therefore, PLE at a

temperature of 120°C and pressure of 110 bar proved to be a suitable method to extract

phenolics, including the compounds with medicinal relevance. The chemical

composition of the extracts had mainly hydrolysable tannins and flavonoids. With

regard to drying, the increase in air-drying temperature reduced the drying time and

increased the effective moisture diffusivity. The best evaluated temperature to obtain a

higher phenolic content for both species was 60°C. The results indicate the importance

of the drying temperature control to maintain the quality of the raw material and the

extraction process in obtaining the compounds of interest.

Keywords: Phenolics. Ultrasound. Pressurized liquid. Chemometrics. Convective

drying.

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LISTA DE FIGURAS

REVISÃO DE LITERATURA

Figura 1 Phyllanthus amarus................................................................................... 16

Figura 2 Phyllanthus niruri...................................................................................... 19

Figura 3 Esquema de sistemas de aplicação de ondas ultrassonoras: a) sonda, b)

banho......................................................................................................... 26

Figura 4 Esquema de funcionamento de um extrator com líquido pressurizado

(ELP)......................................................................................................... 29

ARTIGOS

Ultrasound-assisted and pressurized liquid extraction of phenolic compounds from

Phyllanthus amarus and its composition evaluation by UPLC-QTOF

Figure 1 Estimated effects by Pareto plot and response-surface graphs for the

phenolics content (mg/g plant) in ultrasound-assisted extraction............. 47

Figure 2 Estimated effects by Pareto plot and response-surface graphs for the gallic

acid content (mg/g plant) in ultrasound-assisted extraction…………….. 48

Figure 3 Estimated effects by Pareto plot and response-surface graphs for the

phenolics content (mg/g plant) (a) and (b) and gallic acid content (mg/g

plant) (c) and (d) in pressurized-liquid extraction………………………. 50

Figure 4 LC-ESI(+)/MS and LC-ESI(−)/MS chromatograms of P. amarus aqueous

extracts obtained through UAE (a) and (b), PLE (c) and (d), and CE (e)

and (f), respectively……………………………………………………... 52

Figure 5 Structures of the substances identified in P. amarus extracts…………... 53

Figure 6 Proposal of amariinic acid fragmentation with corilagin formation (m/z

633)……………………………………………………………………… 58

Figure 7 Possible formation of monogalloylhexoside through the loss of the HHDP

group (m/z 301)………………………………………………………….. 58

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Figure 8 Proposal of the loss of the galloyl group by ellagitannins, generating the

fragments observed in the positive mode……………………………….. 60

Supplementary material HPLC chromatograms of the aqueous extracts from P.

amarus obtained through PLE at 120°C (a) and 192.4°C (b) at 272 nm……………… 65

UPLC-QTOF-MSE-based chemometric approach driving the choice of the best

extraction process for Phyllanthus niruri

Figure 1 UPLC-QTOF-MSE chromatograms of P. niruri aqueous extracts obtained

through UAE (a), PLE 120 (b), PLE 192 (c) and CE (d)…….................. 74

Figure 2 PCA score plot generated by Pareto of P. niruri extracts obtained through

CE (conventional extraction), PLE (pressurized liquid extraction in

temperatures of 120°C and 192°C) and UAE (ultrasound assisted

extraction). Ions in negative mode……………………………………..... 79

Figure 3 OPLS-DA (S-plot) (A) PLE 192 and CE, (B) UAE and CE and ion

intensity trend plots (C) of P. niruri extracts in negative mode. 5 (tr 1.78

min, m/z 125.0175), 6 (tr 2.04 min, m/z 247.0224), 10 (tr 2.47 min, m/z

667.0755), 11 (tr 2.64 min, m/z 463.0503), 12 (tr 2.86 min, m/z 649.0686),

13 (tr 2.94 min, m/z 169.0096), 16 (tr 3.23 min, m/z 969.0823), 17 (tr 3.30

min, m/z 951.0721), 20 (tr 3.59 min, m/z 925.0958), 21 (tr 3.70 min, m/z

969.0825), 23 (tr 3.90 min, m/z 951.0732), 25 (tr 4.23 min, m/z 463.0856),

27 (tr 4.74 min, m/z 447.0945) and 28 (tr 4.85 min, m/z 923.0792)……....81

Figure S1 Estimated effects by Pareto plot and response-surface graphs for the

phenolics content (mg/g dry plant) in ultrasound-assisted extraction…... 88

Figure S2 Estimated effects by Pareto plot and response-surface graph for the

phenolics content (mg/g dry plant) in pressurized liquid extraction……. 89

Figure S3 Structures of the substances identified in P. niruri extracts…………...... 90

Figure S4 Major fragments observed in mass spectra of glycosylated flavonoids… 91

Figure S5 Proposal of the loss of the HHDP group by ellagitannins, generating the

fragments observed in the negative mode………………………………. 92

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Drying kinetics and effect of air-drying temperature on chemical composition of

Phyllanthus amarus and Phyllanthus niruri

Figure 1 Variation of moisture ratio of (A) P. amarus and (B) P. niruri as a function

of drying time at temperatures ranging from 50 to 70°C………………..100

Figure 2 Arrhenius-type relationship between effective moisture diffusivity and

temperature for P. amarus and P. niruri samples……………………….101

Figure 3 Effect of air-drying temperature on total phenolic content (mg gallic acid

equivalent/g dry plant) of P. amarus and P. niruri samples. Data are the

mean of three replicates. Different letters above the bars indicate

significant difference (p<0.05)…………………………………………..102

Figure 4 PCA score plot generated by Pareto of Phyllanthus extracts obtained from

P. amarus and P. niruri samples submitted to different drying

temperatures. Ions detected in negative mode………….………………. 103

Figure 5 OPLS-DA (S-plot) of Phyllanthus extracts obtained from samples

submitted to different drying temperatures (A) P. amarus at 50°C and

70°C, (B) P. niruri at 50°C and 70°C. Ions in negative mode. a (tr 4.13

min, m/z 300.9967), b (tr 1.77 min, m/z 125.0233), c (tr 7.16 min, m/z

363.0160), d (tr 4.14 min, m/z 609.1443), e (tr 4.19 min, m/z 463.0852), f

(tr 3.59 min, m/z 925.0939), g (tr 3.22 min, m/z 969.0835), h (tr 3.32 min,

m/z 951.0735), i (tr 3.81 min, m/z 593.1484), j (tr 4.11 min, m/z 577.1544),

k (tr 3.13 min, m/z 291.0126)…………………………………………... 104

High-power ultrasound does not hydrolyze ellagitannins from Phyllanthus amarus

Figure 1 UPLC-QTOF-MS/MS chromatograms of of the extracts (a) control, (b)

treated with 188 W/cm2 for 9 min and (c) treated with 373 W/cm2 for 9

min.…………………….......................................................................... 116

Figure 2 HPLC chromatograms at 272 nm of the extracts (a) control, (b) treated

with 188 W/cm2 for 9 min and (c) treated with 373 W/cm2 for 9

min........................................................................................................... 117

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LISTA DE TABELAS

REVISÃO DE LITERATURA

Tabela 1 Constituintes químicos de P. amarus........................................................ 18

Tabela 2 Constituintes químicos de P. niruri........................................................... 21

Tabela 3 Principais classes de compostos fenólicos................................................. 22

Tabela 4 Estudos realizados sobre extração de compostos fenólicos em plantas

utilizando ultrassom (melhores condições encontradas)........................... 27

Tabela 5 Estudos realizados sobre extração de compostos fenólicos em plantas

utilizando líquido pressurizado (melhores condições encontradas).......... 30

ARTIGOS



Table 1 Experimental design of ultrasound-assisted extraction and results obtained

in the P. amarus extracts……………………………………………….. 46

Table 2 Experimental design of pressurized liquid extraction and results obtained

in the P. amarus extracts……………………………………………….. 49

Table 3 Comparison of different extraction methods of P. amarus…………….. 51

Table 4 Compounds determined by UPLC-ESI-QTOF-MS/MS in the P. amarus

aqueous extracts obtained from UAE, PLE and CE techniques………... 54



Table 1 Compounds tentatively determined by UPLC-QTOF-MS/MS in the P.

niruri aqueous extracts obtained from UAE, PLE and CE techniques…. 75

Table S1 Experimental design of ultrasound-assisted extraction and results obtained

in the P. niruri extracts………………………………………………...... 87

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Table S2 Analysis of variance (ANOVA) of the regression model (Eq. 1)………. 87

Table S3 Experimental design of pressurized liquid extraction and results obtained

in the P. niruri extracts……………………………………………...….. 88

Table S4 Analysis of variance (ANOVA) of the regression model (Eq. 2)………. 89



Table 1 Effective moisture diffusivities 𝐷𝑒𝑓𝑓 and activation energies Ea of P. niruri

and P. amarus at temperatures from 50 to 70 °C at air velocity of 0.5 m/s

…………………………………………………………………………...100

Table 2 The significantly changed components identified by UPLC-QTOF-MS/MS

in the P. niruri and P. amarus extracts………………………………… 105


Table 1 Effects of ultrasonic intensity and exposure time on the gallic acid content

of the control extract (pressurized liquid extraction at 120°C/24 min) of P.

amarus…....…………………………………………………………..... 115

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SUMÁRIO

1 INTRODUÇÃO ........................................................................................... 13

2 REVISÃO DE LITERATURA ................................................................... 16

2.1 O gênero Phyllanthus ................................................................................... 16

2.1.1 Phyllanthus amarus ...................................................................................... 16

2.1.2 Phyllanthus niruri ......................................................................................... 19

2.2 Extração de compostos fenólicos em plantas ............................................. 22

2.2.1 Extração assistida por ultrassom (EAU) ...................................................... 25

2.2.2 Extração com líquido pressurizado (ELP) ................................................... 28

3 ARTIGOS…………………………………………………………………..38

3.1 Ultrasound-assisted and pressurized liquid extraction of phenolic

compounds from Phyllanthus amarus and its composition evaluation by

UPLC-QTOF………………………………………………………………. 38

3.2 UPLC-QTOF-MSE-based chemometric approach driving the choice of

the best extraction process for Phyllanthus niruri….................................. 66

3.3 Drying kinetics and effect of air-drying temperature on chemical

composition of Phyllanthus amarus and Phyllanthus niruri….................. 93

3.4 High-power ultrasound does not hydrolyze ellagitannins from

Phyllanthus amarus…..................................................................................110

4 CONCLUSÃO..............................................................................................119

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1. INTRODUÇÃO

Por milênios, as plantas medicinais têm sido uma fonte valiosa de agentes

terapêuticos, e muitos dos medicamentos encontrados atualmente são derivados de

produtos naturais. Os vegetais representam as maiores fontes de substâncias ativas que

podem ser usadas na terapêutica, devido à grande diversidade estrutural de metabólitos

produzidos. Nos últimos anos tem havido um renascimento do interesse em fármacos

naturais ou à base de plantas. Ao contrário das drogas modernas que geralmente

incluem uma única espécie ativa, os extratos vegetais contêm múltiplos constituintes

bioativos. Esses compostos podem agir de forma combinada ou sinérgica dentro do

corpo humano, e podem fornecer propriedades terapêuticas únicas com efeitos

colaterais indesejáveis mínimos ou inexistentes (ATANASOV et al., 2015; BRANDÃO

et al., 2010; HUIE, 2002).

No Brasil, cerca de 80% da população utiliza produtos à base de plantas

medicinais nos seus cuidados com a saúde, seja pelo conhecimento tradicional, ou nos

sistemas oficiais de saúde, como prática de cunho científico, orientada pelos princípios e

diretrizes do Sistema Único de Saúde (SUS) (RODRIGUES & DE SIMONI, 2010).

Muitos foram os avanços nas últimas décadas com a formulação e implementação de

políticas públicas, programas e legislação com vistas à valorização das plantas

medicinais e derivados. A Relação Nacional de Plantas Medicinais de Interesse ao SUS

(Renisus) apresenta plantas medicinais que possuem potencial para gerar produtos de

interesse ao SUS. A finalidade da lista é orientar estudos e pesquisas que possam

subsidiar a elaboração da relação de fitoterápicos disponíveis para uso da população,

com segurança e eficácia para o tratamento de determinada doença. Dentre as 71

espécies que estão cadastradas no Renisus, constam quatro espécies do gênero

Phyllanthus: P. amarus, P. niruri, P. tenellus e P. urinaria (BRASIL, 2012).

As plantas pertencentes ao gênero Phyllanthus (Phyllanthaceae), conhecidas no

Brasil como quebra-pedra, estão presentes na medicina popular brasileira e de muitos

outros países, onde é comum o uso da infusão de diferentes partes dessas plantas para o

tratamento de um largo espectro de doenças, tais como distúrbios renais urinários,

infecções intestinais e diabetes (CALIXTO et al., 1998). Estudos farmacológicos e

testes pré-clínicos e clínicos confirmam as propriedades medicinais de P. amarus e P.

niruri que têm sido mencionadas na medicina tradicional (BAGALKOTKAR et al.,

2006; PATEL et al., 2011). Essas propriedades medicinais estão associadas a alguns dos

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seus constituintes ativos, como lignanas, alcalóides, taninos, terpenos e flavonóides

(BAGALKOTKAR et al., 2006; CALIXTO et al., 1998; PATEL et al., 2011).

Para a produção de fitoterápicos, a qualidade é um aspecto importante que

envolve todo o processo de produção, englobando tanto o estabelecimento rigoroso de

padrões de qualidade da matéria-prima até os processos de preparação de extratos

vegetais, a fim de se obter produtos com uniformidade química (CALIXTO, 2001). A

composição e a bioatividade de extratos vegetais dependem fortemente do processo de

extração utilizado. Atualmente, tem se buscado a utilização de técnicas e processos que

reduzam ou eliminem os solventes, reagentes e outros produtos químicos que são

perigosos para a saúde humana e para o ambiente. A extração verde baseia-se na

descoberta e design de processos de extração que reduzam o consumo de energia,

permitam o uso de solventes alternativos de menor impacto ambiental ou evitem o uso

de solventes, e a utilização de produtos naturais renováveis e que garantam um extrato

seguro e de alta qualidade (ARMENTA et al., 2015). Dentre as técnicas de extração

verde aplicadas com sucesso temos a extração assistida por ultrassom e a extração com

líquido pressurizado.

O ultrassom de potência é uma técnica que acelera consideravelmente o processo

de extração e pode reduzir o consumo de energia. É um processo que utiliza baixa

temperatura e de execução rápida, que geralmente não degrada o extrato. Também

oferece vantagens em termos de produtividade, rendimento e seletividade, melhora o

tempo de processamento, melhora a qualidade, reduz riscos físicos e químicos e é

ecologicamente correto (ALEXANDRU et al., 2014). A extração com líquido

pressurizado utiliza temperatura e pressão elevadas, que além de melhorar o rendimento

de extração, diminui o tempo e consumo de solvente. É realizada em sistemas

automatizados e possui alta reprodutibilidade (MUSTAFA & TURNER, 2011). Estas

técnicas de extração podem ser aplicadas utilizando água como solvente.

Extratos vegetais brutos constituem matrizes bastante complexas contendo

vários metabólitos, geralmente de diferentes classes químicas, o que torna difícil a

identificação do perfil químico. Por isso, uma eficiente e rápida caracterização tem

papel fundamental na pesquisa de produtos naturais. Nesse sentido, a utilização de

técnicas hifenadas, como o LC-MS/MS (cromatografia líquida acoplada com detetor

seletivo de massas em modo tandem), é de grande valia, pois fornece numerosas

informações estruturais dos metabólitos antes mesmo do seu isolamento. Em estudos

comparativos envolvendo diferentes extratos, a quimiometria é frequentemente utilizada

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para facilitar a interpretação do grande número de informações obtidas (RODRIGUES

et al., 2006).

Um fator importante para a qualidade da matéria-prima vegetal é o processo de

secagem. A secagem diminui a velocidade de deterioração do material, por meio da

redução no teor de água, reduzindo ainda a ação de enzimas, possibilitando a

conservação das plantas por maior tempo. Com a redução da quantidade de água,

aumenta-se, também, a quantidade de princípios ativos em relação à massa seca (MELO

et al., 2004). A secagem ao ar quente ou secagem convectiva é uma técnica amplamente

adotada na indústria (KARAM et al., 2016). Dependendo das condições de secagem,

como temperatura, velocidade do ar e tempo, o teor de fitoquímicos do material vegetal

pode aumentar ou diminuir, por isso é importante se determinarem as melhores

condições de secagem de cada material.

Com base nos fatores citados acima, esta tese de doutorado teve como objetivo

geral contribuir para o estudo fitoquímico de duas espécies de quebra-pedra (P. amarus

e P. niruri), determinando as melhores condições de extração aquosa para obtenção de

extratos padronizados com alto conteúdo fenólico e com perfil de metabólitos com

relevância farmacológica e avaliando o efeito da temperatura de secagem das plantas

sobre a composição química. Os objetivos específicos foram:

• Avaliar o efeito de algumas variáveis de extração sobre a extração assistida por

ultrassom (EAU) e extração com líquido pressurizado (ELP) na obtenção de

compostos fenólicos de P. amarus e P. niruri;

• Comparar conteúdo fenólico e composição química entre os extratos obtidos

pelas técnicas de EAU, ELP e extração convencional nas duas espécies, a fim de

se definir o método de extração mais adequado;

• Determinar dados de cinética de secagem e analisar o efeito da temperatura do ar

de secagem sobre a composição química e sobre o conteúdo fenólico das partes

aéreas de P. amarus e P. niruri.

16

2. REVISÃO DE LITERATURA

2.1. O gênero Phyllanthus

O gênero Phyllanthus pertence à família Phyllanthaceae, táxon desmembrado da

família Euphorbiaceae (APG III, 2009). As plantas pertencentes ao gênero Phyllanthus

estão amplamente distribuídas por países tropicais e subtropicais. Este gênero possui

aproximadamente 750 espécies, sendo 200 delas encontradas nas Américas e cerca de

100 no Brasil. As espécies apresentam hábito variado, sendo principalmente herbáceo,

havendo, contudo, espécies arbóreas de pequeno porte e arbustos (CALIXTO et al.,

1998; SECCO et al., 2010; SILVA & SALES, 2007).

Entre os representantes do gênero utilizados pelo homem destacam-se as

espécies conhecidas no Brasil como quebra-pedra, arrebenta-pedra ou erva-pombinha,

entre elas P. niruri L., P. amarus Schum. & Thonn, P. urinaria L. e P. tenellus Roxb.

Müll. Arg. (TORRES et al., 2003).

2.1.1. Phyllanthus amarus

É uma erva ou subarbusto, de 14-70 cm. Possui ramificação filantóide, com

ramos medindo de 3,2-9 cm, pinatiformes, angulosos. A lâmina foliar é membranácea,

elíptica ou oblonga, base obtusa ou ligeiramente assimétrica, ápice obtuso, em geral

mucronado, margem inteira com 0,4-1,1 cm de comprimento e 0,3-0,5 cm de largura. A

inflorescência apresenta-se em cimeiras proximais onde se encontra uma flor feminina e

outra masculina, todas protegidas por duas bractéolas escariosas, lineares (SILVA &

SALES, 2007).

Figura 1. Phyllanthus amarus

Fonte: PATEL et al. (2011).

17

P. amarus está distribuída pela India e China. Nas Américas é encontrada desde

os Estados Unidos até a Argentina. No Brasil distribui-se da região Norte a Sul,

crescendo em ambientes úmidos, ou ainda como ruderal ou invasora em áreas

agricultáveis. É também comum em jardins e frestas de calçadas. Floresce e frutifica

durante todo o ano (PATEL et al., 2011; SILVA & SALES, 2007).

Diversos estudos farmacológicos com P. amarus têm sido publicados. Lee et al.

(2011) demonstraram que os extratos aquoso e metanólico inibiram o crescimento de

células de câncer de mama e de pulmão. Segundo os autores, a capacidade de P. amarus

exercer atividades antimetastáticas é geralmente associada com a presença de

compostos polifenólicos em seus extratos.

A atividade analgésica e anti-inflamatória do extrato aquoso das folhas de P.

amarus foi investigada através de modelos térmicos e químicos de avaliação da dor em

ratos. O extrato causou uma inibição significativa, de forma dose dependente, do edema

de pata induzido por carragenina em ratos. Esse efeito inibitório produzido pelo extrato

foi significativamente mais elevado do que a droga de referência (ácido acetilsalicílico).

Além disso, o extrato aquoso de P. amarus também apresentou atividade analgésica nas

fases precoce e tardia de modelo de dor induzida por injeção de formalina em pata de

ratos (IRANLOYE et al., 2011).

O extrato aquoso das folhas de P. amarus bloqueou a ação das enzimas do vírus

HIV-1 integrase, transcriptase reversa e protease em diferentes graus, inibindo a

replicação do vírus HIV-1, e os elagitaninos isolados geranina e corilagina mostraram

ser os mediadores mais potentes desta atividade antiviral (NOTKA et al., 2004).

Alli et al. (2011) avaliaram o efeito de extratos das partes aéreas de P. amarus

contra Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi,

Staphylococcus aureus e Candida albicans e os extratos aquoso e metanólico foram

ativos contra todos os microrganismos estudados.

Em um estudo clínico conduzido por Srividya & Periwal (1995) foram avaliadas

as atividades diurética, hipotensora e hipoglicemiante de P. amarus. Nove pacientes

hipertensos (4 dos quais também com diabetes mellitus) foram tratados com uma

preparação da planta inteira de P. amarus durante 10 dias. Foram observados

parâmetros apropriados em amostras de urina e de sangue, juntamente com o perfil

fisiológico e padrão alimentar, antes e após o período de tratamento. Houve um

aumento significativo no volume de urina, junto a uma redução na pressão sistólica de

pacientes hipertensos e não diabéticos. O nível de glicose no sangue também foi

18

significativamente reduzido no grupo tratado. As observações clínicas não revelaram

efeitos colaterais.

Essas atividades farmacológicas se devem à presença de compostos bioativos.

Muitos estudos têm isolado e identificado moléculas de P. amarus e diferentes classes

de compostos orgânicos têm sido relatadas, como alcalóides, flavonóides, esteróis,

terpenos, lignanas e taninos. A tabela 1 apresenta os metabólitos secundários presentes

em P. amarus.

Tabela 1. Constituintes químicos de P. amarus

CLASSE COMPOSTO REFERÊNCIA

ALCALÓIDES 4-metoxi-norsecurinina, dihidrosecurinina, GUO et al. (2015);

epibubbialina, isobubbialina, niruroidina, PATEL et al. (2011);

norsecurinina, securinina QI et al. (2014)

ESTERÓIS amarosterol-A e amarosterol B PATEL et al. (2011);

QI et al. (2014)

FLAVONÓIDES astragalina, galocatequina, kaempferol, GUO et al. (2015);

kaempferol-3-O-rutinosídeo, luteolina, KUMAR et al. (2015);

miricetrina, quercetina, quercitrina, PATEL et al. (2011);

quercetina-3-O-glucosídeo, rutina SPRENGER & CASS,

2013; QI et al. (2014)

LIGNANAS 3-(3,4-dimetoxi-benzil)-4-(7-methoxi-benzo [1,3] GUO et al. (2015);

dioxol-5-il-metil)-dihidrofuran-2-onae KUMAR et al. (2015);

4-(3,4-dimetoxi-fenil)-1-(7-metoxi-benzo PATEL et al. (2011);

[1,3]dioxol-5-il)-2,3-bis-metoximetil-butan-1-ol QI et al. (2014)

5-demetoxinirantina, filantina, filtetralina,

hipofilantina, hinoquinina, isonirtetralina,

lintetralina, nirantina, nirtetralina,

virgatusina, pinoresinol

ÓLEOS VOLÁTEIS linalool e fitol PATEL et al. (2011)

TANINOS 1,6-digaloilglucopiranose, ácido 4-O-galoilquínico, GUO et al. (2015);

HIDROLISÁVEIS ácido amariínico, ácido geraniínico B, ácido KUMAR et al. (2015);

repandusínico A, amariina, amarulona, castalina, PATEL et al. (2011);

corilagina, elaeocarpusina, emblicanina A, furosina, SPRENGER & CASS

filantusiina A, B, C e D, geraniina, melatonina (2013); QI et al. (2014)

TERPENOS ácido oleanólico, ácido ursólico, filantenol, KUMAR et al. (2015);

filantenona, filanteol, lupeol PATEL et al. (2011)

OUTROS ácido carboxílico da brevifolina, GUO et al. (2015);

ácido elágico, ácido elágico-O-hexosídeo, KUMAR et al. (2015);

19

ácido gálico, ácido quínico SPRENGER & CASS

ácido tri-O-metilelágico (2013)

2.1.2. Phyllanthus niruri

Erva ou subarbusto, de 12-73 cm. Possui ramificação filantóide, com ramos

medindo de 3-15,5 cm, angulosos. Limbo foliar 0,5-1,5×0,25-0,6 cm, membranáceo,

oblongo a oblongo-elíptico, oval-oblongo ou oval-elíptico, base oblíqua, ápice obtuso

a arredondado. Flores em címulas unissexuais, as estaminadas proximais com 3-7

flores, as pistiladas distais com uma única flor. Brácteas lineares a lanceoladas (SILVA

& SALES, 2007). P. niruri diferencia-se de P. amarus pelas folhas assimétricas,

inflorescências unissexuais e estiletes capitados, apresentando P. amarus folhas

simétricas, inflorescências bissexuais e estiletes agudos (TORRES et al., 2003).

Figura 2. Phyllanthus niruri

Fonte: LORENZI & MATOS (2002).

P. niruri pode ser encontrada na Ásia, Índia e nas Américas, distribuída do Sul

do Texas (Estados Unidos) à Argentina, incluindo Antilhas. No Brasil ocorre em todas

as regiões, em diferentes tipos vegetacionais, em locais úmidos e sombreados ou em

áreas ruderais. Encontrada florida e com frutos durante todo o ano (BAGALKOTKAR

et al., 2006; SILVA & SALES, 2007).

Muitas atividades farmacológicas da espécie P. niruri tem sido descritas. Barros

et al. (2003) estudaram o efeito do extrato aquoso de P. niruri sobre a cristalização de

oxalato de cálcio (CaOx) in vitro, e os resultados mostraram que o extrato reduziu o

20

crescimento e a agregação dos cristais de CaOx, evidenciando o seu potencial de

interferir nas fases iniciais da formação de cálculos renais. P. niruri também modificou

a estrutura do cálculo em ratos para uma forma mais suave e, possivelmente, mais frágil

que poderia facilitar a remoção ou dissolução dos cálculos (BARROS et al., 2006). Um

estudo clínico demonstrou que cápsulas contendo extrato aquoso liofilizado de P. niruri

reduziram o cálcio urinário em pacientes hipercalciúricos (NISHIURA et al., 2004).

A atividade hepatoprotetora de P. niruri contra a cirrose hepática induzida por

tioacetamida (TAA) em ratos foi avaliada. Os animais receberam injeções

intraperitoneais de TAA três vezes por semana e tratamentos diários com o extrato de P.

niruri por via oral durante oito semanas. Os resultados revelaram que o tratamento com

P. niruri reduziu significativamente o efeito de toxicidade de TAA, apresentando o

extrato uma atividade hepatoprotetora eficaz. Na fração ativa de P. niruri foram

isolados dois compostos: ácido 4-O-cafeoilquínico e quercetina-3-O-ramnosídeo

(AMIN et al., 2013).

Couto et al. (2013) estudaram as atividades anti-inflamatória e antialodínica

(analgésica) de extratos aquosos de diferentes partes de P. niruri. Os extratos das folhas

ou de folhas+caules demonstraram um prolongamento da ação antialodínica. Além

disso, o extrato das folhas diminuiu significativamente a inflamação. Foi observada uma

relação direta entre os efeitos anti-inflamatórios e analgésicos com o teor de ácido

gálico, mas a utilização do extrato de folhas+caules mostrou ser mais eficaz, o que

sugere um efeito sinérgico entre os seus constituintes. A corilagina, que é encontrada

em abundância em extratos de P. niruri, também foi identificada como um tanino anti-

hiperalgésico (analgésico), que deriva a sua atividade a partir de seu envolvimento no

sistema glutamatérgico (MOREIRA et al., 2013).

O potencial hipoglicêmico do extrato metanólico das partes aéreas de P. niruri

foi avaliado em ratos normais e diabéticos. A administração oral do extrato causou uma

significativa redução nos níveis de glicose no sangue de um modo dose dependente,

bem como nos níveis de colesterol total e triglicérides em ratos diabéticos e

normoglicêmicos. Os resultados sugerem que o extrato das partes aéreas de P. niruri

tem grande potencial como fármaco antidiabético (OKOLI et al., 2010).

Estudos fitoquímicos sobre P. niruri têm revelado a presença principalmente de

taninos, flavonóides, alcalóides, terpenos e lignanas, que são responsáveis pelas

atividades farmacológicas desta planta. A Tabela 2 resume os vários compostos que

foram isolados a partir de P. niruri.

21

Tabela 2. Constituintes químicos de P. niruri

CLASSE COMPOSTO REFERÊNCIA

ALCALÓIDES 4-metoxi-norsecurinina, alosecurinina, BAGALKOTKAR et al.

Nirurina, norsecurinina, securinina (2006); CALIXTO et al.

(1998); QI et al. (2014)

ESTERÓIS β-sitosterol, estradiol CALIXTO et al. (1998)

FLAVONÓIDES astragalina, (-)-epigalocatequina, BAGALKOTKAR et al.

(-)-epigalocatequina-3-O-galato, (2006); CALIXTO et al.

eriodictiol-7-O-α-L-ramnopiranosídeo, (1998); QI et al. (2014);

kaempferol-4’-O-α-L-ramnopiranosídeo, SPRENGER & CASS

galocatequina, miricetrina, niruriflavona, (2013)

orientina, orientina-2”-O-ramnosídeo, quercetina,

quercetina-3-O-β-D-glucopiranosil-(1→2)-β-D-

xilopiranosídeo, quercetina-3-O-glucosídeo,

quercitrina, rutina, vitexina-2”-O-ramnosídeo

LIGNANAS 4-hidroxisecolintetralina, filantina, filnirurina, BAGALKOTKAR et al.

filtetralina, hidroxinirantina, hinoquinina, (2006); CALIXTO et al.

hipofilantina, isolintetralina, linantina, lintetralina, (1998); QI et al. (2014);

neonirtetralina, nirantina, nirfilina, nirtetralina,

secoisolariciresinol trimetil eter, sesamin-4-ol

TANINOS ácido repandusínico A, β-glicogalina, BAGALKOTKAR et al.

HIDROLISÁVEIS corilagina, filantusiina D, geraniina, (2006); QI et al. (2014);

Isocorilagina SPRENGER & CASS

(2013)

TERPENOS filantenol,filantenona, filanteol, limoneno, BAGALKOTKAR et al.

lupeol, ρ-cimeno (2006); QI et al. (2014)

OUTROS 1-O-galoil-6-O-luteoil-α-D-glucopiranosídeo, BAGALKOTKAR et al.

ácido carboxílico da brevifolina, ácido elágico, (2006); QI et al. (2014);

ácido gálico, brevifolina, filangina, nirurisídeo, SPRENGER & CASS

metil- brevifolinacarboxilato (2013)

22

2.2. Extração de compostos fenólicos em plantas

Compostos fenólicos são importantes metabólitos secundários sintetizados por

plantas durante o desenvolvimento normal e em resposta a condições de estresse como

infecções, ferimentos, radiações UV, dentre outros. Eles são biossintetizados através de

duas rotas metabólicas: a do ácido chiquímico e a do ácido malônico, que levam a

diferentes classes de compostos que são resumidos na Tabela 3 (AZMIR et al., 2013;

SANTOS-BUELGA et al., 2012). Eles podem ocorrer em suas fontes naturais de forma

livre, como derivados glicosilados, e como estruturas oligoméricas ou polimerizadas,

tais como os taninos hidrolisáveis e condensados. Eles também podem ser encontrados

ligados aos componentes da matriz da planta, como constituintes de parede celular,

carboidratos ou proteínas (SANTOS-BUELGA et al., 2012).

Tabela 3. Principais classes de compostos fenólicos

CLASSE ESQUELETO BÁSICO EXEMPLOS

FENÓLICOS SIMPLES

(C6)

OH

Floroglucinol, catecol,

resorcinol, vanilina,

seringaldeído

ÁCIDOS FENÓLICOS

Ácidos hidroxibenzóicos

(C6-C1) COOH

ácido salicílico, ácido

siríngico, ácido gálico

ácidos hidroxicinâmicos

C6-C3) e derivados COOH

ácido cafeico, ácido

cumárico, ácido ferúlico

CUMARINAS (C6-C3) O O

escopoletina,

umbeliferona,

aesculetin

NAFTOQUINONAS

(C6-C4)

O

O

juglona, pumblagina

XANTONAS (C6-C1-C6) O

O

mangostina, mangiferina

23

ESTILBENOS (C6-C2-C6)

resveratrol, piceid,

e-viniferina

FLAVONÓIDES

(C6-C3-C6)

Flavan-3-óis

O

OH

epicatequina,

epigalocatequina

Flavonas

O

O

apigenina, luteolina,

crisina, escutelareína,

diosmetina, crisoeriol

Flavonóis

O

O

OH

quercetina, kaempferol,

miricetina, galangina,

fisetina, morina

Flavanonas

O

O

hesperidina, naringenina,

taxifolina, eriodictiol,

isosakuranetina

Antocianinas

O

OH

cianidina, delfinidina,

malvidina, peonidina,

pelargonidina,

petunidina

Isoflavonas O

O

genisteína, daidzeína,

gliciteína, puerarina,

formononetina,

biochanina A

Taninos condensados

(proantocianidinas)

(C6-C3-C6)n

O

OH

O

OH

procianidinas,

prodelfinidinas

24

Taninos hidrolisáveis

(galotaninos, elagitaninos)

O

OO

O

O

O

O

OH

O

O

OH

OH

OH

OH

HO

HO

HO

HO

OH

HO

O

O

OH

OH

OH

O

HO

O

ácido amariínico, ácido

repandusínico A,

amariina, amarulona,

castalina, corilagina,

filantusiina D, furosina,

geraniina, emblicanina

A, pentagaloilglucose

Lignanas (C6-C2)2

filantina, hipofilantina,

nirantina, hinoquinina,

nirtetralina, filtetralina,

virgatusina

Ligninas (C6-C3)n

O

O

HOO

HO

OO

O

CH3

H3C

CH3

Fonte: adaptado de SANTOS-BUELGA et al. (2012).

A diversidade estrutural dos compostos fenólicos afeta as suas características

físico-químicas, tal como a solubilidade. A polaridade dos compostos varia

significativamente com a sua estrutura, natureza de conjugação e associação com a

matriz da amostra. Formas ligadas e compostos fenólicos de alta massa molecular

podem ser bastante insolúveis. Além disso, os compostos fenólicos não são

uniformemente distribuídos na planta e sua estabilidade varia significativamente, alguns

sendo relativamente estáveis e outros sendo voláteis, termolábeis e/ou facilmente

propensos à oxidação (AZMIR et al., 2013; SANTOS-BUELGA et al., 2012). Por isso,

não há nenhum procedimento uniforme que seja apropriado para a preparação de

amostra e extração de todos os fenóis ou de uma classe específica de substâncias

fenólicas em plantas. Sendo assim, processos específicos devem ser desenvolvidos,

buscando as melhores condições para cada fonte fenólica.

25

Devido à complexidade da maioria das matrizes, o procedimento de preparação

da amostra é uma etapa crítica de todo o processo. Secagem, moagem e

homogeneização são pré-tratamentos comuns antes da extração. A secagem aumenta a

estabilidade do material e a moagem frequentemente melhora a cinética da extração dos

analitos (ONG, 2004; SANTOS-BUELGA et al., 2012).

Os métodos de extração geralmente indicados em farmacopeias são o

aquecimento sob refluxo, extração por Soxhlet e a maceração. No entanto, tais métodos

podem ser demorados, requerer o uso de grande quantidade de solvente orgânico e

podem apresentar menor eficiência de extração (ONG, 2004). Assim, o uso de

tecnologias verdes para reduzir e/ou eliminar o uso ou a produção de materiais

perigosos e que sejam mais eficientes é altamente desejável. Algumas das técnicas mais

promissoras são a extração assistida por ultrassom e a extração com líquido

pressurizado.

2.2.1. Extração assistida por ultrassom (EAU)

Ultrassom é um tipo especial de onda sonora com frequência entre 20 e 100 KHz

que promove vibrações em um meio líquido e causa o fenômeno de cavitação, onde há a

produção, o crescimento e o colapso de bolhas. Esse colapso gera uma onda de choques

que circulam pelo meio líquido e resultam em impacto e aumento da tensão de

cisalhamento (PESSOA JÚNIOR & KILIKIAN, 2005). O principal benefício da EAU

pode ser observado na amostra vegetal sólida, porque a energia de ultrassom facilita a

lixiviação de compostos orgânicos e inorgânicos da matriz da planta (HERRERA &

LUQUE DE CASTRO, 2005). O provável mecanismo do ultrassom é a intensificação

da transferência de massa e acesso acelerado do solvente a materiais celulares de partes

da planta. O teor de umidade da amostra, o tamanho de partícula e o solvente são fatores

muito importantes para a obtenção de uma extração eficiente. Além disso, a

temperatura, a frequência, a potência e o tempo de sonicação são fatores decisivos para

a ação do ultrassom. As vantagens da EAU incluem a redução no tempo de extração,

energia e utilização de solvente. A energia ultrassônica para a extração também facilita

uma mistura mais efetiva, acelera a transferência de energia, reduz os gradientes

térmicos e temperatura de extração, extração seletiva, reduzido tamanho do

equipamento e aumento da produção (AZMIR et al., 2013).

Existem dois tipos distintos de aparelhos geradores de ondas ultrassonoras: o

banho de ultrassom e a sonda (Figura 3). No banho de ultrassom, o transdutor é

26

diretamente preso no fundo da cuba do aparelho e a energia ultrassonora é transmitida

através de um líquido, usualmente a água. A energia é irradiada verticalmente pelas

ondas sonoras geradas na base do banho e transmitidas através das paredes do vaso para

o frasco com a mistura extratora (TIWARI, 2015; VINATORU, 2001). Apresenta como

vantagens uma melhor distribuição de energia através das paredes do vaso de extração e

o fato de não requerer adaptação especial para o frasco extrator. Apresenta como

desvantagens o fato de que a quantidade de energia fornecida para o frasco extrator não

é facilmente quantificável, porque depende do tamanho do banho, do tipo de recipiente,

da espessura das paredes do recipiente e da posição do frasco de extração no banho. É

difícil controlar a temperatura do sistema, pois o equipamento tende a aquecer quando

usado por longos períodos (a temperatura do meio extrator é mais alta que a temperatura

do líquido no banho) (VINATORU, 2001).

Figura 3. Esquema de sistemas de aplicação de ondas ultrassonoras: a) sonda, b) banho

A sonda, por outro lado, encontra-se fixada na extremidade do amplificador do

transdutor, em contato direto com o sistema extrator. Apresenta como vantagens a

potência totalmente disponível (não há transferência de irradiação ultrassônica pelas

paredes do vaso) e a possibilidade de ser ajustada para fornecer um melhor desempenho

a diferentes potências. Como desvantagens apresenta: frequência fixa e dificuldade de

controle de temperatura em sistemas sem refrigeração. A sonda ultrassônica permite

melhores rendimentos de extração que o banho de ultrassom (TIWARI, 2015;

VINATORU, 2001).

EAU é uma técnica de extração eficiente para a obtenção de compostos

fenólicos de materiais vegetais. Estudos têm mostrado que o ultrassom pode melhorar o

rendimento de extração e diminuir o tempo de extração em comparação com métodos

convencionais. A tabela 4 resume algumas aplicações da sonda ultrassônica na

recuperação de compostos fenólicos em plantas.

27

Tabela 4. Estudos realizados sobre extração de compostos fenólicos em plantas

utilizando ultrassom (melhores condições encontradas)

Material Solvente Frequência

(KHz)

Potência

(W)

Tempo

(min)

Razão L/S

(mL/g) Referência

Pistacia

lentiscus Etanol/

água 24 68 14 40

DAHMOUNE et al.

(2015)

Cassia

auriculata Metanol/

água - 50 5 25

SHARMILA et al.

(2016)

Garcinia

indica Água 24 200 35 10 NAYAK.& RASTOGI

(2013)

Mangifera

indica Etanol/

água 40 200 19 38 ZOU et al. (2014)

Sparganii

rizoma Etanol/

água 25 300 40 19 WANG et al. (2013)

Garcinia

mangostana Etanol 20 100 25 20 CHEOK et al. (2013)

Psidium

Guajava Água - 1100 5 12 LIU et al. (2014)

Euryale ferox Etanol/

água 53 500 21 31 LIU et al. (2013)

Areca catechu Acetona/

água 20 30 50 10 CHAVAN &

SINGHAL (2013)

Origanum

majorana Água 20 1500 15 50

HOSSAIN et al.

(2012)

Nos estudos citados na Tabela 4 foram avaliados diferentes parâmetros

operacionais da extração assistida por ultrassom. A variável tempo de extração foi

analisada em todos os trabalhos, sendo avaliada em uma faixa entre 2 e 50 min. 70%

dos resultados apresentaram o tempo ótimo de extração abaixo de 26 min. Em geral o

tempo de sonicação exibiu efeito positivo. Contudo, segundo alguns autores (CHAVAN

& SINGHAL, 2013; DAHMOUNE et al., 2015; LIU et al., 2013; ZOU et al., 2014), a

exposição de polifenóis às ondas ultrassônicas por um período mais longo pode resultar

na destruição estrutural dos mesmos, diminuindo o rendimento. Além disso, radicais

livres podem ser formados quando são usados tempos e amplitudes elevados. Outra

variável bastante estudada é a razão líquido/sólido. Ela foi avaliada em uma faixa geral

de 2 a 50 mL/g, apresentando melhores resultados de rendimento de fenólicos quando

utilizados valores de razão L/S próximos aos extremos superiores das faixas estudadas.

Geralmente, uma maior razão L/S pode dissolver os constituintes mais eficazmente,

levando a um aumento do rendimento de extração (ZOU et al., 2014). Um parâmetro

também muito estudado é a potência de sonicação. Dependendo do tipo de material a

potência necessária pode ser mais elevada ou mais baixa. No trabalho de Sharmila et al.

28

(2016) quando se elevou a potência de 30 para 50 W a extração de polifenóis aumentou.

De acordo com os autores, a potência de sonicação é um parâmetro chave para aumentar

a eficiência da extração por promover o rompimento da célula da planta, permitindo que

o solvente se difunda mais facilmente e extraia os compostos fenólicos. Wang et al.

(2013) também observaram um aumento, de aproximadamente 40%, no rendimento de

extração de fenólicos quando a potência passou de 150 para 300 W. Já Chavan &

Singhal (2013) verificaram um efeito negativo da potência. O aumento da potência pode

elevar a temperatura do meio de extração devido à geração de calor e diminuir o

rendimento de extração de fenóis, provavelmente devido a decomposição. Outras

variáveis estudadas foram: porporção de solvente (etanol, metanol, acetona) em água,

temperatura, amplitude e ciclo de sonicação.

2.2.2. Extração com líquido pressurizado (ELP)

Este método é conhecido por vários nomes, extração com fluido pressurizado,

extração com solvente acelerado e extração com solvente a alta pressão. A técnica é

referida como extração com água quente pressurizada (PHWE), extração com água

subcrítica ou extração com água superaquecida quando a água é utilizada como o agente

de extração. O conceito de ELP é a aplicação de alta pressão, mantendo o solvente

líquido em temperatura além do seu ponto de ebulição normal. A técnica de ELP requer

pequenas quantidades de solventes, por causa da combinação de alta pressão e

temperatura que permite extrair mais rápido. A temperatura de extração elevada pode

promover uma maior solubilidade dos compostos e aumento da taxa de transferência de

massa, além de diminuir a viscosidade e tensão superficial de solventes, melhorando

assim a taxa de extração (HUIE, 2002; HENG et al., 2013).

Para a extração com líquido pressurizado, dependendo do teor de água, o

material vegetal é normalmente disperso em um adsorvente inerte (por exemplo, sulfato

de sódio, terra diatomácea ou outros). A mistura de adsorvente inerte e amostra vegetal

é acondicionada em uma célula de aço inoxidável e inserida em um sistema de fluxo

fechado. Existem duas estruturas principais para ELP: instrumentos estáticos e

dinâmicos. Para a ELP em modo dinâmico, o solvente de extração é continuamente

bombeado através da célula de extração. A operação envolve o ajuste da taxa de fluxo

durante o tempo estático e a bomba fornece o solvente, a uma taxa de fluxo constante,

durante um determinado período de tempo (por exemplo, 1,0-1,5 mL/min por 20-30

minutos) (MUSTAFA & TURNER, 2011; HENG et al., 2013).

29

Por outro lado, para ELP em modo estático, uma vez que os parâmetros de

temperatura e pressão são atingidos, a extração é realizada por um tempo

predeterminado. A faixa comum é de 5-15 minutos que é feita em ciclos diferentes.

Comparado a ELP em modo dinâmico, o processo de extração em modo estático

compreende um ou vários ciclos de extração com a substituição do solvente entre os

ciclos. A célula contendo a amostra é purgada com um gás inerte para lavar o solvente

da célula. Uma ampla faixa de temperatura de extração desde a temperatura ambiente

até 200C e a faixa de pressão de 35-200 bar podem ser aplicadas para ELP

(MUSTAFA & TURNER, 2011; HENG et al., 2013). A figura 4 apresenta um diagrama

esquemático do funcionamento de um extrator com líquido pressurizado.

Figura 4. Esquema de funcionamento de um extrator com líquido pressurizado (ELP)

Fonte: adaptado de KO et al. (2011)

Aplicações da técnica de ELP para a obtenção de compostos bioativos a partir de

produtos naturais estão disponíveis na literatura (AZMIR et al., 2013; MUSTAFA &

TURNER, 2011; HENG et al., 2013). A ELP tem sido aplicada com sucesso para a

extração de compostos fenólicos de plantas (Tabela 5). Em comparação com a

tradicional extração por soxhlet, a ELP diminui drasticamente o consumo de tempo e

solvente. Hoje em dia, para a extração de compostos polares, ELP também é

considerada uma técnica alternativa potencial à extração com fluido supercrítico (EFS),

30

já que a EFS é mais seletiva para compostos de baixa ou média polaridade (AZMIR et

al., 2013).

Tabela 5. Estudos realizados sobre extração de compostos fenólicos em plantas

utilizando líquido pressurizado (melhores condições encontradas)

Material Solvente Temperatura

(°C)

Pressão

(bar)

Tempo

(min)

Ciclos ou

fluxo Referência

Phyllanthus

amarus Etanol/

água 50 100 60

2,5

mL/min

PEREIRA et al.

(2016)

Phyllanthus

niruri Água 100 100 60 1,5

mL/min

MARKOM et al.

(2010)

Schinus

terebinthifolius

Etanol/

água 100 103 10 1

FEUEREISEN et al.

(2017)

Berberis cretica Água 100 103 15 1 KUKULA-KOCH et

al. (2013)

Tilia

Cordata Metanol/

água 120 60 30

3 de 10

min

ONISZCZUK &

PODGÓRSKI (2015)

Rosmarinus

officinalis Metanol/

água 129 103 5 1

HOSSAIN et al.

(2011)

Coriandrum

Sativum Água 100 88 10 1 ZEKOVIĆ et al.

(2016)

Schisandra

chinensis Etanol/

água 160 103 10 1 ZHAO et al. (2012)

Olea europaea Etanol 190 103 45 3 de 15

min XYNOS et al. (2014)

Heracleum

leskowii Metanol 100 103 10 1

SKALICKA-

WOŹNIAK &

GŁOWNIAK (2012)

Capsicum

annuum Etanol/

água 93 100 5 1 KANG et al. (2016)

Nos estudos sumarizados na Tabela 5 foram analisados diferentes parâmetros

operacionais da extração com líquido pressurizado (ELP). A temperatura de extração foi

avaliada em todos os estudos, em uma faixa geral de 35 a 200°C. Em 10 dos 11

trabalhos a temperatura apresentou efeito positivo, ficando com a condição ótima em

um valor igual ou próximo ao do limite máximo da faixa estudada. A elevação da

temperatura aumenta a solubilidade de muitos compostos. Altas temperaturas também

podem aumentar a taxa de difusão dos compostos extraídos (HOSSAIN et al., 2011;

MARKOM et al., 2010). Curiosamente a ELP oferece uma possibilidade única de usar

alta temperatura a alta pressão, evitando a degradação dos compostos extraídos. Isso

ocorre porque a alta pressão geralmente aumenta a estabilidade das ligações covalentes

dentro das moléculas (HOSSAIN et al., 2011). Em alguns estudos (FEUEREISEN et al.,

2017; SKALICKA-WOŹNIAK & GŁOWNIAK, 2012) foram detectadas degradações

31

térmicas quando se utilizaram temperaturas elevadas, acima de 120°C, em compostos

termosensíveis. A segunda variável mais estudada é o solvente de extração. Os mais

utilizados são: água, etanol, metanol e a mistura de etanol ou metanol com água. 64%

dos trabalhos apresentaram a mistura de álcool com água como melhor solvente para

extração. De acordo com Mustafa & Turner (2011), o uso de uma mistura

hidroalcoólica como solvente melhora a solubilização dos compostos alvo e sua

dessorção da matriz vegetal. A utilização da água como solvente também é adequada

neste sistema porque, com o aumento da temperatura a alta pressão, a água torna-se

menos polar devido à diminuição da sua constante dielétrica, ficando com uma

polaridade semelhante a dos álccois (MARKOM et al., 2010). Outra variável bastante

avaliada é o tempo de extração. Nos estudos que utilizaram o sistema de extração em

modo dinâmico (MARKOM et al., 2010; PEREIRA et al., 2016) o tempo total de

extração foi de 60 min. Já nos trabalhos que utilizaram o processo de extração em modo

estático, o tempo ótimo de extração variou de 5 a 15 min em 1 ou 3 ciclos.

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38

ARTIGO 1



Adriana Dutra Sousa, Isabel Vitorino Maia, Tigressa Helena Soares Rodrigues, Kirley

Marques Canuto, Paulo Riceli Vasconcelos Ribeiro, Rita de Cassia Alves Pereira,

Roberto Fontes Vieira, Edy Sousa de Brito

Artigo publicado em: Industrial Crops and Products, Vol. 79, Páginas 91-103, 2016

doi: 10.1016/j.indcrop.2015.10.045

39



Adriana Dutra Sousaa,b, Isabel Vitorino Maiaa, Tigressa Helena Soares Rodriguesa,

Kirley Marques Canutoa, Paulo Riceli Vasconcelos Ribeiroa, Rita de Cassia Alves

Pereiraa, Roberto Fontes Vieirac, Edy Sousa de Britoa,*

a Embrapa Tropical Agroindustry, R Dra Sara Mesquita, 2270, Fortaleza-CE 60511 110,

Brazil.

b Departamento de Engenharia Química, Universidade Federal do Ceará, Brazil.

c Embrapa Genetic Resources and Biotechnology, Brasília-DF, Brazil.

* corresponding author at: Embrapa Tropical Agroindustry, R Dra Sara Mesquita, 2270,

Pici, Fortaleza-CE, 60511 110, Brazil. Tel +55 85 33917393; Fax +55 85 33917109.

Email address: [email protected] (E.S. de Brito)

40

Abstract

Phyllanthus amarus Schum & Thonn is an herb rich in bioactive compounds, mainly

phenols, and it is widely used for its medicinal properties. In this study, aqueous

extraction from aerial parts of P. amarus was performed using ultrasound-assisted

extraction (UAE), pressurized liquid extraction (PLE), and conventional extraction

(CE). Response surface methodology was used to assess the effect of the time,

ultrasonic intensity, and liquid/solid (L/S) ratio in UAE and of time and temperature in

PLE on total phenolics and gallic acid extraction. The chemical composition of the

extracts obtained through the three techniques was also analyzed using UPLC-ESI-

QTOF-MS/MS. The UAE operational condition that afforded the highest phenolic

content (27.23 mg/g plant) used time of 7 min, ultrasonic intensity of 301 W/cm2, and

L/S ratio of 40 mL/g. This value was lower than the one obtained by the conventional

extraction method (42.78 mg/g plant). However, PLE at 192.4°C and time of 15 min

yielded the highest total phenolic content (52.97 mg/g plant).Regarding the extraction of

gallic acid, the non-conventional methods yielded contents three times higher than the

conventional extraction. The chemical composition of P. amarus extracts had mainly

hydrolysable tannins, flavonoids, and lignans. The most significant difference was

found in UAE, which proved to be inefficient to extract ellagitannins.

Keywords: Ellagitannin, Gallic acid, Lignan, Phyllanthus amarus, Response surface

methodology.

1. Introduction

Plants of the genus Phyllanthus (Euphorbiaceae) are used in popular medicine to

treat several diseases such as kidney stones, intestinal infections, diabetes, and hepatitis

(Patel et al., 2011). Phyllanthus amarus Schum & Thonn (Euphorbiaceae) is widely

distributed in tropical and subtropical regions of the world, particularly in the

Caribbean, Brazil, India, and China (Patel et al., 2011), where it grows so easily that it

can be considered a weed. This herb is known as quebra-pedra in Brazil, bhumi amalaki

in India and yu jae in China (Patel et al., 2011). Given its common traditional use, P.

amarus is described in several pharmacopeias in natura, in powder form, and as a

standardized extract (Farmacopeia Brasileira, 2010; USP, 2015) which are

commercially available for analytical and pharmaceutical purposes.

41

The P. amarus extracts and isolated compounds have shown a wide range of

pharmacological activities, including antiviral, antibacterial, antimalarial, anticancer,

antidiabetes, hypolipidemic, antioxidant, hepatoprotective, and diuretic, among others

(Chopade and Sayyad, 2014; Patel et al., 2011; Ravikumar et al., 2011). These

medicinal properties are associated with some of its active components such as lignans,

alkaloids, triterpenes, and polyphenols, e.g., quercetin, rutin, corilagin, and gallic acid

(Maity et al., 2013; Patel et al., 2011; Yang and Liu, 2014). Pre-clinical and clinical

trials have confirmed the medicinal properties of P. amarus (Gurib-Fakim, 2006; Nikam

et al., 2011; Notka et al., 2004).

Most research on P. amarus focuses on identification, isolation, biological

assays, and pharmacological studies. However, few studies aim to assess the effect of

the extraction methods on the achievement of active compounds (Patil et al., 2013).

Furthermore, the use of water as solvent is altogether fitting to a green chemistry

approach and avoids issues with cost, storage, handling, and recovery that occur with

other types of solvents. There are currently several modern extraction techniques with

significant advantages over conventional methods, such as lower organic solvent

consumption and sample degradation, better extraction efficiency, selectivity and/or

kinetics, easy automation, and shorter operational time (Azmir et al., 2013). Among

these techniques, ultrasound-assisted extraction (UAE) and pressurized liquid extraction

(PLE) stand out, both having been successfully applied to Phyllanthus emblica Linn.

and Phyllanthus niruri Linn. (Markom et al., 2007; Tsai et al., 2014).

Ultrasound is a special type of sound wave that causes physical and chemical

phenomena. Physical effects of ultrasound are associated with lower frequencies of 20–

100 kHz, whereas chemical effects dominate at frequencies of 200–500 kHz (Tiwari,

2015). The major effect of sonication in a liquid is caused by acoustic cavitation. When

mechanical waves are transmitted through a fluid, it induces a series of compressions

and rarefactions in the molecules of the medium. Such alternating pressure changes

cause the production, growth, and collapse of bubbles. This phenomenon is known as

“acoustic cavitation” (Esclapez et al., 2011). Some cavitation bubbles are relatively

stable, but others expand further to an unstable size and undergo violent collapse to

generate temperatures of about 5000 K and pressures of the order of 50 MPa at a

minuscule level (Tiwari, 2015). High temperature and pressure that occur from these

implosions cause tissue rupture, which increases the solvent access to the target

compounds and raises the extraction rate. The chemical effects of ultrasound occur

http://www.scopus.com/authid/detail.url?origin=resultslist&authorId=7404472022&zone=



42

under the extreme temperature and pressure conditions that generate highly reactive

radicals. If water is the medium, H• and OH• radicals are generated by the dissociation

of water (H2O→OH- + H+). These free radicals can induce a wide variety of chemical

reactions in the bulk solution (Ashokkumar et al., 2008).

Pressurized liquid extraction consists in applying high pressure while

maintaining the solvent liquid at a temperature beyond its regular boiling point. The

high extraction temperature lowers the solvent viscosity and surface tension,

accelerating the solubilization of the compounds into this phase, which enables quicker

and more efficient extraction (Mustafa and Turner, 2011). Automation techniques are

the main reason for the greater development of PLE-based techniques along with the

decreased extraction time and solvents requirement (Azmir et al., 2013). When water is

used as solvent, this method is also called pressurized hot water extraction or subcritical

water extraction. What makes water an interesting solvent in this technique is that at

certain temperature and applied pressure, the polarity of water becomes similar to that

of some common alcohols (methanol and ethanol). Thus, it can dissolve a wide range of

medium and low polarity analytes (Plaza and Turner, 2015; Teo et al., 2010).

Several factors can impact the extraction processes and their effects on the

compounds of interest can be analyzed using the response surface methodology (RSM),

which consists in a group of statistical and mathematical techniques employed to

develop and optimize processes in which the response of interest is influenced by

different variables. This study aimed to determine the best conditions for phenolic

compounds extraction from P. amarus by UAE and PLE, using the response surface

methodology, and compare the phenolic content and chemical composition with the

extract obtained by a conventional extraction method.

2. Material and Methods

2.1. Chemicals

Folin-Ciocalteu reagent, sodium carbonate, and ethanol were purchased from

Vetec (Duque de Caxias, RJ, Brazil). Water was obtained using a Milli-Q water purifier

system from Millipore (Billerica, MA, USA). Gallic acid and diatomaceous earth were

purchased from Sigma-Aldrich (St. Louis, MO, USA), methanol and acetonitrile from

Tedia (Rio de Janeiro, RJ, Brazil), and phosphoric and formic acids from Fluka (Buchs,

ZU, Switzerland).

43

2.2. Sample Collection and Preparation for Extraction

Aerial parts of P. amarus (genotype CPQBA-14) were collected in August 2012

from the Embrapa Experimental Field (Paraipaba, Ceará state, Brazil) positioned at

3°26’S latitude, 39°08’W longitude and 31 m above sea level and receives 1238 mm

average rain fall annually. The plant materials were dried in a forced air circulation

drying oven at 40°C for 48 h and ground in a knife mill (Wiley type). The grounded

material was classified using sieves with meshes between 0.25 and 4 mm and the

particles between 0.25 and 2.0 mm were used in the extractions.

2.3. Conventional Extraction (CE)

One and a half gram of dried ground plant was transferred to a round-bottom

flask and 300 mL of deionized water were added. The flask was heated in a water bath

under reflux for 30 min at 85±5°C according to the methodology described by

Farmacopeia Brasileira (2010). After extraction, the flask was cooled under running

water and the extract was vacuum-filtered, concentrated in a rotary vacuum evaporator

at 40°C and freeze-dried.

2.4. RSM design for Ultrasound-Assisted Extraction (UAE)

Five grams of the dried material were extracted with deionized water using a

high-power (500 W) ultrasonic probe (Unique model DES500, Indaiatuba, SP, Brazil)

with a titanium tip (13 mm diameter) according to a central composite rotatable design

with three independent variables: time (X1), ultrasonic intensity (X2), and liquid/solid

ratio (X3), and two dependent variables: phenolics (Y1) and gallic acid (Y2) (Table 1).

The design consisted of eight factorial points, six axial points and three replicates at the

central point (average value of each independent variable), totaling 17 experimental

points. The use of replicates at the center aimed to provide an independent estimate of

the experimental error. All experiments were conducted in a randomized order. A

second degree polynomial equation derived from RSM was used,

𝑌 = 𝑏0 + 𝑏1𝑋1 + 𝑏2𝑋2 + 𝑏3𝑋3 + 𝑏11𝑋12 + 𝑏22𝑋2

2 + 𝑏33𝑋32 + 𝑏12𝑋1𝑋2 + 𝑏13𝑋1𝑋3 +

𝑏23𝑋2𝑋3 (1)

Where Y: response variable; b0, b1, b2, b3, b11, b22, b33, b12, b13 and b23: regression

coefficients; and X1, X2 and X3: independent variables.

44

The energy dissipated by the ultrasonic intensity was calculated according to Eq.

2 (Li et al., 2004). The power levels applied were 132, 200, 300, 400, and 468 W,

corresponding to 99, 151, 226, 301, and 353 W/cm2, respectively.

𝑈𝑙𝑡𝑟𝑎𝑠𝑜𝑛𝑖𝑐 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑊/𝑐𝑚2) =𝑃

𝜋𝑟2 (2)

Where P: ultrasound power (W) and r: tip radius (cm).

The extractions were performed at a constant frequency of 19 kHz. In order to

prevent heating during extraction, 60s breaks were taken for every minute of ultrasound

treatment. The extracts obtained were centrifuged at 1,232 g for 15 min, vacuum-

filtered, concentrated in a rotary vacuum evaporator at 40°C and freeze-dried.

2.5. RSM design for Pressurized-Liquid Extraction (PLE)

The extractions were accomplished in a Dionex ASE 350 system (Sunnyvale,

CA, USA) using deionized water as solvent. Five grams of the dried plant were mixed

with 5 g of diatomaceous earth (dispersing agent) and placed in 66 mL stainless steel

cells. The cells were equipped with a stainless steel filter and a cellulose filter at the

bottom to prevent the presence of particulate matter in the collection flask. The

extractions were performed according to the central composite rotatable design with

temperature (X1) and time (X2) as the independent variables and phenolics (Y3) and

gallic acid (Y4) as the response variables (Table 2). The design consisted of four

factorial points, four axial points and three replicates at the central point, totaling 11

experimental points. All experiments were conducted in a randomized order, and it was

used a second degree polynomial equation derived from RSM to describe the response

variables. The extraction time was divided into three cycles and the system pressure

was 110±7 bar. The extracts obtained were concentrated in a rotary vacuum evaporator

at 40°C, then frozen and freeze-dried.

2.6. Extract Analysis

2.6.1. Total Phenolics

The methodology adapted from Singleton and Rossi (1965) was employed to

determine the total polyphenols content. The extracts were diluted with a solution of

10% ethanol in water and mixed with 0.5 mL of Folin-Ciocalteu reagent, 0.5 mL of

45

20% sodium carbonate, and 3.5 mL of water. After 90 minutes at rest, the absorbances

were read in a UV spectrophotometer (Cary 300, Varian, Palo Alto, CA, USA) at 725

nm. The results were expressed as mg of gallic acid equivalent per g of dried plant.

2.6.2 Gallic Acid

Gallic acid in the aqueous extracts from P. amarus was quantified using the

method of De Souza et al (2002) with minor modifications. The analysis was performed

in a 920LC HPLC (Varian, Palo Alto, CA, USA) equipped with a quaternary pump,

auto sampler, and diode array detector (DAD). A C18 (Microsorb) analytical column (5

µm, 250 x 4.6 mm) was used at a flow rate of 1.0 mL/min. The column oven

temperature was set at 35°C. The mobile phase was composed of methanol and a 0.1%

phosphoric acid (H3PO4) aqueous solution. The UV detector was set at 272 nm. The

injection volume was 20 µL and gradient elution was carried out ranging from 20 to

100% MeOH for 25 min. The results were expressed as mg of gallic acid per g of plant.

The extracts were dissolved in 20% methanol at a concentration of 1 mg/mL and filtered

through a 0.45 µm PTFE syringe filter. Gallic acid was identified based on the

comparison with its retention time and the UV spectrum. Concentration was calculated

based on a standard curve.

2.6.3. UPLC-ESI-QTOF-MS/MS Analysis

It was employed the methodology of Khoza et al. (2014) with slight

modifications. An Acquity UPLC system (Waters, Milford, MA, USA) coupled to a

quadrupole/time-of-flight (QToF) system (Waters, Milford, MA, USA) was used. The

compounds were separated on an Acquity BEH C18 (1.7 µm, 2.1 x 150 mm; Waters,

Milford, MA, USA) column operated at 40°C. The eluent system employed was a

combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile)

at a flow rate of 0.4 mL/min. The gradient varied linearly from 5 to 95% B (v/v) over 15

min. The sample injection volume was 5 µL. Mass spectra were obtained in the positive

and negative modes in a mass range between 50 and 1,180 Da. The spectrometer

operated with MSE centroid programming using tension ramp from 20 to 40 V. Drying

gas pressure was 35 psi and temperature was 370°C, while nebulizer gas pressure was

40 psi. Capillary voltage of 3,500 V for both the positive (PI) and negative (NI) modes

and 600 V spray shield voltage were used. The samples were dissolved in water at a

concentration of 2 mg/mL and filtered on 0.22 µm PTFE membranes.

46

2.7. Statistical Analysis

The experimental designs were generated and their results were analyzed using

the software STATISTICA (Statsoft version 7.0). A one-way analysis of variance

(ANOVA) was performed to compare the extraction methods and the significant

differences on the results were determined by Tukey’s test at 5% significance level.

3. Results and Discussion

3.1. Ultrasound-Assisted Extraction (UAE)

The experimental design and results for different ultrasound extraction

conditions are presented in Table 1.

Table 1. Experimental design of ultrasound-assisted extraction and results obtained in

the P. amarus extracts.

Run Time

(min)

Ultrasonic intensity

(W/cm2)

L/S ratio

(mL/g)

Phenolics (mg/g

dry plant)

Gallic acid

(mg/g dry plant)

1 3 151 20 21.75±0.56 4.09±0.13

2 3 151 40 18.34±0.36 3.62±0.09

3 3 301 20 22.26±0.53 3.54±0.12

4 3 301 40 23.03±0.48 3.23±0.16

5 7 151 20 20.63±0.47 3.15±0.10

6 7 151 40 23.14±0.68 3.96±0.04

7 7 301 20 20.90±0.49 3.08±0.13

8 7 301 40 27.23±0.75 5.55±0.15

9 1.6 226 30 22.30±0.66 3.22±0.07

10 8.4 226 30 26.40±0.78 5.33±0.03

11 5 99 30 24.66±0.69 3.64±0.18

12 5 353 30 22.91±0.54 3.11±0.05

13 5 226 13.2 17.21±0.42 2.93±0.17

14 5 226 46.8 24.13±0.49 3.43±0.08

15 © 5 226 30 25.69±0.71 3.74±0.15

16 © 5 226 30 24.23±0.69 3.87±0.09

17 © 5 226 30 25.24±0.75 3.57±0.12

© central point of the experimental design.

From the linear regression analysis of the results obtained, polynomial models

were formulated to describe the response variables (Eqs. 3 and 4).

𝑌1 = 25.09 + 0.98𝑋1 − 0.39𝑋12 + 0.48𝑋2 − 0.59𝑋2

2 + 1.31𝑋3 − 1.69𝑋32 −

0.10𝑋1X2 + 1.43𝑋1X3+1.00𝑋2X3 (3)

47

𝑌2 = 3.71 + 0.35𝑋1 + 0.23𝑋12 − 0.02𝑋2 − 0.08𝑋2

2 + 0.24𝑋3 − 0.15𝑋32 +

0.31𝑋1X2 + 0.51𝑋1X3+0.23𝑋2X3 (4)

Where Y1: phenolics (mg/g plant), Y2: gallic acid (mg/g plant), X1: time (min), X2:

ultrasonic intensity (W/cm2), and X3: L/S ratio (mL/g).

The coefficients of determination (R2) of fitted models presented in Eqs. 3 and 4

were 0.84 and 0.83, respectively, and the calculated F-values were 4.14 (Eq. 3) and 3.90

(Eq. 4).The models were validated by ANOVA analysis and F-test at 95% of confidence

level. All models were statistically significant since the calculated F-values were higher

than the tabled value (F9.7 = 3.68).

Figure 1. Estimated effects by Pareto plot and response-surface graphs for the phenolics

content (mg/g plant) in ultrasound-assisted extraction.

Regarding the total phenolic content in the extracts obtained using UAE, the L/S

ratio variable and its interaction with time impacted the extraction of these compounds

(Figure 1a). L/S ratio values between 30 and 40 mL/g associated with longer extraction

times yielded approximately 27 mg/g plant (Figure 1c).The higher L/S ratio provides

more solvent to enter the cells, which improves permeation of the phenolic compounds,

while contact time is needed for the complete diffusion of the compounds. However,

48

when the proportion of solvent was further increased to over 40 mL/g, the extraction of

polyphenol decreased. This behavior was also observed in the UAE of polyphenols

from Sparganium stoloniferum Buch.-Ham. (Wang et al., 2013).

With regard to extraction of gallic acid, a chemical marker compound of the

genus Phyllanthus (Farmacopeia Brasileira, 2010), the linear term for time and its

interaction with L/S ratio were positive and significant (Figure 2a), i.e., increasing these

variables resulted in a gallic acid content three times higher. The best condition used the

longest extraction time (8.4 min) with higher L/S ratios (Figure 2c).This condition

yielded approximately 7 mg gallic acid/g plant.

Figure 2. Estimated effects by Pareto plot and response-surface graphs for the gallic

acid content (mg/g plant) in ultrasound-assisted extraction.

3.2. Pressurized Liquid Extraction (PLE)

Table 2 shows the experimental design employed to optimize PLE and their

respective experimental data. The results were analyzed using the response surface

methodology and the fitted models are shown in Eqs. 5 and 6. The R2 of the fitted

models shown in Eqs. 5 and 6 were 0.89 and 0.94, respectively, and the calculated F-

values were 8.03 (Eq. 5) and 14.93 (Eq. 6). The models were validated by ANOVA

49

analysis and F-test at 95% of confidence level. All models were statistically significant

since the calculated F-values were higher than the tabled value (F5.5 = 5.05).

𝑌3 = 38.64 + 5.33𝑋1 + 2.39𝑋12 + 4.16𝑋2 − 2.29𝑋2

2 − 1.20𝑋1X2 (5)

𝑌4 = 2.90 + 1.64𝑋1 − 0.20𝑋12 + 0.59𝑋2 − 0.12𝑋2

2 + 0.34𝑋1X2 (6)

Where Y3: phenolics (mg/g plant), Y4: gallic acid (mg/g plant), X1: temperature (°C),

and X2: time (min).

Table 2. Experimental design of pressurized liquid extraction and results obtained in the

P. amarus extracts.

Run Temperature (C) Time (min) Phenolics (mg/g dry

plant)

Gallic acid

(mg/g dry plant)

1 120 7 31.67±0.87 0.57±0.17

2 120 23 42.23±1.04 0.90±0.35

3 180 7 40.13±0.94 3.90±0.23

4 180 23 45.90±1.10 5.59±0.40

5 107.6 15 31.38±1.07 0.54±0.15

6 192.4 15 52.97±1.23 4.14±0.28

7 150 3.7 26.81±0.78 1.52±0.34

8 150 26.3 38.81±1.15 3.46±0.48

9 © 150 15 39.79±1.12 3.15±0.21

10 © 150 15 38.55±0.99 2.43±0.22

11 © 150 15 37.59±1.08 3.11±0.15


On total phenolic content of the extracts obtained using PLE, the linear terms of

temperature and time were positive and significant (Figure 3a), with temperature

presenting a stronger effect. An increase of these two variables led to a content of

approximately 50 mg/g plant (Figure 3b), which is higher than what was obtained in the

best UAE condition (27.23 mg/g plant). At high temperatures, the compounds solubility

could be increased due to the decrease in water polarity. Moreover, mass transfer also

increases due to the lower viscosity and water surface tension, while the longer contact

time provides time for the compounds to diffuse (Azmir et al., 2013; Rangsriwong et

al., 2009).

For the extraction of gallic acid, the linear terms of temperature and extraction

time were positive and significant (Figure 3c) as observed in the phenolics response,

which suggests that the increase of these independent variables led to a greater release

of this analyte and other compounds into the extracting solvent. Nevertheless, this

50

increase could possibly be due to the hydrolysis reaction of the tannins, which have

gallic acid in their structures, caused by the higher ionization constant of water at

subcritical condition (Rangsriwong et al., 2009).

Figure 3. Estimated effects by Pareto plot and response-surface graphs for the phenolics

content(mg/g plant) (a) and (b) and gallic acid content (mg/g plant) (c) and (d) in

pressurized-liquid extraction.

3.3. Comparison of different Extraction Methods

Since the response surface methodology revealed how the extraction parameters

of PLE and UAE methods influenced on the chemical composition of P. amarus

extracts, we wanted to compare their performances employing the best conditions

predicted in the experimental design. The comparison included the conventional

extraction recommended by the Brazilian pharmacopeia, ultrasound-assisted extraction,

and pressurized-liquid extraction using the conditions that yielded the highest total

phenolic and gallic acid contents obtained in the experimental designs, besides a

pressurized-liquid extraction at a lower temperature from the design.

According to Table 3, the total phenolics content of the extracts obtained by

conventional extraction and PLE at 120°C did not differ significantly. PLE temperature

was higher, but the extraction time was shorter (23 min) and it is worth pointing out that

the PLE technique used eight times less solvent, being more economical in this regard.

51

PLE at 192.4°C had the best total phenolics result compared to other methods. This

technique uses high pressure and high temperatures to promote greater solubility and

extraction efficiency. However, considering the chromatograms at 272 nm

(supplementary material), the extracts produced at higher temperatures had smaller area

in several peaks and a larger area only for the peak referring to gallic acid. The very

high temperature possibly caused degradation of the thermolabile phenolic compounds.

Rangsriwong et al. (2009) studied the extraction of polyphenol with subcritical water

from Terminalia chebula Retz fruits and observed that the corilagin content decreased

and the gallic acid content increased in temperatures above 120 °C. These authors

suggested that gallic acid might have been generated from the thermal hydrolysis of

corillagin. UAE yielded the lowest phenolic content compared to other methods. In this

technique, the extractions were done over shorter times and at room temperature (25°C),

unlike the other methods that used higher temperatures. This shows that the ultrasound

probe was unable to recover all the compounds and higher temperatures are required to

improve the extraction of phenolics from P. amarus. Another possibility is that the high

power might have degraded some phenolic compounds since radicals may be formed

during cavitation and react with the phenolic compounds, which oxidize them. These

radicals are formed due to the dissociation of the water molecule or other gases (Soria

and Villamiel, 2010). Regarding gallic acid extraction, the extract with the highest total

phenolic content in UAE also had the highest gallic acid content, while in PLE the

highest gallic acid content was obtained using 180°C for 23 min. These extracts were

prepared under extreme conditions of the experimental designs, which shows that gallic

acid resists to high temperatures and powers and it is better extracted under these

conditions. Since temperature was lower in the conventional extraction, the gallic acid

content in the extract was lower.

Table 3. Comparison of different extraction methods of P. amarus

Method T (C) P (bar) Time

(min)

L/S ratio

(mL/g)

Ultrasonic

intensity

(W/cm2)

Phenolics

(mg/g dry

plant)*

Gallic acid

(mg/g dry

plant)*

CE 80-90 1 30 200 - 42.78±0.87c 1.72±0.22c

UAE 25 1 7 40 301 27.23±0.75d 5.55±0.15a

PLE 192.4 103-117 15 24 - 52.97±1.23a 4.14±0.28b

180 103-117 23 24 - 45.90±1.10b 5.59±0.40a

120 103-117 23 24 - 42.23±1.04c 0.90±0.35c

*Means with the same letter in the same column do not significantly differ in Tukey test

(p>0.05).

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52

3.4. Effect of Extraction on Chemical Components

Instruments such as ToF-MS mass spectrometer can facilitate the identification

of known and unknown compounds, as well as differentiate isobaric compounds since

compounds with the same nominal mass but different elemental composition would

have different exact masses. The difference in masses with error of up to 5 ppm is

widely accepted to verify elemental composition (Guo et al., 2015). In addition to this

advantage particular to ToF-MS, the usage of tandem mass analyzers such as

quadrupole mass analyzers allows the pre-selected masses to be fragmented, which

contributes to structural elucidation and isomer distinction.

Figure 4. LC-ESI(+)/MS and LC-ESI(-)/MS chromatograms of P. amarus aqueous

extracts obtained through UAE (a) and (b), PLE (c) and (d), and CE (e) and (f),

respectively.

The analysis of P. amarus extracts obtained by PLE, UAE, and CE

methodologies (Figure 4) showed that the analysis carried out in the positive mode were

more appropriate for lignans, whereas the negative mode was more sensitive to the other

phenolic compounds. This difference can be attributed to the behavior of these

53

compounds in liquid phase. This means that the lignans more easily originate positively

charged species, i.e., protonated species ([M+H]+), on the other hand the other phenolic

compounds are more easily found as negatively charged ions.

Figure 5. Structures of the substances identified in P. amarus extracts.

The three methodologies employed permitted the identification of 31 compounds

(Figure 5), which are grouped in Table 4. Kumar et al. (2015) identified 52 compounds

in Phyllanthus amarus, but their conventional extraction methodology required a large

amount of organic solvent (ethanol) and long extraction time, while the present study

used only water as the extraction solvent and the process was carried out over a shorter

period of time.

54

Table 4. Compounds determined by UPLC-ESI-QTOF-MS/MS in the P. amarus aqueous extracts obtained from UAE, PLE and CE techniques.

Nº Rt

(min)

Obsd m/z

ES(+)

Obsd m/z

ES(-)

Molecular

Formula

Calcd

m/z

Error

(ppm) Proposed compound Detected in Reference

Acids

1 0.86 - 209.0281[M-H]-, 191.0202

[M-H-H2O]-

C6H10O8 209.0297 -7.7 mucic acid UAE, PLE, CE Yang et al.

(2012)

3 1.28 - 191.0188 [M-H]- C6H8O7 191.0192 -2.1 Mucic acid lactone UAE, PLE, CE Yang et al.

(2012)

8 1.79 - 169.0136 [M-H]-, 125.0216

[M-COOH]-

C7H6O5 169.0137 -0.6 Gallic acid UAE, PLE, CE Yang et al.

(2012)

11 3.16 293.0282 [M+H]+ 291.0143 [M-H]-, 247.0230

[M-H-CO2]-

C13H8O8 291.0141 0.7 Brevifolin carboxylic acid UAE, PLE, CE Kumar et al.

(2015)

23 4.14 - 300.9967 [M-H]-, 257.0147

[M-H-CO2]-

C14H6O8 300.9984 -5.6 Ellagic acid UAE Kumar et al.

(2015)

34 6.55 - 343.0457 [M-H]-, 328.0175

[M-H-CH3]-

C17H12O8 343.0454 0.9 tri-O-methylellagic acid UAE, PLE Kumar et al.

(2015)

Alkaloids

4 1.30 222.1130 [M+H]+ - C12H15NO3 222.1130 0.0 Niruroidine UAE, PLE, CE Guo et al.

(2015)

5 1.41 222.1118 [M+H]+ - C12H15NO3 222.1130 -5.4 Niruroidine (isomer) UAE, PLE, CE Guo et al.

(2015)

7 1.62 222.1118 [M+H]+ - C12H15NO3 222.1130 -5.4 Niruroidine (isomer) UAE, CE Guo et al.

(2015)

Ellagitannins

12 3.25 - 969.0879 [M-H]-, 633.0690,

247.0252

C41H30O28 969.0845 3.5 Amariinic acid PLE, CE Yeap Foo

(1995)

13 3.26 783.0695 [M+H]+,

355.0329

- C34H22O22 783.0681 1.8 Emblicanin A CE Guo et al.

(2015)

14 3.32 783.0692 [M+H]+,

355.0329

- C34H22O22 783.0681 1.4 Emblicanin A (isomer) CE Guo et al.

(2015)

15 3.37 - 633.0715 [M-H]-, 463.0464

[M-H- galloyl-H2O]-,

300.9953 [M-H-galloyl-

C27H22O18 633.0728 -2.1 Corilagin (isomer) CE Yang et al.

(2012)



55

Hex]-

16 3.40 - 633.0720 [M-H]-, 481.0528

[M-H- galloyl]-, 463.0527

[M-H- galloyl-H2O]-,

331.1237 [M-H-HHDP]-,

300.9981 [M-H- galloyl-

Hex]-

C27H22O18 633.0728 -1.3 Corilagin PLE, CE Kumar et al.

(2015)

18 3.61 757.0912

[M-galloyl-H2O+H]+

925.0978 [M-H]- C40H30O26 925.0947 3.4 Phyllanthusiin C PLE, CE Latté and

Kolodziej

(2000)

19 3.74 - 969.0818 [M-H]- C41H30O28 969.0845 -2.8 Amariinic acid (isomer) PLE, CE Yeap Foo

(1995)

21 3.95 783.0694

[M-galloyl-H2O+H]+

951.0751 [M-H]-, 300.9980,

169.0141

C41H28O27 951.0740 1.2 Geraniin PLE, CE Kumar et al.

(2015)

30 4.90 755.0757

[M-galloyl-H2O+H]+

923.0760 [M-H]- C40H28O26 923.0791 -3.4 Phyllanthusiin U CE Chen et al.

(1999)

Flavonoids

17 3.51 - 305.0685 [M-H]-, 225.1131 C15H14O7 305.0661 7.9 Gallocatechin UAE, PLE Hossain et

al. (2010)

24 4.15 611.1603 [M+H]+ 609.1445 [M-H]-, 300.9947

[M-H-Hex-Rham]-

C27H30O16 609.1456 -1.8 Rutin PLE, CE Hossain et

al. (2010)

25 4.24 - 463.0882 [M-H]-, 301.0003

[M-H-Hex]-

C21H20O12 463.0877 1.1 Quercetin-3-O-hexoside UAE, PLE, CE Hossain et

al. (2010)

26 4.32 303.0503 [M+H]+ - C15H10O7 303.0505 -0.7 Quercetin CE Guo et al.

(2015)

27 4.55 - 579.1706 [M-H]- C27H32O14 579.1714 -1.4 Narirutin UAE, PLE, CE Spínola et al.

(2015)

35 7.07 365.0312 [M+H]+ 363.0183 [M-H]- C16H12O8S 363.0175 2.2 Niruriflavone UAE, PLE, CE Guo et al.

(2015)

Lignans

40 9.29 441.1885 [M+Na]+ - C23H30O7 441.1889 -0.9 Desmethylniranthin UAE, CE Guo et al.

(2015)

41 10.16 439.1733 [M+Na]+,

367.1522, 335.1258,

- C23H28O7 439.1733 0.0 Virgatusin UAE, PLE, CE Shanker et

al. (2011)




56

247.1371

42 10.56 441.2257 [M+Na]+,

355.1933

- C24H34O6 441.2253 0.9 Phyllanthin UAE, PLE, CE Shanker et

al. (2011)

43 11.06 455.2035 [M+Na]+,

369.1703

- C24H32O7 455.2046 -2.4 Niranthin UAE, PLE, CE Shanker et

al. (2011)

Other compounds

6 1.60 - 331.0671 [M-H]-, 271.0481,

211.0213, 169.0146 [M-H-

Hex]-

C13H16O10 331.0665 1.8 Monogalloyl-hexoside PLE, CE Sentandreu

et al. (2013)

10 2.17 166.0861 [M+H]+,

120.0800 [M+H-

HCOOH]+

- C9H11NO2 166.0868 -4.2 Phenylalanine UAE, CE Hanhineva

et al. (2008)

20 3.84 249.0378 [M+H]+ 247.0232 [M-H]- C12H8O6 247.0243 -4.5 Brevifolin UAE, PLE Sentandreu

et al. (2013)

Unknown

2 0.94 499.0711, 144.0990 515.0638, 247.0226 - - - Unknown UAE

9 1.90 150.0892 - - - - Unknown PLE

22 3.98 - 395.1511 - - - Unknown UAE, PLE, CE

28 4.56 291.0997 - - - - Unknown UAE, CE

29 4.85 239.1286 237.1135 - - - Unknown UAE, PLE, CE

31 5.48 - 629.2106, 453.1465 - - - Unknown UAE, PLE

32 5.62 - 497.0748 - - - Unknown UAE

33 5.70 704.3719 - - - - Unknown UAE, CE

36 7.37 - 375.1806, 347.1933 - - - Unknown UAE

37 8.48 427.2057 - - - - Unknown UAE, CE

57

38 8.50 - 389.1983, 347.1782 - - - Unknown PLE

39 8.67 457.2181 - - - - Unknown UAE, CE

44 11.29 341.3537 - - - - Unknown UAE, PLE

45 11.55 284.3323 - Unknown CE

46 12.37 369.3852 - - - - Unknown UAE, PLE

47 14.70 282.2852 - - - - Unknown UAE, PLE

48 14.78 466.5393 - - - - Unknown CE

49 15.13 494.5659 - - - - Unknown UAE, PLE, CE

50 15.42 522.5831 - - - - Unknown UAE, PLE, CE

51 15.76 550.6157 - - - - Unknown CE

58

The chromatograms of the extracts produced by the three extraction methods

used in this study showed to be quite similar (Figure 4). Nevertheless, according to

Table 4, some differences could be detected. One of the main differences was the

presence of ellagitannins 12, 13, 16, 18, 21, and 30 in PLE and/or CE and their absence

in UAE. Moreover, rutin (24) and monogalloylhexoside (6) were also found only in

PLE and CE. Among the ellagitannins, corilagin (16) stands out for being an important

bioactive compound found in several species of the genus Phyllanthus and for having

multiple activities, including potent analgesic action (Moreira et al., 2013), antitumoral

activity (Jia et al., 2013), antioxidant and anti-inflammatory activities (Jin et al., 2013).

Figure 6. Proposal of amariinic acid fragmentation with corilagin formation (m/z 633).

The fragmentation of amariinic acid (12) originated corilagin (16) (Figure 6)

and the latter one generated one fragment at m/z 331 Da matching monogalloylhexoside

(6) (Figure 7).

Figure 7. Possible formation of monogalloylhexoside through the loss of the HHDP

group (m/z 301).

59

The mass spectra of corilagin (16), amariinic acid (12), and geraniin (21) yielded

a typical fragment of this class at m/z 301 Da due to the loss of one HHDP group. The

mass spectra of the ellagitannins phyllanthusiin C (18) and U (30) and geraniin (18)

exhibited a peak due to a loss of one galloyl group + H2O (169 Da) in the positive mode

(Figure 8), which matched the attempt at identifying these substances.

Among the compounds found in all extracts there are gallic acid (8), which is a

marker phenolic compound of Phyllanthus species according to Farmacopeia Brasileira

(2010), brevifolin carboxylic acid (11), mucic acid (1), and mucic acid lactone (3), three

flavonoids (quercetin-3-O-hexoside (25), narirutin (27), and niruriflavone (35)), besides

three alkaloids (niruroidine (4) and two isomers), and three lignans (virgatusin (41),

phyllanthin (42), and niranthin (43)). Phyllanthin, a chemical marker of lignans used by

the American pharmacopeia (USP, 2015), stood out for having a high intensity of

chromatograms in the positive mode in all methods analyzed. Furthermore, phyllantin

have antitumoral, hepatoprotective, and antioxidant biologic activity potential (Patel et

al., 2011).

In UAE, the main difference observed was the presence of ellagic acid (23) and

phenylalanine (10) and the absence of ellagitannins with higher molecular masses. This

can be justified since the UAE process results in a large amount of energy, which may

lead to cleavage or oxidation of molecules, mainly those with higher molecular masses

(Soria and Villamiel, 2010). On the other hand, the main difference found in CE was

the presence of quercetin (26) and the tannoid emblicanin A (13).

60

Figure 8. Proposal of the loss of the galloyl group by ellagitannins, generating the

fragments observed in the positive mode.

61

4. Conclusion

Phenolics extraction by UAE and PLE were optimized using response surface

methodology. The highest total polyphenol content (52.97 mg/g plant) was obtained by

PLE at 192.4°C and time of 15 min, but this combination of temperature and time led to

degradation of some compounds. Nevertheless, the use of PLE at 120°C and time of 23

min presented a reasonable phenolic content (42.23 mg/g plant) without chemical

degradation. PLE consumed less solvent compared to the other methods, and the use of

120°C for 23 min proved to be a suitable method to extract phenolics, including the

compounds with medicinal relevance. In UAE, the highest polyphenol content (27.23

mg/g plant) was obtained at 7 min, ultrasonic intensity of 301 W/cm2 and L/S ratio of

40 mL/g. Regarding gallic acid extraction, the non-conventional methods yielded

contents three times higher than the conventional extraction. The chemical composition

of P. amarus extracts had mainly hydrolysable tannins, flavonoids, and lignans. The

most significant difference was found in UAE, which showed to be inefficient to extract

ellagitannins. It is important to point out that the success of the present study is directly

linked to the use of modern extraction and organic compounds analysis techniques. This

means that matching these methods enables the achievement of quick and efficient

results, which makes them powerful allies to extract and chemically characterize plant

extracts.

Acknowledgements

The Authors are extremely grateful to financial support from Embrapa

(02.10.06.019.00.00), CNPq (402654/2012-9) and FUNCAP, for a PhD scholarship

granted to the first author. To CPQBA-Unicamp for providing P. amarus seeds.

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65

Supplementary material – HPLC chromatograms of the aqueous extracts from P.

amarus obtained through PLE at 120°C (a) and 192.4°C (b) at 272 nm.

66

ARTIGO 2



Adriana Dutra Sousa, Isabel Vitorino Maia, Paulo Riceli Vasconcelos Ribeiro, Kirley

Marques Canuto, Guilherme Julião Zocolo, Edy Sousa de Brito

Artigo publicado em: Separation Science and Technology

doi: 10.1080/01496395.2017.1298612

67



Running title: MS-Chemometrics guided extraction development

Adriana Dutra Sousa1,2, Isabel Vitorino Maia1, Paulo Riceli Vasconcelos Ribeiro1,

Kirley Marques Canuto1, Guilherme Julião Zocolo1, Edy Sousa de Brito1,*

1 Embrapa Tropical Agroindustry, R Dra Sara Mesquita, 2270, Fortaleza-CE 60511 110,

Brazil.

2 Departamento de Engenharia Química, Universidade Federal do Ceará, Brazil.




Abstract

P. niruri extracts obtained by ultrasound-assisted extraction (UAE), pressurized liquid

extraction (PLE), and conventional extraction (CE) were compared. The extracts

produced by PLE had the highest phenolic content. In the principal component analysis,

CE and PLE 120 °C extracts formed a single group, separated from PLE 192 °C and

UAE extracts. The orthogonal partial least square discriminant analysis revealed

geraniin, phyllanthusiin C, repandusinic acid A and phyllanthusiin U as chemical

markers in CE and PLE 120 °C. PLE 192 °C extract presented a high content of gallic

acid and ellagic acid hexose and UAE extract presented virganin and furosin as

characteristic compounds.

Keywords: OPLS-DA; PCA; phenolics; pressurized liquid extraction; tannin;

ultrasound-assisted extraction

68

Introduction

Phyllanthus niruri Linn. (Euphorbiaceae) is a small erect annual herb widely

distributed in tropical and subtropical countries, including South East Asia, Southern

India, Brazil and China [1, 2]. The whole plant has been used in folk medicine for

treatment of a variety of diseases such as dysentery, diabetes, diuretics, kidney stones

and dyspepsia [1, 2]. Pharmacological studies reported that P. niruri has analgesic [3],

hepatoprotective [4], antihepatitis [5], antitumor [6] and antiviral [7] properties. Its

biological activities are attributed to alkaloids, lignans, tannins, terpenes, flavonoids,

saponins and phenylpropanoids, which are found in the leaves, stem and roots [1, 2].

The composition and bioactivity of plant extracts strongly depend on the

extraction process employed. Conventional extraction techniques have some limitations,

such as low extraction selectivity, long extraction time required, and the use of large

amount of organic solvents, which are expensive and require costly disposal [8]. To

overcome these limitations, modern extraction techniques have been introduced. Some

of the most promising techniques are ultrasound-assisted extraction (UAE) and

pressurized liquid extraction (PLE), both having been successfully applied to

Phyllanthus niruri, Phyllanthus amarus and Phyllanthus emblica [9-12]. The present

study is the first one to compare the limitations and advantages of each technique, based

on chemometrics, indicating if the extraction methods have effect on the degradation of

substances, hence affecting the quality of the raw material.

UAE is an interesting process to obtain bioactive compounds. The enhancement

of the mass transfer brought about by acoustic-induced cavitation in a liquid

medium is one of the benefits. When mechanical waves are transmitted through a

fluid, it induces a series of compressions and rarefactions in the molecules of the

medium. Such alternating pressure changes cause the production, growth, and collapse

of bubbles. This phenomenon is known as “acoustic cavitation” [13]. The implosion of

cavitation bubbles generates micro-turbulence, high-velocity inter-particle collisions

and agitation in micro-porous particles of the biomass, which accelerates the diffusion,

increasing the solvent access to the target compounds, therefore raising the extraction

rate [14]. On the other hand, PLE is a technique that combines elevated temperature and

pressure with liquid solvents to achieve fast and efficient extraction of compounds from

solid matrix. The use of higher temperatures implies a reduction in solvent viscosity,

thereby increasing the solvent ability to wet the matrix and to solubilize the target

69

analytes. High temperatures also assist in breaking down compound–matrix bonds and

favor the compound diffusion to the matrix surface [15]. When water is used as solvent,

this method is also called pressurized hot water extraction or subcritical water

extraction. What makes water an interesting solvent in this technique is that at elevated

temperatures and moderate pressures, the polarity of water can be reduced considerably

and becomes similar to that of some common alcohols (methanol and ethanol). Thus, it

can dissolve a wide range of medium and low polarity analytes [15, 16].

Ultra-performance liquid chromatography (UPLC) applied for short run times in

combination with time-of-flight mass spectrometry (TOF-MS), which offers high mass

accuracy, has become a method of choice for unbiased compound screening and

provides a significant source of global constituent and metabolite profiling data [17, 18].

Furthermore, statistical multivariate methods can be used to facilitate data interpretation

when a large number of variables are analyzed. The multivariate method of principal

component analysis (PCA) has been applied to reduce the number of dimensions of the

original data system to visualize whether different groups could be related to specified

compounds or not [19], and orthogonal partial least squared discriminant analysis

(OPLS-DA) is used to identify the compositional variation of groups.

In this study, the phenolic content and chemical composition of P. niruri

aqueous extracts produced by UAE, PLE and conventional extraction method were

compared. The chemical profiling was determined using UPLC-QTOF-MSE in

combination with multivariate data models (PCA and OPLS-DA). The results show

how much the extraction methods affect the extract profile, indicating the most

recommended extraction process using water, a “green solvent”, in order to obtain P.

niruri extracts with high phenolic content and a metabolite profile with pharmacological

relevance.

Materials and methods

Chemicals

Gallic acid and diatomaceous earth were purchased from Sigma-Aldrich (St.

Louis, MO, USA). Water was obtained using a Milli-Q water purifier system from

Millipore (Billerica, MA, USA). Folin-Ciocalteu reagent, sodium carbonate, and

ethanol were purchased from Vetec (Duque de Caxias, RJ, Brazil), acetonitrile from

70

Tedia (Rio de Janeiro, RJ, Brazil), and formic acid from Fluka (Buchs, ZU,

Switzerland).

Sample collection and preparation for extraction

Aerial parts of P. niruri were harvested in July 2013 from the Embrapa

Experimental Field (Paraipaba, Ceará state, Brazil). Voucher specimen was deposited in

the herbarium of the University of Campinas, Brazil, (UEC 112.740). The plant

materials were dried in a forced air circulation drying oven at 40 °C for 48 h and ground

in a knife mill (Wiley type). The grounded material was classified through 0.25-4 mm

mesh sieves, providing 0.25-2.0 mm particles for the extractions.

Extraction

We previously reported the effect of some extraction parameters: time,

ultrasonic intensity and liquid/solid ratio on the ultrasound-assisted extraction and

temperature and time on pressurized liquid extraction of phenolic compounds from

Phyllanthus amarus using the response surface methodology and we determined the

best conditions for phenolic extraction using water as solvent [11]. The same

experimental designs for extraction conditions were performed for P. niruri (Supporting

Information). The best extraction conditions based on phenolic content were selected

and applied on the experiments described below. For comparison, a conventional

extraction was also employed.

Ultrasound-assisted extraction (UAE)

Based on the optimized condition (Table S1, supporting information), 5 g of the

dried material were extracted with 234 mL of deionized water (liquid/solid ratio: 46.8

mL/g) using a high-power (500 W) ultrasonic probe (Unique model DES500,

Indaiatuba, SP, Brazil) with a titanium tip (13 mm diameter), at a frequency of 19 KHz.

The energy dissipated by the ultrasonic intensity was calculated according to Eq. 1 [20].

The power level applied was 300 W, corresponding to 226 W/cm2 or 1282 W/L.


𝜋𝑟2 (1)


71

The extraction was performed for five min. In order to prevent heating during

extraction, 60s breaks were taken for every minute of ultrasound treatment. The extract

obtained was centrifuged at 1,232 g for 15 min, vacuum-filtered, concentrated in a

rotary vacuum evaporator at 40 °C and freeze-dried.

Pressurized-liquid extraction (PLE)


CA, USA) using deionized water as solvent. Following the optimized condition (Table

S3, supporting information), 5 g of the dried plant were mixed with 5 g of diatomaceous

earth (dispersing agent) and placed in 66 mL stainless steel cell. The cell was equipped

with a stainless steel filter and a cellulose filter at the bottom to prevent the presence of

particulate matter in the collection flask. The extraction was performed at 192 °C for 15

min (PLE 192). It was also made an extract at a lower temperature from the

experimental design, PLE 120 (120 °C for 7 min). The extractions were carried out in a

sequence of three cycles and the system pressure was 110±7 bar. The extracts obtained

were concentrated in a rotary vacuum evaporator at 40 °C, then frozen and freeze-dried.

Conventional extraction (CE)

1.5 g of dried ground plant was transferred to a round-bottom flask and 300 mL

of deionized water were added. The flask was heated in a water bath under reflux for 30

min at 85±5 °C. After extraction, the flask was cooled with tap water and the extract

was vacuum-filtered, concentrated in a rotary vacuum evaporator at 40 °C and freeze-

dried.

Determination of total phenolics

The methodology adapted from Singleton and Rossi [21] was employed to



20% sodium carbonate, and 3.5 mL of water. After 90 min at rest, the absorbance was

read in a UV spectrophotometer (Cary 300, Varian, Palo Alto, CA, USA) at 725 nm.

The results were expressed as mg of gallic acid equivalent (GAE) per g of dried plant.

72

UPLC-QTOF-MSE analysis

An Acquity UPLC system (Waters, Milford, MA, USA) coupled to a

quadrupole/time-of-flight (QTOF) system (Waters, Milford, MA, USA) was used. The


Milford, MA, USA) column kept at 40 °C. The eluent system employed was a


at a flow rate of 0.4 mL/min. The gradient varied linearly from 2 to 95% B (v/v) over

0.0-15.0 min, held constant at 100% B over 15.1-17.0 min, and a final wash and

reequilibration at 2% B over 17.1–19.1 min. The sample injection volume was 5 µL.

The spectrometer operated with MSE centroid. Mass spectra were recorded in both

positive and negative polarity electrospray ionization (ESI) modes in a mass range

between 110 and 1180 Da, scan time of 0.1 sec, with leucine enkephalin as a lock mass

standard. The instrument settings were as follows for the ESI- and ESI+ modes,

respectively: collision energy of 5eV, capillary voltage of 2.6 and 3.2 kV, sample cone

voltage of 20 and 32 V, extraction cone voltage of 0.5 and 1 V, source temperature at

120 °C, desolvation temperature at 350 °C, and desolvation gas flow at 500 and 350

L/h. The samples were dissolved in water at a concentration of 2 mg/mL and filtered on

0.22 µm PTFE membranes. The compounds were tentatively identified based on their

exact mass and comparison with published data [18, 22-32].

Multivariate analysis

To identify potential discriminatory compounds of P. niruri extracts obtained by

different extraction techniques, the ESI- raw data from all samples were processed with

the MarkerLynx software (Waters, Milford, MA, USA). The method parameters were

set as follows: retention time range 0.8-6 min, mass range 110-1180 Da, mass tolerance

0.05 Da, and noise elimination level set at 5. For data analysis, a list of the intensities of

the detected peaks was generated using a pair of retention time (tr) and mass data (m/z)

as the identifier of each peak. An arbitrary ID was assigned to each of these tr–m/z pairs

based on their order of elution from the UPLC system. The ion intensities for each

detected peak were normalized against the sum of the peak intensities within that

sample using MarkerLynx. Ions from different samples were considered to be the same

ones when they matched their tr and m/z values. Pareto scaling method was used to

generate the PCA plot. The data comprising the peak number (tr-m/z pair), sample

73

name, and ion intensity were analyzed by principal component analysis (PCA) and

orthogonal partial least squares discriminant analysis (OPLS-DA) using the

MarkerLynx software.

Results and discussion

Total phenolics

Based on the responses obtained from the experimental designs performed for

the PLE and UAE methods (Supporting Information), the best extraction conditions

were selected as follows: PLE extracting at 192 °C and time of 15 min (PLE 192) and

UAE operating with ultrasonic intensity of 226 W/cm2 for 5 min and L/S ratio of 46.8

mL/g. The performances of the optimized PLE and UAE were compared with the

conventional extraction, besides a PLE at a lower temperature from the design, PLE 120

(120 °C for 7 min). The extract obtained by PLE 192 presented the highest phenolic

content (99.0±1.2 mg GAE/g dry plant). This technique uses high pressure combined

with high temperatures to promote greater solubility due to the decrease in water

polarity, besides increasing the mass transfer, improving the extraction efficiency. PLE

120 extract showed the second best result (77.8±1.2 mg GAE/g dry plant). The CE yield

(67.0±3.6 mg GAE/g dry plant) was lower than the obtained by PLE. In this control

method the temperature was lower than in pressurized systems (85 °C), but the

extraction time was longer (30 min) and the L/S ratio used was 8 times higher, with

greater solvent consumption. Ultrasound-assisted extraction had the lowest phenolic

content in relation to other methods (50.6±1.2 mg GAE/g dry plant). In this technique

the extraction occurred in 5 min at 25 °C, unlike the other methods that used higher

temperatures. Furthermore, the high power might have also degraded some phenolic

compounds, since radicals may be formed during cavitation and react with the phenolic

compounds, which oxidize them. These radicals are formed due to the dissociation of

the water molecules or other gases [33]. Our group [11] studied the same extraction

conditions for Phyllanthus amarus and also obtained the best total phenolics result

using PLE at 192 °C for 15 min. In the UAE extracts, the highest total phenolic content

(27.23±0.75 mg GAE/g dry plant) was obtained using ultrasonic intensity of 301 W/cm2

for 7 min and L/S ratio of 40 mL/g, unlike the result for P. niruri. In P. amarus

extraction it was observed a significant influence of the quadratic effect of L/S ratio

variable with maximum for values between 30 and 40 mL/g. In P. niruri result, the L/S

74

ratio presented a linear effect. P. niruri extracts presented higher phenolic content

compared to P. amarus [11].

Identification of chemical components

In order to achieve a more detailed characterization of P. niruri extracts, a

UPLC-QTOF-MSE analysis was carried out, since it is a technique capable of

identifying compounds with high accuracy and differentiating ions with approximately

the same mass. Moreover, it can also provide information about the chemical structure

of the constituents through mass fragmentation (MS/MS) [11, 22]. Table 1 presents a

summary of the compounds identified in P. niruri extracts. The proposed structures for

the main compounds characterized in this study are shown in Fig. S3 (supporting

information). The numbers in parenthesis followed by the names of compounds in Fig.

S3 (supporting information) correspond to the peak numbers in the chromatograms

presented in Fig. 1. The features of mass spectra from some of the major compounds are

discussed in more detail below.

Figure 1. UPLC-QTOF-MSE chromatograms of P. niruri aqueous extracts obtained

through UAE (a), PLE 120 (b), PLE 192 (c) and CE (d).

75

Table 1. Compounds tentatively determined by UPLC-QTOF-MS/MS in the P. niruri aqueous extracts obtained from UAE, PLE and CE

techniques.

Nº tr

(min)

Obsd m/z

ES(+)

MS/MS fragments

m/z, ES(+)

Obsd m/z

ES(-)

MS/MS fragments

m/z, ES(-)

Molecular

Formula

Calcd

m/z

Error

(ppm) Proposed compound Detected in Reference

Acids

1 0.86 - - 209.0292 [M-H]- 191.0204 [M-H-H2O]- C6H10O8 209.0297 -2.4 Mucic acid UAE, PLE 120,

PLE 192, CE

Yang et al.22

2 0.91 - - 191.0548 [M-H]- 127.0332

[M-H-CO-2H2O]-

C7H12O6 191.0556 -4.2 Quinic acid UAE, PLE 120,

PLE 192, CE

Kumar et al.23

3 1.22 357.0464 [M+H]+ 339.0367 [M-H2O+H]+, 247.0266

355.0304 [M-H]- 337.0199 [M-H-H2O]-, 711.0754 [2M-H]-

C14H12O11 355.0301 0.8 Chebulic acid UAE, PLE 120, PLE 192, CE

Yang et al.22

5 1.78 - - 169.0127 [M-H]- 125.0175 [M-H-CO2]- C7H6O5 169.0137 -5.9 Gallic acid UAE, PLE 120,

PLE 192, CE

Yang et al.22

11 2.64 - - 463.0503 [M-H]- 301.0013 [M-H-Hex]- C20H16O13 463.0513 -2.2 Ellagic acid hexose PLE 192 Yang et al.22

15 3.12 293.0304 [M+H]+ 219.0288 291.0140 [M-H]- 247.0190 [M-H-CO2]-,

219.0288 [M-H-CO2-

CO]-, 191.0352

[M-H-CO2-2CO]-

C13H8O8 291.0141 -0.3 Brevifolin carboxylic

acid

UAE, PLE 120,

PLE 192, CE

Kumar et al.23

19 3.51 - - 387.1658 [M-H]- 207.1033 [M-H-C6H10O5-H2O]-

C18H28O9 387.1655 0.8 Tuberonic acid hexoside

UAE, PLE 120, PLE 192, CE

Kumar et al.23

30 5.56 561.1644 [M+H]+ 415.1047, 397.0965 559.1468 [M-H]- 483.1901, 395.0781 C27H28O13 559.1452 2.9 3-O-Sinapoyl-5-O-

caffeoylquinic acid

UAE, PLE 120,

PLE 192, CE

Kuhnert et

al.18

Flavonoids

22 3.82 595.1673 [M+H]+ 449.1098, 431.0995, 413.0957, 329.0662

593.1514 [M-H]- 473.1075, 429.0836 [M-H-Rhamnose-

H2O]-, 357.0600,

327.0480, 309.0415

C27H30O15 593.1506 1.3 Orientin-2″-O-rhamnoside

UAE, PLE 120, PLE 192, CE

Sprenger et al.24

24 4.11 579.1704 [M+H]+ 433.1115, 415.1058,

313.0742, 271.0631

577.1565 [M-H]- 457.1142, 413.0857

[M-H-Rhamnose-

H2O]-, 293.0454

C27H30O14 577.1557 -4.7 Vitexin-2″-O-

rhamnoside

UAE, PLE 120,

PLE 192, CE

Sprenger et

al.24

25 4.23 465.1061 [M+H]+ 433.1154, 319.0444,

303.0193 [M-Hex+H]+

463.0856 [M-H]- 301.0015 [M-H-Hex]- C21H20O12 463.0877 -4.5 Quercetin-3-O-

hexoside

UAE, PLE 120,

PLE 192, CE

Hossain et

al.25

26 4.41 365.0330 [M+H]+ - 363.0180 [M-H]- - C16H12O8S 363.0175 1.4 Niruriflavone UAE, PLE 120,

CE

Guo et al.26

76

27 4.74 449.1051 [M+H]+ 303.0481 447.0945 [M-H]- 301.0348 C21H20O11 447.0927 4.0 Quercitrin UAE, PLE 120,

PLE 192, CE

Kumar et al.23

29 5.30 585.1272 [M+H]+ 433.1175, 415.1093,

397.0933

583.1094 [M-H]- 431.1013, 413.0872,

395.0781, 285.0442, 169.0140

C28H24O14 583.1088 1.0 Kaempferol-O-galloyl-

deoxyhexoside

UAE, PLE 120,

PLE 192, CE

Gu et al.27

Ellagitannins

7 2.21 - - 669.0931 [M-H]- 337.0199 [M-H-

galloyl-Hex-H2O]

C27H26O20 669.0939 -1.2 Neochebuloyl

galloylglucose

UAE, PLE 120,

CE

Yang et al.22

9 2.42 - - 669.0927 [M-H]- 337.0187 [M-H-

galloyl-Hex-H2O]

C27H26O20 669.0939 -1.8 Neochebuloyl

galloylglucose

(isomer)

PLE 120, CE Yang et al.22

12 2.86 704.0013 481.0655, 463.0548, 275.0200, 247.0258,

127.0389

649.0686 [M-H]- 435.0561, 169.0117 C27H22O19 649.0677 1.4 Furosin UAE Lai et al.28

13 2.94 704.0032 481.0642, 463.0551, 275.0223, 247.0252,

127.0387

649.0692 [M-H]- 435.0569, 169.0096 C27H22O19 649.0677 0.5 Furosin (isomer) UAE Lai et al.28

14 3.04 - 801.0847, 783.0759 969.0824 [M-H]- 301.0018, 247.0239, 169.0129

C41H30O28 969.0845 -2.2 Repandusinic acid A PLE 120, CE Ogata et al.29

16 3.23 - 783.0717 969.0823 [M-H]- 633.0740, 301.0053,

247.0244, 169.0151

C41H30O28 969.0845 -2.3 Repandusinic acid A

(isomer)

UAE, PLE 120,

PLE 192, CE

Ogata et al.29

17 3.30 975.0837 [M+Na]+ 783.0704 [M-galloyl-

H2O+H]+, 303.0167

951.0721 [M-H]- 933.0770 [M-H-H2O]-,

300.9990, 169.0144

C41H28O27 951.0740 -2.0 Geraniin PLE 120, CE Kumar et al.23

18 3.36 - 465.0694 [M-galloyl-H2O+H]+, 303.0153,

277.0369

633.0721 [M-H]- 463.0515 [M-H- galloyl-H2O]-,

300.9999

[M-H-galloyl-Hex]-

C27H22O18 633.0728 -1.1 Corilagin UAE, PLE 120, PLE 192, CE

Kumar et al.23

20 3.59 949.1006 [M+Na]+ 757.0907 [M-galloyl-

H2O +H]+, 303.0172

925.0958 [M-H]- 301.0006 C40H30O26 925.0947 1.2 Phyllanthusiin C PLE 120, PLE

192, CE

Latté and

Kolodziej30

21 3.70 - 801.0836, 783.0809 969.0825 [M-H]- 301.0010, 247.0271,

169.0108

C41H30O28 969.0845 -2.1 Repandusinic acid A

(isomer)

UAE, PLE 120,

CE

Ogata et al.29

23 3.90 - 783.0709 [M-galloyl-

H2O+H]+, 303.0168

951.0732 [M-H]- 300.9975, 169.0116 C41H28O27 951.0740 -0.8 Geraniin (isomer) UAE, PLE 120,

PLE 192, CE

Kumar et al.23

28 4.85 947.0850 [M+Na]+ 755.0776 [M-galloyl-H2O+H]+,

433.1146, 303.0214

923.0792 [M-H]- 327.0511, 300.9980 C40H28O26 923.0791 0.1 Phyllanthusiin U PLE 120, CE Chen et al.31

Other compounds

4 1.59 - - 331.0665 [M-H]- 271.0484, 211.0220,

169.0125 [M-H-Hex]-,

C13H16O10 331.0665 0.0 Monogalloyl-hexoside UAE, PLE 120,

PLE 192, CE

Sentandreu et

al.32





77

151.0021

6 2.04 - - 667.0782

[M+HCOO]-

247.0224, 191.0320 C26H22O18 667.0783 -0.1 Virganin UAE, PLE 120,

PLE 192, CE

Guo et al.26

10 2.47 - - 667.0755

[M+HCOO]-

247.0275 C26H22O18 667.0783 -4.2 Virganin (isomer) UAE Guo et al.26

Unknown

8 2.36 - - 447.1163 337.0210, 265.0375,

177.0551

Unknown UAE, PLE 120,

PLE 192, CE

78

Most peaks of acids in P. niruri extracts were observed in negative mode

showing the deprotonated ions [M-H]- with the loss of one mass unit. The mucic, quinic

and chebulic acids showed ions at m/z 209, 191, and 355, respectively. Chebulic acid

was identified based on its fragmentation pattern with ions at m/z 355 [M−H]−, m/z 337

[M−H−H2O]−, and m/z 711 [2M−H]−. For gallic acid, two characteristic ions were

observed at m/z 169 referring to [M−H]− and m/z 125 attributed to the neutral loss of

CO2 [M−H−CO2]−. Ellagic acid was not detected in P. niruri extracts, however, its

derivative ellagic acid hexoside was identified in accordance with characteristic ions at

m/z 463 [M–H]– and 301 [M–H–Hex]–. The ions at m/z 387 [M–H]– and 207 [M–H–

C6H10O5–H2O]− were assigned to tuberonic acid hexoside. Flavonoids present in P.

niruri extracts, mostly glycosylated, showed peaks both in positive and negative mode,

corroborating each other, allowing for unambiguous identification. For example,

quercetin-3-O-hexoside exhibited ions at m/z 465 [M+H]+ and 303 [M-Hex+H]+ in

positive mode, referring to the protonated molecule and the hexoside loss, respectively,

and also, ions at m/z 463 [M-H]- and 301 [M-H-Hex]- in negative mode, concerning the

deprotonated molecule and the hexoside loss, respectively (Fig. S4, supporting

information). Orientin-2″-O-rhamnoside and vitexin-2″-O-rhamnoside are C-

glycosylated flavones, a flavonoid subclass which have a sugar unit linked to the

benzene ring A [24]. In this case, the flavonoids have a rhamnose bound to the aglycone

by an O-glycosidic bond and also have in their mass spectra characteristic ions allowing

accurate identification (Fig. S4, supporting information). The ellagitannins are

hydrolysable tannins and, often, in their mass spectra provide a fragment at m/z 301 due

to the loss of a hexahydroxydiphenoyl (HHDP) group [22]. Some ellagitannins and its

isomers were found in P. niruri extracts, however, the designation of isomers in this

study was tentatively performed, since the MS and/or MS/MS data are not enough for

their unequivocal differentiation. Geraniin, corilagin, phyllanthusiin C and U and

repandusinic acid A are examples of compounds characterized as ellagitannins found in

P. niruri extracts (Fig. S5, supporting information). Furosin and virganin are also

hydrolysable tannins [28] and were identified in the extracts analyzed in this work.

Multivariate statistical analysis

The results of principal component analysis (PCA) of P. niruri extracts obtained

by different extraction methods are shown in Fig. 2. The PC1 versus PC2 accounted for

70.00% of the total variance (PC1 = 44.31%, PC2 = 25.69%). Analysis of each

79

extraction process in five replicates showed a good clustering, confirming the

reproducibility of the UPLC-QTOF-MSE method.

Figure 2. PCA score plot generated by Pareto of P. niruri extracts obtained through CE

(conventional extraction), PLE (pressurized liquid extraction in temperatures of 120 °C

and 192 °C) and UAE (ultrasound assisted extraction). Ions in negative mode.

According to the PCA score plot, samples from conventional extraction (CE),

pressurized liquid extraction (PLE 120) and (PLE 192) and ultrasound assisted

extraction (UAE) were divided into three clusters. This division indicated that the use of

these extraction processes could significantly change the composition of the extracts.

CE and PLE 120 samples clustered in the upper right region and formed a single group,

indicating that there is more similarity between the extracts of these two processes in

comparison with others. The colors of these two extracts were also similar, a light

brown. In both processes there was a heating, 85 °C in CE and 120 °C in PLE 120, even

though the extraction time was lower in the latter one (7 min). The PLE 192 samples

also clustered in the upper region of the graph, but on the left, indicating that the

difference between the two aforesaid groups occurred along PC1. The color of the

extract was brown. In PLE 192 method, the extraction temperature was high (192 °C),

promoting change in the chemical composition of the extract. Samples obtained by

UAE clustered at the bottom left region, far from the other samples. The separation

occurred mainly along PC2. The color of the extract was dark brown. In this technique,

80

the extractions were done at room temperature (25 °C), unlike other methods, but with a

high ultrasonic power.

To find out which components most contributed to the significant differences

among the samples, S-plots were generated (Fig. 3) from the OPLS-DA analysis

(Orthogonal Partial Least squared discriminant analysis) of the molecular ions in the

negative mode. Comparisons were made between the samples obtained by UAE and CE

(Fig. 3A) as well as between PLE 192 and CE (Fig. 3B). In the S-plot, each point

represents an ion (tr–m/z pair). The X-axis represents variable contribution, and the

further the ion tr–m/z pair point departs from zero, the more it contributes to the

difference between the two groups. The Y-axis represents variable confidence, and the

further the ion tr–m/z pair point departs from zero, the higher is the confidence level for

the difference between the two groups [34]. According to the S-plot (Fig. 3A), ions 5 and

11 at the bottom left corner of "S" were the ions from the PLE 192 sample that

contributed most to the difference regarding the CE sample. Likewise, the ions 21, 20,

17, 28, 23, 25, 16 and 27 at the top right corner of the "S" were identified as the most

characteristic ions in CE extract. The gallic acid was identified in the S-plot as the ion 5

(tr 1.78 min, m/z 125.0175). The m/z 125.0175 is a characteristic fragment of the gallic

acid [M-H-CO2]- and presented the highest relative abundance among the ions in the

mass spectra of this compound, therefore it was used for chemometrics. Ellagic acid

hexose was observed in the ion 11 (tr 2.64 min, m/z 463.0503 [M-H]-). This compound

was found only in the PLE 192 extract. Repandusinic acid A corresponds to ion 21 (tr

3.70 min, m/z 969.0825 [M-H]-). Phyllanthusiin C was found in the ion 20 (tr 3.59 min,

m/z 925.0958 [M-H]-). Geraniin was observed in the ion 17 (tr 3.30 min, m/z 951.0721

M-H]-), phyllanthusiin U corresponds to ion 28 (tr 4.85 min, m/z 923.0792 [M-H]-), an

isomer of geraniin was found in the ion 23 (tr 3.90 min, m/z 951.0732 [M-H]-),

quercetin-3-O-hexoside was identified in the ion 25 (tr 4.23 min, m/z 463.0856 [M-H]-),

an isomer of repandusinic acid A was found in the ion 16 (tr 3.23 min, m/z 969.0823

[M-H]-) and quercitrin was identified in the ion 27 (tr 4.74 min, m/z 447.0945 [M-H]-).

Ions 17, 20, 21 and 28 also appeared in Fig. 3B at the top right corner of the "S" and

represent the ions of the CE sample that contributed most to the difference from the

UAE extract. This indicates that the compounds geraniin, phyllanthusiin C,

repandusinic acid A and phyllanthusiin U were extracted in greater amounts by the CE

technique. As PLE 120 and CE samples belonged to the same group in the PCA and

showed the same trend in the intensities of these ions (Fig. 3C), we can assume that

81

PLE 120 samples contain the aforesaid metabolites in similar quantities to the CE

samples.

Figure 3. continued

82

Figure 3. OPLS-DA (S-plot) (A) PLE 192 and CE, (B) UAE and CE and ion intensity

trend plots (C) of P. niruri extracts in negative mode. 5 (tr 1.78 min, m/z 125.0175), 6 (tr

2.04 min, m/z 247.0224), 10 (tr 2.47 min, m/z 667.0755), 11 (tr 2.64 min, m/z 463.0503),

12 (tr 2.86 min, m/z 649.0686), 13 (tr 2.94 min, m/z 169.0096), 16 (tr 3.23 min, m/z

969.0823), 17 (tr 3.30 min, m/z 951.0721), 20 (tr 3.59 min, m/z 925.0958), 21 (tr 3.70

min, m/z 969.0825), 23 (tr 3.90 min, m/z 951.0732), 25 (tr 4.23 min, m/z 463.0856), 27

(tr 4.74 min, m/z 447.0945) and 28 (tr 4.85 min, m/z 923.0792)

UAE extracts presented the ions 13, 5, 6, 10 and 12 as the most characteristic

ones. Furosin was identified in the ion 13 (tr 2.94 min, m/z 169.0096). The m/z 169.0096

is a fragment of this compound. Furosin was observed only in the UAE extract. The

gallic acid, represented by the ion 5 (tr 1.78 min, m/z 125.0175 [M-COOH]-) was also

found in greater quantities in PLE 192 extract. Virganin was identified in the ion 6 (tr

2.04 min, m/z 247.0224). The m/z 247.0224 is a fragment of this compound. An isomer

of virganin was found in the ion 10 (tr 2.47 min, m/z 667.0755 [M+HCOO]-) and an

isomer of furosin was observed in the ion 12 (tr 2.86 min, m/z 649.0686 [M-H]-).

Accordingly, the chemical markers of CE and PLE 120 extracts were geraniin,

phyllanthusiin C, repandusinic acid A and phyllanthusiin U. These compounds are

ellagitannins with mass above 900 Da [30]. Geraniin stands out for being an important

bioactive compound found in some species of the genus Phyllanthus and being related

to multiple activities such as anticancer, antiviral, antihypertensive, antihyperglycaemic,

analgesic, among others [1, 23, 35]. Repandusinic acid A showed strong inhibitory activity

against human immunodeficiency virus type-1 reverse transcriptase [29]. The chemical

markers in the extracts obtained by UAE and PLE 192 are probably degradation

83

products derived from high molecular mass ellagitannins such as geraniin,

phyllanthusiin C, repandusinic acid A and phyllanthusiin U. Ellagic acid hexose, a

chemical marker of PLE 192 extract, was likely produced by thermal hydrolysis [36].

The ellagic acid hexose molecule is the product of a lactonization of HHDP group

found in ellagitannins linked to a hexose. Virganin and furosin, chemical markers of

UAE extract, are also products of a partial degradation by hydrolysis, probably due to

the large amount of energy of the ultrasonic probe.14 Finally, gallic acid found in greater

amounts in UAE and PLE 192 extracts is the final degradation product of hydrolysable

tannins, it is found in ellagitannins as galloyl and can be released by boiling water,

tannase enzyme and acidic or basic environment [36, 37].

Conclusion

Liquid chromatography-mass spectrometry in conjunction with chemometric

techniques were successfully applied in the chemical characterization of the extracts and

allowed to identify chemical markers of each extraction method, including degradation

products. The use of PLE technique at 120 °C, for 7 min, at the pressure of 110 bar,

increased the yield of phenolics extraction, preserving compounds with pharmacological

relevance such as geraniin, repandusinic acid A and corilagin. In this study it was

possible to improve the yield and quality of P. niruri extracts using a quick and solvent-

free method. In a future work, it can be studied the feasible of scaling up this extraction

method. Besides the evaluation of the extract in biological activities.

Funding

The current study was financially supported by Embrapa (No 02.10.06.019.00.00 and

03.14.04.002.00) and Conselho Nacional de Desenvolvimento Científico e Tecnológico

(402654/2012-9).

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87

Supporting Information for



Table S1. Experimental design of ultrasound-assisted extraction and results obtained in

the P. niruri extracts.

Run Time (min) Ultrasonic intensity

(W/cm2)

L/S ratio

(mL/g)

Phenolics

(mg/g dry plant)

1 3.0 151.0 20.0 34.5±0.9

2 3.0 151.0 40.0 32.5±0.8

3 3.0 301.0 20.0 33.0±0.8

4 3.0 301.0 40.0 45.6±1.3

5 7.0 151.0 20.0 33.9±0.9

6 7.0 151.0 40.0 41.9±1.0

7 7.0 301.0 20.0 36.9±0.6

8 7.0 301.0 40.0 45.6±1.0

9 1.6 226.0 30.0 42.8±1.0

10 8.4 226.0 30.0 43.8±1.1

11 5.0 99.0 30.0 37.6±0.8

12 5.0 353.0 30.0 42.8±0.9

13 5.0 226.0 13.2 30.2±0.5

14 5.0 226.0 46.8 50.6±1.2

15© 5.0 226.0 30.0 41.6±1.2

16© 5.0 226.0 30.0 40.0±1.0

17© 5.0 226.0 30.0 40.9±1.1


Regression model for phenolics of UAE:

𝑌1 = 41.07 + 1.04𝑋1 + 0.13𝑋12 + 1.98𝑋2 − 0.95𝑋2

2 + 4.50𝑋3 − 0.91𝑋32 −

0.62𝑋1𝑋2 + 0.75𝑋1𝑋3+1.92𝑋2𝑋3 (1)

Where 𝑌1: phenolics (mg/g dry plant) of UAE, 𝑋1: time (min), 𝑋2: ultrasonic intensity

(W/cm2), and 𝑋3: L/S ratio (mL/g).

Table S2. Analysis of variance (ANOVA) of the regression model (Eq. 1).

Source of variation Sum of

squares

Degrees of

freedom

Mean square F-value

Regression 412.22 9 45.80 3.97

Residual 80.81 7 11.54

Total 493.03 16

Correlation

coefficient

0.8158

F- listed value (95%) F9,7 = 3.68

88

Figure S1. Estimated effects by Pareto plot and response-surface graphs for the

phenolics content (mg/g dry plant) in ultrasound-assisted extraction.

Table S3. Experimental design of pressurized liquid extraction and results obtained in

the P. niruri extracts.

Run Temperature (C) Time (min) Phenolics (mg/g dry plant)

1 120.0 7.0 77.8±1.2

2 120.0 23.0 72.1±0.9

3 180.0 7.0 83.3±1.0

4 180.0 23.0 93.9±1.2

5 107.6 15.0 68.6±0.9

6 192.4 15.0 99.0±1.2

7 150.0 3.7 76.0±1.1

8 150.0 26.3 87.4±1.1

9© 150.0 15.0 81.1±1.4

10© 150.0 15.0 82.7±1.3

11© 150.0 15.0 83.6±1.3


Regression model for phenolics of PLE:

𝑌2 = 82.49 + 8.79𝑋1 + 0.40𝑋12 + 2.61𝑋2 − 0.63𝑋2

2 + 4.09𝑋1𝑋2 (2)

Where 𝑌2: phenolics (mg/g dry plant) of PLE, 𝑋1: temperature (°C), and 𝑋2: time (min).

89

Table S4. Analysis of variance (ANOVA) of the regression model (Eq. 2).

Source of variation Sum of

squares

Degrees of

freedom

Mean square F-value

Regression 743.41 5 148.68 14.30

Residual 51.97 5 10.39

Total 795.38 10

Correlation

coefficient

0.9347

F- listed value (95%) F5,5 = 5.05

Figure S2. Estimated effects by Pareto plot and response-surface graph for the

phenolics content (mg/g dry plant) in pressurized liquid extraction.

90

Figure S3. Structures of the substances identified in P. niruri extracts.

91

Figure S4. Major fragments observed in mass spectra of glycosylated flavonoids.

92

Figure S5. Proposal of the loss of the HHDP group by ellagitannins, generating the

fragments observed in the negative mode.

93

ARTIGO 3



Adriana Dutra Sousa, Paulo Riceli Vasconcelos Ribeiro, Kirley Marques Canuto,

Guilherme Julião Zocolo, Fabiano André Narciso Fernandes, Edy Sousa de Brito

Artigo a ser submetido à revista Drying Technology

94



Adriana Dutra Sousa1,2, Paulo Riceli Vasconcelos Ribeiro1, Kirley Marques Canuto1,

Guilherme Julião Zocolo1, Fabiano André Narciso Fernandes2, Edy Sousa de Brito1,*


Brazil.





Abstract

In this study, the drying kinetics of Phyllanthus amarus and Phyllanthus niruri were

investigated experimentally in an air-drying oven as a function of drying temperature

(50, 60 and 70°C). The effect of air-drying temperature on phenolic content and on LC-

MS profile was also studied. The increase in air-drying temperature reduced the drying

time and increased the effective moisture diffusivity. Effect of temperature on the

diffusivity was expressed by an Arrhenius relation with activation energy values of

22.828 and 43.129 kJ/mol for Phyllanthus niruri and Phyllanthus amarus, respectively.

The use of air-drying at 70°C increased the availability of some phenolic compounds.

However, some sensitive components were negatively affected by the higher

temperature.

Keywords: medicinal plant; moisture diffusivity; phenolics; Phyllanthus

95

1. Introduction

Phyllanthus amarus Schum & Thonn and Phyllanthus niruri Linn

(Phyllanthaceae) are small herbs, widely used in folk medicine to treat various diseases

such as hepatitis, intestinal infections, diabetes, kidney disorders and dyspepsia [1, 2].

These plants contain a series of lignans, alkaloids, triterpenes, flavonoids and tannins,

which have been reported to be hepatoprotective, anti-inflammatory, anticancer,

antiviral, analgesic and diuretic [1-3].

Drying of medicinal herbs is often used to prevent the microbial growth and

hence to preserve the quality of the harvested plant as well as to reduce the weight and

bulk of plants for cheaper transport and storage. Conventional drying, also referred as

hot-air or convective drying, is a widely adopted technique in the food industry [4]. In

air-drying, the hot and dry air meets the surface of the wet material, which transfers heat

into the solid bulk product primarily by conduction. The liquid migrates then onto the

material surface and is transported away by air convection [4]. Transport of moisture

occurs by diffusion mechanisms, especially in the falling drying rate period. It is

generally difficult to predict mass diffusion coefficients. Therefore, experimental

approaches based on sorption/desorption kinetics have been used. Furthermore, Fick’s

second law of diffusion equation is commonly used to describe moisture transport

during drying [4-6]. Data from drying kinetics of biological materials is useful in

design, optimization and control of drying processes. In fact, mathematical modelling

and experimental studies have been conducted on the drying process of plants, such as

rosemary leaves [7], dill and parsley leaves [5], besides nettle and mint leaves [6].

However, no literature on air-drying kinetics of P. amarus and P. niruri has been found.

Regarding the influence of drying on the phytochemicals content, hot-air drying

treatment might produce changes in the structure of plants, which become more open

and interconnected than in fresh material. Due to this alteration, the solvent can

penetrate more easily into the plant tissue and provide a greater surface for mass

transfer, resulting in a more efficient extraction of these compounds [8, 9].

Nevertheless, thermal processing can also affect the phytochemicals by thermal

breakdown from chemical reactions involving enzymes, light and oxygen [8].

Rodríguez et al. [10] reported a decrease by 50% in total phenolic and total flavonoid

contents after air-drying of Aristotelia chilensis berries. However, no significant

difference was observed between the total phenolic contents of P. amarus extracted

96

with boiling water from fresh and hot-air dried plants [11]. The heat treatment

significantly enhanced the total phenolic and total flavonoid contents in Shiitake

mushroom [9], and Martínez-Las Heras et al. [8] described loss of flavonoids during air-

drying of persimmon leaves. However, the total phenol content was higher in dry leaf

extracts as compared to fresh leaf. Thus, hot-air drying might enhance or deplete the

phytochemicals content depending on the matrix and drying conditions, e.g.,

temperature and air velocity.

The aim of this study was to assess the effect of air-drying temperature on the

effective moisture diffusivity, as well as on the phenolic content and on LC-MS

chemical profile from aerial parts of P. amarus and P. niruri.

2. Materials and methods

2.1. Sample collection and preparation

Aerial parts (flowers, leaves and stems) of Phyllanthus amarus and P. niruri

were harvested at the Embrapa Experimental Field (Paraipaba, Ceará state, Brazil). The

plant materials (leaf and stem) were manually cut before the drying experiments.

2.2. Air-drying

Drying kinetics was carried out in a circulating air-drying oven (Tecnal model

TE-394/1). The experiments were performed at three temperatures (50, 60 and 70°C)

and air velocity of 0.5 m/s. The samples (5 g) with a thickness of 0.5 mm were

uniformly spread as a thin layer on a circular steel sample holder and were placed in the

drying chamber. The samples were weighed every 15 min during 2 h and then every 30

min for more 2 h and at 24h. The drying experiments were conducted in triplicate.

2.3. Drying kinetics

Drying curves were constructed using data obtained at different temperatures.

The moisture content (M) was calculated on a dry weight basis using the standard

formula:

𝑀 =𝑊−𝑊𝑑

𝑊𝑑 (1)

Where 𝑊 is the weight of sample and 𝑊𝑑 is the weight of dry matter in the sample.

97

The moisture content data were converted to moisture ratio (MR) given by the

following equation.

𝑀𝑅 =𝑀𝑡−𝑀𝑒

𝑀0−𝑀𝑒 (2)

Where 𝑀𝑡, 𝑀0 and 𝑀𝑒 are moisture content at any time of drying, initial moisture

content and equilibrium moisture content, respectively. The values of 𝑀𝑒 are relatively

small compared to 𝑀𝑡 or 𝑀0, thus, equation (2) may be simplified to equation (3) as

given by Doymaz [12].

𝑀𝑅 =𝑀𝑡

𝑀0 (3)

2.4. Effective moisture diffusivity and activation energy

Most drying processes of plant material occur in the falling-rate period, and the

transfer of moisture during the drying process is controlled by internal diffusion. Fick’s

second law of diffusion has been widely used to describe the drying process during the

falling-rate period of most biological materials [4]. Fick's second law in Cartesian

coordinates and in dimensionless form can be written as in Equation 4 [13].

𝜕𝑀𝑅

𝜕𝑡=

𝜕

𝜕𝑦(𝐷𝑒𝑓𝑓

𝜕𝑀𝑅

𝜕𝑦) (4)

where 𝐷𝑒𝑓𝑓 is the effective moisture diffusivity (m2/s), 𝑡 is time, 𝑦 is space coordinate

measured from center to the board, and MR is moisture ratio. This equation can be

applied for different regularly shaped bodies such as cylindrical, spherical and

rectangular products, while Equation 5 can be used for materials with slab geometry by

assuming uniform initial moisture content, negligible shrinkage, constant temperature

gradients and diffusion coefficients [14].

𝑀𝑅 =8

𝜋2∑

1

(2𝑛+1)2∞𝑛=0 𝑒𝑥𝑝 (−

(2𝑛+1)2𝜋2𝐷𝑒𝑓𝑓 𝑡

4𝐿2 ) (5)

Where 𝐷𝑒𝑓𝑓 is the effective diffusivity (m2/s), L is the thickness of slab in the sample

and t is the drying time (s).

The temperature dependence of effective diffusivity can be represented by an

Arrhenius-type expression (Equation 6) to obtain activation energy (Ea). Activation

98

energy represents the energy level of water molecules for moisture diffusion and

evaporation [15].

𝐷𝑒𝑓𝑓 = 𝐷0exp (−𝐸𝑎

𝑅𝑇) (6)

Where Ea is the activation energy (kJ/mol), D0 is the Arrhenius factor for the drying

process (m2/s); R represents the universal gas constant (8.314 kJ/mol K) and T is the

absolute temperature (K). By plotting ln (𝐷𝑒𝑓𝑓) against 1/T, a straight line was obtained

with slope, −Ea/R and y intercept of In (D0). The activation energy and Arrhenius factor

were obtained from slope and y intercept respectively.

2.5. Pressurized-liquid extraction

To evaluate the influence of the drying process on the product quality, the dried

samples were ground with a knife mill (Wiley type) and extracted in a Dionex ASE 350

system (Sunnyvale, CA, USA) using deionized water as solvent [16]. Two grams of the

dried plant were mixed with 2 g of diatomaceous earth (dispersing agent) and placed in

66 mL stainless steel cell. The extraction was performed at 90°C in a sequence of three

cycles of 5 minutes and the system pressure was 110±7 bar. The extracts obtained were

concentrated in a rotary vacuum evaporator at 40°C, then frozen and freeze-dried.

2.6. Determination of total phenolics

The methodology adapted from Singleton and Rossi [17] was employed to



20% sodium carbonate, and 3.5 mL of water. After 90 minutes at rest, the absorbance

was read in a UV spectrophotometer (Cary 300, Varian, Palo Alto, CA, USA) at 725

nm. The results were expressed as mg of gallic acid equivalent (GAE) per g of dried

plant.

2.7 UPLC-QTOF-MSE and multivariate analysis

To identify potential discriminatory compounds of extracts obtained from

Phyllanthus samples submitted to different drying temperatures, a multivariate analysis

using UPLC-MS data was performed. An Acquity UPLC system (Waters, Milford, MA,

USA) coupled to a quadrupole/time-of-flight (QTOF) system (Waters, Milford, MA,

99

USA) was used. The compounds were separated on an Acquity BEH C18 (1.7 µm, 2.1 x

150 mm; Waters, Milford, MA, USA) column kept at 40°C. The eluent system

employed was a mixture of A (0.1% formic acid in water) and B (0.1% formic acid in

acetonitrile) at a flow rate of 0.4 mL/min. The gradient varied linearly from 2 to 95% B

(v/v) over 0.0-15.0 min, held constant at 100% B over 15.1-17.0 min, and a final wash

and reequilibration at 2% B over 17.1–19.1 min. The sample injection volume was 5

µL. The spectrometer operated with MSE centroid. Mass spectra were recorded in both

positive and negative electrospray ionization (ESI) modes in a mass range between 110

and 1180 Da, scan time of 0.1 sec, with leucine enkephalin as a lock mass standard. The

samples were dissolved in water at a concentration of 2 mg/mL and filtered on 0.22 µm

PTFE membranes.

The ESI- raw data from all samples were processed with the MarkerLynx

software (Waters, Milford, MA, USA). The method parameters were set as follows:

retention time range 0.8-6 min, mass range 110-1180 Da, mass tolerance 0.05 Da, and

noise elimination level set at 5. The ion intensities for each detected peak were

normalized against the sum of the peak intensities within that sample using

MarkerLynx. Ions from different samples were considered to be the same ones when

they matched their tr and m/z values. Pareto scaling method was used to generate the

PCA plot. The data comprising the peak number (tr-m/z pair), sample name, and ion

intensity were analyzed by principal component analysis (PCA) and orthogonal partial

least squares discriminant analysis (OPLS-DA) using the MarkerLynx software.

2.8 Statistical analysis

The phenolics results were expressed as mean ± SD. Statistical analyses were

carried out using the software Statistica (Statsoft version 7.0). A one-way analysis of

variance (ANOVA) was performed and the significant differences on the results were

determined by Tukey test at p˂0.05.

3. Results

3.1. Drying kinetics

Drying curves (moisture ratio versus time) were shown in Figure 1. The total

drying time reduced significantly as drying temperature increased. The time required to

dry aerial parts of P. amarus and P. niruri from an initial moisture content of 2.691 Kg

100

Kg-1 and 3.285 Kg Kg-1 (d.b.), respectively, to the final moisture content equal to or

below 0.05 Kg Kg-1 (d.b.) was 3.5, 2.5 and 1.5 hours at 50, 60 and 70°C of drying air

temperature, respectively, for both species. The increasing temperature increased the

energy of water molecules allowing for their rapid escape from the matrix of the plant.

P. amarus and P. niruri presented the typical drying kinetic behavior observed for other

plants [4, 7], and only the falling rate period was observed during air drying.

Figure 1. Variation of moisture ratio of (A) P. amarus and (B) P. niruri as a function

of drying time at temperatures ranging from 50 to 70°C.

Effective moisture diffusivity (𝐷𝑒𝑓𝑓) represents the conductive term of all

moisture transfer mechanisms, is a key drying parameter [15]. The 𝐷𝑒𝑓𝑓 was determined

by fitting experimental data to the Equation 5. The Table 1 lists the temperature

dependence of the 𝐷𝑒𝑓𝑓.

Table 1. Effective moisture diffusivities 𝐷𝑒𝑓𝑓 and activation energies Ea of P. niruri and

P. amarus at temperatures from 50 to 70 °C at air velocity of 0.5 m/s

Sample Temperature (°C) 𝐷𝑒𝑓𝑓 (m2/s) R2 Ea

(KJ/mol) R2

P. niruri

50 5.871x10-11 0.999

60 7.657x10-11 0.996 22.828 0.999

70 9.631x10-11 0.999

P. amarus

50 2.900x10-11 0.998

60 3.911x10-11 0.997 43.129 0.951

70 7.420x10-11 0.997

101

The 𝐷𝑒𝑓𝑓 of both species increased with the increase of drying temperature. The

effective moisture diffusivity of P. niruri thin layer ranged from 5.871x10-11 to

9.631x10-11 m2/s. While the effective moisture diffusivity of P. amarus ranged from

2.900x10-11 to 7.420x10-11 m2/s. The values lie within the general range of 10-12 to 10-8

m2/s for food materials [18]. The observed increase in diffusivities with increase in

temperature was similar to results obtained for drying of dill and parsley leaves [5] and

some herbal leaves [6].

The effective moisture diffusivity was plotted against inverse of absolute

temperature in Figure 2 for P. amarus and P. niruri, and the relationship was found to

be Arrhenius-type as described in Equation 6. The activation energy for diffusion,

calculated from Eqution 6, was 22.828 kJ/mol for P. niruri and 43.129 kJ/mol for P.

amarus (Table 1). This result shows that lower energy is required to remove water from

P. niruri compared to the energy needed to dry the P. amarus sample. The activation

energy values for P. amarus and P. niruri are similar to those proposed by other authors

for different plant materials: 35.05 and 43.92 kJ/mol for dill and parsley leaves [5];

46.80 for Allium roseum leaves [19] and 21.2 kJ/mol for mulberry fruits [20].

Figure 2. Arrhenius-type relationship between effective moisture diffusivity and

temperature for P. amarus and P. niruri samples.

102

3.2. Total phenolics

The stabilization of herbs by drying processes involves changes in the matrix

that can affect the concentration of chemical components in the dry product and the

extractability thereof. Thus, the total phenolic content of extracts obtained from P.

amarus and P. niruri samples dried at different temperatures was assessed. The higher

phenolic content was recorded at drying temperature of 60°C in both species (Figure 3).

This temperature was reported as the optimum for the retention of phenolic compounds

by some authors: Katsube et al. [21] who tested temperatures of 40-110°C for mulberry

leaves drying, Rodríguez et al. [10] who dried Aristotelia chilensis berries using

temperatures of 40-80°C and Wiriya et al. [22] who used the temperatures of 50-70°C

for chilli drying. According to the authors, the lower air-drying temperatures (˂60°C)

present a longer drying period, resulting in a loss of phenolic compounds due to

oxidation induced by the presence of oxygen. On the other hand, air-drying

temperatures higher than 60°C provided a lower content of phenolic compounds due to

thermal degradation. Comparing the two species, P. niruri extracts presented higher

phenolic contents in relation to P. amarus.

Figure 3. Effect of air-drying temperature on total phenolic content (mg gallic acid

equivalent/g dry plant) of P. amarus and P. niruri samples. Data are the mean of three

replicates. Different letters above the bars indicate significant difference (p<0.05).

103

3.3. Multivariate statistical analysis

The results of principal component analysis (PCA) of P. amarus and P. niruri

extracts obtained by different air-drying temperatures are shown in Fig. 4. The PC1

versus PC2 biplot accounted for 69.79% of the total variance (PC1 = 62.11%, PC2 =

7.68%). In PCA loadings biplot, the X variables represent all the chemical compounds

present in the samples and the plot demonstrates how the X variables in the datasets

correlate with each other. According to Fig. 4, P. amarus samples clustered in the left

side of the graph and P. niruri samples clustered in the right side. This division

indicated that the two species presented significant differences in the composition of

their extracts. The difference between the two aforementioned groups occurred along

PC1. Sprenger et al. [23] compared the chemical profile of four Phyllanthus species,

including P. amarus and P. niruri, and they found vitexin-2″-O-rhamnoside, orientin-

2″-O-rhamnoside and orientin as chemical markers of P. niruri extract and rutin,

quercetin-3-O-glucuronide and Kaempferol-3-O-rutinoside were present in P. amarus

extract and absent in P. niruri extract.

Figure 4. PCA loadings biplot generated by Pareto of Phyllanthus extracts obtained

from P. amarus and P. niruri samples submitted to different drying temperatures. Ions

detected in negative mode.

Considering the drying temperatures, P. amarus samples were divided into three

clusters, indicating that the air-drying temperature could significantly change the

104

chemical profile of the extracts. The separation occurred along PC2, where samples

dried at 50°C clustered in the upper region, samples dried at 60°C clustered in the

middle and samples dried at 70°C clustered at the bottom region. Therefore, samples

dried at 60°C presented an intermediate chemical composition compared to the other P.

amarus samples. The P. niruri samples were also divided into three clusters along PC2,

following the same order found for P. amarus, but the groups were closer, meaning that

the drying temperature promoted a smaller change in the chemical profile of the P.

niruri samples.

To find out which components contributed to the significant differences among

the samples, S-plots were generated (Fig. 5) from the OPLS-DA analysis of the

molecular ions in the negative mode. Comparisons were made between the P. amarus

samples dried at 50 and 70°C (Fig. 5A) as well as between P. niruri samples dried at 50

and 70°C (Fig. 5B). In the S-plot, each point represents an ion (tr–m/z pair). The X-axis

represents variable contribution and the Y-axis represents variable confidence.

Figure 5. OPLS-DA (S-plot) of Phyllanthus extracts obtained from samples submitted

to different drying temperatures (A) P. amarus at 50°C and 70°C, (B) P. niruri at 50°C

and 70°C. Ions in negative mode. a (tr 4.13 min, m/z 300.9967), b (tr 1.77 min, m/z

125.0233), c (tr 7.16 min, m/z 363.0160), d (tr 4.14 min, m/z 609.1443), e (tr 4.19 min,

m/z 463.0852), f (tr 3.59 min, m/z 925.0939), g (tr 3.22 min, m/z 969.0835), h (tr 3.32

min, m/z 951.0735), i (tr 3.81 min, m/z 593.1484), j (tr 4.11 min, m/z 577.1544), k (tr

3.13 min, m/z 291.0126).

As shown in the S-plot (Fig. 5A), the first three molecular ions (a, b and c) at the

bottom left corner of "S" were the molecular ions from the P. amarus sample dried at

50°C that contributed most to the difference regarding the P. amarus sample dried at

70°C. Analogously, the molecular ions d, e, f and g at the top right corner of the "S"

were identified as the most characteristic ions in P. amarus sample dried at 70°C. The

molecular ions e, f and g also appeared at the top right corner of the "S" in Fig. 5B and

represent the molecular ions of the P. niruri sample dried at 70°C that contributed most

to the difference from the P. niruri dried at 50°C. Molecular ions h, i, j and k were the

105

most characteristics in P. niruri sample dried at 50°C. Using an UPLC-QTOF-MSE

analysis, the eleven significantly changed components were identified as (a) ellagic

acid, (b) gallic acid, (c) niruriflavone, (d) rutin, (e) quercetin-3-O-hexoside, (f)

phyllanthusiin C, (g) repandusinic acid A, (h) geraniin, (i) orientin-2″-O-rhamnoside, (j)

vitexin-2″-O-rhamnoside and (k) brevifolin carboxylic acid. The details of the identified

components were summarized in Table 2.

Table 2. The significantly changed components identified by UPLC-QTOF-MS/MS in

the P. niruri and P. amarus extracts

The compounds extracted in greater amounts from P.amarus sample dried at

70°C when compared to dried at 50°C were rutin, quercetin-3-O-hexoside,

phyllanthusiin C and repandusinic acid A. The same compounds were better extracted

from P.niruri sample dried at 70°C compared to 50°C, except for rutin, which,

generally, is not present in P.niruri extracts. Rutin and quercetin-3-O-hexoside are

flavonoids and phyllanthusiin C and repandusinic acid A are ellagitannins. In a study

about the influence of drying methods on total flavonoids and total polyphenols content

of loquat flower, it was observed that in hot-air dried samples the contents of both

components increased as the drying temperature raised from 40 to 80°C [30]. Another

study reported that the rutin content of Aristotelia chilensis berries was increased when

Nº tr

(min)

Obsd m/z

ES(-) [M-H]-

MS/MS fragments

m/z, ES(-)

Molecular

Formula

Calcd

m/z

Error

(ppm) Proposed compound Reference

1 a 4.13 300.9967 257.0134

[M-H-CO2]-

C14H6O8 300.9984 -5.6 Ellagic acid Kumar et al. [24]

2 b 1.77 169.0132 125.0233 [M-H-CO2]

- C7H6O5 169.0137 -3.0 Gallic acid Yang et al. [25]

3 c 7.16 363.0160 - C16H12O8S 363.0175 -4.1 Niruriflavone Guo et al. [26]

4 d 4.14 609.1443 300.9960 [M-H-

Hex-Rham]-

C27H30O16 609.1456 -2.1 Rutin Hossain et al. [27]

5 e 4.19 463.0852 300.9984

[M-H-Hex]-

C21H20O12 463.0877 -5.4 Quercetin-3-O-

hexoside

Hossain et al. [27]

6 f 3.59 925.0939 300.9981 C40H30O26 925.0947 -0.9 Phyllanthusiin C Latté and Kolodziej [28]

7 g 3.22 969.0835 633.0706,

300.9977

C41H30O28 960.0845 -1.0 Repandusinic acid A Ogata et al. [29]

8 h 3.32 951.0735 933.0645 [M-H-

H2O]-, 300.9955

C41H28O27 951.0740 -0.5 Geraniin Kumar et al. [24]

9 i 3.81 593.1484 473.1068,

429.0811 [M-H-Rhamnose-H2O]-

C27H30O15 593.1506 -3.7 Orientin-2″-O-

rhamnoside

Sprenger et al.

[23]

10 j 4.11 577.1544 413.0868 [M-H-

Rhamnose-H2O]-

C27H30O14 577.1557 -2.3 Vitexin-2″-O-

rhamnoside

Sprenger et al.

[23]

11 k 3.13 291.0126 247.0211 [M-H-CO2]

- C13H8O8 291.0141 -5.2 Brevifolin

carboxylic acid

Kumar et al. [24]

106

the drying temperature raised from 40 to 80°C [10]. One reason for this increase might

be consequence of a balance between drying temperature and time. Additionally, part of

polyphenols and flavonoids may have been transformed from combined state to free

state at high temperature [30]. The better extracted compounds from P.amarus sample

dried at 50°C were ellagic acid, gallic acid and niruriflavone, whereas P.niruri sample

dried at 50°C presented geraniin, orientin-2″-O-rhamnoside, vitexin-2″-O-rhamnoside

and brevifolin carboxylic acid as the most characteristic compounds. Ellagic acid, gallic

acid and brevifolin carboxylic acid are phenolic acids. Niruriflavone, orientin-2″-O-

rhamnoside and vitexin-2″-O-rhamnoside are flavonoids, while geraniin is an

ellagitannin. Esparza- Esparza-Martínez et al. [31] studied the effect of air-drying

temperature on some phenolic acids, including ellagic and gallic acid, along with some

flavonoids of lime wastes and they observed that the increase in temperature from 60 to

90°C decreased the content of these compounds. This finding indicates that these

components were more affected by drying conditions and are thus more thermally

sensitive than other phenolic compounds.

4. Conclusion

The increase in air-drying temperature from 50 to 70°C reduced the drying time

in 57% and increased the effective moisture diffusivity of P. amarus and P. niruri. The

activation energy for moisture diffusion of P. amarus (43.129 kJ/mol) was higher than

that for P. niruri (22.828 kJ/mol). 60°C was found to be the best drying temperature to

obtain a higher phenolic content for both species. The use of higher air-drying

temperature increased the availability of some flavonoids and ellagitannins. However,

some phenolic acids and other flavonoids and ellagitannins were negatively affected by

the higher temperature.

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110

ARTIGO 4


Adriana Dutra Sousa, Paulo Riceli Vasconcelos Ribeiro, Kirley Marques Canuto,

Guilherme Julião Zocolo, Brijesh Tiwari, Fabiano Andre Narciso Fernandes, Edy Sousa

de Brito

Short Communication a ser submetida à revista Ultrasonics Sonochemistry

111


Adriana Dutra Sousa1,3, Paulo Riceli Vasconcelos Ribeiro1, Kirley Marques Canuto1,

Guilherme Julião Zocolo1, Brijesh Tiwari2, Fabiano André Narciso Fernandes3, Edy

Sousa de Brito1,*


Brazil.

2 Food Biosciences, Teagasc Food Research Centre, Dublin, Ireland





Abstract

Ultrasound assisted extraction is an efficient technique to obtain phenolic compounds

from medicinal plants. However, free radicals can be generated in cavitation and

degrade the target compounds. The aim of this study was to verify if the ultrasonic

power can degrade ellagitannins from Phyllanthus amarus. Extracts rich in ellagitannins

were treated with a high-power ultrasonic probe, appling two different ultrasonic

intensities: 188 and 373 W/cm2. The chemical profiles were determined by UPLC-ESI-

QTOF-MS/MS analysis and quantitatively determined using HPLC. The control and

treated extracts presented the same chemical profile, and the phenolics were

quantitatively maintained after treatment. Therefore, ultrasound process did not

hydrolise ellagitannins from P. amarus.

Keywords: phenolics; Phyllanthus; ultrasonic intensity

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1. Introduction

Ultrasound has been widely used due to its various applications, such as release

of cellular components and molecular structures, particle modification in liquids, and

de-aeration of liquids and surfaces. According to the ultrasonic intensity, ultrasound

devices can be classified as low intensity (<1 W/cm2) and high intensity (10-1000

W/cm2). The first is generally used as a non-destructive analytical technique for quality

control, while high intensity is used for extraction and processing (Tiwari, 2015).

Several physical and chemical phenomena, including turbulence, shock waves,

pressure, heat, shear forces, compression and rarefaction, cavitation and radical

formation are responsible for ultrasonic effect, being the cavitation the most important.

The ability of ultrasound to cause cavitation depends on some characteristics such as

frequency and intensity, medium viscosity, surface tension, vapor pressure, type and

concentration of dissolved gas, presence of solid particles and temperature and pressure

of the treatment. For extraction applications, solvent properties are also important. For

example, vapor pressure governs intensity of collapse; surface tension and viscosity

govern the cavitation threshold. The chemical reactivity of the solvent dictates the

primary and secondary sonochemical reactions (Esclapez et al., 2011; Soria and

Villamiel, 2010; Tiwari, 2015).

The chemical effects of ultrasound are produced by highly reactive radicals that

are generated in cavitation. The water molecules can be broken (H2O → OH- + H+),

generating H• and OH• radicals (Soria and Villamiel, 2010). These free radicals can

react with easily oxidizable compounds; they can induce a variety of chemical reactions

in the extractive solution, and may degrade the target compounds (Tiwari, 2015).

In a previous study, our group compared the ultrasound assisted extraction with

pressurized liquid extraction and reflux extraction to obtain phenolic compounds of P.

amarus, a medicinal plant, and the extracts produced by ultrasound presented lower

yield of phenolic compounds, besides absence of ellagitannins, present in extracts

obtained by the other extraction techniques (Sousa et al., 2016). Therefore, the objective

of the present study was to verify if the ultrasonic power can degrade phenolic

compounds from P. amarus.





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2. Material and Methods

2.1. Sample Collection and Preparation for Extraction

Aerial parts of P. amarus were collected from the Embrapa Experimental Field

(Paraipaba, Ceará state, Brazil). The plant materials were dried in a forced air

circulation drying oven at 40°C for 48 h and ground in a knife mill (Wiley type). The

grounded material was classified using sieves with meshes between 0.25 and 4 mm and

the particles between 0.25 and 2.0 mm were used in the extractions.

2.2. Control Extract Obtention


CA, USA) using deionized water as solvent. Ten grams of the dried plant were placed in

66 mL stainless steel cells. The cells were equipped with a stainless steel filter and a

cellulose filter at the bottom to prevent the presence of particulate matter in the

collection flask. The extractions were performed at 120°C, the extraction time was

divided into three cycles of 8 min, and the system pressure was 110±7 bar. The extracts

were homogenized and stored frozen for further treatment with ultrasonic probe.

2.3. Ultrasonic Probe Treatment

A part of the control extract was treated with a high-power (500 W) ultrasonic

probe (Unique model DES500, Indaiatuba, SP, Brazil) with a titanium tip (13 mm

diameter) and frequency of 19 kHz. The energy dissipated by the ultrasonic intensity

was calculated according to Eq. 1 (Li et al., 2004). The power levels applied were 250

and 495 W, corresponding to 188 and 373 W/cm2 or 5000 and 9900 W/L, respectively.


𝜋𝑟2 (1)


Fifty milliliter of extract were placed in a 100 mL becker and submitted to the

ultrasonic probe. The application of each ultrasonic intensity was performed during 9

min, and 2 mL aliquots were removed for analysis at times 1, 3, 5, 7 and 9 min. In order

to prevent heating during the treatment, 2 min breaks were taken for every 2 min of

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exposure to the ultrasonic waves. The treatments were done in triplicate. The aliquots of

the treated extracts were frozen for further analysis.

2.4. Determination of Gallic Acid

Gallic acid in the P. amarus samples was quantified using a 920LC HPLC

(Varian, Palo Alto, CA, USA) equipped with a quaternary pump, auto sampler, and

diode array detector (DAD). A C18 (Microsorb) analytical column (5 µm, 250 x 4.6

mm) was used at a flow rate of 1.0 mL/min. The column oven temperature was set at

35°C. The mobile phase was composed of methanol and a 0.1% phosphoric acid

(H3PO4) aqueous solution. The UV detector was set at 272 nm. The injection volume

was 20 µL and gradient elution was carried out ranging from 20 to 100% MeOH for 25

min. The results were expressed as mg of gallic acid per g of plant. The samples were

filtered through a 0.45 µm PTFE syringe filter. Gallic acid was identified based on the

comparison with its retention time and the UV spectrum. Concentration was calculated

based on a standard curve.

2.5. UPLC-ESI-QTOF-MS/MS Analysis

An Acquity UPLC system (Waters, Milford, MA, USA) coupled to a

quadrupole/time-of-flight (QTOF) system (Waters, Milford, MA, USA) was used. The


Milford, MA, USA) column kept at 40°C. The eluent system employed was a


at a flow rate of 0.4 mL/min. The gradient varied linearly from 2 to 95% B (v/v) over

0.0-15.0 min, held constant at 100% B over 15.1-17.0 min, and a final wash and

reequilibration at 2% B over 17.1–19.1 min. The sample injection volume was 5 µL.

The spectrometer operated with MSE centroid. Mass spectra were recorded in negative

polarity electrospray ionization (ESI) mode in a mass range between 110 and 1180 Da.

The instrument settings were as follows: collision energy of 5eV, capillary voltage of

2.6 kV, sample cone voltage of 20 V, extraction cone voltage of 0.5 V, source

temperature at 120°C, desolvation temperature at 350°C, and desolvation gas flow at

500 and 350 L/h. The samples were filtered on 0.22 µm PTFE membranes.

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2.6. Statistical Analysis

The gallic acid results were expressed as mean ± SD. Statistical analyses were

carried out using the software Statistica (Statsoft version 7.0). The results were

evaluated using analysis of variance (ANOVA) at a significance level of 5%.

3. Results and Discussion

The chemical composition of P. amarus presents several hydrolyzable tannins

which have gallic acid in their structures, besides its free form. These tannins could be

degraded when exposed to high temperature, reactive substances, acidic or basic

environment, and release gallic acid (Rangsriwong et al., 2009; Tuominen and

Sundman, 2013). Therefore, the gallic acid content was quantified in the control extract

and in the extracts submitted to the ultrasonic treatment. According to Table 1, the

control extract, obtained by pressurized liquid extraction at 120°C/24 min, showed no

significant difference (p> 0.05) in the gallic acid content when compared to the treated

extracts in any of the intensities studied, even in the longest exposure times,

emphasizing that there was no release of gallic acid with the ultrasonic treatment.

Table 1. Effects of ultrasonic intensity and exposure time on the gallic acid content of

the control extract (pressurized liquid extraction at 120°C/24 min) of P. amarus.

Treatment Control 188 W/cm2

1 min 3 min 5 min 7 min 9 min

Gallic acid*

(mg/g plant) 1.94±0.12 1.62±0.18 1.78±0.40 1.80±0.25 1.78±0.23 1.89±0.23

Treatment Control 373 W/cm2

1 min 3 min 5 min 7 min 9 min

Gallic acid*

(mg/g plant) 1.94±0.12 1.98±0.03 1.93±0.13 1.95±0.05 1.95±0.06 1.93±0.06

*Means do not significantly differ (p>0.05).

To confirm that no chemical degradation occurred in the treated extracts, an

UPLC-QTOF-MS/MS analysis was performed to determine the chemical profile of the

extracts. The control and the treated extracts with ultrasonic probe at the intensities of

188 W/cm2 for 9 min and 373 W/cm2 for 9 min were analyzed. The compounds were

identified based on their exact mass and comparison with published data (Sousa et al.,

2016). The control and treated extracts presented the same chemical profile (Figure 1),

confirming that the ultrasonic probe did not promote degradation of any compound. In

116

order to have a quantitative evaluation, the HPLC chromatograms at 272 nm, which

were obtained in the quantification of gallic acid, from the same extracts were compared

(Figure 2). In the three samples, the corresponding peaks, which have the same retention

time, presented approximately the same area. This shows that the phenolic composition

of the control extract was quantitatively maintained after treatment. Therefore,

ultrasound process did not hydrolise ellagitannins, but its lack of effect on elagitannin

extraction remains to be clarified.

Figure 1. UPLC-QTOF-MS/MS chromatograms of of the extracts (a) control, (b)

treated with 188 W/cm2 for 9 min and (c) treated with 373 W/cm2 for 9 min.

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Figure 2. HPLC chromatograms at 272 nm of the extracts (a) control, (b) treated with

188 W/cm2 for 9 min and (c) treated with 373 W/cm2 for 9 min.

118

References

Esclapez, M.D., García-Pérez, J.V., Mulet, A., Cárcel, J.A., 2011. Ultrasound-assisted

extraction of natural products. Food Eng. Rev. 3, 108-120.

Li, H., Pordesimo, L., Weiss, J. 2004. High intensity ultrasound-assisted extraction of

oil from soybeans. Food Res. Int. 37, 731-738.

Rangsriwong, P., Rangkadilok, N., Satayavivad, J., Goto, M., Shotipruk, A., 2009.

Subcritical water extraction of polyphenolic compounds from Terminalia chebula

Retz. fruits. Sep. Purif. Technol. 66, 51-56.

Soria, A.C., Villamiel, M., 2010. Effect of ultrasound on the technological properties

and bioactivity of food: A review. Trends in Food Sci. Technol. 21, 323-331.

Sousa, A.D., Maia, A.I.V., Rodrigues, T.H.S., Canuto, K.M., Ribeiro, P.R.V., Pereira,

R.C.A., Vieira, R.F., Brito, E.S., 2016. Ultrasound-assisted and pressurized liquid

extraction of phenolic compounds from Phyllanthus amarus and its composition

evaluation by UPLC-QTOF. Ind. Crops Prod. 79, 91-103.

Tiwari, B.K., 2015. Ultrasound: A clean, green extraction technology. Trends in


Tuominen, A., Sundman, T., 2013. Stability and oxidation products of hydrolysable

tannins in basic conditions detected by HPLC/DAD-ESI/QTOF/MS. Phytochem.

Anal., 24(5), 424-435.

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4. CONCLUSÃO

A extração com líquido pressurizado (ELP) a 120ºC e pressão de 110 bar

forneceu extratos de P. amarus e P. niruri com alto conteúdo fenólico e sem degradação

dos compostos com relevância farmacológica. Já os extratos obtidos pela extração

assistida por ultrassom (EAU) apresentaram menor conteúdo fenólico em comparação

com os obtidos com as outras técnicas de extração, mesmo nas condições otimizadas, e

a ausência de alguns elagitaninos. Contudo, foi mostrado que a potência ultrassônica

não promoveu a degradação desses compostos.

Os extratos obtidos por ELP e EAU exibiram maior conteúdo de ácido gálico,

marcador químico de espécies do gênero Phyllanthus, que os extratos obtidos pela

extração convencional. O perfil químico de P. amarus foi composto por alcalóides,

ácidos fenólicos, flavonóides, elagitaninos e lignanas. Já o de P. niruri apresentou

ácidos fenólicos, flavonóides e elagitaninos.

A elevação da temperatura reduziu o tempo de secagem em 57% e aumentou a

difusividade efetiva de umidade. A temperatura de 70°C aumentou a disponibilidade de

alguns compostos fenólicos. Entretanto, alguns compostos sensíveis foram afetados

negativamente pela alta temperatura. A melhor temperatura testada para se obter um

maior conteúdo fenólico para ambas as espécies foi de 60°C.

Neste estudo foi possível melhorar a obtenção de metabólitos de quebra-pedra

utilizando um método de extração rápido e com economia de solvente, além de se ter

determinado a melhor temperatura de secagem para manter a qualidade da matéria-

prima.

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