universidade federal do cearÁ centro de … · “os sonhos são como uma bússola, indicando os...
TRANSCRIPT
1
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
2
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
3
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
4
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
Embrapa Agroindústria Tropical
_________________________________________
Drª. Henriette Monteiro Cordeiro de Azeredo
Embrapa Agroindústria Tropical
_________________________________________
Dr. Kirley Marques Canuto
Embrapa Agroindústria Tropical
5
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.
6
“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)
7
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.
8
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.
9
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
10
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
11
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
12
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
Ultrasound-assisted and pressurized liquid extraction of phenolic compounds from
Phyllanthus amarus and its composition evaluation by UPLC-QTOF
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
UPLC-QTOF-MSE-based chemometric approach driving the choice of the best
extraction process for Phyllanthus niruri
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
13
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
Drying kinetics and effect of air-drying temperature on chemical composition of
Phyllanthus amarus and Phyllanthus niruri
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
High-power ultrasound does not hydrolyze ellagitannins from Phyllanthus amarus
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
14
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
13
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
14
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
15
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.
REFERÊNCIAS
ALEXANDRU, L.; BINELLO, A.; MANTEGNA, S.; BOFFA, L.; CHEMAT, F.;
CRAVOTTO, G. Efficient green extraction of polyphenols from post-harvested agro-
industry vegetal sources in Piedmont. Comptes Rendus Chimie, v. 17, p. 212–217,
2014.
ALLI, A. I.; EHINMIDU, J. O.; IBRAHIM, Y. K. E. Preliminary phytochemical
screening and antimicrobial activities of some medicinal plants used in Ebiraland.
Bayero Journal of Pure and Applied Sciences, v. 4, n. 1, p. 10–16, 2011.
AMIN, Z. A.; ALSHAWSH, M. A.; KASSIM, M.; ALI, H. M.; ABDULLA, M. A.
Gene expression profiling reveals underlying molecular mechanism of hepatoprotective
effect of Phyllanthus niruri on thioacetamide-induced hepatotoxicity in Sprague
Dawley rats. BMC Complementary and Alternative Medicine, 13:160, 2013.
APG III. An update of the Angiosperm Phylogeny Group classification for the orders
and families of flowering plants: APG III. Botanical Journal of the Linnean Society,
v. 161, p. 105–121, 2009.
ARMENTA, S.; GARRIGUES, S.; DE LA GUARDIA, M. The role of green extraction
techniques in green analytical chemistry. Trends in Analytical Chemistry, v. 71, p. 2-
8, 2015.
32
ATANASOV, A. G.; WALTENBERGER, B.; PFERSCHY-WENZIG, E. M.; LINDER,
T.; WAWROSCH, C.; UHRIN, P.; TEMML, V.; WANG, L.; SCHWAIGER, S.;
HEISS, E. H.; ROLLINGER, J. M.; SCHUSTER, D.; BREUSS, J. M.; BOCHKOV, V.;
MIHOVILOVIC, M. D.; KOPP, B.; BAUER, R.; DIRSCH, V. M.; STUPPNER, H.
Discovery and resupply of pharmacologically active plant-derived natural products: A
review. Biotechnology Advances, v. 33, p. 1582-614, 2015.
AZMIR, J.; ZAIDUL, I. S. M.; RAHMAN, M. M.; SHARIF, K. M.; MOHAMED, A.;
SAHENA, F.; JAHURUL, M. H. A.; OMAR, A. K. M. Techniques for extraction of
bioactive compounds from plant materials: A review. Journal of Food Engineering, v.
117, n. 4, p. 426-436, 2013.
BAGALKOTKAR, G.; SAGINEEDU, S. R.; SAAD, M. S.; STANSLAS, J.
Phytochemicals from Phyllanthus niruri Linn. and their pharmacological properties: A
review. Journal of Pharmacy and Pharmacology, v. 58, p. 1559-1570, 2006.
BARROS, M. E.; SCHOR, N.; BOIM, M. A. Effects of an aqueous extract from
Phyllanthus niruri on calcium oxalate crystallization in vitro. Urological Research, v.
30, n. 6, p. 374–379, 2003.
BARROS, M. E.; LIMA, R.; MERCURI, L. P.; MATOS, J. R.; SCHOR, N.; BOIM, M.
A. Effect of extract of Phyllanthus niruri on crystal deposition in experimental
urolithiasis. Urological Research, v. 34, n. 6, p. 351-357, 2006.
BRANDÃO, H. N.; DAVID, J. P.; COUTO, R. D.; NASCIMENTO, J. A. P.; DAVID,
J. M. Química e farmacologia de quimioterápicos antineoplásicos derivados de plantas.
Química Nova, v. 33, n. 6, p. 1359-1369, 2010.
BRASIL. Ministério da Saúde. Secretaria de Atenção à Saúde. Práticas integrativas e
complementares: plantas medicinais e fitoterapia na Atenção Básica. Brasília:
Ministério da Saúde, 2012.
CALIXTO, J. B.; SANTOS, A. R. S.; CECHINEL FILHO, V.; YUNES, R. A. A review
of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic
potential. Medicinal Research Reviews, v.18, n. 4, p. 225-258, 1998.
CALIXTO, J. B. Medicamentos Fitoterápicos. Plantas Medicinais: sob a ótica da
química medicinal moderna. Chapecó, SC, Editora Argos, 2001. p. 500.
CHAVAN, Y. & SINGHAL, R. S. Ultrasound-assisted extraction (UAE) of bioactives
from arecanut (Areca catechu L.) and optimization study using response surface
methodology. Innovative Food Science & Emerging Technologies, v. 17, p. 106-113,
2013.
CHEOK, C. Y.; CHIN, N. L.; YUSOF, Y. A.; TALIB, R. A.; LAW, C. L. Optimization
of total monomeric anthocyanin (TMA) and total phenolic content (TPC) extractions
from mangosteen (Garcinia mangostana Linn.) hull using ultrasonic treatments.
Industrial Crops and Products, v. 50, p. 1-7, 2013.
COUTO, A. G.; KASSUYA, C. A. L.; CALIXTO, J. B.; PETROVICK, P. R.
Antiinflammatory, antiallodynic effects and quantitative analysis of gallic acid in spray
33
dried powders from Phyllanthus niruri leaves, stems, roots and whole plant. Revista
Brasileira de Farmacognosia, v. 23, p. 124-131, 2013.
DAHMOUNE, F.; REMINI, H.; DAIRI, S.; AOUN, O.; MOUSSI, K.; BOUAOUDIA-
MADI, N.; ADJEROUD, N.; KADRI, N.; LEFSIH, K.; BOUGHANI, L.; MOUNI, L.;
NAYAK, B.; MADANI, K. Ultrasound assisted extraction of phenolic compounds from
P. lentiscus L. leaves: Comparative study of artificial neural network (ANN) versus
degree of experiment for prediction ability of phenolic compounds recovery. Industrial
Crops and Products, v. 77, p. 251-261, 2015.
FEUEREISEN, M. M.; GAMERO BARRAZA, M.; ZIMMERMANN, B. F.;
SCHIEBER, A.; SCHULZE-KAYSERS, N. Pressurized liquid extraction of
anthocyanins and biflavonoids from Schinus terebinthifolius Raddi: A multivariate
optimization. Food Chemistry, v. 214, p. 564-571, 2017.
GUO, J.; CHEN, Q.; WANG, C.; QIU, H.; LIU, B.; JIANG, Z.-H.; ZHANG, W.
Comparison of two exploratory data analysis methods for classification of Phyllanthus
chemical fingerprint: unsupervised vs. supervised pattern recognition technologies.
Analytical and Bioanalytical Chemistry, v. 407, p. 1389-1401, 2015.
HENG, M. Y.; TAN, S. N.; YONG, J. W. H.; ONG, E. S. Emerging green technologies
for the chemical standardization of botanicals and herbal preparations. TrAC - Trends
in Analytical Chemistry, v. 50, p. 1-10, 2013.
HERRERA, M. C. & LUQUE DE CASTRO, M. D. Ultrasound-assisted extraction of
phenolic compounds from strawberries prior to liquid chromatographic separation and
photodiode array ultraviolet detection. Journal of Chromatography, v. 1100, n. 1, p.
1–7, 2005.
HOSSAIN, M. B.; BARRY-RYAN, C.; MARTIN-DIANA, A. B.; BRUNTON, N. P.
Optimisation of accelerated solvent extraction of antioxidant compounds from rosemary
(Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and oregano (Origanum
vulgare L.) using response surface methodology. Food Chemistry, v. 126, n. 1, p. 339-
346, 2011.
HOSSAIN, M. B.; BRUNTON, N. P.; PATRAS, A.; TIWARI, B.; O'DONNELL, C. P.;
MARTIN-DIANA, A. B.; BARRY-RYAN, C. Optimization of ultrasound assisted
extraction of antioxidant compounds from marjoram (Origanum majorana L.) using
response surface methodology. Ultrasonics Sonochemistry, v. 19, n. 3, p. 582-590,
2012.
HUIE, C. W. A review of modern sample-preparation techniques for the extraction and
analysis of medicinal plants. Analytical and Bioanalytical Chemistry, v. 373, n. 1-2,
p. 23-30, 2002.
IRANLOYE, B. O.; OWOYELE, V. B.; KELANI, O. R.; OLALEYE, S. B. Analgesic
activity of aqueous leaf extracts of Phyllanthus amarus. African Journal of Medicine
and Medical Sciences, v. 40, p. 47–50, 2011.
KANG, J. H.; KIM, S.; MOON, B. Optimization by response surface methodology of
lutein recovery from paprika leaves using accelerated solvent extraction. Food
Chemistry, v. 205, p. 140-145, 2016.
34
KARAM, M. C.; PETIT, J.; ZIMMER, D.; DJANTOU, E. B.; SCHER, J. Effects of
drying and grinding in production of fruit and vegetable powders: A review. Journal of
Food Engineering, v. 188, p. 32-49, 2016.
KO, M. J.; CHEIGH, C. I.; CHO, S. W.; CHUNG, M. S. Subcritical water extraction of
flavonol quercetin from onion skin. Journal of Food Engineering, v. 102, p. 327–333,
2011.
KUKULA-KOCH, W.; ALIGIANNIS, N.; HALABALAKI, M.; SKALTSOUNIS, A.
L.; GLOWNIAK, K.; KALPOUTZAKIS, E. Influence of extraction procedures on
phenolic content and antioxidant activity of Cretan barberry herb. Food Chemistry, v.
138, n. 1, p. 406-413, 2013.
KUMAR, S.; CHANDRA, P.; BAJPAI, V.; SINGH, A.; SRIVASTAVA, M.; MISHRA,
D. K.; KUMAR, B. Rapid qualitative and quantitative analysis of bioactive compounds
from Phyllanthus amarus using LC/MS/MS techniques. Industrial Crops and
Products, v. 69, p. 143-152, 2015.
LEE, S. H.; JAGANATH, I. B.; WANG, S. M.; SEKARAN, S. D. Antimetastatic
effects of Phyllanthus on human lung (A549) and breast (MCF-7) cancer cell lines.
PLoS ONE, v. 6, n. 6, e20994, doi:10.1371/journal.pone.0020994, 2011.
LIU, C. H.; WANG, Y. C.; LU, H. C.; CHIANG, W. D. Optimization of ultrasound-
assisted extraction conditions for total phenols with anti-hyperglycemic activity from
Psidium guajava leaves. Process Biochemistry, v. 49, n. 10, p. 1601-1605, 2014.
LIU, Y.; WEI, S.; LIAO, M. Optimization of ultrasonic extraction of phenolic
compounds from Euryale ferox seed shells using response surface methodology.
Industrial Crops and Products, v. 49, p. 837-843, 2013.
LORENZI, H. & MATOS, F. J. A. Plantas Medicinais no Brasil: Nativas e Exóticas.
Instituto Plantarum, Nova Odessa, 2002.
MARKOM, M.; HASAN, M.; DAUD, W. R. W. Pressurized water extraction of
hydrolysable tannins from Phyllanthus niruri Linn. Separation Science and
Technology, v. 45, p. 548-553, 2010.
MELO, E. DE C.; RADÜNZ, L. L.; ALVARENGA E MELO, R. C. Influência do
processo de secagem na qualidade de plantas medicinais – revisão. Engenharia na
Agricultura, Viçosa, MG, v.12, n.4, 307-315, 2004.
MOREIRA, J.; KLEIN-JÚNIOR, L. C.; CECHINEL FILHO, V.; DE CAMPOS
BUZZI, F. Anti-hyperalgesic activity of corilagin, a tannin isolated from Phyllanthus
niruri L. (Euphorbiaceae). Journal of Ethnopharmacology, v. 146, n. 1, p. 318-323,
2013.
MUSTAFA, A. & TURNER, C. Pressurized liquid extraction as a green approach in
food and herbal plants extraction: A review. Analytica Chimica Acta, v. 703, p. 8-18,
2011.
35
NAYAK, C. A. & RASTOGI, N. K. Optimization of solid–liquid extraction of
phytochemicals from Garcinia indica Choisy by response surface methodology. Food
Research International, v. 50, n. 2, p. 550-556, 2013.
NISHIURA, J. L.; CAMPOS, A. H.; BOIM, M. A.; HEILBERG, I. P.; SCHOR, N.
Phyllanthus niruri normalizes elevated urinary calcium levels in calcium stone forming
(CSF) patients. Urological Research, v. 32, n.5, p. 362-366, 2004.
NOTKA, F.; MEIER, G. R.; WAGNER, R. Concerted inhibitory activities of
Phyllanthus amarus on HIV replication in vitro and ex vivo. Journal of Antiviral
Research, v. 64, p. 93–102, 2004.
OKOLI, C. O.; IBIAM, A. F.; EZIKE, A. C.; AKAH, P. A.; OKOYE, T. C. Evaluation
of antidiabetic potentials of Phyllanthus niruri in alloxan diabetic rats. African Journal
of Biotechnology, v. 9, p. 248–259, 2010.
ONG, E. S. Extraction methods and chemical standardization of botanicals and herbal
preparations. Journal of Chromatography B, v. 812, n. 1–2, p. 23-33, 2004.
ONISZCZUK, A. & PODGÓRSKI, R. Influence of different extraction methods on the
quantification of selected flavonoids and phenolic acids from Tilia cordata
inflorescence. Industrial Crops and Products, v. 76, p. 509-514, 2015.
PATEL, J. R.; TRIPATHI, P.; SHARMA, V.; CHAUHAN, N. S.; DIXIT, V. K.
Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review.
Journal of Ethnopharmacology, v. 138, p. 286–313, 2011.
PEREIRA, R. G.; GARCIA, V. L.; NOVA RODRIGUES, M. V.; MARTÍNEZ, J.
Extraction of lignans from Phyllanthus amarus Schum. & Thonn using pressurized
liquids and low pressure methods. Separation and Purification Technology, v. 158, p.
204-211, 2016.
PESSOA JÚNIOR, A.; KILIKIAN, B. V. Purificação de Produtos Biotecnológicos. 1.
ed. Barueri, SP: Manole, 2005.
QI, W; HUA, L; GAO, K. Chemical constituents of the plants from the genus
Phyllanthus. Chemistry Biodiversity, v. 11, p. 364-395, 2014.
RODRIGUES, A. G. & DE SIMONI, C. Plantas medicinais no contexto de políticas
públicas. Informe Agropecuário, Belo Horizonte, v. 31, n. 255, p. 7-12, 2010.
RODRIGUES, M. V. N.; REHDER, V. L. G.; SARTORATTO, A.; BOAVENTURA
JÚNIOR, S.; SANTOS, A. S. O emprego de técnicas hifenadas no estudo de plantas
medicinais. MultiCiência: Construindo a história dos produtos naturais, v. 7, 2006.
SANTOS-BUELGA, C.; GONZALEZ-MANZANO, S.; DUEÑAS, M.; GONZALEZ-
PARAMAS, A. M. Extraction and isolation of phenolic compounds. Methods in
Molecular Biology, v. 864, p. 427-464, 2012.
SECCO, R. S.; CORDEIRO, I.; MARTINS, E. R. Catálogo de plantas e fungos do
Brasil, vol. 2 [organização Rafaela Campostrini Forzza... et al.]. - Rio de Janeiro:
36
Andrea Jakobsson Estúdio: Instituto de Pesquisas Jardim Botânico do Rio de
Janeiro, v. 2, p. 1439-1442, 2010.
SHARMILA, G.; NIKITHA, V. S.; ILAIYARASI, S.; DHIVYA, K.; RAJASEKAR,
V.; MANOJ KUMAR, N.; MUTHUKUMARAN, K.; MUTHUKUMARAN, C.
Ultrasound assisted extraction of total phenolics from Cassia auriculata leaves and
evaluation of its antioxidant activities. Industrial Crops and Products, v. 84, p. 13-21,
2016.
SILVA, M. J. & SALES, M. F. Phyllanthus L. (Phyllanthaceae) em Pernambuco,
Brasil. Acta Botânica Brasílica, v. 21, n. 1, p. 79-98, 2007.
SKALICKA-WOŹNIAK, K. & GŁOWNIAK, K. Pressurized liquid extraction of
coumarins from fruits of Heracleum leskowii with application of solvents with different
polarity under increasing temperature. Molecules, v. 17, n. 4, p. 4133-4141, 2012.
SPRENGER, R. F. & CASS, Q. B. Characterization of four Phyllanthus species using
liquid chromatography coupled to tandem mass spectrometry. Journal of
Chromatography A, v. 1291, p. 97-103, 2013.
SRIVIDYA, N. & PERIWAL, S. Diuretic, hypotensive and hypoglycaemic effect of
Phyllanthus amarus. Indian Journal of Experimental Biology, v. 33, p. 861–864,
1995.
TIWARI, B. K. Ultrasound: A clean, green extraction technology. Trends in
Analytical Chemistry, v. 71, p. 100-109, 2015.
TORRES, D. S. C.; CORDEIRO, I.; GIULIETTI, A. M. O gênero Phyllanthus
L.(Euphorbiaceae) na Chapada Diamantina, Bahia, Brasil. Acta Botânica Brasílica, v.
17, n. 2, p. 265-278, 2003.
VINATORU, M. An overview of the ultrasonically assisted extraction of bioactive
principles from herbs. Ultrasonics Sonochemistry, v. 8, p. 303-313, 2001.
WANG, X.; WU, Y.; CHEN, G.; YUE, W.; LIANG, Q.; WU, Q. Optimisation of
ultrasound assisted extraction of phenolic compounds from Sparganii rhizoma with
response surface methodology. Ultrasonics Sonochemistry, v. 20, n. 3, p. 846-854,
2013.
XYNOS, N.; PAPAEFSTATHIOU, G.; GIKAS, E.; ARGYROPOULOU, A.;
ALIGIANNIS, N.; SKALTSOUNIS, A.-L. Design optimization study of the extraction
of olive leaves performed with pressurized liquid extraction using response surface
methodology. Separation and Purification Technology, v. 122, p. 323-330, 2014.
ZEKOVIĆ, Z.; KAPLAN, M.; PAVLIĆ, B.; OLGUN, E. O.; VLADIĆ, J.; CANLI, O.;
VIDOVIĆ, S. Chemical characterization of polyphenols and volatile fraction of
coriander (Coriandrum sativum L.) extracts obtained by subcritical water extraction.
Industrial Crops and Products, v. 87, p. 54-63, 2016.
37
ZHAO, L. C.; HE, Y.; DENG, X.; YANG, G. L.; LI, W.; LIANG, J.; TANG, Q. L.
Response surface modeling and optimization of accelerated solvent extraction of four
lignans from fructus schisandrae. Molecules, v. 17, n. 4, p. 3618-3629, 2012.
ZOU, T. B.; XIA, E. Q.; HE, T. P.; HUANG, M. Y.; JIA, Q.; LI, H. W. Ultrasound-
assisted extraction of Mangiferin from Mango (Mangifera indica L.) leaves using
response surface methodology. Molecules, v. 19, n. 2, p. 1411-1421, 2014.
38
ARTIGO 1
Ultrasound-assisted and pressurized liquid extraction of phenolic compounds from
Phyllanthus amarus and its composition evaluation by UPLC-QTOF
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
Ultrasound-assisted and pressurized liquid extraction of phenolic compounds from
Phyllanthus amarus and its composition evaluation by UPLC-QTOF
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
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
© central point of the experimental design.
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).
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.
References
Ashokkumar, M., Sunartio, D., Kentish, S., Mawson, R., Simons, L., Vilkhu, K.,
Versteeg, C.(K.), 2008. Modification of food ingredients by ultrasound to improve
functionality: A preliminary study on a model system. Innovative Food Sci.
Emerg. Technol. 9 (2), 155-160.
Azmir, J., Zaidul, I.S.M., Rahman, M.M., Sharif, K.M., Mohamed, A., Sahena, F.,
Jahurul, M.H.A., Omar, A.K.M., 2013. Techniques for extraction of bioactive
compounds from plant materials: A review. J. Food Eng. 117, 426-436.
62
Chen, Y.W., Ren, L.J., Li, K.M., Zhang, Y.W., 1999. Isolation and identification of a
novel polyphenolic compound from Phyllanthus urinaria. Yaoxue Xuebao, 34,
526-529.
Chopade, A.R.; Sayyad, F.J., 2014. Antifibromyalgic activity of standardized extracts
of Phyllanthus amarus and Phyllanthus fraternus in acidic saline induced
chronicmuscle pain. Biomed. Aging Pathol. 4, 123-130.
De Souza, T.P., Holzschuh, M.H., Lionço, M.I., González Ortega, G., Petrovick, P.R.,
2002. Validation of a LC method for the analysis of phenolic compounds from
aqueous extract of Phyllanthus niruri aerial parts. J. Pharmaceutical and
Biomedical Anal. 30, 351-356.
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.
Farmacopeia Brasileira, volume 2 / Agência Nacional de Vigilância Sanitária. Brasília:
Anvisa, 2010. 904p, 2v/il. 1. Substâncias farmacêuticas químicas, vegetais e
biológicas. 2. Medicamentos e correlatos. 3. Especificações e métodos de análise.
I Título. ISBN 978-85-88233-41-6.
Guo, J., Chen, Q., Wang, C., Qiu, H., Liu, B., Jiang, Z.-H., Zhang, W, 2015.
Comparison of two exploratory data analysis methods for classification of
Phyllanthus chemical fingerprint: unsupervised vs. supervised pattern recognition
technologies. Anal. Bioanal. Chem. 407, 1389-1401.
Gurib-Fakim, A., 2006. Medicinal plants: traditions of yesterday and drugs of
tomorrow. Mol. Aspects Med. 27, 1-93.
Hanhineva, K., Rogachev, I., Kokko, H., Mintz-Oron, S., Venger, I., Kärenlampi, S.,
Aharoni, A., 2008. Non-targeted analysis of spatial metabolite composition in
strawberry (Fragaria × ananassa) flowers. Phytochemistry 69, 2463-2481.
Hossain, M.B., Rai, D.K., Brunton, N.P., Martin-Diana, A.B., Barry-Ryan, A.C., 2010.
Characterization of phenolic composition in lamiaceae spices by LC-ESI-MS/MS.
J. Agric. Food Chem.58, 10576-10581.
Jia, L., Jin, H., Zhou, J., Chen, L., Lu, Y., Ming, Y., Yu, Y., 2013. A potential anti-
tumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through
blocking the TGF-β signaling pathways. BMC Complementary and Alternative
Med. 13, 33.
Jin, F., Cheng, D., Tao, J.-Y., Zhang, S.-L., Pang, R., Guo, Y.-J., Ye, P., Dong, J.-H.,
Zhao, L., 2013. Anti-inflammatory and anti-oxidative effects of corilagin in a rat
model of acute cholestasis. BMC Gastroenterology13, 79.
Khoza, B.S., Chimuka, L., Mukwevho, E., Steenkamp, P.A., Madala, N.E., 2014. The
effect of temperature on pressurised hot water extraction of pharmacologically
important metabolites as analysed by UPLC-qTOF-MS and PCA. Evidence-based
Complementary and Alternative Med. 914759.
63
Kumar, S., Chandra, P., Bajpai, V., Singh, A., Srivastava, M., Mishra, D.K., Kumar, B.,
2015. Rapid qualitative and quantitative analysis of bioactive compounds from
Phyllanthus amarus using LC/MS/MS techniques. Ind. Crops Prod. 69, 143-152.
Latté, K.P., Kolodziej, H. 2000. Pelargoniins, new ellagitannins from Pelargonium
reniforme. Phytochemistry 54, 701-708.
Li, H., Pordesimo, L., Weiss, J. 2004. High intensity ultrasound-assisted extraction of
oil from soybeans. Food Res. Int. 37, 731-738.
Maity, S., Chatterjee, S., Variyar, P.S., Sharma, A., Adhikari, S., Mazumder, S., 2013.
Evaluation of antioxidant activity and characterization of phenolic constituents of
Phyllanthus amarus root. J. Agric. Food Chem. 61, 3443-3450.
Markom, M., Hasan, M., Daud, W.R.W., Singh, H., Jahim, J.M., 2007. Extraction of
hydrolysable tannins from Phyllanthus niruri Linn.: Effects of solvents and
extraction methods. Sep. Purif. Technol. 52, 487-496.
Moreira, J., Klein-Júnior, L.C., Filho, V.C., Buzzi, F.D.C., 2013. Anti-hyperalgesic
activity of corilagin, a tannin isolated from Phyllanthus niruri L. (Euphorbiaceae).
J. Ethnopharmacol. 146, 318-323.
Mustafa, A., Turner, C., 2011. Pressurized liquid extraction as a green approach in food
and herbal plants extraction: A review. Analytica Chimica Acta 703, 8-18.
Nikam, P.S., Nikam, S.V., Sontakke, A.V., Khanwelkar, C.C., 2011. Role of
Phyllanthus amarus treatment in Hepatitis-C. Biomed. Res. 22, 319–322.
Notka, F., Meier, G.R., Wagner, R., 2004. Concerted inhibitory activities of Phyllanthus
amarus onHIVreplication in vitro and ex vivo. J. Antivir. Res. 64,93–102.
Patel, J.R., Tripathi, P., Sharma, V., Chauhan, N.S., Dixit, V.K., 2011. Phyllanthus
amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review. J.
Ethnopharmacol. 138, 286–313.
Patil, A.A., Bhusari, S.S., Shinde, D.B., Wakte, P.S., 2013. Optimization of process
variables for phyllanthin extraction from Phyllanthus amarus leaves by
supercritical fluid using a Box-Behnken experimental design followed by HPLC
identification. Acta Pharmaceutica 63, 193-207.
Plaza, M., Turner, C., 2015. Pressurized hot water extraction of bioactives. Trends in
Analytical Chem. 71, 39-54.
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.
Ravikumar, Y.S., Ray, U., Nandhitha, M., Perween, A., Naika, H.R., Khanna, N.,
Das, S., 2011. Inhibition of hepatitis C virus replication by herbal extract:
Phyllanthus amarus as potent natural source. Virus Res. 158, 89–97.
64
Sentandreu, E., Cerdán-Calero, M., Sendra, J.M., 2013. Phenolic profile
characterization of pomegranate (Punica granatum) juice by high-performance
liquid chromatography with diode array detection coupled to an electrospray ion
trap mass analyzer. J. Food Compos. Anal. 30, 32-40.
Shanker, K., Singh, M., Srivastava, V., Verma, R.K., Gupta, A.K., Gupta, M.M., 2011.
Simultaneous analysis of six bioactive lignans in Phyllanthus species by reversed
phase hyphenated high performance liquid chromatographic technique. Acta
Chromatographica 23, 321-337.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144-158.
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.
Spínola, V., Pinto, J., Castilho, P.C., 2015. Identification and quantification of phenolic
compounds of selected fruits from Madeira Island by HPLC-DAD-ESI-MSn and
screening for their antioxidant activity. Food Chem.173, 14-30.
Teo, C.C., Tan, S.N., Yong, J.W.H., Hew, C.S., Ong, E.S., 2010. Pressurized hot water
extraction (PHWE). J. Chromatography A 1217, 2484-2494.
Tiwari, B.K., 2015. Ultrasound: A clean, green extraction technology. Trends in
Analytical Chem. 71, 100-109.
Tsai, C.-C., Chou, C.-H., Liu, Y.-C., Hsieh, C.-W., 2014. Ultrasound-assisted extraction
of phenolic compounds from Phyllanthus emblica L. and evaluation of
antioxidant activities. Int. J. Cosmetic Sci. 36, 471-476.
United States Pharmacopeial Convention in
https://hmc.usp.org/monographs/phyllanthus-amarus-aerial-parts-powdered-
extract-0-2. Accessed 11 May 2015.
Wang, X., Wu, Y., Chen, G.,Yue, W., Liang, Q., Wu, Q., 2013. Optimisation of
ultrasound assisted extraction of phenolic compounds from Sparganii rhizoma
with response surface methodology. Ultrasonic. Sonoch. 20, 846-854.
Yang, B., Kortesniemi, M., Liu, P., Karonen, M., Salminen, J.-P, 2012. Analysis of
hydrolyzable tannins and other phenolic compounds in emblic leaf flower
(Phyllanthus emblica L.) fruits by high performance liquid chromatography-
electrospray ionization mass spectrometry. J. Agric. Food Chem. 60, 8672-8683.
Yang, B., Liu, P., 2014. Composition and biological activities of hydrolyzable tannins
of fruits of Phyllanthus emblica.J. Agric. Food Chem. 62, 529-541.
Yeap Foo, L., 1995. Amariinic acid and related ellagitannins from Phyllanthus amarus.
Phytochemistry 39 (1), 217-224.
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
UPLC-QTOF-MSE-based chemometric approach driving the choice of the best
extraction process for Phyllanthus niruri
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
UPLC-QTOF-MSE-based chemometric approach driving the choice of the best
extraction process for Phyllanthus niruri
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.
* 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)
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.
𝑈𝑙𝑡𝑟𝑎𝑠𝑜𝑛𝑖𝑐 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑊/𝑐𝑚2) =𝑃
𝜋𝑟2 (1)
Where P: ultrasound power (W) and r: tip radius (cm).
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)
The extractions were accomplished in a Dionex ASE 350 system (Sunnyvale,
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
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
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
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
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 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).
References
(1) Bagalkotkar, G.; Sagineedu, S.R.; Saad, M.S.; Stanslas, J. (2006)
Phytochemicals from Phyllanthus niruri Linn. and their pharmacological
properties: A review. J. Pharm. Pharmacol., 58(12):1559-1570.
(2) Calixto, J.B.; Santos, A.R.S.; Cechinel Filho, V.; Yunes, R.A. (1998) A review
of the plants of the Genus Phyllanthus: Their chemistry, pharmacology, and
therapeutic potential. Med. Res. Rev., 18(4):225-258.
84
(3) Moreira, J.; Klein-Júnior, L.C.; Cechinel Filho, V.; Buzzi, F.D.C. (2013) Anti-
hyperalgesic activity of corilagin, a tannin isolated from Phyllanthus niruri L.
(Euphorbiaceae). J. Ethnopharmacol., 146(1):318-323.
(4) Syamasundar, K.V.; Singh, B.; Thakur, R.S.; Husain, A.; Kiso, Y.; Hikino, H.
(1985) Antihepatotoxic principles of Phyllanthus niruri herbs. J.
Ethnopharmacol., 14(1):41-44.
(5) Liu, S.; Wei, W.; Li, Y.; Lin, X.; Shi, K.; Cao, X.; Zhou, M. (2014) In vitro and
in vivo anti-hepatitis B virus activities of the lignan nirtetralin B isolated from
Phyllanthus niruri L. J. Ethnopharmacol., 157:62-68.
(6) Sharma, P.; Parmar, J.; Verma, P.; Sharma, P.; Goyal, P.K. (2009) Anti-tumor
activity of Phyllanthus niruri (a medicinal plant) on chemical-induced skin
carcinogenesis in mice. Asian Pac. J. Cancer Prev., 10(6):1089-1094.
(7) Tan, W.C.; Jaganath, I.B.; Manikam, R.; Sekaran, S.D. (2013) Evaluation of
antiviral activities of four local Malaysian phyllanthus species against herpes
simplex viruses and possible antiviral target. Int. J. Med. Sci., 10(13):1817-1829.
(8) Azmir, J.; Zaidul, I.S.M.; Rahman, M.M.; Sharif, K.M.; Mohamed, A.; Sahena,
F.; Jahurul, M.H.A.; Ghafoor, K.; Norulaini, N.A.N.; Omar, A.K.M. (2013)
Techniques for extraction of bioactive compounds from plant materials: A
review. J. Food Eng., 117(4):426-436.
(9) Markom, M.; Hasan, M.; Daud, W.R.W.; Singh, H.; Jahim, J.M. (2007)
Extraction of hydrolysable tannins from Phyllanthus niruri Linn.: Effects of
solvents and extraction methods. Sep. Purif. Technol., 52(3):487-496.
(10) Markom, M.; Hasan, M.; Daud, W. (2010) Pressurized Water Extraction of
Hydrolysable Tannins from Phyllanthus niruri Linn. Separation Science and
Technology, 45(4): 548-553.
(11) 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.
(12) Tsai, C.-C.; Chou, C.-H.; Liu, Y.-C.; Hsieh, C.-W. (2014) Ultrasound-assisted
extraction of phenolic compounds from Phyllanthus emblica L. and evaluation
of antioxidant activities. Int. J. Cosmetic Sci., 36(5):471-476.
(13) Esclapez, M.D.; García-Pérez, J.V.; Mulet, A.; Cárcel, J.A. (2011) Ultrasound-
assisted extraction of natural products. Food Eng. Rev., 3(2):108-120.
(14) Tiwari, B.K. (2015) Ultrasound: A clean, green extraction technology. Trends
Anal. Chem., 71:100-109.
(15) Carabias-Martínez, R.; Rodríguez-Gonzalo, E.; Revilla-Ruiz, P.; Hernández-
Méndez, J. (2005) Pressurized liquid extraction in the analysis of food and
biological samples. J. Chromatogr. A, 1089(1-2):1-17.
85
(16) Plaza, M.; Turner, C. (2015) Pressurized hot water extraction of bioactives.
Trends Anal. Chem., 71:39-54
(17) Guo, M.; Zhao, B.; Liu, H.; Zhang, L.; Peng, L.; Qin, L.; Zhang, Z.; Li, J.; Cai,
C.; Gao, X. (2014) A metabolomic strategy to screen the prototype components
and metabolites of shuang-huang-lian injection in human serum by ultra
performance liquid chromatography coupled with quadrupole time-of-flight
mass spectrometry. J. Anal. Methods Chem., 241505.
(18) Kuhnert, N.; Jaiswal, R.; Eravuchira, P.; El-Abassy, R.; von der Kammer, B.;
Materny, A. (2011) Scope and limitations of principal component analysis of
high resolution LC-TOF-MS data: the analysis of the chlorogenic acid fraction
in green coffee beans as a case study. Anal. Methods, 3(1): 144-155.
(19) Fraige, K.; Pereira-Filho, E.R.; Carrilho, E. (2014) Fingerprinting of
anthocyanins from grapes produced in Brazil using HPLC-DAD-MS and
exploratory analysis by principal component analysis. Food Chem., 145:395-
403.
(20) Li, H.; Pordesimo. L.; Weiss. J. (2004) High intensity ultrasound-assisted
extraction of oil from soybeans. Food Res. Int., 37(7):731-738
(21) Singleton, V.L.; Rossi, J.A. (1965) Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic., 16:144-
158.
(22) Yang, B.; Kortesniemi, M.; Liu. P.; Karonen, M.; Salminen, J.-P. (2012)
Analysis of hydrolyzable tannins and other phenolic compounds in emblic
leafflower (Phyllanthus emblica L.) fruits by high performance liquid
chromatography-electrospray ionization mass spectrometry. J. Agric. Food
Chem., 60(35): 8672-8683.
(23) Kumar, S.; Chandra, P.; Bajpai. V.; Singh, A.; Srivastava, M.; Mishra, D.K.;
Kumar, B. (2015) Rapid qualitative and quantitative analysis of bioactive
compounds from Phyllanthus amarus using LC/MS/MS techniques. Ind. Crops
Prod., 69:143-152.
(24) Sprenger, R.D.F.; Cass, Q.B. (2013) Characterization of four Phyllanthus
species using liquid chromatography coupled to tandem mass spectrometry. J.
Chromatogr. A, 1291:97-103.
(25) Hossain, M.B.; Rai, D.K.; Brunton, N.P.; Martin-Diana, A.B.; Barry-Ryan, A.C.
(2010) Characterization of phenolic composition in lamiaceae spices by LC-ESI-
MS/MS. J. Agric. Food Chem., 58(19):10576-10581.
(26) Guo, J.; Chen, Q.; Wang, C.; Qiu, H.; Liu, B.; Jiang, Z.-H.; Zhang, W. (2015)
Comparison of two exploratory data analysis methods for classification of
Phyllanthus chemical fingerprint: unsupervised vs. supervised pattern
recognition technologies. Anal. Bioanal. Chem., 407(5):1389-1401.
(27) Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H.A. (2013) A LC/QTOF-
MS/MS application to investigate chemical compositions in a fraction with
86
protein tyrosine phosphatase 1B inhibitory activity from Rosa rugosa flowers.
Phytochem. Anal., 24(6):661-670.
(28) Lai, T.N.H.; Herent, M.-F.; Quetin-Leclercq. J.; Nguyen, T.B.T.; Rogez, H.;
Larondelle, Y.; André, C.M. (2013) Piceatannol, a potent bioactive stilbene, as
major phenolic component in Rhodomyrtus tomentosa. Food Chem., 138(2-3):
1421-1430.
(29) Ogata, T.; Higuchi, H.; Mochida, S.; Matsumoto, H.; Kato, A.; Endo, T.; Kaji,
A.; Kaji, H. (1992) HIV-1 reverse transcriptase inhibitor from Phyllanthus
niruri. AIDS Res. Hum. Retroviruses, 8(11):1937-1944.
(30) Latté, K.P.; Kolodziej, H. (2000) Pelargoniins, new ellagitannins from
Pelargonium reniforme. Phytochemistry, 54(7):701-708.
(31) Chen, Y.W.; Ren, L.J.; Li, K.M.; Zhang, Y.W. (1999) Isolation and
identification of a novel polyphenolic compound from Phyllanthus urinaria.
Yaoxue Xuebao, 34:526-529.
(32) Sentandreu, E.; Cerdán-Calero, M.; Sendra, J.M. (2013) Phenolic profile
characterization of pomegranate (Punica granatum) juice by high-performance
liquid chromatography with diode array detection coupled to an electrospray ion
trap mass analyzer. J. Food Compos. Anal., 30(1):32-40.
(33) Soria, A.; Villamiel, M. (2010) Effect of ultrasound on the technological
properties and bioactivity of food: a review. Trends Food Sci. Technol., 21(7):
323-331.
(34) Shang, E.; Zhu, Z.; Liu, L.; Tang, Y.; Duan, J.-A. (2012) UPLC-QTOF-MS with
chemical profiling approach for rapidly evaluating chemical consistency
between traditional and dispensing granule decoctions of Tao-Hong-Si-Wu
decoction. Chem. Cent. J., 6:143.
(35) Perera, A.; Ton. S.H.; Palanisamy, U.D. (2015) Perspectives on geraniin, a
multifunctional natural bioactive compound. Trends Food Sci. Technol.,
44(2):243-257.
(36) Kool, M.M.; Comeskey, D.J.; Cooney, J.M.; McGhie, T.K. (2010) Structural
identification of the main ellagitannins of a boysenberry (Rubus loganbaccus ×
baileyanus Britt.) extract by LC-ESI-MS/MS, MALDI-TOF-MS and NMR
spectroscopy. Food Chem., 119(4):1535-1543.
(37) 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.
87
Supporting Information for
UPLC-QTOF-MSE-based chemometric approach driving the choice of the best
extraction process for Phyllanthus niruri
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
© central point of the experimental design.
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
© central point of the experimental design.
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
Drying kinetics and effect of air-drying temperature on chemical composition of
Phyllanthus amarus and Phyllanthus niruri
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
Drying kinetics and effect of air-drying temperature on chemical composition of
Phyllanthus amarus and Phyllanthus niruri
Adriana Dutra Sousa1,2, Paulo Riceli Vasconcelos Ribeiro1, Kirley Marques Canuto1,
Guilherme Julião Zocolo1, Fabiano André Narciso Fernandes2, 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.
* 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)
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
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
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.
References
1. Patel, J.R.; Tripathi, P.; Sharma, V.; Chauhan, N.S.; Dixit, V.K. Phyllanthus
amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review.
Journal of Ethnopharmacology 2011, 138, 286–313.
2. Bagalkotkar, G.; Sagineedu, S.R.; Saad, M.S.; Stanslas, J. Phytochemicals from
Phyllanthus niruri Linn. and their pharmacological properties: A review. Journal
of Pharmacy and Pharmacology 2006, 58, 1559-1570.
107
3. Calixto, J.B.; Santos, A.R.S.; Cechinel Filho, V.; Yunes, R.A. A review of the
plants of the Genus Phyllanthus: Their chemistry, pharmacology, and
therapeutic potential. Medicinal Research Reviews 1998, 18 (4), 225-258.
4. Karam, M.C.; Petit, J.; Zimmer, D.; Djantou, E.B.; Scher, J. Effects of drying
and grinding in production of fruit and vegetable powders: A review. Journal of
Food Engineering 2016, 188, 32-49.
5. Doymaz, I.; Tugrul, N.; Pala, M. Drying characteristics of dill and parsley
leaves. Journal of Food Engineering 2006, 77 (3), 559-565.
6. Kaya, A.; Aydın, O. An experimental study on drying kinetics of some herbal
leaves. Energy Conversion and Management 2009, 50 (1), 118-124.
7. Arslan, D.; Özcan, M.M. Evaluation of drying methods with respect to drying
kinetics, mineral content and colour characteristics of rosemary leaves. Energy
Conversion and Management 2008, 49 (5), 1258-1264.
8. Martínez-Las Heras, R.; Heredia, A.; Castelló, M.L.; Andrés, A. Influence of
drying method and extraction variables on the antioxidant properties of
persimmon leaves. Food Bioscience 2014, 6, 1-8.
9. Choi, Y.; Lee, S.M.; Chun, J.; Lee, H.B.; Lee, J. Influence of heat treatment on
the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus
edodes) mushroom. Food Chemistry 2006, 99, 381–387.
10. Rodríguez, K.; Ah-Hen, K.S.; Vega-Gálvez, A.; Vásquez, V.; Quispe-Fuentes,
I.; Rojas, P.; Lemus-Mondaca, R. Changes in bioactive components and
antioxidant capacity of maqui, Aristotelia chilensis [Mol] Stuntz, berries during
drying. LWT - Food Science and Technology 2016, 65, 537-542.
11. Lim, Y.Y.; Murtijaya, J. Antioxidant properties of Phyllanthus amarus extracts
as affected by different drying methods. LWT-Food Science and Technology
2007, 40, 1664–1669.
12. Doymaz, I. Convective air drying characteristics of thin layer carrots. Journal of
Food Engineering 2004, 61, 359-364.
13. Martins, M.G.; Martins, D.E.G.; Pena, R.S. Drying kinetics and hygroscopic
behavior of pirarucu (Arapaima gigas) fillet with different salt contents. LWT -
Food Science and Technology 2015, 62, 144-151.
14. Crank, J. The mathematics of diffusion (2nd ed.); Clarendon Press: Oxford, UK,
1975.
15. Chen, D.; Zheng, Y.; Zhu, X. Determination of effective moisture diffusivity
and drying kinetics for poplar sawdust by thermogravimetric analysis under
isothermal condition. Bioresource Technology 2012, 107, 451-455.
16. 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. Ultrasound-assisted and pressurized
liquid extraction of phenolic compounds from Phyllanthus amarus and its
108
composition evaluation by UPLC-QTOF. Industrial Crops and Products 2016,
79, 91-103.
17. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology
and Viticulture 1965, 16, 144-158.
18. Zogzas, N.P.; Marulis, Z.B.; Mariinos-Kouris, D. Moisture diffusivity data
compilation in foodstuffs. Drying Technology 1996, 14, 2225–2253.
19. Ben Haj Said, L.; Najjaa, H.; Farhat, A.; Neffati, M.; Bellagha, S. Thin layer
convective air drying of wild edible plant (Allium roseum) leaves: experimental
kinetics, modeling and quality. Journal of Food Science and Technology 2015,
52 (6), 3739-3749.
20. Maskan, M.; Göğüş, F. Sorption isotherms and drying characteristics of
mulberry (Morus alba). Journal of Food Engineering 1998, 37, 437–449.
21. Katsube, T.; Tsurunaga, Y.; Sugiyama, M.; Furuno, T.; Yamasaki, Y. Effect of
air-drying temperature on antioxidant capacity and stability of polyphenolic
compounds in mulberry (Morus alba L.) leaves. Food Chemistry 2009, 113 (4),
964-969.
22. Wiriya, P.; Paiboon, T.; Somchart, S. Effect of drying air temperature and
chemical pretreatments on quality of dried chilli. International Food Research
Journal 2009, 16, 441-454.
23. Sprenger, R.D.F.; Cass, Q.B. Characterization of four Phyllanthus species using
liquid chromatography coupled to tandem mass spectrometry. Journal of
Chromatography A 2013, 1291, 97-103.
24. Kumar, S.; Chandra, P.; Bajpai, V.; Singh, A.; Srivastava, M.; Mishra, D.K.;
Kumar, B. Rapid qualitative and quantitative analysis of bioactive compounds
from Phyllanthus amarus using LC/MS/MS techniques. Industrial Crops and
Products 2015, 69, 143-152.
25. Yang, B.; Kortesniemi, M.; Liu, P.; Karonen, M.; Salminen, J.-P. Analysis of
hydrolyzable tannins and other phenolic compounds in emblic leafflower
(Phyllanthus emblica L.) fruits by high performance liquid chromatography-
electrospray ionization mass spectrometry. Journal of Agricultural and Food
Chemistry 2012, 60, 8672-8683.
26. Guo, J.; Chen Q.; Wang C.; Qiu H.; Liu B.; Jiang Z.-H.; Zhang W. Comparison
of two exploratory data analysis methods for classification of Phyllanthus
chemical fingerprint: unsupervised vs. supervised pattern recognition
technologies. Anal. Bioanal. Chem. 2015, 407, 1389-1401.
27. Hossain, M.B.; Rai, D.K.; Brunton, N.P.; Martin-Diana, A.B.; Barry-Ryan,
A.C. Characterization of phenolic composition in lamiaceae spices by LC-ESI-
MS/MS. Journal of Agricultural and Food Chemistry 2010, 58, 10576-10581.
109
28. Latté, K.P.; Kolodziej, H. Pelargoniins, new ellagitannins from Pelargonium
reniforme. Phytochemistry 2000, 54, 701-708.
29. Ogata, T.; Higuchi, H.; Mochida, S.; Matsumoto, H.; Kato, A.; Endo, T.; Kaji,
A.; Kaji, H. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri. AIDS
Research and Human Retroviruses 1992, 8 (11), 1937-1944.
30. Zheng, M.; Xia, Q.; Lu, S. Study on drying methods and their influences on
effective components of loquat flower tea. LWT - Food Science and Technology
2015, 63 (1), 14-20.
31. Esparza-Martínez, F.J.; Miranda-López, R.; Guzman-Maldonado, S.H. Effect of
air-drying temperature on extractable and non-extractable phenolics and
antioxidant capacity of lime wastes. Industrial Crops and Products 2016, 84, 1-6.
110
ARTIGO 4
High-power ultrasound does not hydrolyze ellagitannins from Phyllanthus amarus
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
High-power ultrasound does not hydrolyze ellagitannins from Phyllanthus amarus
Adriana Dutra Sousa1,3, Paulo Riceli Vasconcelos Ribeiro1, Kirley Marques Canuto1,
Guilherme Julião Zocolo1, Brijesh Tiwari2, Fabiano André Narciso Fernandes3, Edy
Sousa de Brito1,*
1 Embrapa Tropical Agroindustry, R Dra Sara Mesquita, 2270, Fortaleza-CE 60511 110,
Brazil.
2 Food Biosciences, Teagasc Food Research Centre, Dublin, Ireland
3 Departamento de Engenharia Química, Universidade Federal do Ceará, 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)
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
112
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.
113
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
The extractions were accomplished in a Dionex ASE 350 system (Sunnyvale,
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.
𝑈𝑙𝑡𝑟𝑎𝑠𝑜𝑛𝑖𝑐 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑊/𝑐𝑚2) =𝑃
𝜋𝑟2 (1)
Where P: ultrasound power (W) and r: tip radius (cm).
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
114
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
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
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 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.
115
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.
117
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
Analytical Chem. 71, 100-109.
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.
119
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.