RENORBIO
Programa de Pós-graduação em Biotecnologia
Estudos químicos e farmacológicos da Lippia gracilis Schauer
Sandra Santos Mendes
São Cristóvão – SE
2012
Universidade Federal de Sergipe
Centro de Ciências Biológicas e da Saúde – CCBS
Departamento de Fisiologia
Rede Nordeste de Biotecnologia – RENORBIO
Ponto Focal: Universidade Federal de Sergipe
Sandra Santos Mendes
Tese apresentada ao Programa RENORBIO, como parte dos requisitos necessários à obtenção do título de Doutor em Biotecnologia.
Orientadora: Profa. Dra. Sara Maria Thomazzi
São Cristóvão – Sergipe
2012
FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA CENTRAL
UNIVERSIDADE FEDERAL DE SERGIPE
M538e
Mendes, Sandra Santos
Estudos químicos e farmacológicos da lippia gracilis Schauer / Sandra Santos Mendes; orientador Sara Maria Thomazzi. – São Cristóvão, 2012.
73 f. : il.
Tese (Doutorado em Biotecnologia) – Universidade Federal de Sergipe, 2012.
1. Lippia gracilis. 2. Plantas medicinais. 3. Óleo essencial. 4. Antioxidantes. 5. Anti-inflamatório. l. Thomazzi, Sara Maria, orient. lI. Título.
CDU 542:582.929.4
Estudos químicos e farmacológicos da Lippia gracilis
Schauer
Tese de Doutorado apresentada à Rede Nordeste de Biotecnologia, na área de concentração
em Biotecnologia de produtos naturais, na linha de pesquisa de Bioprospecção, Biodiversidade
e Conservação na Universidade Federal de Sergipe (ponto focal Sergipe) como um dos pré-
requisitos para a obtenção do grau de Doutor em Biotecnologia.
Aprovado em: 23/03/2012.
BANCA EXAMINADORA
_______________________________________________________________
Profa. Dra. Sara Maria Thomazzi Universidade Federal de Sergipe / UFS / DFS - Orientadora
_______________________________________________________________ Prof. Dr. Roberto Rodrigues de Souza
Universidade Federal de Sergipe / UFS / DEQ
_______________________________________________________________ Profa. Dra. Adriana Andrade Carvalho
Universidade Federal de Sergipe / UFS / Campus de Lagarto
________________________________________________________________ Prof. Dr. Marcelo Ferreira Fernandes Embrapa Tabuleiros Costeiros / SE
________________________________________________________________ Prof. Dr. Daniel Pereira Bezerra
Universidade Federal de Sergipe / UFS / DFS
AGRADECIMENTOS
A minha mãe pelo apoio, incentivo e amor incondicional, capazes de me fazer alcançar
todos os meus sonhos; sem ela eu jamais teria chegado até aqui. Meu porto seguro,
minha melhor amiga e companheira. Muito obrigada!
A minha orientadora Sara Thomazzi, pela orientação, pelos conselhos sempre
construtivos e pela paciência de esperar pela minha maturidade emocional e
profissional.
Ao meu orientador holandês, Ric De Vos, pela paciência, amizade e incentivo. Sempre
tratou seus orientandos com muito respeito e disponibilidade procurando formar mão-
de-obra qualificada para o mercado de trabalho.
A minha grande amiga Dayseanne Falcão, pela amizade incondicional, pelo apoio nas
horas difíceis e pelas alegres e proveitosas conversas. Agradeço também pelas leituras e
correções incansáveis desse trabalho. Sem seu apoio eu não teria seguido adiante e nem
concluído a parte escrita desta tese.
Ao meu companheiro, Márcio, pelo apoio, amor e incentivo sempre, fazendo com que a
vida pareça mais leve e simples.
Ao professor Charles Estevam pelo apoio na execução dos ensaios de antioxidantes bem
como pela concessão do material utilizado no estágio que realizei na Holanda
(Doutorado Sanduíche).
Aos professores Arie Fitzgerald Blank e Renata Silva-Mann pelo assessoramento no
envio do material utilizado no meu estágio na Holanda.
Ao professor Renato Delmondez por acreditar em meu trabalho, me incentivar e me
auxiliar na viagem para o exterior, sem o qual essa experiência não teria acontecido.
Aos meus colegas de laboratório, em especial a Kathia Riella, (pelo valioso apoio e
amizade), Jandson e Cliomar pelas correções nas minhas apresentações, sempre muito
pertinentes e Mirabeau pelos momentos agradáveis que me proporcionou com sua
alegria contagiante.
A Fundação de Amparo à Pesquisa e à Inovação Tecnológica do Estado de Sergipe -
FAPITEC/SE, pela concessão da bolsa de estudos.
A Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES pela
concessão da bolsa de estudos do doutorado sanduíche.
Aos membros da banca, pela leitura e exame da presente tese, aos técnicos, professores
do curso, e demais pessoas que contribuíram direta ou indiretamente para a realização
deste trabalho.
RESUMO
O gênero Lippia apresenta grande potencial farmacológico e terapêutico e possui
aproximadamente 200 espécies de ervas, arbustos e pequenas árvores, apresentando um
perfil consistente de composição química e atividades farmacológicas, terapêuticas e
culinárias. O objetivo deste trabalho foi estudar aspectos químicos e farmacológicos da
Lippia gracilis Schauer (Verbenaceae), conhecida popularmente por “alecrim do
campo”, proveniente do banco de germoplasma da Universidade Federal de Sergipe.
Assim, este estudo foi dividido em duas partes: na primeira buscou-se estudar aspectos
relacionados aos constituintes apolares da L. gracilis, presentes no óleo essencial (OE)
obtido das folhas de plantas submetidas ao estresse hídrico (sazonal) e sem estresse. A
composição dos constituintes do OE foi estudada com a utilização da cromatografia
gasosa acoplada à espectrometria de massa (CG/EM) e suas atividades anti-inflamatória
e antinociceptiva estudadas em modelos in vivo. Como resultados, verificamos que
quatro compostos foram encontrados em maior concentração nos OE
(independentemente das plantas terem sido submetidas ou não ao estresse hídrico):
timol, p-cinemo, metil timol e carvacrol. Como a composição química dos OEs foi
muito similar, escolheu-se trabalhar com o OE das plantas submetidas ao estresse
hídrico nos ensaios in vivo. Para tanto, os animais (n=6/grupo) foram inicialmente pré-
tratados com o OE (50, 100 e 200 mg/kg, v.o.), veículo (tween 80 a 0,2% em salina, 10
mL/kg, v.o.) e ácido acetilssalicílico (AAS, 300 mg/kg, v.o.) ou dexametasona (Dexa, 2
mg/kg, s.c.), dependendo do modelo experimental, 1 h antes do início dos experimentos.
A atividade anti-inflamatória foi avaliada utilizando-se os modelos de edema de pata
(ratos Wistar, 120-180 g) e peritonite (camundongos Swiss, 20-30 g) induzidos por
carragenina. O tratamento dos animais com o OE na dose de 200 mg/kg reduziu de
forma significativa (p<0,01) o edema induzido pela carragenina (1%, 100 µL/pata) em
todos os tempos mensurados (1, 2, 3 e 4 h após a indução do edema). De forma similar,
o AAS reduziu (p<0,05) a formação do edema em todos os tempos. No modelo de
peritonite, todas as doses do OE (50, 100 e 200 mg/kg, p<0,01), bem como a
dexametasona (p<0,001), foram capazes de reduzir a migração de leucócitos induzida
por carragenina (1%, 250 µL, i.p.). Para avaliação da atividade antinociceptiva utilizou-
se o modelo de contorções abdominais induzidas por ácido acético (0,6%, 100 µL/10g,
i.p.) em camundongos (Swiss, 20-30g). Observou-se que todas as doses do OE foram
capazes de reduzir (p<0,05) a nocicepção induzida pelo ácido acético, sendo que com a
dose de 200 mg/kg do OE observaram-se efeitos semelhantes ao AAS (p<0,001). Na
segunda parte do presente estudo, os constituintes polares foram estudados in vitro,
utilizando-se os ensaios de avaliação da atividade antioxidante do ABTS (2,2’-azino-bis
(3-etil-benzolina-6-sulfonado), FRAP (poder antioxidante de redução do ferro) e DPPH
(2,2-di(4-tertoctilfenil)-1-picrilhidrazil), com o composto de referência sendo o trolox,
um análogo da vitamina E. A composição química dos extratos aquoso e alcoólico
obtidos das folhas da L. gracilis, foi avaliada por cromatografia líquida de alto
desempenho acoplada a espectrometria de massa (HPLC/EM). Através dos perfis
químicos dos extratos obtidos por HPLC/EM, foi possível identificar três compostos
majoritários com intensa atividade antioxidante: luteolina-C-6-glucosídeo, luteolina-C-
8-glucosídeo e carvacrol. Em ensaios in vitro, verificou-se uma maior atividade
antioxidante (40%) do extrato alcoólico quando comparado ao aquoso, independente do
solvente utilizado para a dissolução. Os três compostos majoritários foram testados
isoladamente, além de terem sido comparados com compostos antioxidantes de ação
conhecida, como quercetina e luteolina. As atividades detectadas dependeram
claramente do ensaio utilizado e da estrutura do composto. O carvacrol apresentou
maior atividade no ABTS, quando comparado com as luteolinas, porém atividade
inferior no DPPH e no FRAP. Em todos os ensaios, a luteolina-C-6-glucosideo
apresentou valores maiores que a luteolina-C-8-glucosideo. A L. gracilis representa
uma fonte potencial de antioxidantes, além de possuir atividades anti-inflamatória e
antinociceptiva. Este é o primeiro relato de detecção das luteolinas C-6 e C-8 glucosídeo
na espécie Lippia gracilis.
Palavras-chave: Lippia gracilis, plantas medicinais, inflamação, dor, antioxidantes.
ABSTRACT
Lippia genus has a great pharmacological and therapeutic potential and encompasses
approximately 200 species of herbs, bushes and small trees with a consistent chemical
composition and pharmacological, therapeutics and culinary activities. This research
aimed to evaluate the chemical and pharmacological aspects of Lippia gracilis Schauer
(Verbenaceae), well-known as “alecrim do campo”, which grew at the germplasm bank
of the Federal University of Sergipe, Brazil. This study was divided in two parts: in the
first one we tried to evaluate the nonpolar compounds from the essential oil (EO) of L.
gracilis plants under hydric stress and without hydric stress. The identification of the
EO was made by using GC/MS and its anti-inflammatory and antinociceptive activities
were studied in animal models. As results, thymol, p-cineme, methyl thymol, and
carvacrol were found in the EO analysis, independent of the hydric stress L. gracilis
leaves were submitted. Although the different hydric stresses the plants pass through,
the chemical composition of the EO was too similar, so it was chosen to study the one
obtained from the plants submitted to hydric stress in the animal models. In the animal
models, we fed 1 hour before the beginning of the experiments with the EO (50, 100,
and 200 mg/kg, p.o.), vehicle (tween 80 at 0.2% in saline, 10 mL/kg, p.o.) and
acetylsalicylic acid (ASA, 300 mg/kg, p.o.) or dexamethasone (Dexa, 2 mg/kg, s.c.). To
evaluate the anti-inflammatory activity, we used paw oedema model (Wistar rats, 120-
180 g) and leukocyte migration into the peritoneal cavity (Swiss mice, 20-30 g). To
evaluate the antinociceptive activity, we used acetic acid-induced abdominal writhes
model in Swiss mice. In all in vivo experiments we used groups of 6 animals. In the paw
oedema assay, could be verified a reduction in inflammation (p<0.01) at the 200 mg/kg
dose in Wistar rats in all measurement time (1, 2, 3 and 4 hours after carrageenan-
induced paw oedema). The positive control (AAS – 300 mg/kg) was also able to reduce
the inflammation (p<0.05). In the leucocyte migration model, all EO doses used (50,
100, and 200 mg/kg) were able to reduces leucocytes migration, caused by carrageneen
(p<0.01). Dexa was also able to reduce the migration (p<0.001). In the acetic acid-
induced abdominal writhes model, all the doses reduced the nociception caused by
acetic-acid, whereas the 200 mg/kg dose showed similar effects to ASS (p<0.001). In
the second part of this study, the main polar compounds of L. gracilis were evaluated
using antioxidant in vitro assays, namely ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-
6-sulphonic acid), FRAP (Ferric Reducing Antioxidant Power) and DPPH (2,2-di(4-
tertoctylphenyl)- 1-picrylhydrazyl), with trolox as reference antioxidant. The chemical
composition of aqueous and ethanolic extracts was also evaluated by HPLC (High
Performance Liquid Chromatography) and LCMS (Liquid Chromatography – Mass
spectrometry). Three major compounds with high antioxidant activity could be
identified: luteolin-C-6-glucoside, luteolin-C-8-glucoside and carvacrol. The
antioxidant activity of the ethanolic extract was higher than the water extract (about
40%), independent of the solvent used. These three compounds were also tested isolate,
besides their comparison with well-known antioxidants as quercetin and luteolin. The
antioxidant activity clearly depends on the assay used and the compound structure.
Carvacrol showed higher activity in ABTS assay than C-6 and C-8-glucosides, but
lower in DPPH and FRAP assays. In all assays, luteolin-C-6 showed higher antioxidant
activity than luteolin-C-8-glucoside. L. gracilis is a good source of compounds with
potential antioxidant, anti-inflammatory and antinociceptive activities. This is the first
time that luteolin C-6 and C-8-glucosides were described in the L. gracilis.
Key words: Lippia gracilis, medicinal plants, inflammation, pain, antioxidants.
LISTA DE ABREVIATURAS
AAS Ácido acetilssalicílico
ABTS 2,2’-azino-bis (3-etil-benzolina-6-sulfonado)
AINES Anti-inflamatórios não esteroidais
CG/EM Cromatografia gasosa aplicada a espectrometria de massa
COX Ciclo-oxigenase
DEXA Dexametasona
DPPH 2,2-di(4-tertoctylphenyl)- 1-picrylhydrazyl
ERN Espécies reativas de nitrogênio
ERO Espécies reativas de oxigênio
ET Transferência de elétrons
FRAP Poder antioxidante de redução do ferro
HAT Transferência de átomo de hidrogênio
HPLC/EM Cromatografia líquida de alta resolução acoplada a espectrometria de massa
LT Leucotrieno
NF-kB Fator de transcrição nuclear kappa-B
NO Óxido nítrico
OE Óleo essencial
PGI2 Prostaciclina
PGs Prostaglandinas
PGE2 Prostaglandina E2
TPTZ 2,4,6-tripiridil-s-triazina
SUMÁRIO
1. Introdução 1
2. Revisão da literatura 3
2.1. O uso de plantas medicinais 3
2.2. A Caatinga 4
2.3. Considerações sobre o gênero Lippia 6
2.4. Inflamação 8
2.5. Dor 10
2.6. Atividade antioxidante e inflamação 13
3. Conclusões
4. Referências
16
17
5. Artigos científicos 27
5.1. Artigo 1 28
5.2. Artigo 2 35
1
INTRODUÇÃO
As plantas medicinais têm sido usadas em países em desenvolvimento como
uma alternativa no tratamento de doenças. Muitos extratos e óleos essenciais obtidos a
partir de plantas têm apresentado atividades biológicas in vitro e in vivo, o que justifica
pesquisas baseadas na medicina popular para as mais diversas atividades das plantas
medicinais (Martínez et al., 1996).
As plantas atuam como fontes de agentes terapêuticos, modelos para novos
medicamentos sintéticos ou ainda como material de partida para a produção semi-
sintética de moléculas de alta complexidade, o que justifica os 25% dos fármacos
utilizados na atualidade serem de origem vegetal (Bruschi et al., 2000).
A região Nordeste do Brasil apresenta uma diversidade de espécies nativas que é
conhecida pelas suas propriedades medicinais e pelo uso contínuo da população na
medicina tradicional. Dentre essas espécies, destacam-se as pertencentes ao gênero
Lippia, sendo utilizadas para os mais diversos fins, como para distúrbios
gastrointestinais, respiratórios, antimaláricos e antibacterianos. Aliado a isso são
também utilizadas na preparação de pratos da culinária regional. Na maioria dos casos,
as partes usadas das plantas são as folhas e flores sob a forma de decocção ou infusão,
sendo administradas oralmente (Pascual et al., 2001).
Algumas espécies desse gênero produzem óleos essenciais cuja atividade
antimicrobiana é bastante acentuada devido à presença de monoterpernos fenólicos,
como timol e carvacrol. Dentre essas espécies, a Lippia gracilis, nativa do nordeste
brasileiro também conhecida como “alecrim do campo” e “alecrim da chapada”, possui
grande quantidade desses compostos (Matos et al., 1999) e um potencial para várias
atividades medicinais.
O presente trabalho buscou contribuir para o conhecimento da espécie Lippia
gracilis Schauer, a partir de informações obtidas da análise química e farmacológica do
óleo essencial e dos extratos aquoso e alcoólico de suas folhas. Para tanto, o estudo foi
dividido em duas partes, onde as atividades anti-inflamatória e antinociceptiva dos
compostos voláteis produzidos pela planta foram analisados através da utilização do
óleo essencial obtido de suas folhas, em modelos animais (primeira parte), e os
compostos não-voláteis (obtidos nos extratos aquoso e alcoólico) foram analisados e
2
testados em relação à atividade antioxidante in vitro, através dos ensaios de ABTS
[2,2’-azino-bis(3-etil-benzolina-6-sulfonado)], DPPH [2,2-di(4-tertoctylphenyl)- 1-
picrylhydrazyl] e FRAP (poder antioxidante de redução do ferro), extensamente
utilizados na avaliação de produtos naturais, bem como pela utilização de HPLC/EM
para a análise de seus constituintes e correlação com as atividades antioxidantes
encontradas.
A segunda parte deste estudo foi desenvolvida no departamento de
metabolômica do instituto de fisiologia de plantas em Wageningen University
(Holanda), sob a supervisão do Dr. Ric de Vos.
3
REVISÃO DA LITERATURA
O Uso de Plantas Medicinais
Planta medicinal é todo vegetal que contém em um de seus órgãos ou em toda a
planta, compostos que podem ser empregados com fins terapêuticos (Amorozo, 2002).
O uso das plantas como medicamento é tão antigo quanto o próprio homem. Em
países nos quais o acesso médico e hospitalar é restrito, os remédios originários de
plantas são a principal fonte terapêutica. Nesse sentido, as plantas estão sendo
preparadas e prescritas por vários profissionais de saúde e vendidas em muitas
farmácias de manipulação (Franco & Fontana, 2003).
Os produtos naturais tem sido a maior fonte de fármacos por séculos, como
demonstrado com o isolamento da morfina a partir do ópio no início do século XIX e o
isolamento de outros fármacos que se seguiram como a cocaína, codeína, digitoxina e
quinina (Butler, 2004; Newman et al., 2000). Estima-se que atualmente 25% a 30% dos
medicamentos utilizados derivam de produtos naturais (Calixto, 2005). O uso de plantas
medicinais vem crescendo substancialmente nos últimos anos, principalmente devido à
facilidade de acesso, o baixo custo e sua compatibilidade cultural. As formas de uso das
plantas medicinais podem variar desde o uso de chás, preparados com plantas frescas,
até o uso de pós, gotas, cápsulas e outros tipos de fitoterápicos (Nogueira et al., 1996).
A fitoterapia surge como uma opção medicamentosa bem aceita e acessível aos
povos de todo o planeta. Em virtude disso, é extremamente importante a investigação do
uso popular de plantas medicinais. A difusão e pesquisa dos fitoterápicos devem ser
amplamente incentivadas e incorporadas aos sistemas de saúde (Eldin & Dunford,
2001).
Várias áreas estão envolvidas na pesquisa de novas substâncias provenientes de
plantas, como a fitoquímica, que trabalha no isolamento, purificação e caracterização de
princípios ativos; a etnobotânica e a etnofarmacologia, que buscam informações a partir
do conhecimento de diferentes povos e etnias; e a farmacologia, que estuda os efeitos
farmacológicos de extratos e dos constituintes químicos isolados (Maciel et al., 2002); a
etnobotânica possui um campo vasto para pesquisa no Brasil, que apresenta grande
diversidade biológica, e a intensificação de trabalhos nessa área leva ao conhecimento
4
de espécies que são utilizadas, podendo servir de instrumento para delinear estratégias
de utilização e conservação das espécies nativas e seus potenciais (Ming et al., 2000).
O Brasil é o país com maior biodiversidade mundial, abrigando cerca de 50 mil
espécies de plantas superiores, distribuídas em grandes biomas (Mata Atlântica,
Cerrado, Pantanal, Amazônia e Caatinga), com características edafoclimáticas distintas,
que conferem uma riqueza e diversidade de vegetação (Skorupa & Vieira, 2004).
A Caatinga
A Caatinga é um mosaico de arbustos espinhosos e florestas sazonalmente secas
que cobre a maior parte dos estados do Piauí, Ceará, Rio Grande do Norte, Paraíba,
Pernambuco, Alagoas, Sergipe, Bahia e a parte nordeste de Minas Gerais, no vale do
Jequitinhonha (Leal et al., 2005). Este bioma apresenta grande variedade de paisagens e
relativa riqueza biológica, se estendendo numa área de 73.683.649 hectares, que
equivale a 6,83% do território nacional. Trata-se de um bioma exclusivamente brasileiro
e uma das vegetações mais ameaçadas do planeta, mas, apesar disto, esta exclusividade
não foi suficiente para direcionar estudos botânicos nesta área (Brasil, 2002). Várias
populações distribuídas dentro deste bioma dependem, na maioria das vezes, dos
recursos vegetais disponíveis para o seu sustento (Albuquerque & Andrade, 2002;
Albuquerque & Lucena, 2004). Comumente a caatinga é descrita como um ecossistema
pobre, mas o que se vê são inúmeros estudos que descrevem e demonstram as riquezas e
potencialidades deste bioma. Tais potencialidades transformam-na em um laboratório
para estudos e pesquisas nos mais diversos campos (Leal et al., 2005).
A caatinga se caracteriza por apresentar um reduzido potencial hídrico no solo,
precipitações escassas e irregulares, com acentuado período de estação seca, entre sete e
dez meses. A flora nativa da caatinga apresenta espécies vegetais com caracteres
anatômicos, morfológicos e funcionais especializados para a sobrevivência destas
plantas às condições adversas de clima e solo, típicos desta fisionomia. A vegetação é
composta por espécies lenhosas e herbáceas, de pequeno porte, muitas dotadas de
espinhos, sendo, geralmente, caducifólias, e cactáceas e bromeliáceas. Das 596 espécies
já registradas para esta formação, 180 são endêmicas, com densidade, frequência e
dominância determinada pelas variações topográficas, tipo de solo e pluviosidade
(Drumond et al., 2000).
5
Espécies nativas da Caatinga são utilizadas com fins terapêuticos, sob a forma de
extrato aquoso, especialmente partes do caule. Essas plantas tem o uso amplamente
difundido entre gerações e são conhecidas como eficientes anti-inflamatórios,
cicatrizantes, adstringentes, antidiabéticos, dentre outras propriedades medicinais (Silva,
2008). Estudos têm comprovado a ação benéfica de muitas espécies ocorrentes neste
bioma, promovendo o uso dos vegetais com efeito comprovado entre a população
economicamente mais carente (Almeida & Albuquerque, 2002).
Dentro deste contexto podemos citar várias espécies encontradas na caatinga. A
Myracrodruon urundeuva, mais conhecida como “aroeira”, é bastante utilizada na
medicina tradicional nordestina, secularmente conhecida por seu uso sob a forma de
“banho-de-assento”, após o parto, com o extrato aquoso do caule (casca). Esta espécie é
indicada como anti-inflamatória e cicatrizante no tratamento de ferimentos, gastrites,
úlceras gástricas, cervicites, vaginites e hemorróidas (Lorenzi & Matos, 2002). A
Sideroxylon obtusifolium, ou “quixabeira”, é amplamente empregada na medicina
caseira devido as suas propriedades adstringente, tônica, anti-inflamatória e
antidiabética (Mors et al., 2000); possui ações anti-inflamatória e antinociceptiva
comprovadas (Araujo-Neto et al., 2010). A Zizyphus joazeiro, popularmente conhecida
como “juá”, é objeto de exploração comercial e altamente valorizada, devido à sua
utilização por importantes indústrias farmacêuticas, na fabricação de cosméticos,
xampus anticaspa e cremes dentais. Sua utilização para assepsia bucal pela população é
anterior à exploração industrial. Na medicina popular é indicada no tratamento de
gastrites, gripes, contusões e ferimentos (Lima, 2000; Matos, 2000). A casca da
Auxemma oncocalyx, mais conhecida como “pau-branco”, é muito utilizada na medicina
popular no tratamento auxiliar de ferimentos (Pessoa, 1994); estudos farmacológicos
mostraram ações antiagregante plaquetária e vasoconstritora (Souza et al., 2002). E
ainda, a Caesalpinia ferrea, utilizada para o tratamento de ferimentos e contusões,
alívio da tosse crônica e asma (Bacchi et al., 1995); possui propriedades anti-
inflamatórias e analgésicas (Carvalho et al., 1996), e seus frutos são utilizados no
tratamento da diabetes e na prevenção do câncer (Nakamura, 2002).
Várias plantas encontradas na Caatinga possuem ações medicinais comprovadas
e, conforme visto anteriormente, muitas delas com propriedades anti-inflamatórias e
analgésicas já descritas.
6
Considerações sobre o gênero Lippia
O gênero Lippia, pertencente à família Verbenaceae, possui mais de 200
espécies de ervas, arbustos e pequenas árvores. A maioria das espécies é medicinal,
sendo usada pela população para tratamento de doenças gastrointestinais e respiratórias
(Morton, 1981). Na maioria dos casos as partes aéreas, como folhas, caules e flores são
utilizadas sob a forma de infusão ou decocção e administradas oralmente ou através de
emplastros ou ainda na lavagem de ferimentos (Calvacanti, 2006). Em geral, o gênero
apresenta composição química constante, com alguns compostos encontrados em várias
espécies apresentando atividades antimalárica, antiviral e citostática (Pascual et al.,
2001).
Muitas espécies do gênero Lippia apresentam folhas aromáticas, cujo aroma está
relacionado aos constituintes predominantes nos óleos essenciais (OE), em função de
diversos fatores, tais como: estação do ano, época de floração, idade da planta,
quantidade de água circulante (resultante da precipitação), fatores geográficos e
climáticos (Corrêa, 1992; Tavares et al., 2005).
Os OE obtidos das espécies do gênero Lippia possuem atividades biológicas
bastante conhecidas e estudadas na literatura. Além dos OE, as plantas do gênero Lippia
possuem compostos não voláteis com atividades medicinais.
Estudos realizados com espécies da Lippia vêm comprovando suas atividades
farmacológicas. O OE da Lippia multiflora possui algumas atividades farmacológicas
(analgésica, anti-inflamatória e antipirética) e é utilizado para tratar insuficiência
hepática e febre, e seu extrato, possui efeitos analgésicos (Abena et al., 2003). A Lippia
dulcis é comumente usada na medicina popular para tratar inflamações, resfriados,
diarréia e dores no estômago, além de possuir efeitos inibitórios sobre o crescimento de
algumas enterobactérias (Cáceres et al., 1993). A Lippia alba, popularmente conhecida
como “cidreira”, é utilizada como sedativa, digestiva, espasmolítica, e suas propriedades
emenagogas têm sido reportadas em estudos etnobotânicos (Corrêa, 1992; Di Stasi &
Hiruma-Lima, 2002; Lorenzi & Matos, 2002).
De acordo com Matos et al. (1996) na composição química do OE de L. alba, os
compostos mais abundantes são geranoil (20,7%), neral (16,4%), mirceno (15,0%) e
nerol (3,8%). Sendo assim, foi possível caracterizar a L. alba com o quimiotipo
7
mirceno-citral-geraniol, sendo o citral (37,1%) uma mistura de isômeros naturais do
neral e geraniol (Matos et al., 1996).
Algumas investigações com os OE produzidos por três diferentes quimiotipos de
L. alba (citral-mirceno, citral-limoneno e carvona-limoneno) mostraram efeitos
ansiolíticos e atividade anticonvulsivante (Viana et al., 2000). Em especial, o
quimiotipo citral-limoneno, que apresentou efeitos em baixas doses, quando comparado
com os demais. Resultados similares foram obtidos quando mirceno e limoneno foram
testados isoladamente, sendo mais ativos na presença de citral (Vale et al., 2002).
Outra espécie da Lippia que tem o uso popular bastante difundido é a Lippia
sidoides, conhecida como “alecrim pimenta”, uma planta aromática usada
principalmente como antisséptica. Seu OE possui efeitos contra fungos e bactérias
(Lemos et al., 1990), atividade antioxidante e seu constituinte principal é o timol
(66,6%), provavelmente o responsável por essas atividades (Monteiro et al., 2007).
A Lippia gracilis Schauer (Figura 1), popularmente conhecida como “alecrim-
de-tabuleiro” ou “alecrim-da-chapada”, é um arbusto caducifólio, ramificado, de até 2 m
de altura, típica da caatinga, de terrenos bem drenados, sendo comum nos Estados da
Bahia, Sergipe e Piauí (Lorenzi & Matos, 2002). Esta espécie possui folhas aromáticas
que são utilizadas juntamente com as flores para a produção de OE. Seu OE possui
atividade bactericida e é utilizado externamente para tratar doenças cutâneas,
queimaduras, injúrias em geral e úlceras. É tradicionalmente utilizada no tratamento de
infecções gastrointestinais, respiratórias e cutâneas (Pascual et al., 2001; Pessoa et al.,
2005). Guimarães et al. (2012) estudaram os extratos aquoso e metanólico da L. gracilis
e constataram o seu potencial antinociceptivo e anti-inflamatório em modelos animais.
8
A B
Figura 1: Lippia gracilis no campo (A) e detalhe das folhas (B). Campus rural da Universidade Federal
de Sergipe. São Cristóvão, Sergipe, Brasil, 2010.
Inflamação
A inflamação é um dos primeiros sinais do corpo na defesa contra lesões e/ou
invasões de microrganismos. É um mecanismo fisiológico que ajuda o corpo a reparar
danos e/ou lesões (Tzoulaki, 2006). Os tecidos inflamados podem responder a estímulos
nocivos através da produção de diferentes mediadores bioativos, os quais interagem
com diversos tipos celulares e moleculares para minimizar a reação flogística (Kumar et
al., 2005; Sherwood & Toliver-kinsky, 2004). Este processo envolve cascatas de
eventos bioquímicos e celulares que incluem extravasamento de fluídos, ativação
enzimática, migração celular, liberação de mediadores, sensibilização e ativação de
receptores, lise tecidual e reparo (Becker, 1983; Piper, 1983).
Um dos fenômenos celulares envolvidos nesse processo é a migração celular. Os
neutrófilos, células leucocitárias, são os primeiros recrutados para o sítio inflamatório e
também os principais efetores da lesão tecidual, através da produção de proteases e
radicais derivados do metabolismo do oxigênio e nitrogênio (Keel et al., 1997). Neste
local, as células podem desempenhar uma grande variedade de funções, dependendo do
seu modo de ativação, tais como: apresentação de antígenos, citotoxicidade celular,
remoção de fragmentos celulares e remodelamento tecidual, regulação da inflamação,
indução de imunidade, trombose e várias formas de endocitose (Al-Sarireh & Eremin,
2000).
9
O recrutamento dos neutrófilos durante a inflamação depende da liberação de
mediadores pró-inflamatórios (Luster et al., 2005). Dentre eles, encontram-se citocinas,
metabólitos do ácido araquidônico (prostaglandinas, prostaciclinas, tromboxanos e
leucotrienos), histamina, fator de ativação plaquetária, bradicinina, NO e
neuropeptídeos (Rang et al., 2007).
Em 1971, pesquisadores estudando a atividade anti-inflamatória da aspirina
conseguiram demonstrar que ela estaria ligada a sua capacidade em inibir a produção de
prostaglandinas (PGs), através de uma provável competição com o sítio ativo da enzima
ciclooxigenase (COX). Em 1990, foi demonstrado que a COX é constituída por duas
isoformas principais, com características químicas e fisiológicas bem definidas, a COX
do tipo 1 (COX-1), constitutiva ou fisiológica, e a COX do tipo 2 (COX-2), induzida ou
inflamatória (Júnior et al., 2007).
As duas isoformas da enzima COX convertem o ácido araquidônico em
endoperóxidos cíclicos instáveis os quais se transformam em PGs, prostaciclina (PGI2)
e tromboxanos (Adams, 1992; Guerra et al., 2001). A COX-1 serve para a formação dos
tromboxanos, PGs e PGI2, expressos numa grande variedade de tecidos e órgãos, com
atividades fisiológicas como proteção da mucosa digestiva, controle do fluxo de sangue
nos rins, dentre outras.
O ácido araquidônico, quando liberado, não tem ação inflamatória; entretanto, os
produtos da sua degradação, formados através da ação das enzimas COX-2 (PGs, PGI2 e
tromboxanos) e lipoxigenase (leucotrienos), são mediadores químicos fundamentais
para o desenvolvimento do processo inflamatório (Tasaka, 2002). As prostaglandinas
(principalmente a PGE2) são majoritariamente expressas em resposta aos estímulos
inflamatórios e contribuem para a formação do edema, hiperalgesia e febre (Radi &
Khan, 2005).
A partir de descobertas que rotulavam a COX-1 como fisiologicamente
constitutiva, agindo como citoprotetora gástrica e mantenedora da homeostase renal e
plaquetária, e COX-2 como induzível, a qual surgia apenas em situação de trauma
tissular e processos inflamatórios, foi possível a introdução de diferentes fármacos para
o tratamento da inflamação. Surgiu a idéia de que inibidores específicos da COX-2
impediriam o processo inflamatório sem causar os efeitos colaterais indesejáveis,
principalmente os distúrbios gastrintestinais, advindos do bloqueio inespecífico da COX
(Kummer & Coelho, 2002). Os anti-inflamatórios não esteroidais (AINES) diminuem a
10
inflamação por bloquearem a COX-2 (Tasaka, 2002), podendo ser inespecíficos
(atuando em ambas as COX) ou seletivos (atuando sobre a COX-1 ou COX-2).
Os AINES apresentam um amplo espectro de ações terapêuticas: analgésica,
anti-inflamatória, antipirética e profilática nas doenças cardiovasculares (Dubois et al.,
1998).
Vários modelos experimentais de inflamação têm sido usados para investigar o
perfil de resposta de extratos e substâncias isoladas de plantas. O uso de modelos
experimentais com animais, para avaliação quantitativa e qualitativa da nocicepção e
inflamação, é considerado muito importante para a comercialização de fitoterápicos,
dando embasamento ao uso popular (Heilborn et al., 2007).
Dor
A dor pode ser considerada como um sintoma ou manifestação de uma doença
ou afecção orgânica, mas também pode vir a constituir um quadro clínico mais
complexo. A definição de dor de acordo com a Associação Internacional para o Estudo
da Dor consiste em “uma experiência emocional e sensorial desagradável associada
com uma lesão tecidual real ou potencial ou descrita em termos de tal lesão” (Loeser &
Melzack, 1999).
A sensação de dor aciona no organismo um mecanismo de alerta, indicando a
presença de um estímulo lesivo. O organismo responde acionando respostas apropriadas
a fim de protegê-lo contra agressões e lesões (Julius & Basbaum, 2001). Dessa maneira,
o funcionamento adequado desse sistema é essencial para proteger o organismo de
danos teciduais. Entretanto, sob condições patológicas este sistema se torna
sensibilizado e a dor transforma-se em uma doença (Zeilhofer, 2005).
A dor é influenciada pela ansiedade, depressão, expectativa e outras variáveis
psicológicas. É uma experiência multifacetada, um entrelaçamento das características
físicas dos estímulos com as funções motivacionais, afetivas e cognitivas do indivíduo
(Abreu, 2001). Além disso, pode manifestar-se entre os indivíduos sob intensidades
diferentes, a depender do sexo, idade, condições físicas e estado de humor (Faucett &
Levine, 1991; Ganong, 1988).
Enquanto a dor envolve a percepção e interpretação de estímulos nocivos, a
nocicepção corresponde apenas às manifestações neurofisiológicas e neuroquímicas
11
geradas pelo estímulo nocivo, ou seja, é a sensação determinada pela estimulação das
fibras aferentes primárias, sem levar em consideração os aspectos psicológicos que
também influenciam a percepção final da dor (Millan, 1999). Como os animais não são
capazes de verbalizar os componentes subjetivos da dor, neles não se avalia dor, mas
nocicepção.
Os nociceptores são extremamente heterogêneos, diferindo quanto aos tipos de
neurotransmissores, receptores e canais iônicos. Eles expressam suas propriedades de
resposta ao estímulo nocivo pela sua capacidade de serem sensibilizados durante a
inflamação, lesão e doença (Stucky et al., 2001).
O entendimento de como funciona esses nociceptores tem sido facilitado pelos
estudos farmacológicos, eletrofisiológicos e anatômicos, que têm contribuído para o
descobrimento de múltiplos mediadores químicos envolvidos na dor, de mecanismos de
ação dos neurotransmissores e das drogas envolvidas na modulação central e periférica
da dor (Wood & Docherty, 1997).
De acordo com os nociceptores envolvidos no estímulo, a dor pode ser
classificada de várias maneiras. Considerando a duração da sua manifestação, ela pode
se apresentar nas formas transitória, aguda ou crônica. Na dor transitória, a ativação dos
nociceptores acontece na ausência de qualquer dano tecidual e contribui para proteger o
organismo de potenciais danos físicos, causados pelo ambiente ou por estresse de
tecidos corporais. A dor aguda é uma resposta causada por uma lesão de tecido com
consequente ativação dos nociceptores, no local da lesão, caracterizando-se por ser de
curta duração, desaparecendo, até mesmo, antes da cura do dano tecidual (Loeser &
Melsack, 1999). Já a dor crônica, causada por uma lesão tecidual ou doença, geralmente
ultrapassa o tempo de recuperação do organismo, ou seja, esse tipo de dor pode não
desaparecer mesmo quando a lesão foi resolvida (Brennan et al., 2007; Tracey &
Mantyh, 2007).
Além da duração, outras variáveis podem caracterizar o tipo de dor, como a
capacidade do organismo de reparar o sítio de lesão e o processamento neural
responsável pela transmissão dolorosa (Hill, 2001).
A dor também pode ser classificada de acordo com a sua origem, em quatro
tipos principais: 1) dor nociceptiva: quando há uma estimulação excessiva dos
nociceptores da pele, vísceras e outros órgãos; 2) dor neurogênica: quando há um dano
no tecido neural no sistema nervoso central ou na periferia; e 3) dor psicogênica:
12
quando não há uma fonte somática detectável, apresentando, em geral, influência dos
fatores psicológicos (Millan, 1999).
A sensibilização dos nociceptores se deve a vários estímulos, tais como lesão
tecidual, mudança de temperatura, isquemia e hipóxia, entre outros. Alguns
nociceptores podem permanecer silenciosos durante estímulos de menor intensidade,
mas são ativados em situações específicas, como na inflamação, ampliando a resposta
dolorosa (Julius & Basbaum, 2001).
A atividade dos nociceptores pode ser mediada pela ação de substâncias
algogênicas liberadas e/ou sintetizadas em elevada concentração, nos tecidos, em
decorrência de processos inflamatórios (Cotran et al., 2001). Substâncias endógenas,
como PGs, neuropeptídeos, cininas, aminoácidos excitatórios, entre outros, são
produzidas e/ou liberadas pelo tecido lesionado e estimulam os receptores presentes na
membrana dos neurônios. Além disso, os mediadores inflamatórios liberados facilitam a
neurotransmissão e sensibilizam o nociceptor (Björkman, 1995).
Estudos em animais de laboratório têm auxiliado bastante na pesquisa de novos
fármacos com função analgésica. Os produtos naturais podem contribuir muito com
moléculas capazes de atuar nos mediadores envolvidos nos processos de transmissão de
dor. Existem vários ensaios capazes de dar início ao processo e descoberta de novos
fármacos analgésicos. Dentre eles destacamos o teste de contorções abdominais em
camundongos, método muito utilizado para a avaliação da atividade analgésica de
substâncias contra a dor de origem inflamatória. Nesse ensaio, o ácido acético na
concentração de 0,6% (v/v) induz uma lesão no abdômen provocando espasmos
traduzidos como contorções (Koster et al., 1959). O ácido acético age induzindo a
liberação de mediadores endógenos que estimulam os nociceptores que são sensíveis
aos AINES e/ou analgésicos opióides (Collier et al., 1968).
Atividade antioxidante e inflamação
Radical livre é qualquer átomo, molécula ou fragmento de molécula contendo
um ou mais elétrons desemparelhados nas suas camadas de valência. Tal característica o
torna altamente instável e quimicamente reativo (Bianchi & Antunes, 1999). Existem,
entretanto, compostos igualmente reativos que não possuem elétron desemparelhado na
última camada e, portanto, não podem ser classificados como radicais livres. Esses
13
compostos são classificados de maneira mais ampla como espécies reativas de oxigênio
(ERO) ou espécies reativas de nitrogênio (ERN) (Dröge, 2002).
As ERO são produzidas, essencialmente, durante a fosforilação oxidativa e/ou
por ativação de células fagocíticas durante uma explosão oxidativa no processo
inflamatório. Essas moléculas participam de várias funções fisiológicas dentre as quais,
na defesa contra microrganismos invasores. Elas são produzidas durante o metabolismo
celular aeróbico normal e têm papel importante na manutenção do estado celular redox
(Surh, 2005). A produção excessiva de ERO pode danificar lipídeos, proteínas,
membranas e ácidos nucléicos, e também serve como um importante sinalizador
intracelular que amplifica respostas inflamatórias. Inúmeros estudos demonstram o
envolvimento de ERO na patogênese das artropatias crônicas inflamatórias. Acredita-se
que as ERO possam atuar como segundos mensageiros para ativação do NF-kB que
orquestra a expressão de vários genes que perpetuam a resposta inflamatória (Filippin et
al., 2008).
O ataque de ERO sobre os lipídeos de membrana gera o processo de peroxidação
lipídica, dando lugar a uma reação em cadeia que perpetua o ciclo de formação desses
agentes agressores, ao mesmo tempo em que origina uma desestruturação da membrana
e a consequente morte celular. Também os radicais livres podem reagir com proteínas,
dando lugar a alterações da funcionalidade normal da célula (Roessner et al., 2008).
Portanto, é essencial que as células e os tecidos possuam um sistema de defesa para
controlar os níveis de radicais livres e evitar esses processos inflamatórios. Para isso as
células dispõem de um sistema de defesa antioxidante que pode ser classificado como
enzimático e não enzimático; entre os antioxidantes não-enzimáticos estão incluídos os
compostos da dieta que são encontrados em vegetais, frutas, extratos de plantas,
vitaminas C e E, flavonóides e zinco (Kruidener & Verspaget, 2002).
A utilização de compostos antioxidantes, encontrados na dieta ou mesmo
sintéticos, é um dos mecanismos de defesa contra os radicais livres, empregado nas
indústrias de alimentos, cosméticos, bebidas e também na medicina, sendo que muitas
vezes os próprios medicamentos aumentam a geração intracelular desses radicais
(Halliwell et al., 1995).
Entre os antioxidantes presentes nos vegetais, os mais ativos e frequentemente
encontrados são os compostos fenólicos, tais como os flavonóides. As propriedades
benéficas desses compostos podem ser atribuídas à sua capacidade de sequestrar os
14
radicais livres (Decker, 1997) e podem inibir o processo de peroxidação lipídica
(Halliwell et al., 1995).
Atualmente, o estudo dos antioxidantes tem despertado interesse de
pesquisadores e indústrias, já que eles contribuem para o controle de doenças
degenerativas causadas por danos oxidativos. Muitas plantas tem mostrado atividade
antioxidante e tem servido de ponto de partida para o desenvolvimento de compostos
sintéticos e semi-sintéticos nessa área (Kumaran & Karunakaran, 2006).
Vários ensaios são capazes de mensurar a atividade antioxidante de compostos;
entretanto, ainda não há um consenso entre os diferentes ensaios disponíveis e nem é
possível realizar uma comparação dos resultados obtidos em diferentes laboratórios,
provavelmente por causa da complexidade do assunto (Huang et al., 2005). Esses
ensaios podem ser classificados em: ensaios baseados em transferência de elétrons (ET)
– ABTS, FRAP – e os baseados em transferência de átomo de hidrogênio (HAT) –
DPPH (Prior et al., 2005). Essas diferenças podem ser causadas por diversos fatores,
como polaridade do solvente utilizado, temperatura e pH do composto testado (Pérez-
Jiménez & Saura-Calixto, 2006).
O DPPH constitui-se num método fácil e acurado de medir a capacidade
antioxidante de frutas, vegetais e extratos de plantas. A molécula do DPPH é
caracterizada como um radical livre estável em virtude da presença de um elétron
desemparelhado por toda a molécula. Este ensaio se baseia na medida da capacidade
antioxidante de uma determinada substância em sequestrar o radical DPPH, reduzindo-o
a hidrazina. Quando uma determinada substância age como doador de átomos de
hidrogênio, a hidrazina é obtida com mudança simultânea na coloração de violeta a
amarelo (Alves et al., 2010).
No método do ABTS+•, o radical monocation verde/azul é gerado pela oxidação
do ABTS com o persulfato de potássio. A adição do antioxidante ao radical cátion pré-
formado o reduz novamente a ABTS, promovendo a supressão da cor da solução. O
grau deste descoramento e usado para avaliar a atividade antioxidante (Re, et al., 1999).
O método FRAP é baseado na habilidade de redução do Fe3+ para Fe2+, em pH
baixo. Quando isso ocorre na presença de 2,4,6-tripiridil- s-triazina (TPTZ), a redução é
acompanhada pela formação de um complexo Fe2+ – TPTZ de cor azul intensa que pode
ser monitorado a 593 nm. É um método barato, os reagentes são de fácil preparo, os
15
resultados são altamente reprodutíveis e o procedimento simples e rápido (Benzie &
Strain, 1996).
16
CONCLUSÕES
Nas condições em que o presente trabalho foi realizado, os resultados permitem concluir que:
• O OE das folhas da Lippia gracilis contém majoritariamente timol, metil timol e carvacrol, ambos com atividade antioxidante comprovada.
• Foi demonstrada, pela primeira vez na literatura, as atividades anti-inflamatória e antinociceptiva do OE da L. gracilis in vivo.
• O solvente orgânico extraiu melhor os compostos fenólicos do extrato da L. gracilis, do que o solvente inorgânico.
• Foi a primeira vez que luteolina-C-6-glicosideo e luteolina-C-8-glicosideo foram relatadas no gênero Lippia.
17
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Artigo 1 Evaluation of the analgesic and anti-inflammatory effects
of the essential oil of Lippia gracilis leaves
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valuation of the analgesic and anti-inflammatory effects of the essentialil of Lippia gracilis leaves
.S. Mendesa, R.R. Bomfima, H.C.R. Jesusb, P.B. Alvesb, A.F. Blankc,.S. Estevama, A.R. Antoniolli a, S.M. Thomazzia,∗
Department of Physiology, Federal University of Sergipe, CEP 49100-000, São Cristóvão, Sergipe, BrazilDepartment of Chemistry, Federal University of Sergipe, CEP 49100-000, São Cristóvão, Sergipe, BrazilDepartment of Agronomic Engineering, Federal University of Sergipe, CEP 49100-000, São Cristóvão, Sergipe, Brazil
r t i c l e i n f o
rticle history:eceived 8 January 2010eceived in revised form 14 March 2010ccepted 10 April 2010vailable online 24 April 2010
eywords:nti-inflammatory activityntinociceptive activityssential oilippia gracilis
a b s t r a c t
Aim of the study: The aim of the present study is to investigate the antinociceptive, anti-inflammatory,and antioxidant activities of essential oil (EO) of Lippia gracilis Schauer (Verbenaceae) leaves to supportthe medicinal uses claimed by folklore practitioners in the caatinga region (semi-arid) of NortheasternBrazil.Materials and methods: The chemical composition and antinociceptive and anti-inflammatory activitiesof the EO of Lippia gracilis leaves (50–200 mg/kg) were investigated. Antinociceptive activity of the EOwas evaluated by writhing test. Anti-inflammatory activity of the EO was evaluated using paw oedemaand peritonitis methods.Results: Oral treatment with the EO of Lippia gracilis leaves elicited inhibitory activity on acetic acid effectat 50, 100, and 200 mg/kg (30.33 ± 2.36, 25.20 ± 1.48, and 21.00 ± 1.54 abdominal writhes, respectively,P < 0.05), as compared with the control group (36.73 ± 1.92 writhes). The compound acetylsalicylic acid(ASA, 300 mg/kg) inhibited the acetic acid-induced writhing (12.67 ± 0.50 abdominal writhes, P < 0.001).Carrageenan-induced oedema formation was reduced with the EO of Lippia gracilis leaves at 200 mg/kg(0.72 ± 0.06 mL h, P < 0.001) and by the reference compound ASA (300 mg/kg, 0.85 ± 0.04 mL h, P < 0.001),
as compared with the control group (1.76 ± 0.06 mL h). Leukocyte migration into the peritoneal cavityinduced by carrageenan was reduced with the EO of Lippia gracilis leaves at 50, 100, and 200 mg/kg(13.81 ± 0.61, 11.77 ± 0.91, and 10.30 ± 0.60 leukocytes × 106/mL, respectively, P < 0.01), and by thecompound dexamethasone (2 mg/kg, 5.34 ± 0.33 leukocytes × 106/mL, P < 0.001), as compared with thecontrol group (16.71 ± 0.54 leukocytes × 106/mL). The analyses of the essential oil allowed the identifi-cation of Lippia gracilis as a thymol-p-cymene chemotype (32.68% and 17.82%, respectively).pia g
Conclusions: The EO of Lip. Introduction
Medicinal plants have been used in developing countries as
lternative treatments to health problems. Many plant extractsnd essential oils isolated from plants have been shown to exertiological activity in vitro and in vivo, which justified research onraditional medicine (Martínez et al., 1996).Abbreviations: ASA, acetylsalicylic acid; AUC, area under the curve;HT, buthylated hydroxytoluene; Dexa, dexamethasone; DPPH, 2,2-diphenyl-1-icrylhydrazyl; EO, essential oil; IC50, inhibiting concentration 50%.∗ Corresponding author at: Department of Physiology, Center of Sciences Biologics
nd of Health, Federal University of Sergipe, CEP 49100-000, São Cristóvão (SE),razil. Tel.: +55 79 21056640; fax: +55 79 21056474.
E-mail addresses: [email protected], [email protected]. Thomazzi).
378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.04.005
racilis leaves shows antinociceptive and anti-inflammatory activities.© 2010 Elsevier Ireland Ltd. All rights reserved.
Essential oils are volatile, natural, complex compounds charac-terized by a strong odour and are formed by aromatic plants assecondary metabolites. Known for their antiseptic, i.e. bactericidal,virucidal and fungicidal, and medicinal properties and their fra-grance, they are used in embalment, preservation of foods and asantimicrobial, analgesic, sedative, anti-inflammatory, spasmolyticand locally anesthesic remedies (Bakkali et al., 2008). Generally,these major components determine the biological properties of theessential oils.
The genus Lippia (Verbenaceae) is widely distributed in tropicaland subtropical America and Africa, and consists of approximately
250 species of herbs, shrubs and small trees (Moldenke, 1965;Jansen-Jacobs, 1988). In Brazil, the genus Lippia is represented bynearly 120 species conspicuous for their flash appearance duringthe blooming period and by its fragrance, in general, strong andpleasant (Bezerra et al., 1981). There have been numerous chem-3 nopha
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cal studies of Lippia spp. which have mostly focused on essentialils (Fricke et al., 1999; Siani et al., 2002; Stashenko et al., 2004;arreto et al., 2008; Chanotiya and Yadav, 2009).Some species of the Lippia gender are characterized by the
resence of essential oils, with antimicrobial activity, due to thehenolic monoterpenes thymol and carvacrol (Matos et al., 1999).he components which were found in the highest frequency inhe Lippia essential oils are: limonene, �-caryophyllene, p-cymene,amphor, linalool, �-pinene and thymol (Pascual et al., 2001). Theost common use of Lippia species is for the treatment of respira-
ory disorders (Pascual et al., 2001).In general, the genus appears to present a consistent profile
f chemical composition, pharmacological activities and folk uses.n most cases, the parts used are leaves or aerial parts, and flow-rs. They are commonly prepared as an infusion or decoction, anddministered orally (Pascual et al., 2001).
Lippia gracilis Schauer (Verbenaceae), known in Brazil by theame “alecrim-da-chapada”, is an herb commonly found in North-astern Brazil vegetation, is highlighted because it presents highontents of these monoterpenes (Matos et al., 1999). This speciesroduces an essential oil which contains as major components: car-acrol, o-cymene, �-terpinene, and �-caryophyllene (Silva et al.,008). Essential oil of Lippia gracilis has antimicrobial activity and
s used externally to treat cutaneous diseases, burns, wounds, andlcers, as reviewed by Pascual et al. (2001). Lippia gracilis is usedo treat influenza, cough, sinusitis, bronchitis, nasal congestion,eadache, jaundice, and paralysis by traditional communities in theaatinga region (semi-arid) of Northeastern Brazil (Albuquerque etl., 2007). More commonly, leaf infusions or decoctions are used astea; however, it is also used as a macerate in alcohol for topical
pplication (Agra et al., 2007; Lorenzi and Matos, 2008). The leafnfusions are also used topically to treat wounds and scabies, as a
outh antiseptic and in baths to treat and prevent general infec-ions of the body (Lorenzi and Matos, 2008). Recently, Silva et al.2008) demonstrated a potent larvicidal activity of the essential oilf Lippia gracilis against the major dengue vector, the Aedes aegyptiarvae.
The goal of the present study was to evaluate the antinocicep-ive, anti-inflammatory, and antioxidant effects of the essential oilEO) from Lippia gracilis leaves.
. Materials and methods
.1. Plant material
Lippia gracilis was cultivated in the Experimental Station “Cam-us Rural” of the Federal University of Sergipe, in the municipalityf São Cristóvão, Sergipe State, Brazil (10◦55′32′′S, 37◦06′08′′W).eaves were collected in February 2008 (under water stress condi-ion) and August 2007 (under non-stress condition). The plant wasuthenticated by Professor Ana Paula Prata, Department of Biology,ederal University of Sergipe, and a voucher specimen under watertress and non-stress conditions deposited in the Federal Universityf Sergipe Herbarium (Av. Marechal Rondon S/N, São Cristóvão-SE9100-000, Brazil, voucher number ASE 9209 and ASE 9205, respec-ively). Prior to extraction, leaves were dried at 40 ◦C in a forced airven (Marconi MA 037) for 5 days.
.2. Extraction of the essential oil
The essential oils of dried leaves under water stress or non-stressonditions were obtained by hydrodistillation on a Clevenger-typepparatus for 4 h using 100 g of dried leaves. The essential oilsEO) obtained were separated from the aqueous phase and keptn freezer until further analysis.
rmacology 129 (2010) 391–397
2.3. Gas chromatography–mass spectrometry
Oil sample analysis was performed in a Shimadzu QP5050A (Shi-madzu Corporation, Kyoto, Japan) gas chromatograph interfacedto a mass spectrometer (GC–MS) system comprised of a AOC-20Iauto-injector (Shimadzu Corporation, Kyoto, Japan) and a gas chro-matograph interfaced to a mass spectrometer (GC–MS) instrumentemploying the following conditions: column J&W Scientific DB-5MS fused silica capillary column (30 m × 0.25 mm i.d., composedof 5% phenylmethylpolysiloxane). Helium (99.999%) was used asthe carrier gas at a constant flow of 1.2 mL/min, and an injectionvolume of 0.5 �L was employed (split ratio of 1:100) with an injec-tor temperature of 250 ◦C, and an ion-source temperature of 280 ◦C.The oven temperature was programmed from 50 ◦C (isothermal for2 min), with an increase of 4 ◦C/min, to 200 ◦C, then 15 ◦C/min to300 ◦C, ending with a 15 min isothermal at 300 ◦C. Mass spectrawere taken at 70 eV, a scan interval of 0.5 s, and fragments from 40to 500 Da.
2.4. Gas chromatography (GC-FID)
Quantitative analysis of the chemical constituents was per-formed by flame ionization gas chromatography (FID), usingShimadzu GC-17A (Shimadzu Corporation, Kyoto, Japan) equip-ment, under the following operational conditions: capillaryZB-5MS column (5%-phenyl–95%-dimethylpolysiloxane) fused sil-ica capillary column (30 m × 0.25 mm i.d. × 0.25 �m film thickness)from Phenomenex (Torrance, CA, USA), under the same conditionsas GC–MS. Quantification of each constituent was estimated by areanormalization (%). Compound concentrations were calculated fromthe GC peak areas and were arranged in order of GC elution.
2.5. Identification of essential oil constituents
Identification of individual components of the essential oils wasperformed by computerized matching of the acquired mass spectrawith those stored in the NIST21 and NIST107 mass spectral librariesof the GC–MS data system. Retention indices (RI) for all compoundswere determined according to Van Den Dool and Kratz (1963) foreach constituent, as previously described (Adams, 2007).
2.6. Animals
Wistar rats (120–180 g) and Swiss mice (20–30 g) of both sexeswere obtained from the Central Biotery of the Federal University ofSergipe (São Cristóvão, Brazil). Animals were randomly assigned togroups and maintained in plastic boxes at controlled room temper-ature (25–28 ◦C) with free access to food and water, under a 12:12 hlight/dark cycle. All the experimental procedures were carried outduring the light period of the day (08:00 a.m. to 05:00 p.m.) andcomplied with the guidelines on animal care of the Federal Uni-versity of Sergipe Ethics Committee for Animal Use in Research(CEPA/UFS 11/08) which was conducted in accordance with theinternationally accepted principles for laboratory animal use andcare. The animals submitted to oral administration of the EO ordrugs were fasted for 12 h before the experiments and acclimatizedfor at least 2 h before the experiments.
2.7. Acetic acid-induced abdominal writhes
Abdominal writhes consisted of a contraction of the abdominal
muscle together with a stretching of the hind limbs, induced byintraperitoneal (i.p.) injection in mice of acetic acid (0.6% solution,0.1 mL/10 g), the nociceptive agent (Koster et al., 1959).The animals were pre-treated with the EO of Lippia gracilisleaves under water stress condition (50, 100, or 200 mg/kg) orally
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per os, p.o.) 60 min before initiating algesic stimulation, or withcetylsalicylic acid (ASA, 300 mg/kg, p.o., 60 min beforehand), useds positive control (n = 6/group). The abdominal writhes werebserved, in separate individual chambers, for a period of 20 min,tarting after administration of acetic acid.
.8. Measurement of paw oedema in rats
The anti-inflammatory activity was studied using the pawedema model induced by 1% carrageenan, administrated at vol-me of 0.1 mL/animal into the subplantar region of the rightindpaw of the rat (Winter et al., 1962).
The volume of the paw was measured by the removal of theater column using a hydroplethysmometer (model 7150, Ugoasile, Varese, Italy), at the time 0 and the intervals of 1, 2, 3, andh immediately after the subplantar injection of carrageenan.
The EO of Lippia gracilis leaves under water stress condition athe doses of 50, 100, or 200 mg/kg, ASA (300 mg/kg) and vehicle0.2% Tween 80 in 0.9% NaCl solution) were administrated p.o. 1 hefore the oedematogenic agent to different groups of animals forach treatment (n = 6/group).
The data obtained for the various groups were reported aseans ± S.E.M. and expressed in milliliters. The percentage inhibi-
ion on oedema experiment was calculated based on the area underhe curves (AUC) after 4 h, starting at the time 0.
.9. Leukocyte migration into the peritoneal cavity in mice
The leukocyte migration was induced by injection of car-ageenan (1%, 250 �L, i.p.) into the peritoneal cavity of mice 1 h afterdministration of the EO of Lippia gracilis leaves under water stressondition (50, 100, or 200 mg/kg, p.o., n = 6) or dexamethasone2 mg/kg, s.c., n = 6) by technique previously described by Matos etl. (2003). The animals were anesthetized with sodium pentobar-ital (50 mg/kg, i.p.) and were euthanized by cervical dislocationh after carrageenan injection. Shortly after, saline containingDTA (1 mM, i.p., 3 mL) was injected. Immediately a brief mas-age was done for further fluid collection, which was centrifuged1000 × g, 5 min) at room temperature. The supernatant was dis-osed and the precipitate was re-suspended in 1 mL of saline. Anliquot of 10 �L from this suspension was dissolved in 200 �Lf Turk solution and the total cells were counted in a Neubauerhamber, under optic microscopy. The results were expressed ashe number of leukocytes/mL. The percentage of the leukocytenhibition = (1 − T/C) × 100, where T represents the treated groupseukocyte counts and C represents the control group leukocyteounts.
.10. Quantitative assay of antioxidant activity
The quantitative analysis of antioxidant activity was based onhe method described by Brand-Williams et al. (1995), with minor
odifications. The scavenging of 2,2-diphenyl-1-picrylhydrazylDPPH) radical was followed by monitoring the decrease inbsorbance at 515 nm, which occurred due to reduction by thentioxidant.
The calibration curve was established by preparing dilutions ofDPPH radical stock solution (40 �g/mL) to obtain final concen-
rations of 5, 10, 15, 20, 25, and 30 �g/mL. The absorbance of eachtandard concentration was then monitored in a spectrophotome-er (UV BEL Photonics 1105) at 515 nm. The measures were carried
ut in triplicate with intervals of 1 min. The equation of the con-entration × absorbance calibration curve for the DPPH radical was= 110,547 − 002,804A, where C is the concentration of the DPPHadical in medium, A is the absorbance at 515 nm. The correlationoefficient was R = 0.9983.
rmacology 129 (2010) 391–397 393
Solution containing 500 �g/mL of the EO of Lippia gracilis leavesunder water stress condition was prepared in methanol, and dilutedin concentrations of 5, 10, 15, 20, 25, and 30 �g/mL. The dis-appearance of DPPH radical was monitored by the decrease inabsorbance at 515 nm, which was recorded after 0, 1, 5, and 10 min,and subsequently every 10 min up to 1 h. The negative control waspure methanol used for dissolving the samples, while the positivecontrol was the buthylated hydroxytoluene (BHT) and gallic aciddissolved in methanol in concentrations of 5, 10, 15, 20, 25, and30 �g/mL. The mixture of methanol and EO of leaves was used asblank.
The concentration of the DPPH radical in the reaction mix-ture was calculated based on calibration curve, where [DPPH]is expressed in �g/mL. The percentage of remaining DPPH(%DPPHREM) was calculated according to Brand-Williams et al.(1995), as follows: %DPPHREM = [DPPH]T /[DPPH]T0
× 100, where Tis the time when absorbance was determined (1–60 min) and T0is the time zero. The amount of antioxidant necessary to decreasethe initial concentration of DPPH radical by 50% (IC50) was calcu-lated by plotting the %DPPHREM at time of 60 min. The results wereexpressed as �g antioxidant/mL DPPH ± standard deviation.
2.11. Chemicals and drugs
The following chemicals and drugs used were: acetylsalicylicacid (ASA), buthylated hydroxytoluene (BHT), carrageenan, dex-amethasone, gallic acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH)from Sigma Chemical Co. (St. Louis, MO, USA). Acetic acid fromMerck (Damstadt, Germany). Solvents from Vetec (Rio de Janeiro,RJ, Brazil). All substances used were dissolved in 0.2% Tween 80in 0.9% NaCl solution, with the exceptions of BHT, gallic acid, andDPPH that were dissolved in methanol. The final concentration ofTween 80 did not exceed 0.2% and did not cause any effect per se.
2.12. Statistical analysis
The results of antinociceptive and anti-inflammatory activitiesare presented as the mean ± S.E.M. of n animals per group. Theantioxidant activity was performed using the Origin version 7.5(Microcal, Northampton, MA, USA) and the values are presentedas the mean ± standard deviation (triplicate). Statistical evalua-tion of the data was performed using one-way analysis of variance(ANOVA) followed by Bonferroni’s test. P values less than 0.05 wereconsidered significant.
3. Results
3.1. Chemical analysis of the essential oils
The essential oils of Lippia gracilis leaves under water stressand non-stress conditions were obtained in 2.10% and 1.12% yield,respectively. Thirty compounds, representing 99.54–99.76% of theessential oils have been identified; their retention indices andpercentage composition, listed in order of elution in the DB-5MScolumn, are given in Table 1. In addition, representative exampleof the essential oil of Lippia gracilis leaves under water stress andnon-stress conditions is shown in Fig. 1.
Major components identified in the essential oils of Lippia gra-cilis under water stress and non-stress conditions are listed inTable 1, according to their retention times. In the essential oil of Lip-pia gracilis under water stress condition thymol (32.68%), p-cymene
(17.82%), methyl thymol (10.83%), carvacrol (7.53%), �-terpinene(7.13%), �-caryophyllene (6.47%), 1,8-cineole (3.45%), and myrcene(3.35%) were the major compounds, while for Lippia gracilis undernon-stress condition thymol (24.08%), p-cymene (15.91%), methylthymol (11.18%), �-terpinene (10.88%), �-caryophyllene (8.18%),394 S.S. Mendes et al. / Journal of Ethnopharmacology 129 (2010) 391–397
Table 1Essential oils composition from the leaves of Lippia gracilis under water stress andnon-stress conditions.
RI Compounds Water stress(%) (Peak no.a)
Non-stress (%)(Peak no.b)
924 �-Thujene 1.28 (1) 1.90 (1)932 �-Pinene 0.71 (2) 1.33 (2)949 Canphene 0.16 (3) 0.49 (3)973 Sabinene 0.17 (4) 0.61 (4)979 �-Pinene 0.24 (5) 0.37 (5)989 Myrcene 3.35 (6) 4.06 (6)
1008 �-Phellandrene 0.37 (7) 0.77 (7)1010 �-3-Carene 0.15 (8) 0.13 (8)1018 �-Terpinene 1.76 (9) 2.31 (9)1026 p-Cymene 17.82 (10) 15.91 (10)1031 Limonene 0.61 (11) 0.85 (11)1034 1,8-Cineole 3.45 (12) 4.80 (12)1060 �-Terpinene 7.13 (13) 10.88 (13)1101 Linalool 0.45 (14) 0.78 (14)1152 Camphor 0.03 (15) 0.54 (15)1184 Terpin-4-ol 0.73 (16) 0.67 (16)1199 �-Terpineol 0.39 (17) 0.28 (17)1232 Methyl thymol 10.83 (18) 11.18 (18)1294 Thymol 32.68 (19) 24.08 (19)1302 Carvacrol 7.53 (20) 5.31 (20)1348 Thymol acetate 0.06 (21) 0.05 (21)1423 �-Caryophyllene 6.47 (22) 8.18 (22)1435 �-trans-B ergamotene 0.19 (23) 0.28 (23)1443 Aromadendrene 0.44 (24) 0.55 (24)1461 �-Humulene 0.48 (25) 0.57 (25)1495 Viridifloreno 0.44 (26) 0.55 (25)1500 Bicyclogermacrene 0.77 (27) 1.95 (27)1510 �-B isabolene – 0.13 (28)1584 Spathulenol 0.34 (28) –1590 Caryophillene oxide 0.73 (29) 0.03 (29)
Total identified 99.76 99.54
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Fig. 2. Influence of the EO of Lippia gracilis leaves in nociceptive behavior of miceevaluated in acetic acid-induced abdominal writhing model. Nociception was reg-istered by the number of writhes, which the animal presented 20 min following i.p.acetic acid injection. Groups of animals were pre-treated with vehicle (C, controlgroup, n = 6, open column), acetylsalicylic acid (ASA, 300 mg/kg, n = 6, cross-hatched
Anti-inflammatory effect of the EO of Lippia gracilis leaves(50–200 mg/kg) was evaluated in the paw oedema model(n = 6/group). As observed in Fig. 3, the single oral treatment of
I, relative retention index calculated against n-alkanes applying the Van Den Doolnd Kratz (1963) equation. %, compound percentage.
a Peak number in Fig. 1A.b Peak number in Fig. 1B.
arvacrol (5.31%), 1,8-cineole (4.80%), and myrcene (4.06%), werehe major ones. According to the results, it is possible to character-ze Lippia gracilis under water stress and non-stress conditions as a
hymol-p-cymene chemotype. Both samples are characterized by aigh content of monoterpenes (Table 1). The chemical compositionf these two oils is very similar. In order to evaluate the activitiesf this plant studies were performed with the EO of Lippia gracilisnder water stress condition.ig. 1. GC chromatograms of the essential oil of Lippia gracilis leaves under watertress (A) and non-stress (B) conditions. AU, arbitrary units.
column) or EO (50–200 mg/kg, n = 6/group, right-hatched columns), p.o., 60 minbefore irritant agent. Each column represents the mean ± S.E.M. Asterisks denotestatistical significance, *P < 0.05 and **P < 0.001, in relation to control group. ANOVAfollowed by Bonferroni’s test.
3.2. Acetic acid-induced writhing in mice
The results in Fig. 2 show that the EO of Lippia gracilis leavesgiven p.o. (50–200 mg/kg, n = 6/group) 1 h beforehand caused aninhibition of 17.4 (P < 0.05), 31.4 (P < 0.001), and 42.8% (P < 0.001) onacetic acid-induced writhes at the doses of 50, 100, and 200 mg/kg,respectively. ASA (300 mg/kg, n = 6) exhibited significant (65.5%,P < 0.001) inhibition of the control writhes in the acetic acid-induced writhing.
3.3. Carrageenan-induced paw oedema in rats
rats with the EO of Lippia gracilis leaves at 200 mg/kg (p.o., 1 h
Fig. 3. Effect of the EO of Lippia gracilis leaves on rat paw oedema induced by car-rageenan. Groups of rats were pre-treated with vehicle (control group, 10 mL/kg,p.o., n = 6), acetylsalicylic acid (ASA, 300 mg/kg, p.o., n = 6), or Lippia gracilis EO atthe doses of 50, 100, and 200 mg/kg (p.o., n = 6/group) 60 min before carrageenan-induced paw oedema. Measurement was performed at the times 0, 1, 2, 3, and 4 hafter the subplantar injection of carrageenan (1%, 100 �L). Each value representsthe mean ± S.E.M. Asterisks denote statistical significance, *P < 0.05, **P < 0.01, and***P < 0.001, in relation to control group. ANOVA followed by Bonferroni’s test.
S.S. Mendes et al. / Journal of Ethnopha
Fig. 4. Effect of the EO of Lippia gracilis leaves on leukocyte migration into the peri-toneal cavity induced by carrageenan in mice. Groups of mice were pre-treatedwith vehicle (C, control group, 10 mL/kg, p.o., open column), dexamethasone (Dexa,2 mg/kg, s.c., cross-hatched column), or leaves EO (p.o., right-hatched columns) atthe doses of 50, 100, and 200 mg/kg 60 min before carrageenan (1%, 250 �L, i.p.)-induced peritonitis. Cell counts were performed at the time 4 h after the injection ofcsf
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eforehand) was capable of reducing (P < 0.01) the oedema forma-ion induced by carrageenan (1%, 100 �L/paw), an effect observedt 1, 2, 3, and 4 h after the administration of this phlogisticgent.
Likewise, ASA (300 mg/kg, p.o., 1 h beforehand, n = 6) signifi-antly inhibited (P < 0.05) the oedematogenic response evoked byarrageenan in rats, at 1, 2, 3, and 4 h (Fig. 3).
In the assay with the EO of leaves the mean AUC found inarrageenan-treated rats was 1.76 ± 0.06 mL h (n = 6). Based on AUCalues, the EO of leaves at 200 mg/kg caused 59.1% (P < 0.001) ofnhibition on the oedema response (n = 6/group). ASA at 300 mg/kgn = 6) caused an inhibition of 51.7% (P < 0.001).
.4. Carrageenan-induced peritonitis in mice
Carrageenan (1%, 250 �L) induced leukocyte migration intohe peritoneal cavity 4 h after stimulus (Fig. 4). Fig. 4 shows thenhibitory effect of the EO of Lippia gracilis leaves (17.4, 29.6, and8.4% at the doses of 50, 100, and 200 mg/kg, respectively, P < 0.01,= 6/group), on carrageenan-induced response.
The results obtained with the control group support the effectf the EO of Lippia gracilis leaves since the vehicle presented noctivity, and the control drug dexamethasone (2 mg/kg, s.c., 1 heforehand, n = 6) inhibited (68.0%, P < 0.001) the carrageenan-
nduced leukocyte migration into the peritoneal cavity (Fig. 4).
.5. Antioxidant activity
According to the IC50 values, the antioxidant concentrationeeded to decrease by 50% the initial concentration of DPPH radi-al is the highest for the EO of leaves (162.22 ± 4.65 �g/mL DPPH,riplicate), as compared with the reference compounds BHT andallic acid (12.33 ± 3.25 and 9.33 ± 0.22 �g/mL DPPH, respectively,riplicate).
. Discussion and conclusions
The present study demonstrates that the EO of Lippia graciliseaves displays antinociceptive and anti-inflammatory properties
ith a moderate antioxidant potential, and provides some evidence
rmacology 129 (2010) 391–397 395
on the mechanisms implicated in these effects. The chemical com-position of the EO of Lippia gracilis leaves under water stress andnon-stress conditions is very similar, with no significant differencesbetween both samples.
For the first time, this work shows that the EO of Lippia gracilisp.o. produces significant antinociception according to assessmentof the abdominal writhes elicited by acetic acid, a model usedto evaluate the potential analgesic activity of drugs. It has beensuggested that acetic acid acts by releasing endogenous media-tors that stimulate the nociceptive neurons (Collier et al., 1968).In the acetic acid-induced abdominal writhing model, which is avisceral pain model, the processor releases arachidonic acid viacyclo-oxygenase (COX); notably, prostaglandins biosynthesis playsan important role in the nociceptive mechanism (Duarte et al.,1988). This method is sensitive to non-steroidal anti-inflammatorydrugs (NSAIDs) and to narcotics and other centrally acting drugs(Collier et al., 1968; Santos et al., 1998).
The anti-inflammatory effect of Lippia gracilis EO was evalu-ated in carrageenan-induced paw oedema, an animal model widelyemployed for the screening of anti-inflammatory compounds andhas frequently been used to assess the anti-oedematogenic effect ofnatural products. The experimental model exhibits a high degreeof reproducibility. In rats, the inflammatory response induced bycarrageenan is characterized by a biphasic response with markedoedema formation resulting from the rapid production of sev-eral inflammatory mediators such as histamine, serotonin andbradykinin (first-phase), which is subsequently sustained by therelease of prostaglandins and nitric oxide (second-phase) withpeak at 3 h, produced by inducible isoforms of COX (COX-2) andnitric oxide synthase (iNOS), respectively (Seibert et al., 1994).In the present work, previous oral treatment with the EO of Lip-pia gracilis leaves was effective in reducing the oedematogenicresponse evoked by carrageenan in rats between the first and thefourth hours after the injection. This evidence allows us to sug-gest that anti-inflammatory actions of the EO of Lippia gracilisleaves are related to the inhibition of one or more intracellular sig-naling pathways involved in the effects of several inflammatorymediators.
Cell recruitment during inflammation depends on the orches-trated release of local mediators which are responsible for localvascular and tissue changes as well as for the recruitment of hostdefense cells. The inflammation induced by carrageenan involvescell migration, plasma exudation and production of mediators,such as nitric oxide, prostaglandin E2, interleukin (IL)-1�, IL-6, andtumour necrosis factor (TNF)-� (Salvemini et al., 1996; Loram etal., 2007). These mediators are able to recruit leukocytes, such asneutrophils, in several experimental models. The EO of Lippia gra-cilis leaves inhibited leukocyte migration induced by i.p. injectionof carrageenan (in peritonitis model). A putative mechanism asso-ciated with this activity may be inhibition of the synthesis of manyinflammatory mediators whose involvement in the cell migrationis well established.
The antioxidant capacity of plants confers a therapeutic poten-tial with antinociceptive and anti-inflammatory properties (Li etal., 2002). Our results showed that the EO of Lippia gracilis leaveshas a moderate antioxidant potential. It is thus suggested that theseantioxidant effects are unrelated to the functional (antinociceptiveand anti-inflammatory) activities of the EO.
Major components identified in the essential oils of Lippiagracilis are thymol (32.68%), p-cymene (17.82%), methyl thymol(10.83%), carvacrol (7.53%), �-terpinene (7.13%), �-caryophyllene
(6.47%), 1,8-cineole (3.45%), and myrcene (3.35%). Interestingly,unlike other study (Silva et al., 2008), the major components in Lip-pia gracilis EO identified were carvacrol (44.43%), o-cymene (9.42%),�-terpinene (9.16%), and �-caryophyllene (8.83%). This may be dueto environmental effects, different analytical conditions, as well as3 nopha
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he genetic variability of this plant species. The variability is soommon that has been suggested the division into different chemo-ypes.
Generally, the major components are found to reflect quite wellhe biophysical and biological features of the essential oils fromhich they were isolated, the amplitude of their effects being justependent on their concentration when they were tested alone.owever, it is possible that the activity of the main components isodulated by other minor components.Thymol, the major component of the EO of Lippia gracilis,
as been credited with a series of pharmacological propertieshat include antimicrobial, antioxidant, antinociceptive, and anti-nflammatory effects (Haeseler et al., 2002; Braga et al., 2006).
arsik et al. (2005) have demonstrated that thymol can partic-pate in the general anti-inflammatory activity of Nigella sativa
ith inhibitory effect on COX-1 and suggest that this agenthould be further studied for possible use as non-steroidal anti-nflammatory drugs. Haeseler et al. (2002) demonstrated thatntinociceptive effect of thymol might be mediated via blockadef voltage-operated sodium channels. In human macrophage-like937 cells, carvacrol suppressed lipopolysaccharide-induced COX-mRNA and protein expression, suggesting that carvacrol regulatesOX-2 expression through its agonistic effect on PPARgammaHotta et al., 2010). Santos and Rao (2000) have showed that 1,8-ineole produces anti-inflammatory and antinociceptive effectsith inhibitory actions on carrageenan-induced oedema, increased
apillary permeability, and granuloma formation. Inhibitory effectsn cytokine production and arachidonic acid metabolism by cineoleere demonstrated by Juergens et al. (1998). A significant inhibi-
ion of gamma-interferon and IL-4 production by monoterpenesas observed by Souza et al. (2003).
The EO of Lippia gracilis leaves shows antinociceptive andnti-inflammatory activities. A discreet antioxidant effect wasemonstrated for the EO of Lippia gracilis. Thereby, the phar-acological actions of Lippia gracilis could be related to the
resence of thymol as major component in the EO. However,he synergistical action of other minor constituents cannot beisregarded.
cknowledgements
This study was supported by Conselho Nacional de Desenvolvi-ento Científico e Tecnológico (CNPq – RENORBIO), Coordenacão
e Aperfeicoamento de Pessoal de Nível Superior (CAPES) andundacão de Apoio à Pesquisa e à Inovacão Tecnológica do Estadoe Sergipe (FAPITEC/SE – Universal 2008).
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Artigo 2 Antioxidants in ethanol and aqueous extracts of the
medicinal plant Lippia gracilis
Imprimir http://br.mg5.mail.yahoo.com/neo/launch?.rand=5vnk7gnf201i2 1
Assunto: Planta Medica - PLAMED-2011-11-1105-OP Editor assignment
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Para: [email protected]; [email protected];
Data: Quarta-feira, 23 de Novembro de 2011 12:28
23-Nov-2011
Dear Dr. Vos,
Thank you for submitting your manuscript PLAMED-2011-11-1105-OP entitled
Antioxidants in ethanol and aqueous extracts of the medicinal plant Lippia gracilis to
Planta Medica. Your manuscript was assigned to Prof. (), Editor for Planta Medica, who
will manage the review process.
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Prof. Luc Pieters
Editor-In-Chief Planta Medica
Antioxidants in ethanol and aqueous extracts of the medicinal plant Lippia gracilis
Sandra Santos Mendes1*, Vitor Araujo Neto1, Charles dos Santos Estevam1, Arie Fitzgerald
Blank2, Renato Delmondez de Castro3, Benyamin Houshyani4, Harry Jonker4,5, Sara Maria
Thomazzi1, Ric C.H. de Vos4,5.
1Department of Physiology, Federal University of Sergipe, CEP 49100-000, São Cristóvão,
Sergipe, Brazil
2Department of Agronomic Engineering, Federal University of Sergipe, CEP 49100-000, São
Cristóvão, Sergipe, Brazil
3Department of Biofunction, Federal University of Bahia, CEP 40160-100, Salvador, Bahia,
Brazil
4Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands
5Centre for Biosystems Genomics, PO Box 98, 6700 AA Wageningen, The Netherlands
*Corresponding author:
Dr. Sandra Santos Mendes
Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe.
CEP 49100-000, Brazil
Email: [email protected]
Tel: +55-79-9126-5214
ABSTRACT
This research aimed to identify the major antioxidants in the pharmaceutically active plant
Lippia gracilis, which is used as folk medicine in Brazil since centuries to treat, for instance,
inflammation related disorders. Dried leaf material was extracted with either hot-water or
ethanol to obtain aqueous-extracted (AE) and ethanol-extracted (EE) powders, respectively.
The antioxidant activity of these powders was evaluated using three different in vitro assays,
namely ABTS, FRAP and DPPH. In all assays the EE powder showed significantly higher
(about 40%) antioxidant activity than the AE powder, independent of solvent solution or
temperature used for re-dissolving the extracted powders. Identification of antioxidant
compounds was performed using both HPLC-PDA with online antioxidant detection and
accurate mass HPLC-PDA-QTOF MS analyses. The major antioxidants, together contributing
for more than 50% to the total antioxidant activity of extracts, were unambiguously identified
as the flavonoids luteolin-C-6-glucoside and luteolin-C-8-glucoside, both described for the
first time in the Lippia genus, and the terpenoids carvacrol and thymol. The luteolin-C-
glucosides were about 2.5-fold higher in EE than in AE, while carvacrol, being the most
apolar antioxidant in the extracts, was even more than 20-fold higher. In the EE preparation,
the amount of carvacrol was 5.3% of total dry weight (DW), while both luteolin-C-glucosides
each contributed for 0.6%.
Keywords: Lippia gracilis, trolox equivalent antioxidant capacity, luteolin-C-glucoside,
carvacrol
INTRODUCTION
Reactive oxygen species (ROS) can cause numerous pathophysiological processes ranging
from cancer to toxic effects of drugs. This involvement of oxidative molecules, including
radicals and non-radicals such as hypochlorous acid and peroxynitrile, is the base for
antioxidant research and identifying new sources of natural antioxidants 1. Antioxidants
provide protection for living organisms against damage caused by an uncontrollable
production of ROS, resulting in oxidative damage to proteins, membranes and DNA 2.
Plants as well as products derived thereof are particularly rich in natural antioxidants,
especially flavonoids and phenolic acids are attractive phytochemicals, as they are known to
exhibit various beneficial pharmacological properties, such as vasoprotective,
anticarcinogenic, antineoplastic, antiviral, anti-inflammatory, as well as antiproliferative
activity on tumor cells. At least a part of these beneficial properties of these compounds have
been related to their action as antioxidants, free radical scavengers, quenchers of singlet and
triplet oxygen and/or inhibitors of lipid peroxidation processes 3.
Many medicinal plants are known to possess antioxidant activity, mostly resulting
from biosynthesis of flavonoids and other phenolic compounds 4 and it is generally believed
that these antioxidant compounds play a key role in the pharmacological activity of extracts
prepared from medicinal plants. The Lippia genus (Verbenaceae) includes approximately 200
species of herbs, shrubs and small trees. Most species exhibit pharmacological activities and
have been used as folk medicines for many centuries. In practice, mostly aerial parts such as
leaves and flowers of Lippia plants are used and extracts are commonly prepared as a hot
water infusion or decoction (boiling water), and administered orally 5. In addition, alcohol
macerates can be prepared and used as topical application6. Extracts from various species of
the Lippia genus and are reported to possess antioxidant activity7 8 9 10. Lippia sidoides, for
instance, is an aromatic plant mainly used as source of anti-septic extracts. Its essential oil
shows antimicrobial effects against fungus and bacteria11 and also exhibits both
pharmacological and antioxidant activities. Its main constituent thymol, a terpenoid that can
contribute up to 67 % of the oil, is thought to be responsible for these activities9. Likewise,
essential oil from Lippia gracilis contains several terpenoids, including thymol, carvacrol, p-
cymene, 4-terpenil acetate, α-copaene, and β-cubebene 5 12. Thymol and carvacrol are
phenolic monoterpenes with known in vitro antioxidant activity13. Moreover, we recently
showed that L. gracilis essential oil exhibits both anticinoceptive and anti-inflammatory
activities in animals12. With regard to non-essential oils, it was recently showed that a
methanolic extract of L. gracilis exhibited antinociceptive and anti-inflammatory activities in
animal assays and suggested that the flavonoid naringenin was one of the bioactive
compounds14.
The aim of this study was to compare the antioxidant capacity of hot water and ethanol
extracts from L. gracilis and to identify their main antioxidants. It is well-known that the
antioxidant activity of plant extracts depends on the chosen assay method, on the
concentration of antioxidants and on the physicochemical properties of the antioxidants
studied15. It is therefore advised to perform more than one type of antioxidant capacity
measurements16. In this study, we therefor applied the 2,2′-azinobis-3-ethyl-benzothiazoline-
6-sulfonic acid (ABTS), the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-
2-picrylhydrazyl hydrate (DPPH) assays to evaluate the total antioxidant capacity of extracts.
Moreover, we used both HPLC with photodiode array (PDA) detection coupled to an on-line
antioxidant detection system based on post-column scavenging of ABTS•+ radicals, and
HPLC-PDA coupled to accurate mass detection (QTOF-MS) to identify and quantify the
major antioxidants in the Lippia gracilis extracts.
EXPERIMENTAL SECTION
Preparation of plant extracts
Fresh leaves of L. gracilis were collected from plants growing at the germplasm bank
of the Federal University of Sergipe, Brazil (10º55’32”S, 37º06’08”W), in January 2009 and
one exsiccate was deposited at the Herbarium (ASE) of the same University (Voucher number
ASE 9205). Leaves were dried in a sterilizer (Model MA-037) with hot air circulation and
renewal at 37ºC until complete dehydration. Dried leaves were ground until a fine powder
was obtained. The aqueous extract (AE) was prepared by adding distilled hot water to the
dried plant powder in a 1:5 (w/v) ratio. This solution was vacuum-filtered (with filter paper)
and dried in a rotary evaporator at 50ºC, yielding 8.2 % of the initial plant dry weight. The
ethanol extract (EE) was prepared by exhaustive extraction with 90 % ethanol in water for 5
days, using a Soxhlet apparatus20. The obtained EE was filtered and dried using a rotary
evaporator at 50ºC, to give the EE powder (yield: 8.1 %). The dry powders were stored in
closed containers at room temperature until use.
For testing their antioxidant activity, the two extract powders (EE and AE) were re-
dissolved in two different solvents, i.e. in A) MeOH/water (75:25 v/v) containing 0.1 %
formic acid and B) Milli Q-water. A range of temperatures, i.e. 22, 30, 45, 60 and 90ºC was
tested in order to elucidate the effect of temperature on the efficiency to re-dissolve
antioxidants from the dried AE and EE powders. The extracts were sonicated for 20 min and
then filtered over a 0.2 µm PTFE filter before use.
Determination of antioxidant capacity
The ABTS and FRAP assays were performed as previously described by18; while the
DPPH procedure followed the method of21, both using trolox as a standard. Solutions and
reagents were prepared freshly. The extract to be analyzed was first adequately diluted to fit
within the linearity range of the trolox calibration curves. All antioxidant assays were carried
out in triplicate. Trolox was used as reference compound at a range of 0.2 – 1.6 mM.
Ferric Reducing Antioxidant Power (FRAP)
The FRAP reagent was prepared from acetate buffer (300 mM, pH 3.6), 10 mM TPTZ
solution in 40 mM HCl in MeOH and 20 mM ferric (III) chloride in aqueous solution, at a
ratio of 10:1:1 (v/v/v), respectively. In a 96-well plate 200 µL of FRAP reagent and 50 µL of
sample were added sequentially. After 10 min of incubation at room temperature, the
absorbance was read at 590 nm against MeOH/water or water as a blank.
ABTS-cation radical scavenging assay
The 2,2′-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS)-cation radical was
produced by oxidation of a ABTS solution (7 mM in water) with ammonium persulfate
(K2S2O8) at a final concentration of 2.45 mM. The mixture was allowed to stand in the dark at
room temperature for 16 h before use, and was then diluted with phosphate buffered saline
(pH 7.4) to an absorbance of about 1.0 at 415 nm. 100 µL of diluted ABTS solution was
pipetted into a 96-well plate and mixed with 10 µL of the extracts or standard solution per
assay. After 1 min incubation at 37oC the absorbance at 415 nm was read.
DPPH radical scavenging assay
Radical scavenging activity of plant extracts against stable 2,2-diphenyl-2-
picrylhydrazyl hydrate radicals (DPPH•) was determined at 590 nm. A 0.1 mM DPPH
solution in pure MeOH was prepared and 200 µL of this DPPH solution was mixed with 10
µL of each sample into a 96-well plate. The samples were allowed to react for 30 min at room
temperature in the dark before measuring the remaining DPPH radicals.
LC-PDA-antioxidant and LC-PDA-MS analyses
In order to detect individual antioxidants, 10 mg of EE powder was dissolved in 2 mL
of MeOH/water using 30 min sonication. After centrifugation the supernatant was filtered
through a 0.45µm filter, and 10 µl was subsequently injected into an HPLC system (Alliance;
Waters) with both a photodiode array (PDA) detector (Waters 996) and a post-column ABTS-
cation radical reaction system17 18. The column used was a Luna C18 (2), 4.6 x 150 mm (3 µm
particle size; Phenomenex, CA). Separation of compounds in the extracts was conducted in a
55 min linear gradient from 5 to 50 % acetonitrile in Milli Q (acidified with 0.1% FA) at a
flow rate of 1 mL/min at 40°C, followed by column washing (in 5 min to 75% acetonitrile
followed by 5 min washing) before 10 min equilibration. Compounds eluting from the column
passed firstly through the PDA detector, set at an absorbance range of 240-700 nm, and were
then allowed to react for 30 s with a 0.2 M Na-phosphate buffered solution (pH 7.5) of
ABTS-cation radicals at 40ºC. The decrease in ABTS-radicals by the reaction with
antioxidants was monitored using a dual-wavelength UV-Vis detector (Waters 2487) at 412
nm.
Extracts were subjected to accurate mass LC-MS using a high resolution time-of-flight
mass spectrometer (QTOF MS) with lock mass correction, in combination with absorbance
spectra analysis using a PDA detector online19. Samples were injected and separated on a
Luna C18 (2) column (2.1 x 150 mm; 3 µm particles), using a Waters Alliance 2795 HT
HPLC system providing the same gradient from 5 to 50 % acetonitrile (acidified with 0.1 %
FA) as described above for the HPLC-PDA system, but at a flow rate of 0.19 mL/ min
(comparable with a flow rate of 1 mL/min on the 4.6 x 150 mm column described above).
Eluting compounds were first detected online at 240-600 nm using a Waters 2996 PDA,
before entering a QTOF Ultima API mass spectrometer (Waters) equipped with an
electrospray ionization (ESI) source and a separate LockSpray for online accurate mass
correction. The following settings were applied during LC-MS runs: desolvation temperature
of 250°C with a nitrogen gas flow of 600 L/h, capillary spray at 3 (ESI+) or 2.5 (ESI-) kV,
source temperature of 120°C, and cone at 35 eV with 50 L/h nitrogen gas flow. Ions in the m/z
range of 100-1500 were detected using a scan time of 0.9 s and an interscan delay of 0.1 s.
Statistical analysis
Total antioxidant activities of extracts were calculated as means ± standard deviation
(SD) of three parallel measurements, and expressed in nmol of trolox per g of powder dry
weight. The data were subjected to 3-way ANOVA using the PASW® Statistics Program 17.0.
Differences among treatments were separated by Tukey’s multiple range tests, considering a
significant threshold of 0.05.
RESULTS AND DISCUSSION
A variety of in vitro antioxidant assays has been developed to determine total
antioxidant capacity of food and vegetable extracts, beverages and biological fluids. These
antioxidant capacity assays may be broadly classified as either electron-transfer (ET)-based
assays or hydrogen atom transfer (HAT)-based assays22. The principle of the antioxidant
assay, the radical that is generated, the end-point detection, and the allocated reaction time
vary considerably from one test to another, and each method has its advantages and
disadvantages17. We therefore chose three different antioxidant assays, namely based on
ABTS, FRAP and DPPH, to assess the antioxidant potential of extracts from the medicinal
plant L. gracilis, using the water-soluble vitamin E analogue trolox as a reference and
calculating their antioxidant activities as Trolox Equivalent Antioxidant Capacity (TEAC) in
all three assays.
In order to test the antioxidant activity of the original ethanol extract (EE) and water
extract (AE) prepared from L. gracilis leaves, the derived powders were re-dissolved freshly
and tested using the three different in vitro assays. In view of a possible differential
metabolite composition of the original extracts (as ethanol usually extracts apolar metabolites
more efficiently than hot water), the efficiency of re-dissolving the compounds present in the
two powders may be different between EE and AE powders and depending upon solvent and
temperature during re-dissolving. We therefor re-dissolved the powders in pure water (MQ) at
different temperatures, i.e. 22°C, 30ºC, 45ºC, 60ºC and 90°C, and also in a less polar solvent,
i.e. acidified aqueous methanol (75 % MeOH+0.1 % formic acid (FA)), at 22°C, 30ºC, 45ºC
and 60ºC. We did not find any significant effect of extraction temperature on the total
antioxidant capacity of either MQ of MeOH/FA re-dissolved extracts, as measured in the
ABTS assay (data not shown), suggesting that differences observed between EE and AE were
not due to possible differential temperature-dependent solubility or stability of compounds.
The subsequent antioxidant assays and analyses were therefore carried out with powders re-
dissolved at 30ºC.
In both FRAP and ABTS assay, re-dissolving AE and EE powders in MeOH/FA
resulted in higher TEAC values than re-dissolving them in MQ water, while in the DPPH
assay such solvent effect was not observed in EE (Table 1). In both AE and EE extracts,
highest antioxidant activities were detected with ABTS and lowest activities with DPPH.
These results indicate that I) the EE sample exhibited significantly higher antioxidant
activities than the AE sample using either MeOH/FA or MQ as solvent, II) different
antioxidant assays result in different TEAC values for the same extract, even though the same
trolox stock solution was used as a reference in each assay, and III) the antioxidants
detectable by ABTS and FRAP can be different, at least partly, from those detectable by
DPPH, as was concluded from the differential ratios of the TEAC values of EE in MeOH/FA
versus MQ water.
The antioxidant values of our L gracilis extracts were higher in the ABTS assay than
in the two other assays, despite the relative short reaction time in the ABTS assay (1 min).
Besides the differential chemical reactions involved, the ABTS assay is carried out at higher
pH than the FRAP and DPPH assays: pH 7.4 versus pH 3.6 and 3.0, respectively. As it is
known that the pH of the reaction mixture is one of the key factors affecting the radical
scavenging activity of compounds in in vitro antioxidant assays23, our results suggest that the
antioxidants of L. gracilis extractable with hot water or ethanol react better at physiological
pH than at lower pH values. Some researchers have examined the influence of the extraction
solvent on different antioxidant assays24 25. It appeared that the choice of the solvent is an
essential parameter on the chemical behaviour of antioxidant compounds, as the solvent
polarity can have a significant effect on the performance of HAT- and ET-based antioxidant
reactions 25 26. In the present study, we also noticed a marked effect of solvent on the
antioxidant capacity of L. gracilis extracts, since in nearly all assays the TEAC values were
significantly lower in the powders re-dissolved in MQ as compared to re-suspension in the
more organic solvent (75% MeOH/FA), with both EE and AE powders. This higher activity
in MeOH/FA was likely not due to a better solubility of extracted antioxidants in this solvent,
as dissolving in MQ at higher temperatures, up to 90°C and including sonication, did not
result in a higher antioxidant activities. More likely, the observed difference between the two
solvents was due to a facilitated electron transfer between antioxidants and radicals in the
presence of MeOH as solvent. In fact, MeOH is the alcohol that best supports ionization of
compounds27.
After determining the total antioxidant activities of the EE and AE preparations, we
aimed to identify their main antioxidants. Therefore, all four types of samples used in the
spectrophotometric assays for total antioxidant capacity (Table 1) were also analyzed on a
dedicated HPLC-PDA with an online ABTS-radical based antioxidant detection system17. The
profiles of the four extracts were quite similar, although the relative amounts of antioxidant
compounds were different (not shown). As an example, the HPLC-PDA chromatograms
(recorded at 280 nm) and the corresponding antioxidant profiles of EE and AE, both re-
dissolved in MeOH/FA, are shown in figure 1. Antioxidants appear as negative peaks, due to
a decrease in the ABTS-cation radical absorbance, resulting from the antioxidant reaction, and
the areas under curve are directly related to the compounds antioxidant activity in the extract,
which is a combination of its actual concentration and its specific antioxidant capacity. The
EE extract contained the highest levels of a variety of antioxidants, with two major
antioxidants eluting at around 17 min of chromatographic retention, while its main
antioxidant eluted at 54 min. Generally, the antioxidant profiles were comparable between the
two different solvents used to re-dissolve the extracted powders (data not shown), indicating
that their antioxidant composition was qualitatively similar. However, the level of most
individual antioxidants was higher in the EE preparation, especially the more apolar
compound eluting at 54 min, as could be deduced from their larger antioxidant peaks, which
fits with its higher total antioxidant activity in the spectrophotometric assays.
Since the same type of PDA detector was placed between the column and the MS, and
the entire HPLC system including column type, solvent gradient and eluent flow were exactly
similar between the HPLC-PDA-antioxidant system and the HPLC-PDA-QTOF MS system,
the major ABTS-antioxidants detected in the extracts (Figure 1) could be traced back in the
LC-MS system based on their specific absorbance spectra and relative retention times. By
coupling this HPLC-PDA-QTOF MS system to electrospray ionization in both negative
(Figure 2) and positive mode, followed by accurate mass determination of the
pseudomolecular ions, we aimed to elucidate the chemical structures of the main antioxidants
present in the L. gracilis extracts. The first two major antioxidants, eluting at 16.8 min and
17.2 min, respectively, appeared to exhibit similar UV/Vis-absorbance spectra with a primary
absorbance maximum at 349 nm and a second absorbance maximum at around 265 nm
(Figure 3A), suggesting a flavone backbone. In addition, both compounds had a similar
accurate mass of m/z 447.0927 ([M-H]-), i.e. within 1.5 ppm corresponding to an elemental
formula of C21H20O11. Thus, these antioxidants were both putatively annotated as luteolin-
hexosides. However, in contrast to luteolin-7-O-glucoside staddard, both L. gracilis luteolin-
hexosides were insensitive to acid hydrolysis, at 90-95°C in 50% methanol + 1.2 M HCl for
60 min (data not shown) and did not show the characteristic loss of the glucose ester (-162)
resulting in a major fragment ion of 285.0404 from the luteolin C15H10O5 backbone upon
MS/MS (cf. www.massbank.jp). In contrast, MS/MS of the L. gracilis compounds indicated
neutral losses of 18 (-H2O) and 30 (-CH2O) and the m/z 285 of the luteolin fragment was
observed at high collision energy only (Figure 3B; cf. www.massbank.jp). Based on the
correspondence of both their chromatographic retentions and UV/Vis-absorbance spectra with
authentic standards, in both the HPLC-PDA and the HPLC-PDA-QTOF MS systems, as well
as their matching accurate masses and MS/MS fragmentation patterns, these L. gracilis
antioxidants could be unambiguously identified as luteolin-6-C-glucoside (C6; isoorientin; rt
= 16.8 min) and luteolin-8-C-glucoside (C8, orientin; rt = 17.2 min), respectively.
The third major antioxidant, eluting at 54 min (Figure 1B), which was most abundant
in EE, exhibited a PDA absorbance maximum at about 280 nm but was not detectable by MS,
neither in negative mode (Figure 2B) nor in positive mode. Since phenolic terpenoid
antioxidants, including thymol and carvacrol have been reported as major compounds in L.
gracilis essential oils12, authentic standards of thymol and carvacrol were injected
simultaneously with the AE and EE extracts in both the HPLC-PDA-antioxidant system and
the HPLC-PDA-MS. In both systems, the retention time and absorbance spectrum of the
carvacrol standard (with optimum at 280nm) corresponded exactly to those of the antioxidant
peak at 54 min (Fig. 1B), which was thus identified as carvacrol. The antioxidant peak eluting
at retention time 56 min (Fig. 1B), which like carvacrol was clearly higher in EE than in AE,
corresponded to a thymol standard.
We subsequently determined the in vitro antioxidant activities of luteolin-6-C-
glucoside, luteolin-8-C-glucoside and carvacrol, and compared them with the known
flavonoid antioxidants quercetin and luteolin in the three antioxidant assays (Table 2). The
antioxidant activity of each compound, relative to trolox, clearly depended upon both the
compound structure and the antioxidant assay used. For instance, the terpenoid carvacrol was
3.3-fold more active than trolox in the ABTS assay (TEAC of 3.3), while being 5.5-fold less
active in the DPPH assay (TEAC of 0.18). Compared with the two luteolin-C-glucosides,
carvacrol showed superior activity in the ABTS assay, but inferior activity in both FRAP and
DPPH assays. In the ABTS and DPPH assays, the aglycon form of luteolin showed similar
activity as its 6-C-glucoside, while in the FRAP assay it was comparable with its 8-C-
glucoside. In each assay, the antioxidant activity of the luteolin-6-C-glucoside form was
higher than the 8-C form, ranging from 193% in the ABTS assay, 164% in the FRAP assay
and 33% in DPPH assay. Thus, differences in the conjugation site influence the antioxidant
activity of the flavones in an assay-dependent manner.
Based on a 6-points dilution series of authentic standards, the amounts of both
luteolin-C-glucosides and carvacrol were determined using HPLC-PDA with peak integration
at 280 nm (Table 3). The amounts of all three compounds were significantly higher in the EE
extract than in the AE extract. However, the ratios between EE and AE preparations differed
between the compounds: both luteolin-C-glucosides were about 2.5-fold higher in EE than in
AE, while carvacrol, being the most apolar antioxidant in the extracts, was more than 20-fold
higher in EE. The amount of carvacrol was 5.3% of the total leaf dry weight in the EE
preparation, while the luteolin-glucosides each contributed for 0.6%.
Based on the specific antioxidant activity of compounds (Table 2) and their amounts
in the extracted plant powder (Table 3), the contribution of each compound to the total
antioxidant capacity of the extract was calculated. For instance, carvacrol had a TEAC of 3.31
and its amount was 352 µmol/g EE powder. Thus, carvacrol in EE represented 1165 µmol of
Trolox per g of powder, which was about 40% of the total activity of EE (re-dissolved in
MeOH). Likewise, luteolin-6-C-glucoside and luteolin-8-C-glucoside each represented about
5% of the total activity in EE-MeOH extracts. For AE powder re-dissolved in MeOH,
carvacrol and both luteolin-glucosides each contributed for about 3% of the total ABTS
antioxidant activity.
Flavonoids are common constituents of plant extracts used in traditional medicine to
treat a wide range of diseases. The flavone luteolin is a common flavonoid species and its
glycosides are widely distributed among the plant kingdom. Preclinical studies have shown
that luteolin possesses a variety of pharmacological activities, including antioxidant, anti-
inflammatory, antimicrobial and anticancer activities29. Using LCMS, various 7-O-
derivatives of luteolin and apigenin, including glucuronides and glucosides, have previously
been reported in extracts of Lippia plants30 31. In fact, the present study, in which we identified
luteolin-6-C-glucoside and luteolin-8-C-glucoside as the major flavonoid antioxidants in L.
gracilis extracts, is the first report indicating the presence of flavone-C-conjugation within the
Lippia genus. Direct evidence for C-conjugation of these compounds, rather than O-
conjugation, was derived from the insensitivity of both luteolin-C-conjugates to acid
hydrolysis and their MS/MS spectra, in agreement with their authentic standards and in
contrast to the luteolin-7-O-glucoside standard that were used as comparison. In fact, in our
LCMS system the luteolin-7-O-glucoside standard (C21H20O11; calculated [M-H]- of
447.0933) co-eluted with a L. gracilis peak of m/z 447.0929 at rt = 20.5 min (Figure 2).
Altogether, these data suggest that in L. gracilis leaves flavone C-glucosides exist next to
their O-glucosides. Together with carvacrol, these two luteolin-C-glucosides accounted for
about 50% of the total antioxidant capacity of the L. gracilis EE sample. Other researchers14
recently reported the presence of naringenin, a flavonone-type of flavonoid, in methanolic
extracts of L. gracilis leaves and suggested that this compound is, at least partly, responsible
for the observed antinociceptive and anti-inflammatory activities of solvent extracts. By
generating the accurate mass-specific chromatogram of naringenin at m/z 271.0611 (allowing
5 ppm mass deviation), in combination with its typical flavonone UV/Vis spectrum showing
an absorbance maximum at 280 nm and a small shoulder at 320nm, we were also able to
identify naringenin in our L. gracilis AE and EE extracts as well, eluting at a retention time of
33.5 min. However, the contribution of naringenin to the total antioxidant activity was only
small in comparison with both luteolin-C-glucosides and carvacrol (Figure 1), thus making its
supposed key contribution to the observed antinociceptive and anti-inflammatory activities of
L. gracilis methanol extracts14 less likely, although differences in flavonoid composition
between original leaf materials may exists. Next to carvacrol, thymol was also present in the
L. gracilis EE extract, indicating that these essential oil constituents12 can also be extracted by
ethanol, at least partly. In contrast to the EE and AE extracts, essential oil from L. gracilis
leaves does not contain the relative polar luteolin-C-glycosides (S. Santos Mendes and RCH
de Vos, unpublished data). Generally, the major components in plant extracts or essential oils
are found to reflect quite well the pharmacological features of the herbal preparations.
Nevertheless, it is possible that the bioactivity of the main components is modulated by one or
more minor components.
In conclusion, we have established that both hot water-based and ethanol-based
preparations from leaves of the plant L. gracilis exhibited significant antioxidant activity in
vitro, and were able to identify luteolin-C-glucosides and carvacrol as its main antioxidants.
These antioxidants which may at least partly contribute to its pharmacological effects in vivo
and its traditional use as folk medicine. Ethanol was superior over hot water in quantitatively
extracting these antioxidants, especially carvacrol, from the original plant material. Clearly,
more research is needed to elucidate the role of plant genotype, growth and development on
the content of these antioxidants in L. gracilis leaves, to optimize the extraction procedure for
pharmaceutical preparations, and to determine the medicinal effect of its extracts and key
bioactive constituents in vivo.
Acknowledgements
This study was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq - RENORBIO), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) and Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de
Sergipe (FAPITEC/SE – Universal 2008). The authors kindly thank Bert Schipper (PRI) for
this excellent technical assistance in LCMS. Ric C.H. de Vos acknowledges additional
financing from the Centre for Biosystems Genomics, which is under the auspices of the
Netherlands Genomics Initiative, and by the European Commission (EU-project TerpMed,
227448).
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Table 1. Antioxidant capacity of L. gracilis aqueous (AE) and ethanol (EE) extracted powders, re-dissolved in
75 % MeOH/0.1 % FA (MeOH) or MilliQ water (Water) at 30oC, as determined by the ABTS, FRAP and DPPH
assays. Data represent means ± standard deviation of 3 independent sample preparations from the dry AE and EE
powders and are expressed in µmol Trolox per g of powder. Different letters in the rows represent statistically
significant differences between assays (p<0.05), using ANOVA/Tukey.
Assay EE (MeOH) EE (Water) AE (MeOH) AE (Water)
ABTS 2638 ± 207a 1095 ± 103c 1563 ± 42b 859 ± 99c
FRAP 1631 ± 37a 902 ± 41c 1191 ± 59b 604 ± 29d
DPPH 733 ± 71a 787 ± 16a 658 ± 84a 504 ± 137b
Table 2: Trolox equivalent antioxidant capacities of standard compounds, as determined in the ABTS, FRAP
and DPPH antioxidant assays. Values are means and standard deviations of different concentrations of standards
(at 30 up to 320 nmol/mL) analyzed during 3 independent analyses series.
Assay Luteolin-
C-6-
glucoside
Luteolin-
C-8-
glucoside
Carvacrol Quercetin Luteolin
ABTS 1.93 ± 0.16 1.00 ± 0.17 3.31 ± 0.24 4.36 ± 0.38 1.90 ± 0.27
FRAP 2.36 ± 0.34 1.44 ± 0.10 0.44 ± 0.06 2.81 ± 0.24 1.34 ± 0.10
DPPH 0.89 ± 0.13 0.67 ± 0.10 0.18 ± 0.26 1.32 ± 0.15 0.87 ± 0.12
Table 3. Levels of luteolin-C-glucosides and carvacrol in the EE and AA powders, after re-dissolving in
MeOH/FA. Values are means and standard deviations of 3 independent extractions and subsequent analyses
using HPLC with PDA detection at 280nm, using calibration curves of authentic standards.
compound EE (µmol/g DW) AE (µmol/g DW)
Luteolin-C-6-glucoside 72.3 ± 13.8 26.7 ± 1.1
Luteolin-C-8-glucoside 77.3 ± 14.6 34.5 ± 1.2
Carvacrol 352.3 ± 49.7 15.0 ± 1.8
Legends to Figures
Figure 1. HPLC-PDA with on-line antioxidant detection, using ABTS-cation radicals, in L.
gracilis leaf powders derived using A) hot-water extraction, i.e. AE, or B) ethanol extraction,
i.e. EE. Powders were re-dissolved in 75% MeOH containing 0.1% formic acid before
analysis. Upper traces, with peaks pointing upwards, represent PDA absorbance (280 nm),
while the lower traces, with peaks pointing downwards, represent ABTS-cation radical
absorbance (412 nm) remaining after post-column reaction; a high negative value of ABTS-
absorbance indicates a high antioxidant activity. The time difference between the two
detectors is 30 seconds resulting from the ABTS post-column reaction. Arrows indicate the
three main antioxidants.
Figure 2. HPLC-PDA-QTOF MS analysis, in negative ionization mode, of L. gracilis leaf
powders derived using A) hot-water extraction (AE) or B) ethanol extraction (EE). Samples
were re-dissolved in 75% MeOH containing 0.1% formic acid before analysis by the LCMS
system. Note: y-axes are set at the same scale (100% corresponds to 2.08x104 ions per
second). Labels at peaks indicate retention time (in min) and their accurate mass. Arrows
indicate the peaks of the two luteolin-C-glucoside antioxidants.
Figure 3. Identification of luteolin-C-6-glucoside in L. gracilis using HPLC-PDA-QTOF MS
and MS/MS. A) UV/Vis-absorbance spectra of L. gracilis compound eluting at 16.8 min
(upper trace) and of luteolin-C-glucoside standard (lower trace); B) ESI-negative mode
MS/MS spectra of the same L. gracilis compound at a collision energy of 10 eV (upper trace)
and 25 eV (lower trace). Detected parent ion 447. 0928 deviates 1.3 ppm from the expected
elemental formula of C21H20O11.
Figure 1. Mendes et al.
Figure 2. Mendes et al
12-Feb-2010 17:23:24AA 0.5mg/ml 60 MeOH
Time10.00 20.00 30.00 40.00 50.00
%
-1
99
10.00 20.00 30.00 40.00 50.00
%
-1
99
M19249 1: TOF MS ES- BPI
2.08e4
17.19447.0911.96
341.10822.04
623.199
M19248 1: TOF MS ES- BPI
2.08e416.83
447.088
12.57609.146
1.96341.107
17.15447.089
20.39623.193
24.49881.212 35.50
299.054
A
B
Figure 3. Mendes et al.