Sílvia Lopes Ferreira Martinsrepositorium.sdum.uminho.pt/bitstream/1822/23111/1...que se está a desenvolver. Como qualquer outra etapa da vida, o Doutoramento é recheado de momentos

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Maio de 2012

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Universidade do Minho

Escola de Engenharia

Tese desenvolvida no âmbito da bolsa de doutoramento de referência

SFRH/BD/40439/2007, financiado pela Fundação para a Ciência e a Tecnologia (FCT),

co-financiado pelo Programa Operacional Potencial Humano (POPH) do Quadro de

Referencia Estratégico Nacional (QREN), comparticipado pelo Fundo Social Europeu e

por fundos nacionais.

Governo da República Portuguesa

União Europeia

Fundo Social Europeu

Programa Doutoral em Engenharia Química e Biológica


Maio de 2012

Bioactive compounds recovery from Larrea tridentata leaves and their potential benefits for human health

Universidade do Minho

Escola de Engenharia

Trabalho realizado sob a orientação do

Professor Doutor José António Couto Teixeira

e da

Doutora Solange Inês Mussatto Dragone

II

Autor


Email: [email protected], [email protected]

Telefone: +351 253 604 400

Título da tese

Bioactive compounds recovery from Larrea tridentata leaves and their potential benefits

for human health

Orientadores

Professor Doutor José António Couto Teixeira

Doutora Solange Inês Mussatto Dragone

Ano de conclusão 2012

Programa Doutoral em Engenharia Química e Biológica

É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTA TESE/TRABALHO

APENAS PARA EFEITOS DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO

ESCRITA DO INTERESSADO, QUE A TAL SE COMPROMETE.

Universidade do Minho, Maio de 2012

III

AGRADECIMENTOS

Mais do que definir um projecto, do que as horas passadas num laboratório ou em

frente ao computador, um Doutoramento é uma escolha, uma etapa na vida de quem

decide aventurar-se no mundo da investigação. É uma aventura que exige empenho,

dedicação, autonomia, algum espírito de sacrifício e, principalmente, gosto pelo trabalho

que se está a desenvolver. Como qualquer outra etapa da vida, o Doutoramento é recheado

de momentos bons e maus, mas o sabor de uma ideia concretizada, a conquista de um

artigo publicado, a finalização de um trabalho bem fundamentado e coerente, compensam

tudo o que de menos positivo se enfrenta nesse caminho. Caminho que integra a presença

de pessoas que fazem também valer todo o esforço e crença nesse percurso. A essas

pessoas dedicarei, de todo o coração, algumas palavras de agradecimento da forma mais

simples e sincera.

Ao meu Orientador, Professor José Teixeira, agradeço a oportunidade e liberdade

de definir um projecto e desenvolvê-lo de forma dinâmica e autónoma, prestando sempre o

seu apoio e orientação nos momentos em que necessitei. À minha Co-Orientadora, Dra.

Solange Mussatto, agradeço todas as palavras de apoio pessoal e académico, toda a

motivação, confiança, Amizade e partilha.

Ao Dr. Cristóbal Aguilar, Universidade Autónoma de Coahuila (Saltillo, México),

pela oportunidade de iniciar o meu projeto de Doutoramento no México, proporcionando-

me todas as condições experimentais adequadas ao desenvolvimento do trabalho de

investigação. À Professora Elba Amorim, Universidade Federal de Pernambuco (Recife),

pela possibilidade de me integrar num grupo de investigação dedicado única e

exclusivamente ao estudo de produtos naturais oriundos de plantas medicinais, dando-me

uma motivação e incentivo extras para o desenvolver do meu plano de trabalho.

Aos meus colegas e amigos, tanto do DEB como fora do DEB, sem mencionar

nomes porque não é necessário, agradeço o apoio, os sorrisos, a partilha, os puxões de

orelha, os conselhos, a força, e a presença deles durante este percurso.

À minha família dedico todo o meu esforço e agradeço o Amor e apoio dados.

IV

Aos meus dois Amores, Lucas Pai e Lucas Filho, agradeço a inspiração e o

empurrão final para concretizar esta etapa.

Agradeço à Vida e à Natureza.

De tudo, ficaram três coisas:

A certeza de que estamos sempre começando...

A certeza de que precisamos continuar...

A certeza de que seremos interrompidos antes de terminar....

Portanto devemos:

Fazer da interrupção um caminho novo ...

Da queda um passo de dança...

Do medo, uma escada...

Do sonho, uma ponte...

Da procura, um encontro...

Fernando Pessoa

V

ABSTRACT

Plants are one of the most important sources of compounds with biological properties of

great interest to human health. Larrea tridentata (Sessé & Moc. Ex DC.) Coville

(Zygophyllaceae), commonly known as gobernadora or creosote bush, is a plant that

grows in semiarid areas of Southwestern United States and Northern Mexico. This plant

was traditionally used for centuries by North American Indians to treat a wide range of

medical conditions and illnesses including genitor-musculoskeletal urinary and respiratory

tract infections, inflammation of the system, damage to the skin, kidney problems, arthritis,

diabetes and cancer, among other diseases. Among several valuable bioactive phenolic

compounds present in L. tridentata leaves, the natural occurring lignan

nordihydroguaiaretic acid (NDGA) has been pointed out as the most important, presenting

antioxidant, antiviral, antimicrobial, and antitumorgenic activities. Other important

bioactive compounds, such as kaempferol (K) and quercetin (Q), are also present at

considerable high concentrations in this plant.

Extraction of bioactive compounds from plants is conventionally performed by heat-reflux

method. Nevertheless, different techniques including ultrasound-assisted extraction,

microwave-assisted extraction, supercritical fluid extraction, and accelerated solvent

extraction have been developed in order to decrease the extraction time, as well as the

solvent consumption, increasing the extraction yield and enhancing the extracts quality.

Solid-state fermentation (SSF) is another interesting technology that can be used for the

extraction and/or production of plant metabolites, able to provide extracts with both high

quality and biological activity, while precluding any toxicity associated to the use of

organic solvents.

Based on the reasons mentioned before, the main purpose of this thesis was to recover

bioactive compounds from L. tridentata leaves and evaluate their potential benefits for

human health. The research consisted of a sequence of tasks, which started by the study of

the nordihydroguaiaretic acid recovery by microwave-assisted extraction technique. The

maximum recovery of bioactive compounds was determined under specific conditions of

solvent concentration, solid-liquid ratio and extraction time. In the sequence, SSF was

evaluated as an environmentally friendly alternative method for the extraction of bioactive

VI

compounds from L. tridentata leaves. Finally, some studies were performed with the

objective of verifying the effects of the produced extracts for human health. The

antibacterial activity of the crude methanolic extract and fractions (hexane,

dichloromethane ethyl acetate, and ethanol) from L. tridentata leaves, as well as of the

pure NDGA against different bacteria species were studied. The cytotoxic activity of the

crude methanolic extract, fractions and pure compounds from L. tridentata leaves against

human cancer cell lines was also determined.

This work revealed that microwave-assisted extraction using methanol as extraction

solvent was a faster and more efficient method for NDGA recovery from L. tridentata

leaves when compared to the conventional heat-reflux method. Methanol in a

concentration of 90% (v/v) was the most efficient organic solvent to recover bioactive

compounds (NDGA, kaempferol and quercetin) from L. tridentata leaves by solid-liquid

extraction, comparing with other solvents used (ethanol, acetone and distilled water). This

plant has the particularity of having a high content of lignin (approximately, 36% w/w),

but when submitted to SSF with the fungus Phanerochaete chrysosporium (which has

ability to degrade lignin), neither a significant liberation nor an improvement of chemical

extraction of NDGA, K and Q occurred. However, some increase of the total phenolic,

flavonoids and protein contents in the extracts were obtained after the plant fermentation.

In terms of biological properties of the produced extracts, crude methanolic extract and

fractions, in particular ethyl acetate and dichloromethane fractions showed promising

results concerning antibacterial and cytotoxic activities, respectively. Further toxicological

and pharmacological studies will be useful to confirm the hypothesis of using

phytochemicals from L. tridentata leaves.

VII

RESUMO

As plantas são uma das principais fontes de compostos com propriedades biológicas de

grande interesse para a saúde humana. Larrea tridentata (Sessé & Moc. Ex DC.) Coville

(Zygophyllaceae), vulgarmente conhecida por gobernadora ou creosote bush, é uma planta

que cresce nas áreas semi-áridas no Sudoeste dos Estados Unidos da América e norte do

México. Esta planta era tradicionalmente usada durante séculos pelos povos indígenas

norte-americanos para o tratamento de diversas condições médicas e doenças, como

infecções do sistema urinário e respiratório, inflamações gerais, problemas de pele e rins,

artrite, diabetes, cancro, entre outras. Entre vários compostos fenólicos bioativos de valor

acrescentado presentes nas folhas de L. tridentata, o ácido nordihidroguaiarético (NDGA)

que surge naturalmente nesta planta é um lignano ao qual se atribui importantes atividades

biológicas como, por exemplo, atividade antioxidante, antiviral, antimicrobiana e

antitumoral. Outros compostos bioativos presentes nas folhas da L. tridentata em

concentrações consideráveis e com notáveis atividades biológicas são o kaenferol (K) e a

quercetina (Q).

A extração de compostos bioativos de plantas é convencionalmente realizada por um

sistema de aquecimento com refluxo. No entanto, diferentes métodos, incluindo extração

assistida por microondas e ultra-som, extração com fluído supercrítico, e extração

acelerada por solvente, entre outros, têm sido desenvolvidos com o objetivos de diminuir o

tempo de extração e o volume de solvente usado, aumentar os rendimentos de extração e

melhorar a qualidade dos extratos. A fermentação em estado sólido (FES) é uma outra

tecnologia interessante que pode ser usada na extração e/ou produção de metabolitos de

plantas capazes de proporcionar extratos com elevada qualidade e atividade biológica,

evitando o uso de solventes orgânicos potencialmente tóxicos.

Com base nas razões anteriormente mencionadas, o principal objetivo desta tese foi a

recuperação de compostos bioativos de folhas de L. tridentata e avaliação dos potenciais

benefícios desses compostos e extratos para a saúde humana. O trabalho de investigação

consistiu numa sequencia de tarefas, iniciando-se pelo estudo da recuperação de NDGA

através da técnica de extração assistida por microondas. O valor máximo recuperado de

compostos bioativos foi determinado sob condições específicas de concentração de

VIII

solvente, relação sólido-líquido e tempo de extração. Em sequência, foi avaliada a

fermentação em estado sólido como técnica alternativa e ecológica para a extração de

compostos bioativos de folhas de L. tridentata. Finalmente, alguns estudos foram

realizados no âmbito de verificar o efeito de extratos produzidos na saúde humana. Para

isso foi estudada a atividade antibacteriana de extrato metanólico bruto de folhas de L.

tridentata, de diversas frações deste mesmo extrato (hexano, diclorometano, acetato de

etilo e etanol), e do composto NDGA puro, contra diferentes espécies bacterianas. A

atividade citotóxica destas amostras, frente a várias linhas celulares cancerígenas, foi

igualmente determinada.

Através deste trabalho foi possível comprovar que a extração assistida por

microondas usando metanol como solvente foi mais rápida e eficiente na recuperação de

NDGA de folhas de L. tridentata quando comparada com a técnica convencional de

aquecimento com refluxo. Metanol a 90% (v/v) demonstrou ser o solvente orgânico mais

eficiente para a extração de compostos bioativos (NDGA, kaenferol e quercetina) de folhas

de L. tridentata por extração sólido-líquido, comparativamente a outros solventes (etanol,

acetona e água destilada). Esta planta possui a particularidade de possuir uma concentração

elevada de lignina (aproximadamente, 36 % p/p) mas quando submetida a FES com o

fungo Phanerochaete chrysosporium (reconhecido pela sua capacidade de degradar

lignina), não se observou uma libertação ou recuperação química significativas de NDGA,

K e Q. No entanto, verificou-se um aumento nas concentrações de fenólicos totais,

flavonóides e proteínas nos extratos obtidos após FES do material vegetal. Em relação às

propriedades biológicas dos extratos produzidos, o extrato bruto metanólico e frações, em

particular a fração de diclorometano e acetato de etilo demonstraram resultados

promissores no que diz respeito a atividade antibacteriana e citotóxica, respectivamente.

Outros estudos toxicológicos e farmacêuticos são necessários de modo a confirmar a

hipótese de usar os fitoquímicos de folhas de L. tridentata.

IX

LIST OF PUBLICATIONS

This thesis is based on the work presented in the following publications:

Sílvia Martins, Diego Mercado, Marco Mata-Gómez, Luis Rodriguez, Antonio Aguilera-

Carbo, Raul Rodriguez and Cristóbal N. Aguilar (2010). Microbial production of potent

phenolic-antioxidants through solid state fermentation. In: Sustainable Biotechnology:

sources of renewable energy. Singh Om V and Steven P (Eds), Biomedical Sciences,

Springer: Germany.

S. Martins, Aguilar C.N., Garza-Rodriguez I., Mussatto S.I., Teixeira J.A. (2010). Kinetic

Study of Nordihydroguaiaretic Acid Recovery from Larrea tridentata by Microwave-

assisted Extraction. Journal of Chemical Technology and Biotechnology, 85 (8), 1142-

1147.

Sílvia Martins, Solange I. Mussatto, GuillermoMartínez-Avila, JulioMontañez-Saenz,

Cristóbal N. Aguilar, Jose A. Teixeira (2011). Bioactive phenolic compounds: Production

and extraction by solid-state fermentation. A review. Biotechnology Advances, 29 (3), 365-

373.

Sílvia Martins, Solange I. Mussatto, Cristóbal N. Aguilar, Jose A. Teixeira (2012).

Bioactive compounds (phytoestrogens) recovery from Larrea tridentata leaves by solvents

extraction. Separation and Purification Technology, 88, 163-167.

Solange I. Mussatto, Lina F. Ballesteros, Silvia Martins & José A. Teixeira (2012). Use of

agro-industrial wastes in solid-state fermentation processes. In: Industrial Waste. Intech:

Croatia, ISBN 979-953-307-543-2.

Sílvia Martins, Elba L.C. Amorim, Tadeu J.S. Peixoto Sobrinho, Antonio M. Saraiva,

Maria N.C. Pisciottano, Cristóbal N. Aguilar, José A. Teixeira, Solange I. Mussatto

(2012). Antibacterial activity of crude methanolic extract and fractions obtained from

X

Larrea tridentata leaves. Industrial Crops and Products, Accepted.

(http://dx.doi.or/10.1016/j.indcrop.2012.04.037)

Sílvia Martins, Elba L.C. Amorim, Tadeu J.S. Peixoto Sobrinho, Teresinha G. da Silva,

Gardénia Militão, José A. Teixeira, Solange I. Mussatto (2012). In vitro cytotoxic activity

of crude extract and fractions obtained from Larrea tridentata leaves against cancer cell

lines. Submitted.

Sílvia Martins, Cristóbal N. Aguilar, José A. Teixeira, Solange I. Mussatto (2012).

Chemical characterization and solid state fermentation of Larrea tridentata leaves by

Phanerochaete chrysosporium. Submitted.

Sílvia Martins, Cristina Pereira-Wilson C, Cristovão F. Lima, José A. Teixeira, Solange I.

Mussatto (2012). Phytochemicals from Larrea tridentata leaves has inhibitors of

proliferation and inducers of apoptosis in human colorectal cancer cells. Submitted.

XI

TABLE OF CONTENTS

Agradecimentos III Abstract V Resumo VII List of Publications IX Table of Contents XI List of Figures XIV List of Tables XVI List of General Nomenclature XVIII Chapter 1. Motivation and Outline 1.1 Thesis Motivation 3 1.2 Research Aims 4 1.3 Outline of the thesis 5 1.4 References 6 Chapter 2. Bioactive phenolic compounds: Production and extraction by solid-state fermentation

2.1 Introduction 11 2.2 Bioactive compounds 11 2.3 Solid-state fermentation (SSF) 14 2.4 Uses of SSF for bioactive compounds production 19

2.4.1 Phenolic content increase in food products 19 2.4.2 Production and extraction of bioactive phenolic compounds from agro-industrial residues

20

2.4.3 Production and extraction of bioactive phenolic compounds from plants 24 2.5 Concluding remarks and future perspectives 25 2.6 References 25 Chapter 3. Kinetic study of nordihydroguaiaretic acid recovery from Larrea tridentata by microwave-assisted extraction

3.1 Introduction 38 3.2 Materials and methods 39

3.2.1 Plant material and chemicals 39 3.2.2 Extraction methodologies 40 3.2.3 HPLC analysis 42 3.2.4 Determination of kinetic parameters and extraction time 42 3.2.5 Scanning electron microscopy 43 3.2.6 Free radical scavenging effectiveness of Larrea tridentata extracts 43 3.2.7 Statistical analysis 44

3.3 Results and discussion 44 3.3.1 Parameters affecting the NDGA extraction 44

3.3.2 Comparison of NDGA extraction by MAE and HRE 47 3.3.3 Effectiveness of Larrea tridentata extracts on free radical scavenging 51

XII

3.4 Conclusion 52 3.5 References 52 Chapter 4. Bioactive compounds (phytoestrogens) recovery from Larrea tridentata leaves by solvents extraction


4.2.1 Plant material and chemicals 59 4.2.2 Extraction methodology 60 4.2.3 Bioactive compounds quantification 60 4.2.4 Determination of total phenols content 60 4.2.5 Determination of total flavonoids content 61 4.2.6 Determination of protein content 61 4.2.7 Free radical scavenging activity 61 4.2.8 Ferric reducing antioxidant power assay (FRAP assay) 62 4.2.9 Statistical analysis 62

4.3 Results and discussion 63 4.3.1 Effect of organic solvents on the extraction of phytoestrogens 63 4.3.2 Effect of organic solvents on total phenols, total flavonoids and protein

contents

64 4.3.3 Antioxidant potential of Larrea tridentata extracts 66

4.4 Conclusion 67 4.5 References 68 Chapter 5. Solid state fermentation of Larrea tridentata leaves by Phanerochaete chrysosporium


5.2.1 Plant material and chemicals 74 5.2.2 Chemical characterization 74 5.2.3 Solid-state fermentation process 75

5.2.3.1 Fungi and spores collection 75 5.2.3.2 Solid-state fermentation conditions 75

5.2.4 Fourier transform infrared (FTIR) assay 78 5.2.5 Scanning electron microscopy analysis 78 5.2.6 Bioactive compounds quantification 78 5.2.7 Determination of total phenols content 79 5.2.8 Determination of total flavonoids content 79 5.2.9 Determination of protein content 80 5.2.10 Total antioxidant capacity 80 5.2.11 Statistical analysis 80

5.3 Results and discussion 80 5.3.1 Chemical characterization of Larrea tridentata leaves 80 5.3.2 FTIR and SEM measurements 83 5.3.3 Bioactive compounds extraction by SSF 85

5.4 Conclusion 88 5.5 References 89

XIII

Chapter 6. Antibacterial activity of crude methanolic extract and fractions obtained from Larrea tridentata leaves


6.2.1 Plant material and chemicals 96 6.2.2 Extraction methodology and fractioning 96 6.2.3 Antibacterial activity assays 97 6.2.3.1 Bacterial strains 97

6.2.3.2 Antibacterial test using the agar diffusion method (well) 97 6.2.4 Determination of minimal inhibitory concentration (MIC) 98

6.2.5 Bioactive compounds quantification 99 6.3 Results and discussion 100

6.3.1 Antibacterial activity by the agar diffusion method 100 6.3.2 Evaluation of minimal inhibitory concentration (MIC) 103 6.3.3 HPLC analysis of tested samples 104

6.4 Conclusion 107 6.5 References 108 Chapter 7. In vitro cytotoxic activity of crude extract and fractions obtained from Larrea tridentata leaves against human cancer cell lines


7.2.1 Plant material and chemicals 114 7.2.2 Extraction methodology and fractioning 115 7.2.3 Phytochemical study by thin layer chromatography 115 7.2.4 Measurement of cytotoxic activity 116

7.2.4.1 Culture of cell lines 116 7.2.4.2 Cell viability/proliferation assay 116 7.2.4.3 Apoptotic nuclear condensation assay 117

7.2.5 Bioactive compounds quantification 118 7.2.6 Statistical analysis 118

7.3 Results and discussion 119 7.3.1 Phytochemical profile of extract and fractions from Larrea tridentata leaves 119 7.3.2 Cytotoxicity of Larrea tridentata leaves extract and fractions on cancer cell

lines 120

7.4 Conclusion 128 7.5 References 128 Chapter 8. General Conclusions 133 8.1 Conclusions 135 8.2 Recomendations 123

XIV

LIST OF FIGURES

CHAPTER 2

Fig. 2.1. Examples of naturally occurring flavonoids. 12 Fig. 2.2. Examples of naturally occurring phenolic acids. 13

CHAPTER 3

Fig. 3.1. Chemical structure of NDGA. 38

Fig. 3.2. Development of the water-bath temperature at 70°C during the conventional heat-reflux extraction of NDGA from Larreatridentata leaves. The symbols (●) represent the experimental values of temperature, and the solid line represents the fitted temperature course using equation (1). .

41

Fig. 3.3. Effect of methanol concentration on NDGA extraction from Larrea tridentata leaves by MAE under the following conditions: 1 g plant/ 30 mL solvent, 70°C,800W, for 4 min. abc

Values in a column with the same superscripts are not significantly different at p<0.05.

45

Fig. 3.4. Effect of methanol concentration on NDGA extraction from Larrea tridentata leaves by HRE under the following conditions: 1 g plant/ 30 mL solvent, 70°C, for ( ) 1 and ( ) 3 h.abcd


46

Fig. 3.5. Effect of solid/liquid ratio on NDGA extraction from Larrea tridentata leaves by MAE using methanol 50% (v/v) as solvent, at 70°C,800W, for 4 min. ab


47

Fig. 3.6. Kinetic study of NDGA extraction from Larrea tridentata leaves by MAE (●) and HRE () using 1 g plant material/ 10 mL methanol 50 % (v/v), at 70 ºC and 800 W. The symbols represent the experimental NDGA values and the solid line represents the fitted data to a first-order kinetic model (equation (2)).

48

Fig. 3.7. Micrographs, by scanning electron microscopy of Larrea tridentata samples in the following forms: (A) untreated; (B) after MAE; and (C) and after conventional HRE. Magnification: 500-fold.

50

Fig. 3.8. Effect of different concentrations of extracts obtained by MAE and HRE from Larrea tridentata leaves in free radical DPPH scavenging activity ( NDGA positive control, extract obtained by HRE, ● extract obtained by MAE).

51

XV

CHAPTER 4 Fig. 4.1. Chemical structure of NDGA (A), kaempferol (B) and quercetin (C). 58

CHAPTER 5 Fig. 5.1. Schematic flow diagram of experimental steps proposed for SSF of L. tridentata leaves using P. chrysosporium.

77

Fig. 5.2. FTIR spectra of Larrea tridentata samples before (A) and after 21 days of SSF (B) with P. chrysosporium at pH 5.0, at temperature 37 ºC and humidity 70%.

83

Fig. 5.3 Micrographs by scanning electron microscopy of L. tridentata samples in the following forms: (A,C) untreated and (B,D) fungal treated (after 21 days SSF). Magnification: 300-fold (A and B) and 1000-fold (C and D).

84

CHAPTER 6 Fig. 6.1. HPLC chromatograms of crude methanolic extract, CME (A), dichloromethane, DCM (B) and ethyl acetate, EA (C) fractions from L. tridentata leaves (Q: quercetin; K: kaempferol; NDGA: nordihydroguaiaretic acid).

106

CHAPTER 7 Fig. 7.1. Effect on cell viability/ proliferation of different concentrations of (A) crude methanolic extract, (B) dichloromethane fraction (DCM), and (C) pure nordihydroguaiaretic acid (NDGA), for 48 h of treatment, in HCT116 colon carcinoma cells, using MTT assays. Results are presented as mean ± standard deviation of at least 3 independent experiments. *p≤ 0.05, *** p≤ 0.01, and *** p≤ 0.001.

125

Fig. 7.2. Dose-response curves for IC50

determination for the (A) crude methanolic extract (CME), (B) dichloromethane fraction (DCM), and (C) pure nordihydroguaiaretic acid (NDGA), for 48 h of treatment, in HCT116 colon carcinoma cells, using MTT assays. Results are presented as mean ± standard deviation of at least 3 independent experiments. * p≤0.05, *** p≤ 0.01, and *** p≤0.001.

126

Fig. 7.3. Effect on nuclear condensation of different concentrations of crude methanolic extract, dichloromethane fraction (DCM), and pure nordihydroguaiaretic acid (NDGA), for 48 h, in HCT116 colon carcinoma cells. The control used was dimethyl sulfoxide (DMSO) and quercetin as a reference coumpound. Results are presented as mean ± standard deviation of at least 3 independent experiments. * p≤0.05, *** p≤0.01, and *** p≤0.001.

127

XVI

LIST OF TABLES

CHAPTER 2 Table 2.1. Examples of secondary metabolites produced with higher yield by solid-state fermentation than by submerged fermentation (Hölker et al., 2004).

16

Table 2.2. Recent studies of solid-state fermentation using different microorganisms and solid supports.

18

Table 2.3. Enzymes produced during solid-state fermentation by lignocellulolytic fungi in several agro-industrial residues.

23

CHAPTER 3 Table 3.1. Kinetic parameters and extraction times obtained for NDGA extracted from L. tridentata leaves by HRE and MAE.

49

CHAPTER 4 Table 4.1. Phytoestrogens extraction from L. tridentata leaves using different organic solvents.

64

Table 4.2. Total phenols, flavonoids and protein contents in L. tridentata leaves extracts obtained by using different organic solvents.

65

Table 4.3. Effect of different organic solvents on antioxidant capacity of L. tridentata leaves extracts.

67

CHAPTER 5 Table 5.1. Chemical characterization of L. tridentata leaves. 81

Table 5.2. Mineral and nonmineral contents in L. tridentata leaves.

82

Table 5.3. Effect of SSF with P. chrysosporium during 21 days on the recovery of some bioactive compounds from L. tridentata leaves extracts.

86

Table 5.4. Total phenols, flavonoids and protein contents, and total antioxidant capacity in L. tridentata leaves extracts obtained after SSF with P. chrysosporium during 21 days, and in the extracts obtained by methanolic extraction of fermented plant material.

87

XVII

Table 5.5. Effect of SSF on total organic carbon (TOC) present in L. tridentata leaves. 88

CHAPTER 6 Table 6.1. Antibacterial activity of crude methanolic extract and fractions obtained from L. tridentata leaves.

101

Table 6.2. Minimum inhibitory concentration (MIC, in µg/mL) of crude methanolic extract and fractions obtained from L. tridentata leaves on growth of different bacteria strains.

103

Table 6.3. Quantification of quercetin, NDGA and kaempferol (in mg/g of plant material) in crude methanolic extract and fractions from L. tridentata leaves.

107

CHAPTER 7 Table 7.1. Phytochemical analysis of L. tridentata leaves using thin layer chromatography.

120

Table 7.2. Cytotoxic activity screening (inhibition of cell viability, in %) of the crude methanolic extract and fractions obtained from L. tridentata leaves on three tumor cell lines measured by the MTT assay.

122

Table 7.3. Quantification of quercetin, NDGA and kaempferol (in mg/g of plant material) in crude methanolic extract and fractions from L. tridentata leaves.

123

XVIII

LIST OF GENERAL NOMENCLATURE

SYMBOL DESCRIPTION

A Absorbance of the control c

A Absorbance of the sample S

C Reference values obtained in a previous research work (Martins et al., 2012)

0

C Control samples (without inoculation with P. chrysosporium) after 10 days under SSF conditions

10

C Control samples (without inoculation with P. chrysosporium) after 21 days under SSF conditions

21

EC Half maximal effective concentration 50

IC Inhibition of cell viability/proliferation by 50 % 50

k Heat transfer coefficient

r Correlation coefficient xy

Time t

T Real water-bath temperature

T Theoretical water-bath temperature b

ABBREVIATIONS

ABBREVIATION DESCRIPTION

ANOVA Analysis of variance

ATCC American type culture collection

CFU Colony forming unit

CLSI Clinical and Laboratory Standards Institute

XIX

DCM Dichloromethane

DMSO Dimetilsufoxide

DPPH 1,1-diphenyl-2-picrylhydrazyl

DW Dry weight

EA Ethyl acetate

Ec1 Escherichia coli standard strain ATCC 10536

Ec2 Escherichia coli standard strain ATCC 30218

Ef1 Enterococcus faecalis standard strain ATCC 51299

Ef2 Enterococcus faecalis isolated from urine

Et Ethanol

ER Estrogen receptors

FE Fermentative extract

FRAP Ferric reducing/antioxidant power

FTIR Fourier transform infrared

GAE Gallic acid equivalent

HPLC High performance liquid chromatography

HRE Heat-reflux extraction

K Kaempferol

Kp1 Klebsiella pneumonia isolated isolated from secretion

Kp2 Klebsiella pneumonia from surgical wound secretion

MAE Microwave-assisted extraction

MIC Minimal inhibitory concentration

MRSA methicillin-resistant Staphylococcus aureus

MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NDGA Nordihydroguaiaretic acid

Pa1 Pseudomonas aeruginosa standard strain ATCC 14502

Pa2 Pseudomonas aeruginosa isolated from blood

XX

PE Plant extract

PTFE Polytetrafluoroethylene

Q Quercetin

QE Quercetin equivalent

Sa1 Staphylococcus aureus standard strain ATCC 6538

Sa2 MRSA strain isolated from secretion

Se Staphylococcus epidermidis isolated from sperm

SEM Scanning electron microscopy

SPSS Statistical Package for Social Sciences

Ss Staphylococcus saprophyticus standard strain LACEN

SSF Solid-state fermentation

TAA Total antioxidant activity

TLC Thin layer chromatography

TN Total nitrogen

TOC Total organic carbon

TPTZ 2,4,6-tris(1-pyridyl)-5-triazine

CHAPTER 1

Context, Aim and Thesis Outline

The motivation and outline, and research aims of this work are approached in this chapter,

where a general overview of the thesis is provided.

CHAPTER 1

CONTEXT, AIM AND THESIS OUTLINE

3

1.1 THESIS MOTIVATION

Plants are one of the primordial sources of bioactive phytochemicals and have been

used since ancient times by human being for health purposes. The traditional use of

medicinal plants provides essential information about plant’s therapeutic potential,

allowing the development of clearer and focused studies about the biological activity of

plants extracts. Larrea tridentata (Sessé & Moc. Ex DC.) Coville (Zygophyllaceae),

commonly known as creosote bush, is a plant traditionally used for centuries by North

American Indians to treat medical conditions and illnesses including genitor-urinary and

respiratory tract infections, inflammation of the musculoskeletal system, damage to the

skin, kidney problems, arthritis, diabetes, cancer, among other diseases (Brinker, 1993;

Ross, 2005). Among several interesting bioactive phenolic compounds found in this

plant (such as quercetin, kaempferol and nordihydroguaiaretic acid (NDGA)), the natural

occurring lignan NDGA has been point out as the most important since it presents

biological activities of large interest in the health area, such as antiviral, antifungic,

antimicrobial, and antitumorgenic (Hwu et al., 2008; Fujimoto et al., 2004; Lambert et

al., 2004).

Extraction of bioactive compounds from plants is conventionally performed by

heat-reflux method. Nevertheless, different techniques including ultrasound-assisted

extraction, microwave-assisted extraction, supercritical fluid extraction, and accelerated

solvent extraction have been developed in order to decrease the extraction time, as well

as the solvent consumption, increasing the extraction yield, and enhancing the extracts

quality (Pascual-Martí et al., 2001; Pinelo et al., 2008; Ma et al., 2009). Solid-state

fermentation (SSF) processes can also be an interesting technology for the extraction

and/or production of plant metabolites, , able to provide extracts with both high quality

and biological activity, while precluding any toxicity associated to the use of organic

solvents (Kumar et al., 2006).

L. tridentata is an outstanding source of natural compounds with approximately

50% of the leaves (dry weight) being extractable matter (Arteaga et al., 2005). Besides

the interest in maximizing the extraction of bioactive compounds from this plant using

different techniques, it becomes important to find environmentally friendly technologies

CHAPTER 1


4

able to extract and/or produce extracts with low or none environmental impact, as is the

case of the SSF process. Another important goal of this work is the achievement of

scientific data related to the biologic activities of the produced extract.

1.2 RESEARCH AIMS

The main purpose of this thesis was to maximize the extraction of bioactive compounds

from L. tridentata leaves using different techniques, and to evaluate the biological

activities of the produced extracts. The main focus areas were:

• Physicochemical characterization of L. tridentata leaves;

• Evaluation of different extraction methodologies in order to maximize the

bioactive compounds recovery from L. tridentata leaves;

• Evaluation of the possibility of using solid-state fermentation as an alternative

technique for the extraction of bioactive compounds from L. tridentata leaves;

• Determination of the antibacterial activity of crude methanolic extract and

fractions obtained from L. tridentata leaves;

• Determination of the in vitro activity of crude methanolic extract and fractions

obtained from L. tridentata leaves against human cancer cell lines.

CHAPTER 1


5

1.3 OUTLINE OF THE THESIS

This thesis comprises eight chapters. In this chapter the motivation, research aims and

the thesis outline are described. CHAPTER 2 presents an overview about bioactive

compounds and solid-state fermentation (SSF) systems, focusing on the production and

extraction of bioactive phenolic compounds from natural sources. The characteristics of

SSF and variables that affect the product formation by this process, as well as the variety

of substrates and microorganisms that can be used in SSF for the production of bioactive

phenolic compounds are reviewed and discussed. The Chapters 3 to 7 contain the main

experimental results, distributed as follows:

In CHAPTER 3 a rapid and effective microwave-assisted extraction (MAE) method for

nordihydroguaiaretic acid (NDGA) recovery from L. tridentata leaves was established,

and the obtained results were compared with those achieved by using the conventional

heat-reflux extraction (HRE). Micrographs of plant material samples (untreated and

treated by MAE and HRE) were obtained with the objective of verifying if the

improvement of NDGA extraction by MAE could be related to a greater extent of cell

rupture of the plant material. The antioxidant potential of the L. tridentata extracts

produced by MAEwas also evaluated.

CHAPTER 4 shows the effect of different organic solvents on the extraction of bioactive

compounds from L. tridentata leaves, namely, NDGA, kaempferol and quercetin. The

antioxidant potential of the produced extracts, as well as the contents of total phenols,

flavonoids and proteins, were also determined and discussed.

CHAPTER 5 explores the potential of the basidiomycete Phanerochaete chrysosporium,

known by its ability to degrade lignin, to recover or enhance the extraction of bioactive

compounds (nordihydroguaiaretic acid, kaempferol and quercetin) from L. tridentata

leaves by solid-state fermentation. A chemical characterization, as well as a mineral

profile of L. tridentata leaves were previously determined and considered. The contents

of total phenolic, flavonoids, and proteins, and the antioxidant activity of the produced

extracts were analyzed. Finally, micrographs by scanning electron microscopy and

Fourier transform infrared (FTIR) spectra were obtained in order to evaluate the capacity

of P. chrysosporium to degrade lignin from L. tridentata leaves.

CHAPTER 1


6

CHAPTER 6 presents the antibacterial activity of the crude methanolic extract (CME) and

fractions (hexane, dichloromethane, ethyl acetate and ethanol) obtained from L.

tridentata leaves. Quantification of bioactive compounds (NDGA, kaempferol and

quercetin) in CME and fractions by high performance liquid chromatography was

performed to underlie their antibacterial characteristics.

CHAPTER 7 was designed to evaluate the in vitro cytotoxic activity of the crude

methanolic extract (CME) and fractions (hexane, dichloromethane, ethyl acetate and

ethanol) obtained from L. tridentata leaves against cancer cell lines. A phytochemical

study by thin layer chromatography and high performance liquid chromatography of the

CME and fractions was also performed in order to obtain a more extended knowledge

about these samples.

Finally, CHAPTER 8 presents the overall conclusions, recommendations and suggestions

for future works.

1.4 REFERENCES

Arteaga S., Andrade-Cetto A., Cárdenas R. (2005). Larrea tridentata (Creosote bush), an

abundant plant of Mexican and US-American deserts and its metabolite

nordihydroguaiaretic acid. Journal of Ethnopharmacoly, 98, 231–239.

Brinker F. (1993). Larrea tridentata (D.C.) Coville (Chaparral or Creosote Bush). British

Journal of Phytotherapy, 3, 10–30.

Fujimoto N., Kohta R., Kitamura S., Honda H. (2004). Estrogenic activity of an

antioxidant, nordihydroguaiaretic acid (NDGA). Life Sciences, 74, 1417-1425.

Hwu J.R., Hsu M.H., Huang R.C. (2008). New nordihydroguaiaretic acid derivates as

anti-HIV agents. Bioorganic and Medicinal Chemistry Letters, 18, 1884–1888.

Kumar A.G., Sekaran G., Krishnamoorthy S. (2006). Solid state fermentation of Achras

zapota lignocellulose by Phanerochaete chrysosporium. Bioresource Technology,

97, 1521-1528.

CHAPTER 1


7

Lambert J.D., Dorr R.T., Timmermann N. (2004) Nordihydroguaiaretic acid: a review of

its numerous and varied biological activities. Pharmaceutical Biology, 42, 149-

158.

Ma Y.Q., Chen J.C., Liu D.H., Ye X.Q. (2009). Simultaneous extraction of phenolic

compounds of citrus peel extracts: effect of ultrasound. Ultrasonic Sonochemistry,

16, 57-62.

Pascual-Martí M.C., Salvador A., Chafer A., Berna A. (2001). Supercritical fluid

extraction of resveratrol from grape skin of Vitis vinifera and determination by

HPLC. Talanta, 54, 735-740.

Pinelo M., Zornoza B., Meyer A.S. (2008). Selective release of phenols from apple skin:

mass transfer kinetics during solvent and enzyme-assisted extraction. Separation

and Purification Technology, 63, 620-627.

Ross I.A. (2005). Medicinal plants of the world - Chemical constituents, traditional and

modern medicinal uses (Vol. 3). Humana Press, New Jersey.

9

CHAPTER 2

Bioactive phenolic compounds: Production and extraction by

solid-state fermentation

This chapter provides a general overview about bioactive compounds with the ability to

promote benefits to human health, pointing out solid-state fermentation systems as an

alternative to produce or extract these compounds from natural sources.

10

CHAPTER 2

BIOACTIVE PHENOLIC COMPOUNDS: PRODUCTION AND EXTRACTION BY SOLID-STATE FERMENTATION

11

2.1 Introduction

Interest in the development of bioprocesses for the production or extraction of

bioactive compounds from natural sources has increased in recent years due to the

potential applications of these compounds in food and pharmaceutical industries. In this

context, solid-state fermentation (SSF) has received great attention because this

bioprocess has potential to successfully convert inexpensive agro-industrial residues, as

well as plants, in a great variety of valuable compounds, including bioactive phenolic

compounds. The aim of this review, after presenting general aspects about bioactive

compounds and SSF systems, is to focus on the production and extraction of bioactive

phenolic compounds from natural sources by SSF. The characteristics of SSF systems

and variables that affect the product formation by this process, as well as the variety of

substrates and microorganisms that can be used in SSF for the production of bioactive

phenolic compounds are reviewed and discussed.

2.2 Bioactive compounds

Bioactive compounds are extra nutritional constituents that naturally occur in small

quantities in plant and food products (Kris-Etherton et al., 2002). Most common

bioactive compounds include secondary metabolites such as antibiotics, mycotoxins,

alkaloids, food grade pigments, plant growth factors, and phenolic compounds (Hölker et

al., 2004; Kris-Etherton et al., 2002; Nigam, 2009). Phenolic compounds comprise

flavonoids, phenolic acids, and tannins, among others. Flavonoids constitute the largest

group of plant phenolics, accounting for over half of the eight thousand naturally

occurring phenolic compounds (Harborne et al., 1999). Variations in substitution patterns

to ring C in the structure of these compounds result in the major flavonoid classes, i.e.,

flavonols, flavones, flavanones, flavanols, isoflavones, and anthocyanidins. Fig. 2.1

shows examples of the most common naturally occurring flavonoids. Similarly to the

flavonoids, phenolic acids constitute also an important class of phenolic compounds with

bioactive functions, usually found in plant and food products. Phenolic acids can be

divided in two subgroups according to their structure: the hydroxybenzoic and the

hydroxycinnamic acids (Fig. 2.2).

CHAPTER 2


12

Fig. 2.1. Examples of naturally occurring flavonoids.

ISOFLAVONES FLAVANOLS

FLAVONES FLAVONOLS

Kaempferol

OH O

OOH

OH

OH

Quercetin

OH O

OOH

OH

OH

OH

Myricetin

OH O

OOH

OH

OH

OH

OH

OH O

OOH

Daidzein

OH

O

O

OOH

CH3

Glycitein

OH

OH

O

OOH

Genistein

Daidzin

O

OH

OH

OH

O

OH

O

OOH

Glycitin

O

OH

OH

OH

O

OH

O

OOH

O

CH3

Genistin

O

OH

OH

OH

O

OH

O

OOH

OH

OH

OH

O

OH

OH

OH

Catechin

OH

OH

O

OH

OH

OH

Epicatechin

OH

OH

O

O

OH

OH

O

OH

OH

OH

Catechin gallate

OH

OH

O

OH

OH

OH

OH

Epigallocatechin

OH

OH

O

O

OH

OH

O

OH

OH

OH

Epicatechin gallate

OH

OH

O

O

OH

OH

O

OH

OH

OH

OH

Epigallocatechin gallate

Chrysin

OH O

OOH

Apigenin

OH O

OOH

OH Luteolin

OH O

OOH

OH

OH

FLAVANONES

Naringin

O

O

OHO

OH

O

O

OH OH

OH

OOH

OH

OH OH

Naringenin

O

OHO

OH

OH

Taxifolin

O

O

OH

OH

OH

OH

OH

ANTHOCYANIDINS

Cyanidin

O+

OH

OH

OH

OH

OH

Malvidin

O+

OH

OH

OH

OH

O

OCH3

CH3

Rutin

O

OHOH

OH

O

O

OH

OH O

OH

OH

OOH

OH

OH OH

O

CHAPTER 2


13

The most commonly found hydroxybenzoic acids include gallic, p-

hydroxybenzoic, protocatechuic, vanillic and syringic acids, while among the

hydroxycinnamic acids, caffeic, ferulic, p-coumaric and sinapic acids can be pointed out

(Bravo, 1998).

Fig. 2.2. Examples of naturally occurring phenolic acids.

In the last few years, greatly attention have been paid to the bioactive compounds

due to their ability to promote benefits for human health, such as the reduction in the

incidence of some degenerative diseases like cancer and diabetes (Conforti et al., 2009;

Kim et al., 2009), reduction in risk factors of cardiovascular diseases (Jiménez et al.,

2008; Kris-Etherton et al., 2002), antioxidant, anti-mutagenic, anti-allergenic, anti-

inflammatory, and anti-microbial effects (Balasundram et al. 2006; Ham et al. 2009;

Parvathy et al. 2009), among others. Due to these countless beneficial characteristics for

HYDROXYBENZOIC ACIDS HYDROXYCINNAMIC ACIDS

OH

OH

OH

OH

O

Gallic acid Protocatechuic acidOH

OH

O

OH

Vanill ic acid

O

OH

CH3

OH

O

p-Hy droxybenzoi c acid

OH

O

OH

Ferulic acid

O

OH

OH

O

CH3

Caffeic acidOH

OH

OH

O

p-Coumaric acid

OH

OH

O

Sinapic a cid

OH

OH

O

O

O

CH3

CH3

Ellagic acid

O

O

OH

OH

O

OH

OH

O

Gentisic acid

OH

O

OH

OH

Syringic acid

OH

O

O

O

OH

CH3

CH3

Salicylic acid

O H

O

OH

Cinnamic acid

O H

O

Chlorogenic acid

O

O

OH

OH

OHOHOH

OOH

OH

OHOH

OHOH

O

Quinic a cid

CHAPTER 2


14

human health, researches have been intensified aiming to find fruits, vegetables, plants,

agricultural and agro-industrial residues as sources of bioactive phenolic compounds.

Usually, bioactive compounds are recovered from natural sources by solid-liquid

extraction employing organic solvents in heat-reflux systems. However, other techniques

have been recently proposed to obtain these compounds including the use of supercritical

fluids, high pressure processes, microwave-assisted extraction and ultrasound-assisted

extraction (Cortazar et al. 2005; Markom et al. 2007; Wang and Weller, 2006).

Extraction/production of bioactive compounds by fermentation is also an interesting

alternative that merits attention, since it is able to provide high quality and high activity

extracts while precluding any toxicity associated to the organic solvents. In this process,

bioactive compounds are obtained as secondary metabolites produced by

microorganisms usually during the later stage of microbial growth, after the microbial

growth is completed (Nigam, 2009). Studies on liquid culture show that the production

of these compounds starts when growth is limited by the exhaustion of one key nutrient:

carbon, nitrogen or phosphate source (Barrios-González et al., 2005).

The purpose of this article is to provide an overview of the bioactive phenolic

compounds extraction and production by fermentation, more specifically by the solid-

state fermentation technique. The current status of this technology, the microorganisms,

substrates and cultivation conditions affecting the phenolic compounds formation are

summarized and discussed.

2.3 Solid-state fermentation (SSF)

Fermentation processes may be divided into two systems: submerged fermentation

(SmF), which is based on the microorganisms cultivation in a liquid medium containing

nutrients, and solid-state fermentation (SSF), which consists in the microbial growth and

product formation on solid particles in absence (or near absence) of water; however,

substrate contains the sufficient moisture to allow the microorganism growth and

metabolism (Pandey, 2003). In recent years, SSF has received more interest from

researchers since several studies have demonstrated that this process may lead to higher

yields and productivities or better product characteristics than SmF. In addition, due to

the utilization of low cost agricultural and agro-industrial residues as substrates, capital

CHAPTER 2


15

and operating costs are lower compared to SmF. The low water volume in SSF has also a

large impact on the economy of the process mainly due to smaller fermenter-size,

reduced downstream processing, reduced stirring and lower sterilization costs (Holker

and Lenz, 2005; Nigam, 2009; Pandey, 2003; Raghavarao et al., 2003). The main

drawback of this type of cultivation concerns the scaling-up of the process, largely due to

heat transfer and culture homogeneity problems (Di Luccio et al., 2004; Mitchell et al.,

2000). However, research attention has been directed towards the development of

bioreactors that overcome these difficulties.

Although many bioactive compounds are still produced by SmF, in the last

decades, there has been an increasing trend towards the utilization of the SSF technique

to produce these compounds since this process has been shown more efficient than SmF

(Nigam, 2009). Table 1 shows several examples of bioactive secondary metabolites that

were demonstrated to be obtained with significantly higher yield by SSF than by SmF.

Besides the higher yields, SSF has also been reported as a technique able to produce

secondary metabolites in shorter times than SmF, without the need of aseptic conditions,

and with capital costs significantly lesser.

Several important factors must be considered for the development of a successful

bioprocess under SSF conditions. Some of the most important include the selection of a

suitable microorganism strain and the solid support to be used. A variety of

microorganisms, including fungi, yeasts and bacteria may be used in SSF processes;

however, due to the low moisture content in the fermentation media, fungi and yeasts are

the most commonly used microorganisms due to their ability to growth in environments

with this characteristic. However, the choice of the microorganism to be used in SSF

depends on the desired end product. Filamentous fungi have great potential to produce

bioactive compounds by SSF, and therefore, they are the most commonly used

microorganisms for this purpose (Aguilar et al., 2008; Nigam, 2009; Topakas et al.,

2003a). Filamentous fungi have also received great attention due to their ability in

producing thermostable enzymes of high scientific

and commercial value

(Christakopoulos et al., 1990; Martins et al., 2002).

CHAPTER 2


16

Table 2.1 Examples of secondary metabolites produced with higher yield by solid-state

fermentation than by submerged fermentation (Hölker et al., 2004).

Product Microorganism 6-pentyl-alpha-pyrone Trichoderma harzianum Bafilomycin B1 + C1 Streptomyces halstedii K122 Benzoic acid Bjerkandera adusta Benzyl alcohol Bjerkandera adusta Cephamycin C Streptomyces clavuligerus Coconut aroma Trichoderma sp. Ergot alkaloids Claviceps fusiformis Giberellic acid Giberella fujikuroi Iturin Bacillus subtillis Ochratoxin Aspergillus ochraceus Oxytetracycline Streptomyces rimossus Penicillin Penicillium chrysogenum Rifamycin-B Amycolatopsis mediterranei Tetracycline Streptomyces viridifaciens

The right selection of the solid substrate is also of great importance for an efficient

and economical production of the compound of interest. Mostly the production yields of

secondary metabolites can be improved with a right choice of substrate or mixture of

substrates with appropriate nutrients (Nigam, 2009). As a whole, the support material

must present characteristic favorable for the microorganism development and be of low

cost. These characteristics are easily found in many natural materials proceeding from

agricultural and agro-industrial activities. In addition, the use of agricultural and agro-

industrial residues as carbon sources through SSF provides an important way to reduce

the fermentation cost and avoid environmental problems caused by their disposal, being

an economical and interesting solution for countries with abundance of these materials.

Several of these residues, including coffee pulp and husk, sugarcane and agave bagasses,

fruit pulps and peels, corn cobs, among others, have been used as supports and/or

substrates for the production of valuable compounds by SSF, such as enzymes

(Guimarães et al., 2009; Mamma et al., 2008; Oliveira et al., 2006; Sabu et al., 2005),

organic acids (John et al., 2006; Sharma et al., 2008; Vandenberghe et al., 2000),

antibiotics (Adinarayana et al., 2003; Ellaiah et al., 2004), flavor and aroma compounds

CHAPTER 2


17

(Medeiros et al., 2006; Rossi et al., 2009; Sarhy-Bagnon et al., 2000), and bioactive

compounds (Hernández et al., 2008; Vattem and Shetty, 2003). Table 2 summarizes

some of the most recent studies in SSF, the microorganisms and solid supports

employed. Note that different products have been obtained by SSF using a large variety

of solid supports. Fungi have been the most used microorganisms.

The process variables including pretreatment and particle-size of substrates,

medium ingredients, supplementation of growth medium, sterilization of SSF medium,

moisture content, inoculum density, temperature, pH, agitation and aeration, have a

significant effect on the efficiency of SSF processes (Nigam and Pandey, 2009).

Therefore, the establishment of the most suitable conditions for use of these variables is

of relevance to achieve elevated process yields. The use of experimental design statistical

methodology may be a useful tool to define such conditions performing a minimal

number of experiments. Recently, several works report the use of statistical analysis to

maximize the product formation through the establishment of the best SSF operational

conditions. Such works include the production of enzymes such as α-amylase (Reddy et

al., 2003), inulinase (Xiong et al., 2007), phytase (Singh and Satyanarayana, 2008b),

protease (Reddy et al., 2008), xylanase (Senthilkumar et al., 2005), and laccase (Liu et

al., 2009), biosurfactants (Mukherjee et al., 2008) and organic acids such as citric acid

(Imandi et al., 2008).

Finally, the selection of the most appropriate downstream process for the obtained

product is also crucial when SSF processes are performed. The product obtained by SSF

may be recovered from the solid fermented mass by extraction with solvents (aqueous or

other solvents mixtures). The type of solvent and its concentration, as well as the ratio of

solvent to the solid and pH are important variables that influence in the product

extraction. In addition, since the metabolites diffuse throughout the solid mass during the

culturing, long extraction-times may be required for complete product recovery. The cost

of purification depends on the quality of the obtained extract. For example, the presence

and concentration of inert compounds in the extract increase the cost of purification and

therefore the cost of recovery is increased. Particularly those secondary metabolites

which are used in bulk in the pharmaceutical and health industry and whose purity is

governed by stringent regulations need to go through specific purification strategy

(Nigam, 2009).

18

Table 2.2 Recent studies of solid-state fermentation using different microorganisms and solid supports.

Microorganism Solid support Reference Fungi Aspergillus niger Creosote bush leaves, variegated Caribbean agave, lemon peel, orange peel, apple pomace,

pistachio shell, wheat bran, coconut husk, pecan nutshell, bean residues Orzua et al., 2009

Aspergillus niveus Sugarcane bagasse Guimarães et al., 2009 Aspergillus oryzae Red gram plant waste Shankar and Mulimani, 2007 Aspergillus sojae Crushed maize, maize meal, corncob Ustok et al., 2007 Bjerkandera adusta Ganoderma applanatum Phlebia rufa Trametes versicolor

Wheat straw Dinis et al., 2009

Phanerochaete chrysosporium Rice straw Yu et al., 2009 Penicillium sp. Soybean bran Wolski et al., 2009 Rhizopus chinensis Combination of wheat bran and wheat flour Sun et al., 2009 Sporotrichum thermophile sesame oil cake Singh and Satyanarayana, 2008a Trichosporon fermentans Rice straw Huang et al., 2009

Yeasts

Baker yeast AF37X Sweet sorghum Yu et al., 2008 Saccharomyces cerevisiae Mahula flowers

Corn stover Mohanty et al., 2009 Zhao and Xia, 2009

Bacteria Nocardia lactamdurans Wheat bran, rice, soybean oil cake, soybean flour Kagliwal et al., 2009 Bacillus sphaericus Wheat bran El-Bendary et al., 2008

Bacillus subtilis Wheat bran Gupta et al., 2008

Pseudomonas aeruginosa Jatropha curcas seed cake Mahanta et al., 2008 Streptomyces sp. Coffee pulp Orozco et al., 2008

CHAPTER 2


19

2.4 Uses of SSF for bioactive phenolic compounds production

2.4.1 Phenolic content increase in food products

Food quality is not only a function of nutritional values but also of the presence of

bioactive compounds exerting positive effects on human health (Cassano et al., 2008).

Phenolic compounds, also referred as polyphenols, are considered to be natural

antioxidants and represent an important group of bioactive compounds in foods (Dueñas

et al., 2005). These compounds are present in all plant foods but their type and levels

vary enormously depending on the plant, genetic factors and environmental conditions

(Kris-Etherton et al., 2002).

In the last years, SSF has been employed to increase the content of phenolic

compounds in certain food products, thus enhancing their antioxidant activity. For

example, black beans are well known for their high nutritional value containing

isoflavones, vitamin E, saponins, carotenoids and anthocyanins (Choug et al., 2001). In a

recent study on the bioprocessing of these beans to prepare koji using SSF with different

food-grade filamentous fungi (in particular Aspergillus sp. and Rhizopus sp.), an

enhancement of the antioxidant properties of the beans was observed, which might be

related to the increase of phenols and anthocyanins content (Lee et al., 2008).

Nevertheless, the enhancement of the antioxidant activity of the black bean koji varied to

each microorganism used. Similarly, SSF of grass peas cooked seeds using Rhizopus

oligosporus caused an increase in the phenolic compounds content, which significantly

improved the antiradical properties of the seeds (Starzynska-Janiszewska et al., 2008).

Two different filamentous fungi (Aspergillus oryzae and Aspergillus awamori)

used in SSF were very effective for the improvement of phenolic content and antioxidant

properties of wheat grains. In this study, fermented wheat grains were considered to be

antioxidant richer and healthier food supplement compared to non-fermented wheat

grains (Bhanja et al., 2009). Soybean products fermented by SSF with Trichoderma

harzianum showed stronger antioxidant activity than unfermented products, which was

probably related to the markedly higher contents of phenolic acids, flavonoids and

aglycone isoflavone with freer hydroxyl groups achieved during SSF (Singh et al., 2010).

CHAPTER 2


20

Chemical composition and bioactivity of stale rice were also improved by SSF with

Cordyceps sinensis (Zhang et al., 2008).

Besides to increase the antioxidant activity of certain foods, bioconversion of

phenolic compounds by SSF may also promote other alterations in the food properties,

with influence on human health. An example of this is the SSF of mung beans (also

known as green beans) with Rhizopus oligosporus. This process has been demonstrated

as being able to mobilize the conjugate forms of phenolic precursors naturally found in

mung beans and improves their health-linked functionality. According to Randhir and

Shetty (2007), SSF of mung beans significantly increased the phenolic content enhancing

the antioxidant activity of the beans. This antioxidant activity enhancement contributed

to the α-amylase inhibition (which is relevant for the diabetes controlling), as well as for

the inhibition of the Helicobacter pylori growth (linked to peptic ulcer management).

2.4.2 Production and extraction of bioactive phenolic compounds from agro-industrial

residues

Another valuable application of SSF is for the production or extraction of bioactive

phenolic compounds from agro-industrial residues. Large amounts of these materials,

including seeds, peels, husks, whole pomace, among others, are generated every year in

the form of wastes, and are poorly valorized or left to decay on the land. Recently,

increased attention has been given to these materials as abundantly available and cheap

renewable feedstocks for the production of value-added compounds. In this sense, a

number of them have been used as solid substrate in SSF processes for the production of

different bioactive phenolic compounds (Hernández et al., 2008; Robledo et al., 2008;

Vattem and Shetty, 2003; Zheng and Shetty, 2000).

Pomegranate wastes are an example of agro-industrial residue containing

significant amount of phenolic compounds, among of which anthocyanins (derived from

delphinidin, cyanidin and pelargonidin), hydrolysable tannins (catechin, epicatechin,

punicalin, pedunculagin, punicalagin, gallic and ellagic acid esters of glucose)

(Cuccioloni et al., 2009; Gil et al., 2000), and several lignans (isolariciresinol,

medioresinol, matairesinol, pinoresinol, syringaresinol, and secoisolariciresinol)

CHAPTER 2


21

(Bonzanini et al., 2009) can be mentioned. These phenolic compounds confer

antioxidant, anti-mutagenic, anti-inflammatory and anticancer activities to the

pomegranate wastes (Gil et al., 2000; Naveena et al., 2008; Negi et al., 2003). In recent

studies, pomegranate husks were successfully used as support and nutrient source for

ellagic acid production by SSF with Aspergillus niger GH1 (Aguilar et al., 2008;

Hernández et al., 2008). This process is economically interesting since from each ton of

pomegranate husks, it is possible to produce 8 kg of ellagic acid by SSF (Robledo et al.,

2008). This process is also quite profitable from an industrial point of view, considering

the commercial price of this acid and the low cost and abundance of the husks.

Cranberry pomace, the by-product of the cranberry juice processing industry, has

also been pointed out as a good source of ellagic acid and other phenolic compounds

(Vattem and Shetty, 2003; Zheng and Shetty, 1998; Zheng and Shetty, 2000).

Bioprocessing of this waste by SSF with Lentinus edodes was useful to increase the

ellagic acid content, being also an interesting alternative for the production of bioactive

compounds (Vattem and Shetty, 2003). In India, Teri pod (Caesalpinia digyna) cover,

the solid residue obtained during processing of the pod for recovery of oil, is a readily

available agro-industrial by-product. This material contains tannin that can be used as

substrate for microbial conversion to gallic acid. Bioconversion of tannin to gallic acid

from powder of Teri pod cover was successfully performed by SSF with the fungus

Rhizopus oryzae (Kar et al., 1999).

Green coconut husk, an abundant agro-industrial residue in Brazil, is a potential

source of ferulic acid, from which vanillin can be obtained via microbial conversion. In a

recent study, the cultivation of the basidiomycete Phanerochaete chrysosporium under

SSF in this agro-industrial residue caused the production of lignolytic enzymes that

released ferulic acid from the coconut husk cell wall and subsequently, vanillin was

obtained with high yield by the ferulic acid conversion (Barbosa et al., 2008). In fact, the

action of enzymes such as α-amylase, laccase and β-glycosidase, tannin acyl hydrolase,

ellagitanin acyl hydrolase, among others, plays an important role in the mobilization of

bioactive phenolic compounds during SSF (Cho et al., 2008; Robledo et al., 2008; Zheng

and Shetty, 2000). The enzymes responsible for the degradation of lignocellulosic

residues are mainly produced by fungi, since these microorganisms have two

extracellular enzymatic systems: a hydrolytic system that produces hydrolases able to

CHAPTER 2


22

degrade polysaccharides, and an oxidative ligninolytic system, which degrades lignin

and opens phenyl rings, increasing the free phenolic content (Sánchez, 2009). Table 3

summarizes some enzymes produced during SSF by lignocellulolytic fungi in several

agro-industrial residues.

The enzyme β-glycosidase (β-D-glycoside glucohydrolase) catalyzes the

hydrolysis of glycosidic linkages in alkyl and aryl β-D-glycosides, as well as glycosides

containing only carbohydrate residues (Vattem and Shetty, 2003). This enzyme has been

described as able of hydrolyzing phenolic glycosides to release free phenolic acids. Some

studies have suggested that crude Lentinus edodes β-glycosidase has higher capacity to

release free phenolic acids from cranberry pomace than the commercial β-glycosidase

(Vattem and Shetty, 2003; Zheng and Shetty, 2000). Such capacity was related to the

possible presence of other enzymes such as esterases, in the crude β -glycosidase

solution. These enzymes might help the cleavage of inter-sugar linkages, releasing the

corresponding glycosides that were hydrolyzed liberating phenolic aglycon moieties.

During SSF of soybean with Bacillus pumilus HY1, Cho et al. (2009) reported a

significant increase in the contents of flavanols and gallic acid, and a decrease in the

amounts of isoflavone glycosides, malonylglycosides and flavanol gallates. This

phenomenon was associated with bacterial β-glycosidase and esterase activities.

Similarly, the improvement in the antioxidant potential of fermented rice has been

associated with the phenolic compounds increase by β-glycosidase and α-amylase

activities during SSF (Bhanja et al., 2008). Recently, elagitannin acyl hydrolase has been

related with the bioconversion of elagitannin into ellagic acid during SSF of

pomegranate husks (Robledo et al., 2008).

23

Table 2.3 Enzymes produced during solid-state fermentation by lignocellulolytic fungi in several agro-industrial residues.

Enzyme (s) Substrate Microorganism Reference β-glycosidase Lentinus edodes

Rhizopus oligosporus Aspergillus oryzae

Cranberry pomace Flour-supplemented guava waste Rice

Zheng and Shetty, 2000 Correia et al., 2004 Bhanja et al., 2008

α-amylase Aspergillus oryzae Rice Bhanja et al., 2008 Polygalacturonase Aspergillus niger Wheat and soy brans Castilho et al., 2000 Xylanase Aspergillus niger

Sporotrichum thermophile

Apple pomace and cotton seed powder Corn cobs

Liu et al., 2008 Topakas et al., 2003b

Cellulase Hemicellulase Glucoamylase Pectinase Acidic proteinase

Aspergillus niger Bran and cotton seed powder Wang et al., 2006

Laccase Lentinus edodes Pleurotus pulmonarius Pleurotus sp. Pleurotus ostreatus

Corn Wheat bran and wheat straw Wheat straw Wheat straw

D’Annibale et al., 1996 Marques de Souza et al., 2002 Lang et al., 1996 Baldrian and Gabriel, 2002

Glycosidase Aspergillus niger Grape Huerta-Ochoa et al., 2003

CHAPTER 2


24

2.4.3 Production and extraction of bioactive phenolic compounds from plants

Plants produce a wide variety of bioactive compounds with significant applications

in the health and food areas (Sarikaya and Ladisch, 1999; Ventura et al., 2008). Such

compounds include a variety of flavonoids, phenolic acids, lignans, sallicylates, stanols,

sterols, and glucosinolates, among others (Hooper and Cassidy, 2006). In fact, plants are

considered to be excellent sources of phenolic compounds with very interesting

nutritional and therapeutic applications (Li et al., 2008; Trouillas et al., 2003). Among

these compounds, a strong correlation between antioxidant activity and the total phenolic

content in the plants has been observed, suggesting that phenolic compounds could be

the major contributor of their antioxidant capacity (Li et al., 2008).

Phenolic compounds are widely distributed in plants, being usually found in higher

concentrations in leaves and green steams (Bennett and Wallsgrove, 1994; Hyder et al.,

2002). These compounds are considered natural defense substances, and their

concentration in each plant may be influenced by several factors including physiological

variations, environmental conditions, geographic variation, genetic factors and evolution

(Figueiredo et al., 2008). The large biodiversity of plants existent, provides a great

exploration field for researches on bioactive phenolic compounds and their biological

properties (Shetty and McCue, 2003; Skerget et al., 2005; Tellez et al., 2001; Yesil-

Celiktas et al., 2009).

Mexico is one of the world’s richest countries in plant biodiversity, with a variety

estimated between 22,000 and 30,000 species (Villaseñor, 2003; Villaseñor et al., 2007).

The scientific and most common names of some plants that have been studied in SSF

processes include Larrea tridentata (gobernadora or creosote bush), Flourensia cernua

(hojasén or tarbush), Jatropha dioica (sangre de drago or dragon’s blood), Euphorbia

antisyphylitica (candelilla) and Turnera diffusa (damiana). These plants dominate some

semiarid areas of the northern Mexico and southwest in the United States, as well as

some desert regions of Argentina (Rzedowski and Huerta, 1994). Extracts from Larrea

tridentata using organic solvents have shown a great potential regarding biological

properties, namely, antioxidant and antifungal activities (Abou-Gazar et al., 2004;

Vargas-Arispuro et al., 2005). These biological properties were related to the presence of

certain lignans, which are phenolic compounds characterized by having a diphenolic ring

CHAPTER 2


25

containing a 2,3-dibenzylbutane structure formed from the oxidative dimerization of two

cinnamic acid residues. Larrea tridentata has also been used as a source of a valuable

lignan named nordihydroguaiaretic acid (Hyder et al., 2002), known for its biological

properties including anticancer and antiviral activities (Cui et al., 2008; Hwu et al., 2008;

Vargas-Arispuro et al., 2005). It has been demonstrated in a recent study that Larrea

tridentata was a potential source for gallic acid and tannase production by SSF using

Aspergillus niger Aa-20 (Treviño-Cueto et al., 2007). High concentrations of gallic and

ellagic acids were also obtained by Aspergillus niger PSH during SSF of tannin-rich

aqueous extracts from Larrea tridentata impregnated in polyurethane foam (Ventura et

al., 2008). Aspergillus niger GH1 has also been reported as being a fungi with great

ability to hydrolyze ellagitannins into ellagic acid during SSF using Larrea tridentata as

substrate (Aguilera-Carbo et al., 2009).

2.5 Concluding remarks and future perspective

SSF is an environmentally clean technology with great potential for application on

the production or extraction of biologically active compounds from natural sources. The

agro-industrial residues reuse in this area is of particular interest due to their availability,

low cost, and characteristics that allow obtaining different bioactive compounds, besides

to be an environmentally friend alternative for their disposal. Another interesting

application for SSF is to increase the bioactive phenolic compounds content in food

products. This area has great potential to expand in a near future due to the increased

consumer desire to improve health through food.

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CHAPTER 3

Kinetic study of nordihydroguaiaretic acid recovery from Larrea

tridentata by microwave-assisted extraction

This chapter presents the development of a rapid and effective microwave-assisted extraction

(MAE) method for the recovery of nordihydroguaiaretic acid from Larrea tridentata leaves,

comparing the obtained results with those found by using the conventional heat-reflux

extraction. Optimum conditions for NDGA extraction using MAE were defined, and the

antioxidant potential of the produced extracts was evaluated.

CHAPTER 3

KINETIC STUDY OF NORDIHYDROGUAIARETIC ACID RECOVERY FROM Larrea tridentata BY MICROWAVE-

ASSISTED EXTRACTION

37

OH

OH

OH

OH

3.1 Introduction

Nordihydroguaiaretic acid (NDGA) is a lignan found in several plants, like Larrea

tridentata (Zygophyllaceae), also known as creosote bush, which grows in semidesert

areas of Southwestern United States and Northern Mexico (Ross, 2005). NDGA (Fig.

3.1) can be found in flowers, leaves, green stems and small woody stems. In Larrea

tridentata it is mainly concentrated in the leaves (38.3 mg/g) and green stems (32.5

mg/g) (Hyder et al., 2002). The higher concentrations of these compounds in leaves and

green stems is because lignans are considered natural defense substances of

photosynthetic tissue in plants, which are more exposed to UV radiation, climatic

changes, herbivores and pathogens attacks (Bennett and Wallsgrove, 1994; Hyder et al.,

2002; Buranov and Mazza, 2008). In addition, the concentration of secondary

metabolites (like NDGA) in plants might be influenced by several other factors namely,

physiological variations, environmental conditions, geographic variation, genetic factors,

and evolution (Figueiredo et al., 2008). NDGA is well known as being a powerful

antioxidant (Moody et al., 1998); however, recent studies have shown other very

important biological activities for this compound, such as antiviral, cancer

chemopreventive, and antitumorgenic activities (Toyoda et al., 2007; Cui et al., 2008;

Hwu et al., 2008).

Fig. 3.1. Chemical structure of NDGA.

Extraction of bioactive compounds from plants is conventionally performed by

heat-reflux systems, which usually are time consuming and require large amounts of

CHAPTER 3


ASSISTED EXTRACTION

38

solvent (Wang and Weller, 2006). Therefore, the increased need for an ideal extraction

method that allows the maximum bioactive compound recovery from a plant, in the

shortest processing time with low costs, represents an important challenge. Different

techniques for bioactive compounds extraction have been proposed, including

ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid

extraction, and high pressure processing (Pascual-Martí et al., 2001; Lianfu and Zelong,

2008; Ma et al., 2009; Jun, 2009). Among these, microwave-assisted extraction (MAE)

has been proved to significantly decrease extraction time and increase extraction yields

in several plants (Guo et al., 2001; Pan et al., 2003; Rostagno et al., 2007; Proestos and

Komaitis, 2008). When MAE is applied, the solvent choice is determined by the

solubility of the extracts of interest, the interaction between solvent and plant matrix, and

the microwave absorbing properties of the solvent determined by its dielectric constant

(Brachet et al., 2002).

There is little information available on the NDGA extraction from Larrea

tridentata. To our knowledge, no studies on MAE method for NDGA recovery from

Larrea tridentata leaves have been reported. Thus, the aim of this work was to develop a

MAE technique for an efficient NDGA extraction from Larrea tridentata leaves and

compare the obtained results with those found by using the conventional heat-reflux

extraction.

3.2 Materials and methods

3.2.1 Plant materials and chemicals

Plant material (Larrea tridentata) was collected from the Chihuahuan semidesert

(North Coahuila, Mexico) during Spring season (April, 2008).

Nordihydroguaiaretic acid (high purity) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)

were purchased from Sigma-Aldrich (Saint Louis, MO, USA). HPLC-grade methanol

and acetonitrile were purchased from Fermont (Monterrey, NL, Mexico). Reagent-grade

CHAPTER 3


ASSISTED EXTRACTION

39

methanol was purchased from Jalmek (Monterrey, NL, Mexico) and acetic acid from

CTR (Monterrey, NL, Mexico).

3.2.2 Extraction methodologies

Air-dried leaves of Larrea tridentata were ground to fine powder and stored in

dark bottles at room temperature for further analysis. Conventional heat-reflux extraction

was performed mixing 1 gram of dried powdered plant with the solvent (solid/liquid ratio

of 1/10 g/mL), in 250-mL Erlenmeyer flasks, which were covered with foil paper to

prevent light exposure and subsequent oxidation (Makkar, 2003). Reactions were

performed in a water-bath at 70 ± 2 °C, using different methanol concentrations as

solvent (25 to 100% v/v) during 1 or 3 h. During the conventional extraction by reflux,

the temperature was monitored using a thermocouple data logger (USB TC-08, Pico

Technology, UK), which was placed inside the flask containing the sample, and after

achieved the desired temperature (70 °C), the extraction time started. Data were

registered by a PC and a temperature profile was obtained (Fig. 2). The temperature data

were fitted using a simple dynamic enthalpy balance (Milinska et al., 2007):

)TT(kdtdT

b −= Equation (1)

where Tb is the theoretical water-bath temperature, T is the real water-bath temperature

at time t, and k is a proportionality factor including the overall heat transfer coefficient.

The heat transfer coefficient k was calculated, obtaining value of 4.17 × 10-3 ± 1.4 × 10-4

s-1

Microwave-assisted extraction was carried out in a microwave apparatus using a

multimode closed vessel system with pressure (Microwave Digestion Unit, CEM MARS

Express, USA). For reactions, 1 gram of dried powdered plant was mixed with the

desired amount of solvent and placed into 100 mL polytetrafluoroethylene (PTFE)

extraction vessels. The suspensions were irradiated with microwaves at a power of 800

W in a pre-setting procedure where after each period of 1 min the sample was allowed to

.

CHAPTER 3


ASSISTED EXTRACTION

40

0

10

20

30

40

50

60

70

80

0 500 1000 1500 2000 2500 3000 3500 4000

Tem

pera

ture

(ºC

)

Time (s)

cool at room temperature. Different methanol concentrations as solvent (25 to 100 %

v/v) and solid/liquid ratios (1/5 to 1/30 g/mL) were tested. The extraction temperature

was 70 ± 2 °C.

Fig. 3.2. Development of the water-bath temperature at 70 °C during the conventional

heat-reflux extraction of NDGA from Larrea tridentata leaves. The symbols (●)

represent the experimental values of temperature, and the solid line represents the fitted

temperature course using equation (1).

The extracts obtained by both methods were filtered using a muslin cloth and filter

paper to remove macro particles. Before HPLC analysis all the extracts were filtered

through a 0.2 µm membrane filter. A total of three extracts were prepared and all

analyses were performed in triplicate. NDGA yield (w/w) was defined as the ratio

between mass of NDGA in the extracts and mass of plant material, × 100%.

CHAPTER 3


ASSISTED EXTRACTION

41

3.2.3 HPLC analysis

NDGA concentration in the obtained extracts was determined by high performance

liquid chromatography (HPLC) (Mercado-Martínez, 2008) using a Varian ProStar 3300

system (Chicago, IL, USA), equipped with a pump (ProStar 230 SDM), an auto sampler

(ProStar 410 AutoSampler), and a UV-photodiode array detector (PDA ProStar 350) at

280 nm. Data acquisition was made using the LC Workstation software (Version 6.2).

Chromatographic separation was carried out in an Optisil ODS reversed-phase column (5

µm; 250×4.6 mm) at a temperature of 31 °C, using a mobile phase consisted of

acetonitrile (solvent A) and 0.3% acetic acid in water (v/v) (solvent B) under the

following gradient profile: 30% A/ 70% B (0-2 min), 50% A/ 50% B (2-11 min), 70% A/

30% B (11-17 min), 100% A (17-22 min), and 30% A/ 70% B (22-40 min). The mobile

phase was eluted in a flow rate of 1.0 mL/min, and samples of 10 µL were injected.

3.2.4 Determination of kinetic parameters and extraction time

The experimental data were fitted to a first-order kinetic model to describe the

NDGA extraction process:

( )t.ke1NDGANDGA −∞ −×= Equation (2)

where k (min-1

−×−=

∞NDGANDGA1ln

k1t

) is the first order extraction rate constant, and t (min) the time. By

rearranging the equation (2), it was possible to determine the time at which the extraction

process reaches the equilibrium by the following equation:

Equation (3)

Considering that 99.0NDGANDGA

≅∞

, the extraction time for both HRE and MAE methods

was determined.

CHAPTER 3


ASSISTED EXTRACTION

42

3.2.5 Scanning electron microscopy

Micrographs of plant material samples (untreated and treated by MAE and HRE)

were obtained by scanning electron microscopy using a Leica Cambridge S360

microscope. To be examined, the samples were prepared as described by Zhang et al.

(2008) with some modifications. Briefly, after the solvent removal, the plant material

was plunged in liquid nitrogen and then cut with a scalpel. The sectioned pieces were

fixed on a specimen holder with aluminum tape and then sputtered with platinum in a

sputter-coater under high vacuum condition. All the specimens were examined at 500-

fold magnification.

3.2.6 Free radical scavenging effectiveness of Larrea tridentata extracts

The free radical effectiveness of Larrea tridentata extracts obtained by MAE and

HRE was determined and compared by measuring the ability of the extracts to scavenge

the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl). The DPPH radical scavenging

activity was determined as described by Szabo et al. (2007) with slight modifications.

One hundred microliters of each extract, duly diluted in methanol at concentrations

ranging from 5 to 100 mg/L, was added to 2.9 mL of DPPH solution (6 × 10-5

The radical scavenging activity was expressed as the inhibition percentage using

the following equation:

M in

methanol). The resulting solutions were vortexed, and allowed to stand for 30 min in

darkness at room temperature. The absorbance was measured at 517 nm in a

spectrophotometer (Biomate 3, UV-Visible Spectrophotometer, NY, USA), using

methanol as blank. The control solution consisted in using methanol instead of the

sample. All the analyses were performed in quadruplicate.

% DPPH radical scavenging = (1 – AS/AC

where A

) × 100 Equation (4)

C and AS are the absorbance of the control solution and the absorbance of the

sample solutions, respectively. The effectiveness of the extracts of Larrea tridentata

CHAPTER 3


ASSISTED EXTRACTION

43

leaves obtained by MAE and HRE in scavenging free radicals was evaluated as the

concentration (mg/l) of extract in the reaction mixture required to scavenge 50% of

DPPH free radical, defined has EC50 (“effectiveness concentration” value). This

parameter (EC50

) was calculated from the inhibition curve plotting the DPPH radical

scavenging percentage versus the extracts concentration. NDGA was used as positive

control.

3.2.7 Statistical analysis

Results were analyzed by one-way analysis of variance (ANOVA) in the general

linear model of SPSS (Statistical Package for Social Sciences, version 16.0), employing

a significance level of p<0.05. Difference among samples was verified by using the

Tukey’s range test.

3.3 Results and discussion

3.3.1 Parameters affecting the NDGA extraction

NDGA is characterized for its insolubility in water and solubility in organic

solvents such as ethanol and methanol (supplier specifications). It is well known that

extracting solvents with high dielectric constant have a greater ability to absorb

microwave energy (Hemwimon et al., 2007); and high microwave energy absorption

results in a fast dissipation of energy into the solvent and solid plant matrix, which

generates an efficient and homogenous heating (Zhang et al., 2008). Therefore, since

methanol has a higher dielectric constant than ethanol (32.6 and 24.3, respectively), it

was chosen as the extraction solvent for NDGA recovery.

Extraction temperature, solvent concentration and solid/liquid ratio are parameters

that play an important role in the extraction of bioactive compounds (Wang and Weller,

2006). In the present study, 70 ºC was selected as extraction temperature considering the

CHAPTER 3


ASSISTED EXTRACTION

44

boiling point of methanol (64.7 ºC), and because no problems with the pressure in the

extraction vessel occurred, which could damage the microwave equipment safety

(Mandal et al., 2007).

Methanol concentration was a parameter of great influence on NDGA extraction

from Larrea tridentata leaves using MAE (Fig. 3.3); the NDGA yield significantly

increased (p<0.05) when a methanol concentration of 50% was used instead of water or a

methanol concentration of 25%. However, there was no significant difference (p<0.05)

in NDGA yields when extractions were performed with methanol concentrations higher

than 50%. These findings showed that the addition of some water resulted in an

enhancement of the extraction efficiency, possibly due to the increase in plant material

swelling in the presence of water, increasing the contact surface area between the plant

matrix and the solvent (Li et al., 2004; Sun et al., 2008).

Fig. 3.3. Effect of methanol concentration on NDGA extraction from Larrea tridentata

leaves by MAE under the following conditions: 1 g plant/ 30 mL solvent, 70 °C, 800W,

for 4 min. abc

Values in a column with the same superscripts are not significantly

different at p<0.05.

The effect of methanol concentration on NDGA extraction from Larrea tridentata

leaves during HRE was also evaluated (Fig. 3.4). In this case, the process was performed

c

b

a a a

CHAPTER 3


ASSISTED EXTRACTION

45

during 1 or 3 h to verify if a larger extraction time could have any effect on NDGA

recovery, but no significant differences (p<0.05) in NDGA yields were observed.

Notwithstanding, NDGA yield was affected by the methanol concentration, the values

being significantly higher when a methanol concentration of 50% was used during 1 h,

compared to water or a methanol concentration of 25%. There was no significant

difference in NDGA yields when methanol concentrations of 75 or 100% were used

compared to a methanol concentration of 50%. In brief, methanol in water at 50 % v/v

was the best extraction solvent for both, HRE and MAE techniques.

Fig. 3.4. Effect of methanol concentration on NDGA extraction from Larrea tridentata

leaves by HRE under the following conditions: 1 g plant/ 30 mL solvent, 70 °C, for ( )

1 and ( ) 3 h. abcd

Values in a column with the same superscripts are not significantly

different at p<0.05.

Some studies report that the solid/liquid ratio affects the bioactive compounds

yield during MAE (Proestos and Komaitis, 2008; Zhang et al., 2008). The effect of

solid/liquid ratio on the NDGA yields during MAE from Larrea tridentata leaves is

d d

c c

b b ab

a b b

CHAPTER 3


ASSISTED EXTRACTION

46

shown in Fig. 3.5. In fact, it can be observed that using a solid/liquid ratio of 1/10 (g/mL)

resulted in significantly higher (p<0.05) NDGA yields compared to a solid/liquid ratio of

1/5. However, there was no significant difference on NDGA yields using solid/liquid

ratios of 1/20 or 1/30. Therefore, 1/10 (g dried plant material/ mL extraction solvent) was

considered the ideal solid/liquid ratio to be used during MAE of NDGA from Larrea

tridentata leaves for 4 min at 70 ºC.

Fig. 3.5. Effect of solid/liquid ratio on NDGA extraction from Larrea tridentata leaves

by MAE using methanol 50% (v/v) as solvent, at 70 °C, 800W, for 4 min. ab

Values in a

column with the same superscripts are not significantly different at p<0.05.

3.3.2 Comparison of NDGA extraction by MAE and HRE

Fig. 3.6 shows the kinetic behavior of NDGA extraction from Larrea tridentata

leaves by MAE and HRE carried out for 4 and 60 min, respectively. Kinetic parameters

and extraction time for both MAE and HRE methods are presented in Table 3.1. Note

that MAE method was more advantageous than HRE since it reduced the extraction time

from 18 to 1 min only, presenting, as a consequence, a higher extraction rate constant

(4.61 ± 0.45 min-1). Moreover, higher NDGA yields were found when using MAE as an

b a

ab ab

CHAPTER 3


ASSISTED EXTRACTION

47

extracting technique. These results are consistent with those reported by other authors

using different plant materials. For example, Zhang et al. (2008) showed that MAE

significantly reduced the extraction time of chlorogenic acid from flower buds of

Lonicera japonica to 5 min in comparison to 30 min by the conventional HRE, and gave

higher extraction efficiency.

Fig. 3.6. Kinetic study of NDGA extraction from Larrea tridentata leaves by MAE (●)

and HRE () using 1 g plant material/ 10 mL methanol 50% (v/v), at 70 ºC and 800 W.

The symbols represent the experimental NDGA values and the solid line represents the

fitted data to a first-order kinetic model (equation (2)).

Zhou and Liu (2006) reported MAE as a faster extraction technique for solanesol

extraction from tobacco leaves than conventional heat-reflux, since it reduced to 40 min,

the 180 min required by the conventional HRE. MAE was also a faster and more

efficient technique for the extraction of flavonoids from Radix Astragali compared to

conventional HRE, reducing the extraction time from two 2 h cycles to two 25 min

cycles, and increasing the percentage flavonoids extraction (Xiao et al., 2008).

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Yie

ld o

f ND

GA

(% w

/w)

Time (min)

CHAPTER 3


ASSISTED EXTRACTION

48

Table 3.1 Kinetic parameters and extraction times obtained for NDGA extracted from

Larrea tridentata leaves by HRE and MAE.

Extraction method

NDGA∞ (%, w/w)

(a) K (min-1)

(b) R

2 (c) Extraction time (min)

HRE

3.42 ± 0.19

0.26 ± 0.02

0.9987

18

MAE

3.79 ± 0.65

4.61 ± 0.45

0.9948

1

NDGA recovered after the extraction process; (b) first order extraction rate constant values; (c) Correlation

factor for the adjustment of experimental NDGA values to the first-order kinetic model.

A possible explanation for the best results of MAE compared to HRE, could be an

efficient dissipation and absorption of microwave energy through the solvent and plant

material, which increases temperature inside the plant cells. This might result in cell

walls breaking, allowing the bioactive compounds release into the surrounding solvent.

Therefore, in order to understand the mechanism of MAE and HRE, samples of plant

material treated by these two techniques were examined by scanning electron

microscopy, and compared with an untreated plant material sample. Analysis of these

micrographs clearly revealed a major destruction of the material surface treated by MAE

(Fig. 7C) than by HRE (Fig. 7B). In the original form (Fig. 7A) the material was a rigid

structure, which was affected by the HRE treatment. However, MAE treatment was able

to strongly destroy the plant structure, probably due to the sudden temperature rise and

the internal pressure increase. Similar results were also found in other studies with MAE

of solanesol from tobacco leaves (Zhou and Liu, 2006), scutellarin from Erigeron

breviscapus (Gao et al., 2007), and chlorogenic acid from flower buds of Lonicera

japonica (Zhang et al., 2008). It was thus concluded that the improvement of NDGA

extraction by MAE when compared to HRE might be related to the greater extent of cell

rupture of the plant material.

CHAPTER 3


ASSISTED EXTRACTION

49

Fig. 3.7. Micrographs, by scanning electron microscopy of Larrea tridentata samples in

the following forms: (A) untreated; (B) after MAE; and (C) and after conventional HRE.

Magnification: 500-fold.

CHAPTER 3


ASSISTED EXTRACTION

50

3.3.3 Effectiveness of Larrea tridentata extracts on free radical scavenging

DPPH assay is a test able to evaluate the antioxidant potential of extracts (Szabo et

al., 2007), and was thus used in the present work for evaluation of the effectiveness of

Larrea tridentata leaves extracts obtained by MAE and HRE, on free radical scavenging

(Fig. 3.8). According to the results, the EC50 (extract in the reaction mixture required to

scavenge 50% of DPPH free radical) for the extract obtained by MAE was slightly

higher than that of the extract obtained by HRE (15.57 ± 0.16 and 12.52 ± 0.31 mg/L,

respectively), and consequently, the antiradical activity was slightly lower (Fig. 3.8).

Such results could be due to the microwave irradiation, which might degrade the

antiradical activity of the respective extracts. These findings are consistent with those of

Hemwimon et al. (2007) who reported that extracts obtained of anthraquinones by MAE

from roots of Morinda citrifolia had slightly higher EC50

values than those obtained by

conventional soxhlet extraction method.

Fig. 3.8. Effect of different concentrations of extracts obtained by MAE and HRE from

Larrea tridentata leaves in free radical DPPH scavenging activity ( NDGA positive

control, extract obtained by HRE, ● extract obtained by MAE).

0

10

20

30

40

50

60

70

80

90

100

5 10 20 25 30 50 70 100

% D

PPH

rad

ical

scav

engi

ng

Concentration of extract (mg/L)

CHAPTER 3


ASSISTED EXTRACTION

51

While extracts obtained by MAE presented the highest DPPH radical scavenging

activity of 91.44% at 50 mg/L, extracts obtained with HRE had a lower antiradical

activity of 85.76% for the same concentration (Fig. 8). When a 70 mg/L concentration

was applied to evaluate the antiradical activity, both extracts obtained by MAE and HRE

exhibited similar DPPH radical scavenging activities (92.25 and 92.94%, respectively).

Additionally, the EC50

for the solution of NDGA used as positive control was higher

than those obtained for the extracts obtained by MAE and HRE (32.53 ± 0.39 mg/L).

Such findings were expected since extracts obtained by MAE and HRE are composed by

several other polyphenolic compounds than NDGA that possess antiradical activity.

3.4 Conclusion

Microwave-assisted extraction was proved to be a faster and more efficient method

for NDGA extraction from Larrea tridentata leaves when compared to the conventional

heat-reflux extraction, since it significantly reduced the extraction time and gave higher

NDGA yields. Under the optimal MAE conditions (50% methanol in water (v/v) as

extraction solvent, solid/liquid ratio of 1/10 (g/mL), 70 ºC, during 1 min), maximum

NDGA yield of 3.79 ± 0.65% was achieved. The best results of NDGA extraction by

MAE might be related to a greater extent of cell rupture of the plant material, which was

observed by scanning electron microscopy. Finally, extracts obtained from Larrea

tridentata leaves using MAE technique appear to have antioxidant potential, since they

presented antiradical activity. However, further studies are needed in order to support

this idea and evaluate the ability of purified fractions of NDGA from MAE extracts to

scavenge other free radicals.

3.5 References

Bennett N., Wallsgrove R.M. (1994). Secondary metabolism in plant defence mechanisms. New

Phytologist, 127, 617-633.

Brachet A., Christen P., Veuthey J.-L. (2002). Focused microwave-assisted extraction of cocaine and

benzoylecgonine from Coca leaves Phytochemical Analysis, 13, 162-169.

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52

Buranov A.U., Mazza G. (2008). Lignin in straw of herbaceous crops. Industrial Crops and Products, 28,

237-259.

Cui Y., Lu C., Liu L., Sun D., Yao N., Tan S., Bai S., Ma X. (2008). Reactivation of methylation-silenced

tumor suppressor gene p161NK4a by nordihydroguaiaretic acid and its implication in G1 cell cycle

arrest. Life Sciences, 82, 247-255.

Figueiredo A.C., Barroso J.G., Pedro L.G., Scheffer J.J.C. Factors affecting secondary metabolite

production in plants: volatile components and essential oils. Flavour and Fragrance Journal, 23,

213-226.

Gao M., Huangb W., RoyChowdhury M., Liu C. (2007). Microwave-assisted extraction of scutellarin from

Erigeron breviscapus Hand-Mazz and its determination by high-performance liquid

chromatography. Analytica Chimica Acta, 591, 161-166.

Guo Z., Jin Q., Fan G., Duan Y., Qin C., Wen M. (2001). Microwave-assisted extrcation of effective

constituents from a Chinese herbal medicine Radix puerariae. Analytica Chimica Acta, 436, 41-47.

Hemwimon S., Pavasant P., Shotipruk A. (2007). Microwave-assisted extraction of antioxidantive

anthraquinones from roots of Morinda citrifolia. Separation and Purification Technology, 54, 44-

50.

Hwu J.R., Hsu M.H., Huang RC. (2008). New nordihydroguaiaretic acid derivates as anti-HIV agents.

Bioorganic and Medicinal Chemistry Letters, 18, 1884-1888.



(Larrea tridentata). Biochemical Systematics and Ecology, 30, 905-912.

Jun X. (2009). Caffeine extraction from green tea leaves assisted by high pressure processing. Journal of

Food Engineering, 94, 105-109.

Li H., Bo C., Zhang Z., Yao S. (2004). Focused microwave-assisted solvent extraction and HPLC

determination of effective constituents in Eucommia ulmodies Oliv. (E. ulmodies). Talanta, 63, 659-

665.

Lianfu Z., Zelong L. (2008). Optimization and comparison of ultrasound/microwave assisted extraction

(UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes. Ultrasonics

Sonochemistry, 15, 731-737.

Ma Y.Q., Chen J.C., Liu D.H., Ye W.Q. (2009). Simultaneous extraction of phenolic compounds of citrus

peel extracts: effect of ultrasound. Ultrasonics Sonochemistry, 16, 57-62.

Makkar H. (2003). Quantification of Tannins in Tree and Shrub Foliage: A Laboratory Manual, Kluwer

Academic Publishers: Dordrecht.

Mandal V., Mohan Y., Hemalatha S. (2007). Microwave assisted extraction – An innovative and

promising extraction tool for medicinal plant research. Pharmacognosy Reviews, 1, 7-18.

Mercado-Martínez D. (2008). Estudio de la recuperación de ácido nordihidroguayarético por cultivos

fúngicos de Larrea tridentata, Master thesis, Autonomous University of Coahuila, Satillo, Mexico.

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ASSISTED EXTRACTION

53

Milinska A., Bryjak J., Illeová V., Polakovic M. (2007). Kinetics of thermal inactivation of alkaline

phosphatase in bovine and caprine milk and buffer. International Dairy Journal, 17, 579-586.

Moody T.W., Leyton J., Martinez A., Hong S., Malkinson A., Mulshine J.L. (1998). Lipoxygenase

inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth. Experimental

Lung Research, 24, 617-628.

Pan X., Niu G., Liu H. (2003). Microwave-assisted extraction of tea polyphenols and tea caffeine from

green tea leaves. Chemical Engineering and Processing, 42, 129-133.

Pascual-Martí M.C., Salvador A., Chafer A., Berna A. (2001). Supercritical fluid extraction of resveratrol

from grape skin of Vitis vinifera and determination by HPLC. Talanta, 54, 735-740.

Proestos C., Komaitis M. (2008). Application of microwave-assisted extraction to the fast extraction of

plant phenolic compounds. LWT - Food Science and Technology, 41, 652-659.

Ross I.A. (2005). Medicinal Plants of the World - Chemical Constituents, Traditional and Modern

Medicinal Uses (Volume 3), Humana Press: New Jersey.

Rostagno M.A., Palma M., Barroso C.G. (2007). Microwave assisted extraction of soy isoflavones.

Analytica Chimica Acta, 588, 274-282.

Sun Y., Wang W. (2008). Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong. Journal of the

Chinese Institute of Chemical Engineers, 39, 653-656.

Szabo M.R., Iditoiu C., Chambre D., Lupea A.X. (2007). Improved DPPH determination for antioxidant

activity spectrophotometric assay. Chemical Papers, 61, 214-216.

Toyoda T., Tsukamoto T., Mizoshita T., Nishibe S., Deyama T., Takenaka Y., Hirano N., Tanaka H.,

Takasu S., Ban H., Kumagai T., Inada K.I., Utsunomiya H., Tatematsu S. (2007). Inhibitory effect

of nordihydroguaiaretic acid, a plant lignan, on Helicobacter pylori-associated gastric

carcinogenesis in Mongolian gerbils. Cancer Science, 98, 1689-1695.

Wang L., Weller C.L. (2006). Recent advances in extraction of nutraceuticals from plants. Trends in Food

Science and Technology, 17, 300-312.

Zhang B., Yang R., Liu C.-Z. (2008). Microwave-assisted extraction of chlorogenic acid from flower buds

of Lonicera japonica Thunb. Separation and Purification Technology, 62, 480-483.

Zhou H.-Y., Liu C.-Z. (2006). Microwave-assisted extraction of solanesol from tobacco leaves. Journal of

Chromatography A, 1129, 135-139.

Xiao W., Han L., Shi B. (2008). Microwave-assisted extraction of flavonoids from Radix Astragali.

Separation and Purification Technology, 62, 614-618.

CHAPTER 4

Bioactive compounds (phytoestrogens) recovery from Larrea

tridentata leaves by solvents extraction

In this chapter the effect of different organic solvents on the extraction of bioactive

compounds from Larrea tridentata leaves, namely, nordihydroguaiaretic acid, kaempferol and

quercetin, was evaluated. The antioxidant potential of the produced extracts, as well as the

contents of total phenols, flavonoids and proteins, were also determined and discussed.

CHAPTER 4

BIOACTIVE COMPOUNDS (PHYTOESTROGENS) RECOVERY FROM Larrea tridentata LEAVES BY SOLVENTS EXTRACTION

57

4.1 Introduction

Phytoestrogens including flavonoids (comprising isoflavonoids and flavonols

derivatives), lignans and coumestanes, are secondary plant metabolites that have

attracted great attention due to their protective action against several health disorders

such as cardiovascular diseases, cancer, brain function disorders, menopausal symptoms

and osteoporosis (Cornwell et al., 2004). Such compounds have the ability to imitate or

modulate the effectiveness of endogenous estrogens. This biological response is based on

their structural and/or functional similarity to estradiol and their capacity to bind to the

human estrogen receptors (ER). Some studies have shown that selective ER modulators,

including phytoestrogens, inhibit cell proliferation in vitro (Kim et al., 2002) and in vivo

(Steiner et al., 2003).

Larrea tridentata (Zygophyllaceae), commonly known as creosote bush, is a plant

that grows in semidesert areas of Southwestern United States and Northern Mexico

(Ross, 2005). This plant was traditionally used for centuries by North American Indians

as a medicine for several illnesses including infections, kidney problems, gallstones,

rheumatism and arthritis, diabetes and to treat tumors (Navarro et al., 1996). L. tridentata

is an outstanding source of natural compounds with approximately 50% of the leaves

(dry weight) being extractable matter (Arteaga et al., 2005). Among several bioactive

compounds present in this plant, nordihydroguaiaretic acid (NDGA), kaempferol and

quercetin can be found at considerable high concentrations (Hyder et al., 2002).

NDGA (Fig. 4.1A) is phenolic lignan with biological activities of large interest in

the health area, such as antiviral, antifungic, antimicrobial, and antitumorgenic (Hwu et

al., 2008). The therapeutic potential of this compound for the treatment of tumors and

cancer has been demonstrated, being related to an inhibition on cancer cells growth via

an apoptotic mechanism (Zavodovskaya et al., 2008). Kaempferol and quercetin are

flavonols that exist as a variety of glycosides or in aglycone form. The aglycone forms of

kaempferol and quercetin are structurally similar, differing only by one hydroxyl group

in the B-ring (Figs 4.1B and 4.1C). Research on cell culture models has shown important

biochemical effects of both compounds, which are relevant to carcinogenesis, including

increase of differentiation and gap junction function (Nakamura et al., 2005), metal

chelation (Brown et al., 1998), antioxidant properties (Boots et al., 2008), the inhibition

of hepatic enzymes involved in carcinogen activation (Labbé et al., 2009), the induction

CHAPTER 4


58

of Phase II (conjugating) enzymes (Uda et al., 2008), and the influence of ER-

transcriptional activity of ERE-reporter systems (Tang et al., 2008). Despite the

anticarcinogenic capacity of kaempferol and quercetin, these compounds are also known

for their anti-inflammatory and antinociceptive capacities (Melo et al., 2009).

(A)

(B)

(C)

Fig. 4.1. Chemical structure of NDGA (A), kaempferol (B) and quercetin (C).

OH

OH

OH

OH

OH O

OH

OH

OH

O

OH O

OH

OH

OH

O

OH

CHAPTER 4


59

Nowadays, bioactive compounds with potential health benefits have attracted great

interest for use in several industrial areas, and researches on this topic have been strongly

encouraged. Extraction is the first step in the isolation of compounds from natural

sources. Among the variety of techniques that can be used for this purpose, solid-liquid

extraction has been widely employed to extract bioactive compounds from plant

materials and agro-industrial residues (Mussatto et al., 2011). However, the efficiency of

this extraction process is greatly affected by the type of solvent and its concentration

(Mussatto et al., 2011; Chirinos et al., 2007), and therefore, studies to define the best

conditions for these variables are necessary to maximize the extraction yields to each

different plant material. Despite several studies evaluating the best solvents to extract the

maximum content of phenolic compounds from plant matrices and the antioxidant

potential of the produced extracts are reported in the literature, to the best of our

knowledge, no detailed study has been developed with L. tridentata. Thus, the purpose of

this study was to evaluate the effect of different organic solvents on the extraction of

phytoestrogens, in particular, NDGA, kaempferol and quercetin, from Larrea tridentata

leaves. The antioxidant potential of the produced extracts, as well as the contents of total

phenols, flavonoids and proteins were also determined and are discussed.


4.2.1 Plant material and chemicals


(North Coahuila, Mexico) during Spring season (April, 2009). Nordihydroguaiaretic acid

(NDGA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), quercetin, kaempferol, aluminum

chloride, 2,4,6-tris (1-pyridyl)-5-triazine (TPTZ), sodium acetate, ferrous sulfate and iron

(III) chloride were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Reagent-

grade methanol, ethanol, acetone, acetic acid and Folin-Ciocalteau were from Panreac

(Barcelona, Spain). Potassium acetate was purchased from AppliChem (Darmstadt,

Germany). HPLC-grade acetonitrile was obtained from Fisher Scientific (Leicestershire,

UK). Ultrapure water from a Milli-Q System (Millipore Inc., USA) was used.

CHAPTER 4


60

4.2.2 Extraction methodology

Air-dried leaves of Larrea tridentata were ground to fine powder and stored in

dark bottles at room temperature for further use. Extractions were performed by mixing 1

g of plant material with 20 ml of organic solvent (methanol, ethanol or acetone, in a

concentration of 90, 70, 50, or 30% v/v) or distilled water. The mixtures were heated

during 30 min in a water-bath at 70 ºC when using methanol, ethanol, or water, and at 60

ºC when using acetone, due to its lower boiling point. After this time, the produced

extracts were filtered through qualitative filter paper and stored at -20 ºC until further

analysis.

4.2.3. Bioactive compounds quantification

NDGA, kaempferol and quercetin concentrations were determined by high

performance liquid chromatography (HPLC) on an equipment LC-10 A (Jasco, Japan)

with a C18

5 µm (3.9 × 300 mm) column at room temperature, and a UV detector at 280

nm. The response of the detector was recorded and integrated using the Star

Chromatography Workstation software (Varian). The mobile phase consisted of




phase was eluted in a flow rate of 1.0 ml/min, and samples of 10 µl were injected.

Previous the analysis, all the extracts were filtered through 0.2 µm membrane filters.

NDGA, kaempferol and quercetin were expressed as the ratio between mass of the

compound in the extracts and mass of plant material (dry weight).

4.2.4. Determination of total phenols content

Total phenols content was determined by the Folin-Ciocalteu method with

modifications. Briefly, 5 µl of the filtered extracts duly diluted were mixed with 60 µl of

sodium carbonate solution (7.5% w/v) and 15 µl of Folin–Ciocalteu reagent in a 96-well

microplate. Then 200 µl of distilled water were added and solutions were mixed. After

CHAPTER 4


61

standing for 5 min at 60 ºC samples were allowed to cool down at room temperature. The

absorbance was measured using a spectrophotometric microplate reader (Sunrise Tecan,

Grödig, Austria) set at 700 nm. A calibration curve was prepared using a standard

solution of gallic acid (200, 400, 600, 800, 1000, 2000, 3000 mg/l, r2

= 0.9987). The total

phenols content determined according to the Folin-Ciocalteau method are not absolute

measurements of the phenolic compounds amounts, but are in fact based on their

chemical reducing capacity relative to an equivalent reducing capacity of gallic acid.

Thus, total phenols content was expressed as milligram gallic acid equivalent (mg

GAE)/g DW plant material (dry weight).

4.2.5. Determination of total flavonoids content

Total flavonoids content was quantified by colorimetric assay. Briefly, 30 μl of the

diluted and filtered extracts was added to 90 μl of methanol in a 96-well microplate.

Subsequently, 6 μl of aluminum chloride (10 % w/v), 6 μl of potassium acetate (1 mol/l)

and 170 μl of distilled water were added to the mixture. The absorbance of the mixture

was measured after 30 min at 415 nm against a blank prepared with distilled water, using

a spectrophotometric microplate reader (Sunrise Tecan, Grödig, Austria). A calibration

curve was prepared using a standard solution of quercetin (25, 50, 100, 150, 200 mg/l,

r2

= 0.9994). Total flavonoids content was expressed as milligram quercetin equivalent

(mg of QE)/gDW plant material (dry weight).

4.2.6. Determination of protein content

Total protein content was estimated using the Bradford assay.

4.2.7. Free radical scavenging activity

The free radical activity of Larrea tridentata extracts was determined by

measuring the ability of the extracts to scavenge the free radical 1,1-diphenyl-2-

picrylhydrazyl (DPPH). The DPPH radical scavenging activity was determined

according to Hidalgo et al. (2010) with modifications. Ten microliters of each extract,

CHAPTER 4


62

duly diluted in methanol, was added to 290 μl of DPPH solution (6 × 10-5

% inhibition of DPPH = (1 – A

M in methanol

and diluted to an absorbance of 0.700 at 517 nm) in a 96-well microplate. The resulting

solutions were vortexed, and allowed to stand for 30 min in darkness at room

temperature. Then the absorbance was measured at 517 nm in a spectrophotometric

microplate reader (Sunrise Tecan, Grödig, Austria), using methanol as blank. The control

solution consisted in using methanol instead of the sample. The radical scavenging

activity was expressed as the inhibition percentage using the following equation:

S/AC

where A

) × 100

C and AS

are the absorbance of the control solution and the absorbance of the

sample solutions, respectively.

4.2.8. Ferric reducing/antioxidant power assay (FRAP assay)

Briefly, 10 µl of duly diluted and filtered extract was mixed with 290 ml of FRAP

reagent in a 96-well microplate. Then, the reaction mixture was incubated at 37 ºC for 15

min. After that, the absorbance was determined at 593 nm against a blank prepared using

distilled water. FRAP reagent should always be freshly prepared by mixing a 10 mM

2,4,6-tris (1-pyridyl)-5-triazine (TPTZ) solution in 40 mM HCl with a 20 mM FeCl3

solution and 0.3 M acetate buffer (pH 3.6) in a proportion 1:1:10 (v/v/v). A calibration

curve was prepared using an aqueous solution of FeSO4.7H2O (200, 400, 600, 800 and

1000 µM, r2

= 0.9992). FRAP values were expressed as millimoles of ferrous equivalent

(mM Fe (II))/g DW plant material (dry weight).

4.2.9. Statistical analysis

All the experimental conditions and determinations were performed in triplicate,

and mean values ± standard errors are presented. Results were analyzed by one-way

analysis of variance (ANOVA) using the general linear model of SPSS (Statistical

Package for Social Sciences, version 16.0) for a significance level of p<0.05. Difference

among samples was verified by using the Tukey’s range test. Linear regression analysis

was performed quoting the correlation coefficient rxy.

CHAPTER 4


63


4.3.1. Effect of organic solvents on the extraction of phytoestrogens

NDGA, kaempferol and quercetin extraction from Larrea tridentata leaves varied

considerably according to the used solvent (Table 4.1), probably due to the polarity of

each solvent and the solubility of the compounds in them (Wang and Weller, 2006). Low

concentration levels of all the three phytoestrogens were observed on the aqueous

extracts, which can be explained by their low solubility in water (Martins et al., 2010).

The highest NDGA, kaempferol and quercetin contents (46.96 ± 3.39, 87.00 ± 6.43 and

10.46 ± 1.01 mg/g dry wt plant, respectively) were recovered using 90% (v/v) methanol

as extraction solvent. These results are in agreement with those obtained by Lin and

Giusti (2005) who reported that extracting solvents with higher polarity extracted a

significantly higher amount of bioactive compounds (isoflavones) from soybeans. In the

present study, the highest amount of phytoestrogens were extracted from Larrea

tridentata leaves using methanol, which has the highest polarity compared to the other

extracting solvents evaluated.

It is worth mentioning that heating has also played an important role in the

recovery of these compounds, particularly when using methanol (data not shown), but

did not influence the extraction with ethanol or acetone. Some studies have demonstrated

the influence of temperature on the extraction of phytochemicals (Bimakr et al., 2009;

Karacabey et al., 2009). Razmara et al. (2010) evaluated the effect of temperature, from

19.8 to 60.8 ºC, on the solubility of quercetin in different solvent mixtures (water +

methanol and water + ethanol), and concluded that raising the solvent temperature

increased the solubility of quercetin.

CHAPTER 4


64

Table 4.1 Phytoestrogens extraction from Larrea tridentata leaves using different

organic solvents.

Solvent (% v/v)

NDGA (mg/g dry wt plant)

Kaempferol (mg/g dry wt plant)

Quercetin (mg/g dry wt plant)

H2 2.12 ± 0.25O 8.00 ± 0.94h 2.28 ± 0.17e

Methanol

f

90 46.96 ± 3.39 87.00 ± 6.43a 10.46 ± 1.01a

70

a 33.57 ± 0.88 65.78 ± 3.00b 8.68 ± 0.38b

50

b 22.53 ± 0.66 42.37± 3.85c 5.91 ± 0.47c

30

c 13.31 ± 1.58 30.26 ± 3.66d 5.00 ± 0.38d

Ethanol

de

90 7.69 ± 0.15 48.96 ± 2.17f 5.54 ± 0.21cd

70

cd 7.74 ± 0.10 49.82 ± 0.93f 5.96 ± 0.50c

50

c 7.18 ± 0.24 47.52 ± 2.27f 5.25 ± 0.25cd

30

d 5.25 ± 0.17 38.29 ± 1.14g 4.99 ± 0.29cd

Acetone

de

90 10.82 ± 1.80 50.93 ± 1.74de 5.71 ± 0.12cd

70

c 8.78 ± 0.11 47.98 ± 1.28ef 5.54 ± 0.14cd

50

cd 6.71 ± 0.10 39.04 ± 1.28f 5.00 ± 0.41cd

30

d 6.20 ± 0.28 37.97 ± 2.19fg 4.95 ± 0.32d

Different letters mean values statistically different at 95% confidence level.

e

4.3.2. Effect of solvents on total phenols, total flavonoids and protein contents

Concentration of total phenols, total flavonoids and protein in the produced

extracts are shown in Table 4.2. As can be seen, the total phenols content ranged from

68.55 ± 5.81 mg GAE/g dry wt plant when distilled water was used as extraction solvent,

to 487.13 ± 27.68 mg GAE/g dry wt plant when using 90% (v/v) acetone. Although it

has been reported that the total phenol contents is increased when the solvent polarity is

increased (Tunalier et al., 2007), the present finding do not show such a trend concerning

the solvent polarity, since acetone-water mixtures were proved to be good solvent

systems for the extraction of phenolic compounds from L. tridentata leaves. In fact,

acetone is commonly used and considered quite efficient for the extraction of phenolic

substances (Arts et al., 2002).

CHAPTER 4


65

There was also a large variation in the total flavonoids content depending on the

extraction solvent used, ranging from 4.49 ± 0.30 to 19.29 ± 0.79 mg QE/g dry wt plant

for 30 and 90% (v/v) methanol extracts, respectively. It is know that flavonoids can bind

proteins, and that their interaction might influence the antioxidant capacity of an extract

(Arts et al., 2002). Therefore, the effect of the extraction solvent on the protein content

was also examined (Table 4.2). Protein content ranged from 5.79 ± 0.69 to 131.84 ± 6.23

mg/g dry wt plant for aqueous and 90% (v/v) methanol extracts, respectively. A

significant linear correlation (p<0.05) was found (r = 0.8977) between total flavonoids

and protein contents. These results support the idea that the flavonoids present on the

plant extracts might have a high potential to bind proteins, which could mask the

antioxidant capacity of the extracts.

Table 4.2 Total phenols, flavonoids and protein contents in Larrea tridentata leaves

extracts obtained by using different organic solvents.

Solvent (%)

Total phenols

(mg GAE/g dry wt plant) Total flavonoids

(mg QE/g dry wt plant) Protein

(mg/g dry wt plant)

H2 68.55 ± 5.81O 6.15 ± 0.72g 5.79 ± 0.69d

Methanol

i

90 263.60 ± 25.78 19.29 ± 0.79e 131.84 ± 6.23a 70

a 336.70 ± 32.61 12.23 ± 0.54c 113.88 ± 2.24b

50

b 227.85 ± 8.88 7.95 ± 0.72e 57.72 ± 5.36d

30

f 216.35 ± 6.18 4.49 ± 0.30e 40.01 ± 0.87e

Ethanol

h

90 201.98 ± 9.91 12.09 ± 1.05f 77.33 ± 3.46b 70

d 237.60 ± 11.58 12.39 ± 0.55e 81.11 ± 1.50b

50

d 334.10 ± 5.80 11.54 ± 0.54c 90.26 ± 1.64bc

30

c 285.35 ± 8.77 7.32 ± 0.16de 47.33 ± 3.49d

Acetone

g

90 487.13 ± 27.68 12.87 ± 1.33a 92.95 ± 2.16b 70

c 409.20 ± 35.54 13.32 ± 1.22b 84.73 ± 3.04b

50

cd 315.60 ± 21.35 9.77 ± 0.27cd 67.85 ± 1.60c

30

e 311.35 ± 44.32 8.26 ± 0.28d 59.69 ± 1.33cd


ef

CHAPTER 4


66

4.3.3. Antioxidant potential of Larrea tridentata extracts

Two different techniques based on fundamentally different approaches were used

to determine the antioxidant potential of the plant extracts, including 1,1-diphenyl-2-

picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power

(FRAP), which are highly sensitive methods with reproducible results. All the produced

extracts showed antioxidant potential with similar results for DPPH radical scavenging

activity (Table 4.3). Nevertheless, different behavior was observed for FRAP results

where extracts obtained using 70% and 90% (v/v) methanol had significantly higher

(p<0.05) values (2.55 ± 0.09 and 2.73 ± 0.11 mM FE(II)/g dry wt plant, respectively)

than the remaining ones. Several studies have examined the type of linear correlation

between antioxidant activities and phenolic contents in whole plant extracts, fruits,

vegetables, and beverages (Alothman et al., 2009; Tawaha et al., 2007). Despite the

considerable number of literature data reporting significant linear correlations,

antioxidant activity might not always correlate with phenolic contents (Kahkonen et al.,

1999; Heinonen et al., 1998). In the present study, FRAP results presented good

correlation with the levels of NDGA and quercetin (r = 0.71 and 0.88, respectively), and

in particular with kaempferol (r = 0.91). However, total phenols content was poorly

correlated with FRAP (r = 0.60). Such results indicate that the antioxidant potential of

the plant extract might be related to the presence of specific bioactive compounds, as

well as by their interaction.

Hidalgo et al. (2010) evaluated flavonoid-flavonoid interactions and their effect on

the antioxidant capacity by DPPH and FRAP methods. Among several flavonoids, the

interaction between kaempferol and quercetin was studied, being concluded that when

these compounds were paired an increase in antioxidant activity of about 20% was

achieved compared with their individual theoretical values. According to these authors,

the antioxidant potential of a compound is closely related to its structural characteristics,

the nature of the radical and its specific reaction mechanism; which can be influenced by

the presence of glycosidic moieties, the number and position of hydroxyl and methoxy

groups, and the reactions that promote structural changes. Thus, the high antioxidant

potential of the extracts obtained using 90% (v/v) methanol could be explained by the

high concentrations of kaempferol and quercetin and due to their interaction. On the

CHAPTER 4


67

other hand, a significant correlation (p<0.05) was observed between FRAP and the

protein content, showing that the known interaction between flavonoids and proteins did

not affected the antioxidant capacity of the extracts.

Table 4.3 Effect of different organic solvents on antioxidant capacity of Larrea

tridentata leaves extracts.

Solvent (%)

DPPH inhibition (%)

FRAP (mM FE(II)/g dry wt plant)

H2 93.20 ± 0.40O 0.77 ± 0.02e

Methanol

g

90 94.81 ± 0.33 2.73 ± 0.11ab 70

a 94.06 ± 0.43 2.55 ± 0.09c

50 a

94.52 ± 0.12 1.92 ± 0.18b 30

d 94.19 ± 0.33 1.43 ± 0.02c

Ethanol

f

90 94.97 ± 0.22 1.52 ± 0.12a 70

f 94.28 ± 0.26 1.90 ± 0.08b

50 d

93.71 ± 0.21 2.13 ± 0.06de 30

bc 94.22 ± 0.20 1.74 ± 0.06c

Acetone

e

90 95.08 ± 0.17 1.89 ± 0.22a 70

d 94.28 ± 0.24 2.16 ± 0.05b

50 b

94.28 ± 0.33 1.81 ± 0.05bc 30

de 94.01 ± 0.43 1.96 ± 0.06cd


cd

4.5 Conclusion

In brief, extraction with 90% (v/v) methanol can be considered as an efficient way

to recover phytoestrogens (NDGA, kaempferol and quercetin) from L. tridentata leaves.

The extract obtained under this condition is also a valuable source of natural products

with antioxidant capacity, and might find a number of industrial applications, particularly

in the food and medicinal fields. However, because of the toxicity of methanol, serious

issues are pointed out when the purpose of the compounds extracted with this solvent is

CHAPTER 4


68

the application in food and pharmaceutical industries. In order to overcome this problem,

the next step of our research work will be focused on finding other less or non-toxic

solvents for the extraction of these bioactive compounds, able to promote high extraction

results as methanol, or even using bioprocesses such as the solid-state fermentation that

do not require the use of any organic solvent. The application of methanol in the present

study was useful to establish the maximum amount of phenolic compounds present in L.

tridentata leaves, as well as to evaluate the antioxidant potential of the obtained extracts.

This extraction solvent is one of the most commonly used extraction solvents due to its

high polarity, being also recognized for its efficiency to extract phenolic compounds

from plant materials.

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varying polarities on polyphenols of Orthosiphon stamineus and evaluation of the free radical-

scavenging activity. Food Chemistry, 93, 311–317.

Alothman M., Bhat R., Karim A.A. (2009). Antioxidant capacity and phenolic content of selected tropical

fruits from Malaysia, extracted with different solvents. Food Chemistry, 115, 785–788.

Arteaga S., Andrade-Cetto A., Cárdenas R. (2005). Larrea tridentata (Creosote bush), an abundant plant of

Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid. Journal of

Ethnopharmacology, 98, 231–239.

Arts M.J.T.J., Haenen G.R.M.M., Wilms L.C., Beetstra S.A.J.N., Heijnen C.G.M., Voss H.-P., Bast A.

(2002). Interactions between flavonoids and proteins: effect on the total antioxidant capacity.

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carbon dioxide (SC-CO2

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CHAPTER 5

Chemical composition and solid-state fermentation of Larrea

tridentata leaves by Phanerochaete chrysosporium

This chapter explores the potential of the basidiomycete Phanerochaete chrysosporium to

recover or enhance the extraction of bioactive compounds from L. tridentata leaves by solid-

state fermentation. A chemical characterization and a mineral profile of L. tridentata leaves

were previously determined and considered. The total phenolic, flavonoids, and proteins

contents, as well as the antioxidant activity of the produced extracts were analyzed.

72

CHAPTER 5 CHEMICAL COMPOSITION AND SOLID-STATE FERMENTATION OF Larrea tridentata LEAVES BY Phanerochaete

chrysosporium

73

5.1 Introduction

Extraction of plant-derived bioactive compounds has usually been accomplished

by conventional extraction processes such as solid-liquid extraction employing organic

solvents. Recently, there has been an increasing interest in the use of environmentally

clean technologies able to provide extracts with high quality and high biological activity,

while precluding any toxicity associated to the solvents. In this sense, fermentation

processes, in particular, the solid-state fermentation (SSF) has become an interesting

alternative technology for the production/extraction of plant bioactive compounds

(Pandey, 2003; Holker and Lenz, 2005).

Larrea tridentata (Zygophyllaceae), commonly known as creosote bush, is a plant

that grows in semidesert areas of Southwestern United States and Northern Mexico

(Ross, 2005). It was traditionally used for centuries by North American Indians as a

medicinal plant for treatment of several illnesses including infections, kidney problems,

gallstones, rheumatism, arthritis, diabetes, and to tumors (Navarro et al., 1996). Larrea

tridentata is an outstanding source of natural compounds with approximately 50% of the

leaves (dry weight) being extractable matter (Arteaga et al., 2005). Nordihydroguaiaretic

acid (NDGA), kaempferol and quercetin can be found at considerable concentrations in

this plant (Martins et al., 2012), and several studies have demonstrated the potential of

these bioactive compounds in the health area (Nakamura et al., 2005; Zavodovskaya et

al., 2008; Chen et al., 2010).

The white rot fungi Phanerochaete chrysosporium has ability of producing lignin

and manganese peroxidases extracellularly (Martin et al., 1999; Kumar et al., 2006), and

is known for its potential to degrade lignin. The biodegradation of lignin by ligninolytic

enzymes is a non specific free radicals linked reaction in the lignin polymer, resulting in

the destabilization of bonds and finally into breakdown of macromolecules (Barr and

Aust, 1994). Therefore, the purpose of the present study was to evaluate the potential of

the basidiomycete P. chrysosporium to recover or enhance the extraction of bioactive

compounds from L. tridentata leaves. The experimental assays were performed under

SSF conditions and the concentrations of quercetin (Q), kaempferol (K),

nordihydroguaiaretic acid (NDGA), total phenols compounds, flavonoids and proteins in

the extracts were taken as responses of these experiments. Total antioxidant activity of

the produced extracts was also determined.


chrysosporium

74



Plant material (L. tridentata) was collected from the Chihuahuan semidesert (North

Coahuila, Mexico) during Spring season (April, 2009). Nordihydroguaiaretic acid

(NDGA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), quercetin, kaempferol, aluminum

chloride, 2,4,6-tris (1-pyridyl)-5-triazine (TPTZ), sodium acetate, ferrous sulfate and iron

(III) chloride were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Reagent-

grade methanol, ethanol, acetone, acetic acid and Folin-Ciocalteau were from Panreac

(Barcelona, Spain). Potassium acetate was purchased from AppliChem (Darmstadt,

Germany). HPLC-grade acetonitrile was obtained from Fisher Scientific (Leicestershire,

UK). Ultrapure water from a Milli-Q System (Millipore Inc., USA) was used.

5.2.2 Chemical characterization

The content of cellulose, hemicellulose, lignin and acetyl groups in L. tridentata

leaves was determined according to the procedure reported by Browning (1967). Briefly,

the plant material was subjected to a quantitative acid hydrolysis with 72% (w/w)

H2SO4 at 45 ºC during 7 min. Afterwards, distilled water was added to the mixture to

dilute the H2SO4 to 1 N, and the samples were autoclaved at 121 ºC for 45 min.. The

solid residue after hydrolysis was recovered by filtration and considered as Klason lignin

(after subtracting the content of ashes in this residual material). The monosaccharides

and acetic acid contained in the hydrolysates were determined by HPLC in order to

estimate (after corrections for stoichiometry and sugar decomposition) the contents of

samples in cellulose (as glucan), hemicellulose (xylan, arabinan, galactan and mannan)

and acetyl groups (Irick et al., 1988). To determine the amount of acid-soluble lignin,

hydrolysate samples had their pH adjusted to 12 by addition of NaOH 6M. Then, the pH-

adjusted samples were diluted with distilled water and analyzed in a spectrophotometer

at 280 nm. Hydrolysate samples were also analyzed by HPLC in order to quantify the

amounts of furfural and hydroximethilfurfural, which were used to calculate the

percentage of acid-soluble lignin. To quantify the total amount of ashes, 1 g of the

ground air-dried plant material, accurately weighed, was placed in a previously ignited

and tared crucible and heated at 550 °C for 4 h. The content of total ashes was calculated


chrysosporium

75

by the difference of weight before and after incineration of the sample. The protein

content was determined by quantification of the total nitrogen using the Kjeldahl method.

A conversion factor of 6.25 was used. The extractives were calculated by difference, i.e.,

by subtracting the sum of cellulose, hemicelluloses, total lignin, ashes, proteins and

acetyl groups from the dry weight of the plant sample.

-Organic carbon and total nitrogen contents were determined by combustion using

a Thermo Scientific Flash 2000 Elemental Analyzer. The mineral content was

determined by inductively coupled plasma atomic emission spectrometry (ICP-AES).

Samples (200 mg) were digested with HNO3 (5 ml) and H2O2

(3 ml) in closed vessels

(XF100, Anton Paar) using a Multiwave 3000 microwave (Anton Paar). For the

digestion, the microwave power was increased from 0 to 1150 W during 9 min, and was

then maintained at 1150 W during 10 min. After cooled to room temperature, the final

volume of the samples was adjusted to 100 ml, and they were analyzed by ICP-AES in a

Thermo Scientific iCAP 6300 equipment.

5.2.3 Solid-state fermentation process

5.2.3.1 Fungal strain and spores collection

Phanerochaete chrysosporium MUM 9415 (from Micoteca of the Centre of

Biological Engineering, University of Minho) was the fungus used in the experiments.

The strain was maintained at 4 ºC on Petri plates containing potato dextrose agar (PDA,

Difco). For the production of spores the cultures were maintained at 37 ºC on fresh PDA

medium (pH 4.5) for 7 days. The inoculum for use in the experiments was obtained by

suspension of the produced spores in sterilized solution of 0.1 % (w/v) Tween 80 and

adjustment to the desired concentration by counting in a Neubauer chamber.

5.2.3.2 Solid-state fermentation conditions

SSF cultivations were performed in 250 mL Erlenmeyer flasks containing 10.0 g

of sterilized powdered plant. The plant material was moistened with the following


chrysosporium

76

culture medium in order to attain 70% moisture content (g/L): K2HPO4 (1.0), NaNO3

(3.0), MgSO4 (0.5), FeSO4.7H2O (0.01), and KCl (0.5), adjusted to pH 5 and sterilized at

121 °C for 15 min. The moistened material was inoculated with 2×107

spores/g plant,

and statically incubated at 37 °C. Samples for analysis were collected after 7, 10, 14, 18

and 21 days of cultivation, and the humidity was regularly checked every three days and

adjusted to 70% by addition of culture medium. The total content of each Erlenmeyer

was collected as a sample. The fermented broth was recovered by filtration through 0.2

µm membrane filters (fermentative extract) and stored at -20 ºC until further analysis.

The fermented plant was dried and subjected to extraction with 90% (v/v) methanol (1 g

of fermented plant to 20 mL methanol) during 30 min in a water-bath at 65-70 ºC. The

produced extracts were separated by filtration through 0.2 µm membrane filters and

stored at -20 ºC until further analysis.


chrysosporium

77

Fig. 5.1 Schematic flow diagram of experimental steps proposed for SSF of L. tridentata

leaves using P. chrysosporium.

Dehydration and pulverization

Sterilization of plant material (15 min at 121ºC)

Preparation of SSF with Czapek-Dox medium (pH = 5.0) with a humidity adjusted to 70%

Inoculation of P. chrysosporium 2 x 107 spores/ g of solid support

Solid-State Fermentation (incubation at 37 ºC for 21 days)

Liquid extract

Sieve to particle size of 300-600 µm

Total phenols FTIR SEM

Humidity control

Flavonoids

Total Antioxidant Activity

Organic solvent extracts HPLC Proteins

Larrea tridentata plant biomass

TOC

Solid extract


chrysosporium

78

5.2.4 Fourier transform infrared spectroscopy (FTIR) assays

FTIR measurements were carried out to investigate the changes in surface

functional groups of the plant material after 21 days of SSF. The analyses were carried

out in a Jasco infrared spectrometer (FT/IR-4100 Type A) using a frequency rangefrom

4000 to 500 cm-1

. For FTIR measurement, the dried plant biomass samples were mixed

with spectroscopic grade KBr and then pressed using a hydraulic pressing system at 10

Ton to form pellets. The pellets were about 10 mm in diameter and 1 mm thickness. The

vibration transition frequencies of the spectra were subjected to base line correction.

5.2.5 Scanning electron microscopy analyses

Micrographs of plant material samples (untreated and fungal treated after 21 days

of SSF) were obtained by scanning electron microscopy using a Leica Cambridge S360

microscope. For the analyses, the samples were fixed on a specimen holder with

aluminum tape and then sputtered with gold in a sputter-coater under high vacuum

condition. Images were obtained at 500-fold magnification.

5.2.6 Bioactive compounds quantification

NDGA, kaempferol and quercetin concentrations in the extracts were determined

by high performance liquid chromatography (HPLC) on an equipment LC-10 A (Jasco,

Japan) with a C18 5 µm (3.9 × 300 mm) column at room temperature, and a UV detector

at 280 nm. The response of the detector was recorded and integrated using the Star





phase was eluted in a flow rate of 1.0 ml/min, and samples of 10 µl were injected.


NDGA, kaempferol and quercetin contents in the extracts were expressed as the ratio

between mass of the compound in the extracts and mass of plant material (dry weight).


chrysosporium

79

5.2.7 Determination of total phenols content

Total phenols content was determined by the Folin-Ciocalteu method with

modifications. Briefly, 5 µl of the filtered extracts duly diluted were mixed with 60 µl of

sodium carbonate solution (7.5% w/v) and 15 µl of Folin–Ciocalteu reagent in a 96-well

microplate. Then 200 µl of distilled water were added and the solutions were mixed.

After standing for 5 min at 60 ºC samples were allowed to cool down at room

temperature. The absorbance was measured using a spectrophotometric microplate

reader (Sunrise Tecan, Grödig, Austria) set at 700 nm. A calibration curve was prepared

using a standard solution of gallic acid (200, 400, 600, 800, 1000, 2000, 3000 mg/l, r2

=

0.9987). The total phenols content determined according to the Folin-Ciocalteau method

is not an absolute measurement of the phenolic compounds amount, but is in fact based

on their chemical reducing capacity relative to an equivalent reducing capacity of gallic

acid. Thus, total phenols content was expressed as milligram gallic acid equivalent (mg

GAE)/g DW plant material (dry weight).

5.2.8 Determination of total flavonoids content

Total flavonoids content was quantified by colorimetric assay. Briefly, 30 μl of the

diluted and filtered extracts was added to 90 μl of methanol in a 96-well microplate.

Subsequently, 6 μl of aluminum chloride (10 % w/v), 6 μl of potassium acetate (1 mol/l)

and 170 μl of distilled water were added to the mixture, which was maintained during 30

minutes in the dark at room temperature. The absorbance of the mixture then readat 415

nm against a blank prepared with distilled water, using a spectrophotometric microplate

reader (Sunrise Tecan, Grödig, Austria). A calibration curve was prepared using a

standard solution of quercetin (25, 50, 100, 150, 200 mg/l, r2

= 0.9994). Total flavonoids

content was expressed as milligram quercetin equivalent (mg of QE)/g DW plant

material (dry weight).


chrysosporium

80

5.2.9 Determination of protein content

Total protein content was estimated using the Bradford assay.

5.2.10 Total antioxidant capacity

To evaluate the total antioxidant capacity of the extracts an aliquot of 0.1 ml of

each sample was mixed in a tube with 1 ml of a reagent solution containing 0.6 M

sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The tubes

were closed with lids and incubated in a water-bath at 95°C for 90 min. After the

samples had cooled to room temperature, the absorbance was measured at 695 nm

against a blank. The blank solution contained 1 ml of reagent solution and 0.1 ml of the

same solvent present in the sample (water or methanol), and it was incubated under the

same conditions used for the other samples. A calibration curve was prepared using a

standard solution of α-tocopherol (25, 75, 125, 250, 375, 500 µg/ml, r2

= 0.9961). The

total antioxidant capacity of the samples was expressed as equivalents of α-tocopherol/ g

of plant material (dry weight).


All the experimental assays and determinations were performed in triplicate, and

mean values ± standard errors are presented. Results were analyzed by one-way analysis

of variance (ANOVA) using the general linear model of SPSS (Statistical Package for

Social Sciences, version 16.0) for a significance level of p<0.05. Difference among

samples was verified by using the Tukey’s range test.


5.3.1 Chemical characterization of L. tridentata leaves

Knowing the chemical composition of L. tridentata leaves is of great importance

if it is desired to use this plant as substrate for solid-state fermentation processes.


chrysosporium

81

Chemical characterization analyses revealed that L tridentata is composed of 2.27% total

nitrogen and 46.30% organic carbon. The contents of cellulose, hemicellulose, lignin,

acetyl groups, proteins, extractives and ashes in the plant leaves are presented in Table

5.1.. As can be seen in this table, the lignin fraction is the most abundant in the leaves’

composition (35.96% w/w), followed by the extractives (17.31% w/w), hemicellulose

(13.10% w/w) and proteins (13.01% w/w), respectively. The lignin content in L.

tridentata leaves is still greater than the sum of the fractions containing sugars (cellulose

and hemicelluloses), revealing the importance of this fraction in the constitution of the

plant. Lignin is a heterogeneous and optically inactive polymer structurally formed by

phenyl propanoid units linked by several covalent bonds like aryl–ether, aryl–aryl, and

carbon-carbon (Brunow, 2001). This biopolymer is closely bound to cellulose and

hemicellulose in cell walls of plants, conferring water impermeability of xylem vessels,

and forming a physic–chemical barrier against microbial attack (Fengel and Wegener,

1989). Due to its complex and heterogeneous structure, lignin is extremely difficult to be

chemically and enzymatically degraded (Martin, 2002).

Table 5.1 Chemical composition of L. tridentata leaves.

Fraction % Dry weight (g/ 100g)

Cellulose (glucan) 10.09

Hemicellulose 13.10

Xylan 4.42

Arabinan 4.68

Galactan 2.05

Mannan 1.95

Total lignin 35.96

Klason lignin 31.06

Acid-soluble lignin 4.90

Ashes 7.91

Protein 13.01

Acetyl groups 2.62

Extractives 17.31


chrysosporium

82

Ashes represent also an important fraction in L. tridentata leaves (7.91% w/w).

The minerals present in the ashes are listed in Table 5.2. Among them, phosphorus,

potassium, calcium, magnesium, sulfur, boron, iron, manganese, zinc, copper,

molybdenum, and nickel are considered mineral elements essential for plant growth.

Besides these essential mineral elements, there are also some beneficial elements that

promote growth in many plant species but are not absolutely necessary for completion of

the plant life cycle, such as aluminium, sodium, cobalt, and selenium (Pilon-Smits et al.,

2009). Besides the essential and beneficial minerals, other elements, in particular,

barium, strontium, tin, iodine, and gallium were also quantified in L.tridentata leaves in

order to have a more detailed chemical characterization of the plant material

composition. Finally, total organic carbon and total nitrogen contents in L. tridentata

leaves were also measured (46.30 ± 0.10 and 2.27 ± 0.10 %, respectively).

Table 5.2 Mineral elements in L. tridentata leaves.

Mineral element Concentration (a) Phosphorus 0.10 ± 0.00 % Potassium 1.11 ± 0.01 % Calcium 2.27 ± 0.01 % Magnesium 0.14 ± 0.00 % Sulfur 0.39 ± 0.00 % Iron 304.8 ± 3.1 mg/kg Manganese 41.0 ± 0.7 mg/kg Boron 52.3 ± 1.5 mg/kg Copper 5.8 ± 0.4 mg/kg Zinc 23.9 ± 0.4 mg/kg Molybdenum 1.02 ± 0.05 mg/kg Sodium 593.0 ± 2.8 mg/kg Aluminum 275.1 ± 6.1 mg/kg Barium 18.39 ± 0.05 mg/kg Strontium 109.3 ± 0.5 mg/kg Chromium < 0.54 mg/kg Tin < 1.3 mg/kg Cobalt <0.59 mg/kg Iodine 51.5 ± 13.1 mg/kg Nickel 1.17 ± 0.29 mg/kg Selenium <1.6 mg/kg Gallium <1.47 mg/kg Vanadium 1.07 ± 0.05 mg/kg

(a)Values are mean ± standard deviation; % in g/100g.


chrysosporium

83

5.3.2 FTIR and SEM measurements

FTIR spectra of fungal treated (21 days) and untreated samples of L. tridentata

leaves are shown in Fig. 5.2. A strong hydrogen bonded (O–H) stretching absorption is

present at 3400 cm-1 and a (C–H) stretching absorption around 2960 cm-1 is also

detected. There are also many well-defined peaks in the finger print region between 1800

and 600 cm-1, being assigned as follows: 1640 cm-1 for absorbed O–H and conjugated C–

O, 1460 cm-1 and 1440 cm-1 for C–H deformation in lignin, 1380 cm-1 for C–H

deformation in cellulose and hemicellulose, 1320 cm-1 for C–H vibration in cellulose and

C–O vibration in syringyl derivatives, 1100 cm-1

for aromatic skeletal and C–O stretch in

cellulose and hemicellulose (Hergert, 1971; Schultz and Glasser, 1986; Faix, 1992;

Collier et al., 1992; Pandey and Theagarajan, 1997). When comparing the FTIR spectra

of the untreated and treated samples it can be observed that after 21 days of SSF the

intensities of absorption decreased, which suggest solubilization of constituents of the

lignocellulose fraction.

Fig. 5.2 FTIR spectra of L. tridentata samples before (A) and after 21 days of SSF (B)

with P. chrysosporium.

500 1000 1500 2000 2500 3000 3500 4000

% T

rans

mitt

ance

Wavenumber range (cm-1)

(B)

(A)


chrysosporium

84

The scanning electron microscopy (SEM) was then used to verify possible

morphological changes of the lignin matrix after SSF of L. tridentata leaves with P.

chrysosporium (Fig. 5.3). SEM micrographs clearly revealed a distinct cellular

organization between samples of untreated plant material (Fig. 5.3A and C) and fungal

treated (21 days) plant material (Fig. 5.3B and D) observing in the latter case a major

disorganization and porosity of the material structure.

Fig. 5.3 Micrographs by scanning electron microscopy of L. tridentata samples in the

following forms: (A,C) untreated and (B,D) fungal treated (after 21 days SSF).

Magnification: 300-fold (A and B) and 1000-fold (C and D).

A

B

C

D


chrysosporium

85

5.3.3 Bioactive compounds extraction by SSF

P. chrysosporium is a white-rot fungus known by its ability to produce extracellular

oxidative enzymes, in particular lignin and manganese peroxidases, which degrade plant

wood compounds such as lignin. Due to the high content of lignin (36% w/w) in L.

tridentata leaves, a hypothesis had been formulated considering that this fungal strain

could liberate bioactive compounds from cellular degradation of this plant material

during SSF. In order to evaluate the efficiency of SSF with P. chrysosporium in

extracting bioactive compounds from L. tridentata leaves, several experimental

responses were considered and distinct quantifications were performed.

NDGA, kaempferol and quercetin were quantified in the fermentative extracts and

also in the plant extracts that were obtained by extraction of the fermented material with

methanol. These results are summarized Table 5.3. As can be seen, the concentration of

these three bioactive compounds in the fermentative extracts was quite low.

Additionally, no significant effect (p<0.05) of the SSF on their extraction with methanol

was observed when comparing the plant extracts with the results obtained in a previous

study for solid-liquid extraction of L. tridentata, which were considered as reference

values (Martins et al., 2012).

Total phenolic compounds, flavonoids and protein contents in the extracts were also

determined in order to verify the effect of SSF on the recovery of bioactive compounds

(Table 5.4). After 7 days of SSF some increase in the total phenolic and flavonoids

contents was observed considering the sum of the concentrations obtained in both

fermentative and plant extracts, and comparing this result with the reference

concentration. However, controls after 10 and 21 days showed similar results, which

suggest that only the mixture of the culture medium with the plant material under the

conditions used for cultivation could have provided the recovery of these

phytochemicals. Similar results were observed for the protein content. Total antioxidant

activity (TAA) of fermentative and plant extracts was determined and results are also

shown in Table 5.4. As observed for the other responses, no significant increment

(p<0.05) was also observed on the TAA of the extracts when compared with the control

assays.

Table 5.3 Effect of SSF with P. chrysosporium during 21 days on the recovery of bioactive compounds from L. tridentata leaves extracts.

Fermentation

period (days)

Quercetin

(mg /g dry wt plant)

Kaempferol

(mg/g dry wt plant)

NDGA

(mg/g dry wt plant)

FE PE FE PE FE PE

C0 nd 9.43 ± 0.77 nd a 11.90 ± 0.58 nd a 20.11 ± 1.04

7

a

0.10 ± 0.01 b 2.43 ± 0.44 0.29 ± 0.00b a 12.42 ± 1.06 0.16 ± 0.01a a 14.05 ± 1.05

10

b 0.16 ± 0.02 a 1.62 ± 0.04 0.27 ± 0.01bc a 9.02 ± 0.18 0.15 ± 0.12b a 8.11 ± 0.11

14

c

0.09 ± 0.01 b 1.80 ± 0.08 0.19 ± 0.02b bc 8.69 ± 0.38 0.08 ± 0.01b b 6.17 ± 0.41

18

c

0.08 ± 0.02 b 1.19 ± 0.01 0.14 ± 0.05c c 7.09 ± 0.11 0.07 ± 0.02c b 4.59 ± 0.02

21

cd

0.09 ± 0.01 b 1.06 ± 0.04 0.21 ± 0.01c b 5.65 ± 0.10 0.07 ± 0.01c b 3.15 ± 0.19

C10

d

0.11 ± 0.03 ab 1.80 ± 0.02 0.21 ± 0.02bc b 9.41 ± 0.43 0.10 ± 0.01b ab 8.51 ± 0.05

C21

c

0.10 ± 0.00 b 1.12 ± 0.05 0.21 ± 0.01c b 6.88 ± 0.52 0.08 ± 0.01c b 5.00 ± 0.04

cd

C0: reference values obtained in a previous research work (Martins et al., 2012); C10 and C21: control samples (without inoculation with P. chrysosporium) after 10 and 21

days under SSF conditions, respectively; FE: fermentative extracts; PE: plant extract; nd: not determined.


Table 5.4 Total phenols, flavonoids and protein contents, and total antioxidant capacity in L. tridentata leaves extracts obtained after SSF with

P. chrysosporium during 21 days, and in the extracts obtained by methanolic extraction of fermented plant material.

Fermentation

period (days)

Total phenols

(mg GAE/g dry wt plant)

Total flavonoids

(mg QE/g dry wt plant)

Protein

(mg BSA/g dry wt plant) Total antioxidant capacity

(nmol α-tocopherol/g dry wt plant)

FE PE FE PE FE PE FE PE

C0 nd 260.60 ± 25.78 nd a 19.29 ± 0.79 nd b 131.80 ± 6.23 nd b nd

7 70.43 ± 5.85 a 280.80 ± 27.83 4.57 ± 0.55a a 24.08 ± 1.72 7.01 ± 0.76a ab 112.00 ± 14.64 103.20 ± 5.52b ab 1330.0 ± 114.6

10

a 71.59 ± 3.56 a 245.60 ± 11.76 4.35 ± 0.35b ab 24.11 ± 1.17 7.56 ± 0.51a a 147.10 ± 14.19 114.00 ± 6.76a a 1315.0 ± 22.8

14

a

65.61 ± 9.11 ab 267.30 ± 28.16 3.51 ± 0.37a b 25.00 ± 1.22 6.76 ± 0.69a bc 133.70 ± 11.18 88.90 ± 9.02a b 1223.0 ± 155.8

18

ab

37.07 ± 9.73 c 281.60 ± 14.78 1.80 ± 0.28a d 22.19 ± 1.12 4.28 ± 0.36ab d 123.80 ± 7.24 63.71 ± 14.43b c 995.5 ± 577.8

21

b

56.88 ± 7.10 b 255.60 ± 26.52 2.81 ± 0.65a c 23.05 ± 1.56 5.06 ± 0.66a cd 121.20 ± 9.87 84.64 ± 6.13b b 577.8 ± 80.19

C10

c

59.56 ± 8.79 b 223.00 ± 14.42 3.73 ± 0.63b b 25.10 ± 1.77 6.49 ± 0.75a c 122.00 ± 5.64 111.00 ± 5.03b a 1185.0 ± 116.2

C21

ab

63.51 ± 6.46 ab 274.80 ± 15.12 3.40 ± 0.25a b 23.30 ± 1.41 6.17 ± 0.61a c 118.70 ± 17.14 87.24 ± 6.61b b 688.0 ± 67.5

c

C0: reference values obtained in a previous research work (Martins et al., 2012); C10 and C21: control samples (without inoculation with P. chrysosporium) after 10 and 21

days under SSF conditions, respectively; FE: fermentative extracts; PE: plant extract; nd: not determined.



chrysosporium

88

Finally, analyzes of the total organic carbon in the samples made possible to

evaluate the release of low molecular weight compounds during SSF. Table 5.5 shows

the TOC values obtained during SSF of L. tridentata leaves with P. chrysosporium. No

significant differences (p<0.05) in TOC values were observed during SSF, when

comparing with the value obtained in the original plant material (46.30 ± 0.10 %, Table

5.2). In brief, despite the FTIR and SEM measurements demonstrated a cellular rupture

that could have facilitated the recovery of phytochemicals from L tridentata; TOC results

might explain the inefficiency of this fungal strain to liberate bioactive compounds from

plant matrix.

Table 5.5 Effect of SSF on total organic carbon (TOC) present in L. tridentata leaves.

Fermentation period

(days)

TOC

(%)

7 46.86 ± 0.10

10 47.19 ± 0.10

14 47.00 ± 0.10

18 46.58 ± 0.10

21 47.60 ± 0.10

C10 46.54 ± 0.10

C21 46.88 ± 0.10 C10 and C21: control samples (without inoculation with P. chrysosporium) after 10 and 21 days under SSF

conditions, respectively.

5.4 Conclusion

High content of lignin (36% w/w) was found in L. tridentata leaves and a hypothesis

was formulated considering that P. chrysosporium, a fungal strain known to produce

ligninolytic enzymes, could liberate bioactive compounds from cellular degradation of


chrysosporium

89

this plant material during SSF. SEM micrographs clearly revealed a major

disorganization of the material structure after fermentation with P. chrysosporium, and

FTIR spectra also indicated a possible solubilization of the constituents of lignocellulose

fraction. However, results showed neither significant liberation nor an improvement of

chemical extraction of NDGA, Q and K by submitting the plant to SSF. No significant

effect was also observed concerning the total antioxidant activity of the produced

extracts. On the other hand, some increase of the total phenols, flavonoids and protein

contents in the extracts were obtained after the plant fermentation.

In general, it was concluded that even being ability to degrade lignin structures, P.

chrysosporium was not an efficient fungal strain to extract bioactive compounds from L.

tridentata leaves, and the mixture of the culture medium with the plant material under

the conditions used for solid cultivation could have been the main responsible for the

recovery of these phytochemicals from the plant structure. In any way, maintaining the

material moistened to 70% with a nutrients solution during 10 days at 37 ºC could be

considered an alternative to extract total phenolic compounds, flavonoids and proteins

without requiring the use of organic solvents; which, despite the longer time required,

would be a more environmentally friendly technology when compared to solid-liquid

extraction with organic solvents. On the other hand, this extraction procedure was not

very efficient to extract NDGA, quercetin and kaempferol from L. tridentata, and further

studies are required in order to establish a low-cost technology able to extract these

compounds with efficiency similar or higher than solid-liquid extraction with methanol.

SSF with other fungal strains could also be evaluated for this purpose.

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Pandey K.K., Theagarajan K.S. (1997). Analysis of wood surfaces by diffuse reflectance (DRIFT) and

photoacoustic (PAS) Fourier transform infrared spectroscopic techniques. Holz. Roh. Werkstoff, 55,

383–390.

Pilon-Smits E.A.H., Quinn C.F., Tapken W., Malagoli M., Schiavon M. (2009). Physiological functions of

beneficial elements. Current Opinion in Plant Biology, 12, 267-274.


uses (Volume 3), Humana Press, New Jersey.


chrysosporium

91

Schultz T.P., Glasser W.G. (1986). Quantitative structural analysis of lignin by diffuse reflectance Fourier

transfer spectrometry. Holzforschung, 40, 37–44.





92

93

CHAPTER 6

Antibacterial activity of crude methanolic extract and fractions

obtained from Larrea tridentata leaves

This chapter shows the antibacterial activity of the crude methanolic extract and fractions

(hexane, dichloromethane, ethyl acetate and ethanol) obtained from L. tridentata leaves.

Quantification of different bioactive compounds in crude methanolic extract and fractions was

performed underlying their antibacterial characteristics.

94

CHAPTER 6

ANTIBACTERIAL ACTIVITY OF CRUDE METHANOLIC EXTRACT AND FRACTIONS OBTAINED FROM Larrea tridentata LEAVES

95

6.1 Introduction

Since human being existence, plants have been used for medicinal purposes and

are the primary source of phytochemicals present in conventional medicaments.

Ethnobotanical studies have described and explained the relationships between cultures

and the traditional use of plants. These studies are of great importance and provide

essential information that allows the development of scientific research more oriented to

explore and prove the therapeutic potential of plants. Larrea tridentata (Sessé & Moc.

Ex DC.) Coville (Zygophyllaceae), commonly known as creosote bush, is a plant that

grows in semiarid areas of Southwestern United States and Northern Mexico (Ross,

2005). This plant was traditionally used for centuries by North American Indians to treat

a wide range of medical conditions and illnesses including genitor-urinary and


skin, kidney problems, arthritis, diabetes and cancer, among other diseases (Brinker,

1993; Ross, 2005). Over the past several years, the increase of bacteria drug-resistance

and the rapid emergence of new infections have intensely decreased the efficiency of the

drugs to treat pathologies caused by certain microorganisms. This situation rises up the

urgent need for the development of new antibacterial agents, preferentially, from natural

sources (Sánchez-Medina et al., 2001; Weckesser et al., 2007).


50% of the leaves (dry weight) being extractable matter (Arteaga et al., 2005). Among

several valuable bioactive phenolic compounds found in this plant, the natural occurring

lignan nordihydroguaiaretic acid (NDGA) has been pointed out as the most important,

since it presents biological activities of large interest in the health area, such as antiviral,

antimicrobial, and antitumorgenic (Hwu et al., 2008; Lambert et al., 2004). Other

secondary metabolites identified in L. tridentata include lignans (dihydroguaiaretic acid,

hemi-norisoguaiacin and norisoguaiacin), flavonoids (aglycones: apigenin and

kaempferol; glycosides: chrysoeriol and quercetin), saponins (larreagenin A and larreic

acid), triterpenes and triterpenoids (Brinker, 1993; Hui-Zheng et al., 1988; Jitsuno and

Mimaki, 2010), among others. Some studies have demonstrated antimicrobial capacity of

extracts from L. tridentata, such as antiviral (Brent, 1999), antifungal (Mojica-Marín et

al., 2011; Tequida et al., 2002), and antibacterial activities (Verástegui et al., 1996).

Nevertheless, to the best of our knowledge, no studies about the antibacterial activity of

CHAPTER 6


96

crude methanolic extract and specific fractions from L. tridentata leaves have been

reported.

The purpose of this study was to evaluate the antibacterial activity of the crude

methanolic extract and fractions (hexane, dichloromethane, ethyl acetate, and ethanol)

from L. tridentata leaves against different bacteria species. High performance liquid

chromatography analyses to identify and quantify some phytocomponents in the samples

were also performed and are discussed.


6.2.1. Plant material and chemicals


(North Coahuila, Mexico) during Spring season (April, 2010). Nordihydroguaiaretic acid

(NDGA) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Reagent-grade

methanol, hexane, dichloromethane, ethyl acetate and ethanol were from Vetec (Rio de

Janeiro, Brazil). HPLC-grade acetonitrile was obtained from Fisher Scientific

(Leicestershire, UK). Ultrapure water from a Milli-Q System (Millipore Inc., USA) was

used.

6.2.2. Extraction methodology and fractioning

Air-dried leaves of L. tridentata were ground to fine powder and stored in dark

bottles at room temperature for further analysis. Extraction was performed by mixing 1 g

of plant material with 20 mL of 90% methanol and heating in a water-bath at 60-65 ºC

for 20 min. The obtained extract was filtered through qualitative filter paper and the

solvent was removed by rotary evaporation under reduced pressure at temperatures

below 45 °C. The resulting crude extract was then stored at 4 °C until further analysis. A

portion of the crude methanolic extract (10 g) was fractioned by filter column

chromatography over 100 g silica gel 60 (S) (Santos et al., 2009), and eluted with

approximately 1 L of the solvents hexane, dichloromethane, ethyl acetate, and ethanol, in

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97

the order of increasing polarity, until a clear extract was obtained at the end of the

elution. Pump pressure at approximately 5 bar was applied to accelerate the elution of the

solvents. Eluates were collected in 1-L Erlenmeyer flasks and each fraction was

subjected to evaporation under reduced pressure in a rotary evaporator. Fractions were

stored at 4 °C until assayed.

6.2.3 Antibacterial activity assays

6.2.3.1 Bacterial strains

The organisms tested in these assays were obtained from the collection of the

Laboratory of Microbiological Analysis (Federal University of Permambuco, Brazil).

Antibacterial evaluations were performed against six Gram-positive bacteria strains:

Staphylococcus aureus - standard strain ATCC 6538 (Sa1) and a methicillin-resistant S.

aureus (MRSA) strain isolated from secretion (Sa2), S. saprophyticus - standard strain

LACEN (Ss), S. epidermidis - isolated from catheter secretion (Se), Enterococcus

faecalis - standard strain ATCC 51299 (Ef1) and a strain isolated from urine (Ef2); and

six Gram-negative bacteria strains: Pseudomonas aeruginosa - standard strain ATCC

14502 (Pa1) and a strain isolated from blood (Pa2), Klebsiella pneumoniae - isolated

from surgical wound secretion (Kp1) and from secretion (Kp2), Escherichia coli -

standard strain ATCC 35218 (Ec1) and a strain isolated from secretion (Ec2).

6.2.3.2 Antibacterial test using the agar diffusion method (well)

A preliminary evaluation of the antibacterial activity of the crude methanolic

extract and fractions from L. tridentata leaves was determined by the agar diffusion

method using the well technique proposed by the Clinical and Laboratory Standards

Institute (CLSI, 2009a). Briefly, all the samples were dissolved in dimethyl sulfoxide

(DMSO 40% v/v) in order to obtain concentrations of 500, 1000, 2000 and 4000 µg/mL.

Inoculum of the bacterial strains (108 CFU/mL) were then plated using sterile swabs into

Petri dishes (90 mm) with 20 mL of Mueller-Hinton agar, where 6 mm wells were cut

and filled with 100 µL of sample (50, 100, 200 and 400 µg/well). Tetracycline (100 µL

at a concentration of 300 µg/mL, equivalent to 30 µg/well) was used as positive control

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98

and DMSO (40%, v/v) as negative control. The Petri dishes were pre-incubated for 3 h at

room temperature, allowing the complete diffusion of the samples (Das et al., 2010;

Möller, 1966) and, then, incubated at 37 ± 1 °C for 24 h. The agar diffusion method

using the well technique is known for its advantage of allowing the use of adjuvants to

improve the solubility of the extract constituents, as well as permitting its radial and

superficial diffusion (Caetano et al., 2002). The complete diffusion of samples into the

Mueller-Hinton agar was visually perceptible and confirmed. The antibacterial activity

was determined by measuring of inhibition zone diameters (mm) and was evaluated

according the parameters suggested by Alves et al. (2000): inhibition zones < 9 mm,

inactive; 9-12 mm, less active; 13-18 mm, active; > 18 mm, very active.

6.2.4. Determination of minimal inhibitory concentration (MIC)

The evaluation of MICs was performed for the samples that showed an inhibition

zone ≥ 13 mm, using the micro -dilution methodology described by the Clinical and

Laboratory Standards Institute (CLSI, 2009b). In these assays, other bacterial strains

were also tested, including S. aureus MRSA strain isolated from tracheal secretion (Sa3),

S. aureus MRSA strain isolated from secretion (Sa4), S. coagulase-negative isolated

from catheter secretion (Scn1), and S. coagulase-negative isolated from abdominal

wound (Scn2). The bacterial cell number was adjusted to approximately 108

The crude methanolic extract and fractions (100 mg/mL) or pure compound

(NDGA, 50 mg/mL) were serially two-fold diluted to obtain the following

concentrations (µg/mL): 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9. Each 20 µL

of bacterial suspensions was added to 90 µL of physiologic serum and 80 µL of Mueller-

Hinton broth in a sterile 96-well microplate. Afterwards, 10 µL of the crude methanolic

extract, DCM and EA fractions, and NDGA were added and the microplate was

incubated at 37 ± 1 °C for 24 h. Then, 50 µL of 2.3.5-triphenyltetrazolium chloride (2.5

mg/mL) were added and incubated again at 37 ± 1 °C for 30 min in the dark (Klančnik et

al., 2010). The growth or no-growth was assessed by the naked eye, and the MIC value

was determined as being the lowest sample concentration that prevents viable bacteria to

CFU

(colony forming unit)/mL (0.5 on the McFarland scale).

CHAPTER 6


99

reduce the yellow dye into a pink color and exhibit complete inhibition of bacterial

growth.

Several controls were considered: physiologic serum + Muller-Hinton broth +

bacterial suspensions to verify microbial growth; physiologic serum + Muller-Hinton

broth to control the sterility; aq. tetracycline or gentamicin solutions (at concentrations

0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64 µg/mL) as positive control;

physiologic serum + Mueller-Hinton broth + bacterial suspensions + DMSO as negative

control. All the assays were performed in triplicate for each sample against all bacterial

strains. MIC values were determined as a mean value of each assay and evaluated as

follows: ≤ 64 µg/mL was judged to show high activity, while 125-500 and 1000 µg/mL

were considered to show moderate and with no antibacterial activity, respectively

(Yasunaka et al., 2005).

6.2.5. Bioactive compounds quantification

NDGA, kaempferol and quercetin concentrations were determined by high


with a C18 5 µm (3.9 × 300 mm) column at room temperature, and a UV detector at 280






phase was eluted in a flow rate of 1.0 mL/min, and samples of 10 µL were injected.


NDGA, kaempferol and quercetin concentrations were expressed as the ratio between

mass of the compound in the extracts and mass of plant material (dry weight).

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100


6.3.1. Antibacterial activity by the agar diffusion method

Agar diffusion techniques have been widely used to assay antimicrobial activity of

plant extracts (Das et al., 2010; Perez et al., 1990; Rojas et al., 2006). In the present

study, the use of this technique was useful to perform a screening study of the samples

evaluating their antibacterial potential. The results of antibacterial activity obtained for

the crude methanolic extract (CME) and fractions from L. tridentata leaves by the agar

diffusion method are shown in Table 6.1. Overall, the antibacterial activity of the tested

samples was noticeable more effective against the growth of Gram-positive bacteria

strains compared to the Gram-negative bacteria strains. In fact, Gram-negative bacteria

are typically more resistant to antimicrobial agents than Gram-positive bacteria, and this

occurrence has been explained by the presence of an outer-membrane permeability

barrier, which limits access of the antimicrobial agents to their targets in the bacterial cell

(Vaara, 1992). In the present study, the highest concentration (400 µg/well) of the ethyl

acetate (EA) extract was active inhibiting the growth of Pa2, and the highest

concentration of NDGA was active against the growth of Ec2 and Kp1, the growth of the

last one strain being also inhibited by a lower NDGA concentration, in the order of 200

µg/well. No further relevant results were observed concerning the antimicrobial effect of

the tested samples against Gram-negative bacteria strains. Verástegui et al. (1996)

reported no inhibiting effect on the growth of Gram-negative Escherichia coli and

Salmonella thyphimurium using an ethanolic extract (80%) from L. tridentata leaves;

nevertheless this extract inhibited the growth of Shigella dysenteriae and Yersinia

enterocolitica strains with MIC values of 14 ± 1 and 10 ± 1 mg/mL, respectively.

Regarding to the antibacterial activity against Gram-positive bacteria, interesting

results were found, in particular, for the CME, dichloromethane (DCM) and EA extracts,

and the reference compound NDGA. All these samples were considered active or very

active, depending on the concentration used, against Sa1, Sa2, Se and Ss bacteria strains,

with exception of CME and DCM at concentration of 50 µg/well for Ss and Se strains,

respectively, presenting a less active effect. For the Enterococcus faecalis strains (Ef1

and Ef2) the most significant findings were observed using NDGA and EA fraction,

which were very active inhibiting its growth at a concentration of 400 µg/well.

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101

Table 6.1 Antibacterial activity of crude methanolic extract and fractions obtained from L. tridentata leaves.

Tested samples Tested microorganisms a b Gram-positive bacteria Gram-negative bacteria Sa1 Sa2 Se Ss Ef1 Ef2 Pa1 Pa2 Ec1 Ec2 Kp1 Kp2

CME

[µg/well] Growth inhibition zone c

400 (mm)

22 21 21 20 16 16 13 11 - - 11 - 200 19 18 18 17 13 14 11 - - - - - 100 16 15 16 14 11 12 - - - - - - 50 13 13 14 12 - - - - - - - - TT 33 31 29 28 - - 14 - 25 25 28 28

H

400 10 - - - - - - - - - 11 - 200 - - - - - - - - - - - - 100 - - - - - - - - - - - - 50 - - - - - - - - - - - - TT 36 31 30 32 32 12 16 12 30 27 26 28

DCM

400 22 21 20 20 16 17 - - - - 9 - 200 20 18 17 17 14 15 - - - - - - 100 17 16 14 15 12 13 - - - - - - 50 15 14 12 13 - 11 - - - - - - TT 34 31 28 29 - - 14 - 25 25 28 27

EA 400 24 22 22 22 20 20 12 14 11 - 12 - 200 20 19 19 19 17 17 - 12 - - 10 - 100 17 17 16 16 15 14 - - - - - -

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102

50 14 14 13 13 13 12 - - - - - - TT 32 29 34 31 34 12 18 12 27 26 27 28

Et

400 11 12 9 10 - - - - - - - - 200 9 9 - - - - - - - - - - 100 - - - - - - - - - - - - 50 - - - - - - - - - - - - TT 32 31 30 29 - - 18 - 27 27 28 29

NDGA

400 25 24 22 22 22 20 11 11 12 14 15 12 200 23 22 21 21 18 18 - - 9 12 13 10 100 21 19 19 19 15 16 - - - - 11 - 50 18 16 16 16 12 13 - - - - - - TT 32 31 28 28 - - 15 10 26 25 27 28

DMSO 20% - - - - - - - - - - - - a The tested samples were CME: crude methanolic extract; fractions: (H: hexane; DCM: dichloromethane; EA: ethyl acetate; Et: ethanol); NDGA: reference compound; DMSO: dimethyl sulfoxide, used as dilution solvent and negative control; TT: tetracycline, reference antibiotic used as positive control (30 µg/well).

b Microorganisms: Sa1: Staphylococcus aureus standard strain ATCC 6538; Sa2: methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from secretion; Se: Staphylococcus epidermidis isolated from sperm; Ss: Staphylococcus saprophyticus standard strain LACEN; Ef1: Enterococcus faecalis standard strain ATCC 51299; Ef2: Enterococcus faecalis isolated from urine; Pa1: Pseudomonas aeruginosa standard strain ATCC 14502; Pa2: Pseudomonas aeruginosa isolated from blood; Ec1: Escherichia coli standard strain ATCC 10536; Ec2: Escherichia coli standard strain ATCC 30218; Kp1: Klebsiella pneumonia isolated from secretion; Kp2: Klebsiella pneumonia from surgical wound secretion.

c

(-) no growth inhibition zone observed

Including the diameter of the hole (6 mm).

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103

Some variability was observed in the activity of tetracycline against Enterococcus

faecalis strains, which could be explain by some degree of heterogeneity of this

antibiotic (Bismuth et al., 1990). No relevant results were obtained for the hexane (H)

and ethanol (Et) fractions.

6.3.2. Evaluation of minimal inhibitory concentration (MIC)

Since the CME, DCM and EA fractions, and NDGA showed antibacterial activity

against the tested gram-positive bacteria strains, the real extend of their inhibitory

activity was evaluated by determining MIC values, which are shown in Table 6.2. As can

be seen, the MIC values significantly varied to each sample, from 62.5 to 250 µg/mL for

CME, from 62.5 to 375 µg/mL for DCM fraction, from 31.3 to 125 µg/mL for EA

fraction, and from 125 to 500 µg/mL for the reference pure compound NDGA.

Table 6.2 Minimum inhibitory concentration (MIC, in µg/mL) of crude methanolic

extract and fractions obtained from L. tridentata leaves on growth of different bacteria

strains.

Tested samples Tested microorganismsa

Sa1

b

Sa2 Sa3 Sa4 Se Ss Scn1 Scn2 Ef1 Ef2

CME 125 125 62.5 62.5 125 125 250 62.5 187.5 250 DCM 62.5 125 62.5 62.5 125 125 62.5 62.5 250 375 EA 62.5 62.5 62.5 31.3 125 125 62.5 62.5 125 125 NDGA 125 125 125 125 125 250 250 125 500 375 TT 1.0 1.0 64 64 1.0 4.0 0.5 64 - - GEN - - - - - - - - 64 64

a The tested samples were CME: crude methanolic extract; fractions: (DCM: dichloromethane; EA: ethyl acetate); NDGA: reference compound; Reference antibiotics: TT (tetracycline) and GEN (gentamicin).

b Microorganisms: Sa1: Staphylococcus aureus standard strain ATCC 6538; Sa2: methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from secretion; Sa3 Staphylococcus aureus MRSA strain isolated from tracheal aspirates; Sa4: Staphylococcus aureus MRSA strain isolated from secretion; Se: Staphylococcus epidermidis isolated from sperm; Ss: Staphylococcus saprophyticus standard strain LACEN; Scn1: Staphylococcus coagulase-negative isolated from catheter secretion; Scn2: Staphylococcus coagulase-negative isolated from abdominal wound; Ef1: Enterococcus faecalis standard strain ATCC 51299; Ef2: Enterococcus faecalis isolated from urine.

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104

The results obtained in these assays revealed that Sa4 (S. aureus - MRSA strain

isolated from secretion) was the most sensitive bacteria to EA fraction, with a MIC value

of 31.3 µg/mL, which was lower than the reference antibiotic tetracycline (64 µg/mL).

Low MIC values (62.5 µg/mL) for this strain were also obtained for CME and DCM

fraction, compared to tetracycline. The strains Sa3 (S. aureus - MRSA strain isolated

from tracheal aspirates) and Scn2 (S. coagulase-negative isolated from abdominal

wound) presented similar values of MIC (62.5 µg/mL) for the CME, DCM and EA

fractions, which were also lower than that of the reference antibiotic (64 µg/mL). These

results demonstrate that CME, DCM and EA fractions obtained from L. tridentata leaves

present a high antibacterial activity. On the other hand, the MIC values obtained for the

above mentioned bacteria strains using NDGA were higher than the values observed for

CME, DCM and EA fractions, revealing that NDGA alone has a poor antibacterial

activity. The efficiency of natural drugs might be explained by the synergistic or additive

effects of several phytochemicals rather than arising from a single compound. Different

bioactive compounds in a mixture can interact to provide a combined effect which is

similar to the sum of the effects of the individual components (additive), or the

combinations of bioactive compounds can exert effects that are greater than the sum of

the individual components (synergistic) (Ginsburg and Deharo, 2011). The results

previously described for the crude methanolic extract, fractions and the pure compound

NDGA confirm the latter statement.

6.3.3. HPLC analyses of tested samples

In order to underlie the antibacterial activity of the crude methanolic extract and

fractions from L. tridentata leaves, as well as to have a deepen knowledge about these

samples, HPLC analyses were carried out and the chromatograms of CME, DCM and

EA fractions are shown in Fig. 6.1. No relevant peaks were identified in HPLC

chromatograms for H and Et fractions. Nevertheless, a previous phytochemical study

performed by this research team showed the presence of several classes of chemical

compounds in H and Et fractions, such as phenolic compounds, saponins, triterpenes and

steroids, among others (data not shown). Despite the detection of different

phytochemicals in these fractions, it is possible that these compounds have no

antibacterial activity, which might explain the poor results observed with these fractions.

CHAPTER 6


105

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200,0

0,1

0,2

0,3

0,4

0,5Ab

sorb

ance

(AU)

Retention Time (min)

Q K

NDGA

A

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200,0

0,1

0,2

0,3

0,4

Abso

rban

ce (A

U)


K

B

NDGA

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106

Fig. 6.1 HPLC chromatograms of crude methanolic extract, CME (A), dichloromethane,

DCM (B) and ethyl acetate, EA (C) fractions from L. tridentata leaves (Q: quercetin; K:

kaempferol; NDGA: nordihydroguaiaretic acid).

On the other hand, several relevant peaks were observed for CME, DCM and EA

fractions during HPLC analyses, among of which, three bioactive compounds were

identified and quantified, namely, quercetin, kaempferol, and NDGA. These compounds

are well known for their biological activities of great importance in the health area, such

as antiviral, antimicrobial, antitumorgenic, anti-inflammatory, and antinociceptive

capacities (De Melo et al., 2009; García-Mediavilla et al., 2007; Hwu et al., 2008;

Lambert et al., 2004). These three bioactive compounds were found at different

concentrations in the samples (Table 6.3), which could explain some of the differences

on their antibacterial potential. The highest concentrations of quercetin, kaempferol and

NDGA were observed in CME (8.67, 21.52 and 35.75 mg/g plant, respectively);

nevertheless, EA fraction also showed considerable levels of these compounds compared

with the remaining fractions, as it can be seen in Table 6.3.

Another important aspect observed through HPLC analyses was a group of other

non-identified phytochemicals (short dots area in Fig. 6.1), which are clearly present at

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Abso

rban

ce (A

U)


Q

K

NDGA

C

CHAPTER 6


107

different concentration levels depending on the sample. These unidentified compounds

could also be responsible for the biological characteristics, in particular the antibacterial

activity, of the CME (Fig. 6.1A), and DCM and EA fractions (Fig. 6.1B and 6.1C,

respectively). These observations indicate the need of additional research in order to

identify all the phytocomponents comprised in each sample.

Table 6.3 Quantification of quercetin, NDGA and kaempferol (in mg/g of plant material)

in crude methanolic extract and fractions from L. tridentata leaves.4

Tested samples Quercetin a Kaempferol NDGA

CME 8.67 21.52 35.75

H - - -

DCM 0.26 6.89 3.19

EA 8.45 11.89 16.51

Et 0.40 0.40 0.19 a

The tested samples were CME: crude methanolic extract; fractions: (H: hexane; DCM: dichloromethane; EA: ethyl acetate; Et: ethanol).

6.4 Conclusion

The findings of the present study demonstrated the potential of phytochemicals

from L. tridentata leaves, a natural source, in the pathway of developing a novel

antibacterial agent able of treating bacterial infections. Ethyl acetate fraction showed

promising results against a methicillin-resistant S. aureus, which represents an important

step for the search and development of a new antibacterial agent. Further toxicological

and pharmacological studies will be useful to confirm the hypothesis of using

phytochemicals from L. tridentata leaves.

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Rojas J.J., Ochoa V.J., Ocampo S.A., Muñoz J.F. (2006). Screening for antimicrobial activity of ten

medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment of

non-nosocomial infections. BMC Complementary and Alternative Medicine, 6, 2.

Ross I.A., (2005). Medicinal plants of the world - Chemical constituents, traditional and modern medicinal

uses (Volume 3), Humana Press: New Jersey.

Santos A.K.L., Magalhães T.S., Monte F.J.Q., Mattos M.C., Oliveira M.C.F., Almeida M.M.B., Lemos

T.L.G., Braz-Filho R. (2009). Alcalóides iboga de Peschiera affinis (Apocynaceae) – atribuição

inequívoca dos deslocamentos químicos dos átomos de hidrogênio e carbono. Quimica Nova, 32,

1834–1838.

Sánchez-Medina A., García-Sosa K., May-Pat F., Peña-Rodríguez L.M. (2001). Evaluation of biological

activity of crude extracts from plants used in Yucatecan Traditional Medicine Part I. Antioxidant,

antimicrobial and β-glucosidase inhibition activities. Phytomedicine, 8, 144–151.

Tequida M., Cortez R., Rosas B., Lopez S., Corrales M. (2002). Effect of alcoholic extracts of wild plants

on the inhibition of growth of Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum,

Penicillium expansum, Fusarium moniliforme and Fusarium poae moulds. Revista Iberoamericana

de Micologia, 19, 84–88 (in Spanish).

Vaara M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Reviews,

56, 395–411.

Verástegui M.A., Sánchez C.A., Heredia N.L., García-Alvarado J.S. (1996). Antimicrobial activity of

extracts of three major plants from the Chihuahuan desert. Journal of Ethnopharmacology, 52, 175–

177.

Yasunaka K., Abe F., Nagayama A., Okabe H., Lozada-Pérez L., López-Villafranco E., Muñiz E.E.,

Aguilar A., Reyes-Chilpa R. (2005). Antibacterial activity of crude extracts from Mexican medicinal

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Weckesser S., Engel K., Simon-Haarhaus B., Wittmer A., Pelz K., Schempp C.M. (2007). Screening of

plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance.

Phytomedicine, 14, 508–516.

111

CHAPTER 7

In vitro cytotoxic activity of crude extract and fractions obtained

from Larrea tridentata leaves against human cancer cell lines

In this chapter the cytotoxic activity of the crude methanolic extract and fractions (hexane,

dichloromethane, ethyl acetate and ethanol) obtained from L. tridentata leaves was evaluated

against cancer cell lines. A phytochemical study by thin layer chromatography and high

performance liquid chromatography was performed in order to have a more extended

knowledge about these samples.

112

113

7.1 Introduction

Plants are one of the primordial sources of phytochemicals present in conventional

medicaments. Larrea tridentata (Sessé & Moc. Ex DC.) Coville (Zygophyllaceae),

commonly known as creosote bush, is a plant traditionally used for centuries by North

American Indians to treat medical conditions and illnesses including genitor-urinary and


skin, kidney problems, arthritis, diabetes, cancer, among other diseases (Brinker, 1993;

Ross, 2005). The traditional use of medicinal plants provides essential information about

their therapeutic potential, allowing the development of clearer and focused studies about

the biological activity of plants extracts. The search for bioactive compounds with

cytotoxic activity and potential as anticancer drugs from plant extracts represents a huge

challenge for cancer treatment and prevention. Nevertheless, not all the bioactive

compounds that exhibit cytotoxicity are of interest, because specific requirements are

needed, such as, concentrations and mediation by a mechanism that allows healthy cells

to survive but not tumor cells (Lindholm, 2005).


50% of the leaves (dry weight) being extractable matter (Arteaga et al., 2005). Among

several interesting bioactive phenolic compounds found in this plant, the natural

occurring lignan nordihydroguaiaretic acid (NDGA) has been point out as the most

significant compound with biological activities of large interest in the health area, such

as antiviral, antifungic, antimicrobial, and antitumorgenic (Hwu et al., 2008; Fujimoto et

al., 2004; Lambert et al., 2004). The therapeutic potential of this compound for the

treatment of tumors and cancer cells has been studied. In vitro assays demonstrated that

the 5-lipoxygenase inhibitor NDGA inhibits the proliferation of human small cell lung

cancer NCI-H209 cells (Avis et al., 1996), non-small cell lung cancer NCI-H1264 cells

(Moody et al., 1998), SW 850 human pancreatic cancer and C4-I cervical human cancer

cells (Seufferlein et al., 2002), and in MCF-7 human breast cancer cells (Youngren et al.,

2005). In vivo studies have also shown that NDGA inhibits IGF-1 and c-

erbB2/HER2/neu receptors suppressing growth in breast cancer cells (Youngren et al.,

2005), tumor growth in esophageal adenocarcinoma (Chen et al., 2002), and significantly

slows NCI-H1264 xenograft growth in nude mice (Moody et al., 1998).

CHAPTER 7

IN VITRO CYTOTOXIC ACTIVITY OF CRUDE EXTRACT AND FRACTIONS OBTAINED FROM Larrea tridentata LEAVES AGAINST HUMAN CANCER CELL LINES

114

Besides NDGA, several secondary metabolites have also been identified in L.

tridentata including other lignans (dihydroguaiaretic acid, hemi-norisoguaiacin and

norisoguaiacin), flavonoids (apigenin, kaempferol, and quercetin), saponins (larreagenin

A and larreic acid), triterpenes, and triterpenoids (Brinker, 1993; Hui-Zheng et al., 1988;

Jitsuno and Mimaki, 2010), among others. Studies on cell culture models have

demonstrated important biochemical effects of some of these compounds (among of

which quercetin, saponin and kaempferol) in cancer therapy and treatment (Soria et al.,

2007; Kim et al., 2008; Labbé et al., 2009). However, to the best of our knowledge, no

studies about cytotoxic activity of crude methanolic extract and fractions obtained from

L. tridentata leaves, neither a phytochemical profile of these samples, have been

reported. Therefore, the purpose of this study was to evaluate the cytotoxic activity of

methanolic crude extract and fractions (hexane, dichloromethane, ethyl acetate, and

ethanol) from L. tridentata leaves against human cancer cell lines HT29 (colon

carcinoma cells), NCI-H292 (lung cancer cells), and HEp-2 (laryngeal carcinoma cells).

Then, further studies were developed using the CME and DCM fraction against a

different human colon carcinoma cell line, HCT116, testing the cell

viability/proliferation and apoptosis by nuclear condensation assay. A phytochemical

study of the crude methanolic extract and fractions was also performed and is discussed.



Plant material (L. tridentata) was collected from the Chihuahuan semidesert (North

Coahuila, Mexico) during Spring season (April, 2009). Nordihydroguaiaretic acid

(NDGA), quercetin and kaempferol were purchased from Sigma-Aldrich (Saint Louis,

MO, USA). Reagent-grade methanol, hexane, dichloromethane, ethyl acetate and ethanol

were from Panreac (Barcelona, Spain). Silica gel 60 F254 was purchased from Merck

(Darmstadt, Germany) and silica gel 60 (S) from Vetec (Rio de Janeiro, Brazil). For the

cytotoxic assay, Dulbecco's Modified Eagle Medium (DMEM), and fetal bovine serum

were purchased from Gibco (Grand Island, NY, USA); glutamine, streptomycin, and 3-

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(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were from

Sigma (St. Louis, MO, USA); and penicillin was from Fluka (Buchs, AG, Switzerland).

7.2.2 Extraction methodology and fractioning

Air-dried leaves of L. tridentata were ground to fine powder and stored in dark

bottles at room temperature for further analysis. Extraction was performed by mixing 1 g

of plant material with 20 mL of 90% methanol and subsequent heating of the mixture in

a water-bath at 60-65 ºC for 20 min. The obtained extract was filtered through qualitative

filter paper and the solvent was removed by rotary evaporation under reduced pressure at

temperatures of approximately 45 °C. The resulting crude extract was then stored at 4 °C

until further analysis. A portion of the crude methanolic extract (10 g) was fractioned by

filter column chromatography over 100 g silica gel 60 (S) (Santos et al., 2009), and

eluted with approximately 1 L, of the solvents hexane, dichloromethane, ethyl acetate,

and ethanol, in the order of increasing polarity, until a clear extract was obtained at the

end of the elution. Pump pressure was applied to accelerate the elution of the solvents.

Eluates were collected in 1 L Erlenmeyer flasks and each fraction was subjected to

evaporation under reduced pressure in a rotary evaporator. Fractions were stored at 4 °C

until assayed.

7.2.3 Phytochemical study by thin layer chromatography

Thin layer chromatography (TLC) of the crude methanolic extract and respective

fractions was carried out. Different solvent systems described by Wagner and Bladt

(1996) were used to identify different classes of compounds based on the polarity of the

organic solvents. The presence or absence of alkaloids, anthocyanins, anthracene

derivatives, lignans, anthraquinone aglycones, coumarins, mono, sesqui and diterpenes,

naftoquinone, phenolic compounds, saponins, condensed and hydrolysable tannins,

triterpenes, steroids and xanthines, was verified using known metabolites standards as

reference (Table 7.1). TLC was performed in chromatographic plates (stationary phase)

pre-coated with silica gel 60 F254 (0.2 nm thickness). The samples were dissolved in

methanol (10 mg/mL) and 15 µL were deposited as a spot on the stationary phase. The

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116

bottom edge of the plate was placed in a closed container with the solvent, which was

moved up the plate by capillary action. Plate was removed from the container when the

solvent front reached the other edge of the stationary phase. TLC spots were visualized

under UV light (254 or 365 nm) and adequate TLC reagents were used to detect the

phytoconstituents. The chemical groups were evaluated as described by Kuete et al.

(2006) using a scale as follows: absent (-), present at low levels (+), abundant (++), and

very abundant (+++).

7.2.4. Measurement of cytotoxic activity

7.2.4.1 Culture of cell lines

The cancer cell lines HT29 (human colon carcinoma cells), NCI-H292 (human

lung cancer cells), and HEp-2 (human laryngeal carcinoma cells) were obtained from the

Cell Bank from Rio de Janeiro (Brazil). Cells were cultured in Dulbecco's modified

Eagle's medium (DMEM) with 10% (v/v) fetal calf serum, 1% antibiotics (penicillin

1000 U/mL + streptomycin 250 mg/mL) and 1% L-glutamine (200 mM), and maintained

at 37 °C under 5% CO2

The HCT116 human colon carcinoma cells were kindly provided by Raquel Seruca

from IPATIMUP (University of Porto). The cell line was maintained 37 °C in a

humidified 5% CO

atmosphere.

2

atmosphere in RPMI-1640 medium (Sigma-Aldrich) supplemented

with 10 mM HEPES, 0.1 mM pyruvate, 1% antibiotic/antimycotic solution (Sigma-

Aldrich), and 6% heat-inactivated fetal bovine serum (FBS; Biochrom, Berlin,

Germany).

7.2.4.2 Cell viability/proliferation assay

Cytotoxic activity was determined by measuring the cancer cell

viability/proliferation through the MTT reduction assay, which quantifies the ability of

living cells to reduce the yellow dye 3-(4,5-dimethiol-2-thioazolyl)-2,5-diphenyl

tetrazolium bromide (MTT) to a blue formazan product (Mosmann, 1983). Cells

suspensions at a concentration of 1 × 105 cells/mL were prepared in specific medium for

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each cancer cell line. Then 225 µL of each suspension of cells were seeded in 96-well

plates and incubated for 24 h (HT29, NCI-H292, and HEp-2 cells) or 48 h (HCT116

cells) at 37 °C under 5% CO2 atmosphere. The crude extract and fractions were diluted

in dimethyl sulfoxide (DMSO) at a concentration of 50 µg/mL, and 25 µL of each

sample were added to the cells seeded in the 96-well plates, and incubated for 72 h

(HT29, NCI-H292, and HEp-2 cells) or 48 h (HCT116 cells) at 37 °C under 5% CO2

A criterion similar to that used by Mesquita et al. (2009), where samples that

inhibited at least 85% of cell growth of two of the cancer cell lines evaluated, were

considered to determine the concentration of the test sample required to inhibit cell

viability/proliferation by 50% (IC

atmosphere (Costa and Nascimento, 2003; Xavier et al., 2009). Standard compounds,

namely, NDGA, quercetin and kaempferol, were used as positive (cytotoxic) controls,

and tested at a concentration of 25 µg/mL. Afterwards, 25 µL of MTT solution (5

mg/mL in phosphate buffer saline) were added and the plates were incubated again for 2

h. Finally, the culture medium together with the excess of MTT was removed, and 100

µL of DMSO were added to each well-plate to dissolve the formazan crystals (Alley et

al., 1998). The absorbance was measured at 450 nm using a MultisKan plate reader. The

effect of the treatment was determined as percentage of viability compared to untreated

cells. An intensity scale was used as follows: no activity (1 to 20% of growth inhibition),

low activity (20 to 50% of growth inhibition), moderated activity (50 to 70% of growth

inhibition), and high activity (70 to 100% of growth inhibition).

50). The IC50 values were determined using the dose-

response curve, and were calculated by the GraphPad Prism 5.0 software, with a 95%

confidence range. The criteria of the American National Cancer Institute, recognizing an

active extract for further studies based at an IC50

lower than 30 µg/mL (Suffness and

Pezzuto, 1990), was followed. All the analyses were performed in triplicate.

7.2.4.3 Apoptotic nuclear condensation assay

After incubating cells with different concentrations of tested extracts/compounds

for 48h, cells were collected (both floating and attached cells), fixed with 4%

paraformaldehyde for 15 min room temperature, and attached onto a polylysine-treated

slide using a Shandon Cytospin. Cells were then washed in PBS and DNA stained with

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Hoechst. The percentage of apoptotic cells was calculated from the ratio between cells

presenting nuclear condensation and the total number of cells, from a count higher than

500 cells per slide from photos taken under a fluorescent microscope. Results are

presented as mean ± standard deviation of at least three independent experiments.

7.2.5 Bioactive compounds quantification

NDGA, quercetin and kaempferol concentrations were determined by high


with a C18 5 µm (3.9 x 300 mm) column at room temperature, and a UV detector at 280






phase was eluted in a flow rate of 1.0 ml/min, and samples of 10 µL were injected.


NDGA, quercetin and kaempferol concentrations were expressed as the ratio between

mass of the compound in the extracts and mass of plant material (dry weight).


Results were analyzed by one-way analysis of variance (ANOVA) using the

GraphPad Prism 5.0 software (San Diego, CA, USA) for a significance level of p<0.05.

Difference among samples was verified by using the Tukey’s range test.

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7.3.1 Phytochemical profile of extract and fractions from L. tridentata leaves

Several phytochemicals, including lignans, glycosylated flavonoids, sapogenins,

alkaloids, among others, have been extracted and identified from distinct parts of L.

tridentata (Argueta, 1994; Hyder et al., 2002; Jitsuno and Mimaki, 2010; Lara and

Márquez, 1996;). Table 7.1 shows the phytochemical profile of the crude extract and

fractions of L. tridentata leaves obtained in the present study. The crude methanolic

extract (CME) contained low levels of anthocyanins, coumarins, lignans, saponins,

condensed tannins, triterpenes and steroids; but abundant levels of anthraquinone

aglycones, mono and sesqui diterpenes, and very abundant levels of phenolic

compounds. On the other hand, only traces of hydrolysable tannins were observed, and

the presence of alkaloids, anthracene derivatives, naftoquinones, xanthines was not

detected.

Concerning the fractions obtained from L. tridentata, several differences on their

composition were observed (Table 7.1). Alkaloids, naftoquinones, condensed tannins and

xanthines were not detected in any of the fractions. Anthocyanins and hydrolysable

tannins were abundant in the dichloromethane (DCM) and ethyl acetate (EA) fractions,

but they were present at low levels in the ethanolic fraction (Et), and were not found in

the hexane fraction (H). Meanwhile, coumarins and mono, sesqui diterpenes were

observed in hexane fraction at abundant levels, but were absent in the remaining

fractions. Anthracene derivatives were found in hexane and dichloromethane fractions

(at abundant and low levels, respectively), as well as triterpenes and steroids (at very

abundant and abundant levels, respectively), Nevertheless, these compounds were not

detected in the ethyl acetate and ethanolic fractions. Anthraquinone aglycones were

detected at different levels in the hexane, dichloromethane and ethanolic fractions

(abundant, very abundant and low levels, respectively) but not in the ethyl acetate

fraction. Saponins compounds were present in the ethanolic fraction at abundant levels,

but absent in the other fractions. Lignans were abundant in the ethyl acetate fraction, but

were not detected in the other fractions. Finally, phenolic compounds were present in a

very abundant level in the ethanolic fraction, and at low level in the ethyl acetate

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120

fraction. The knowledge of the main groups of natural compounds in each fraction is

valuable to explain further results concerning the cytotoxic potential of these fractions.

Table 7.1 Phytochemical analysis of L. tridentata leaves using thin layer

chromatography.

Phytocompounds CME H DCM EA Et

Alkaloids - - - - -

Anthocyanins + - ++ ++ +

Anthracene derivatives - ++ + - -

Anthraquinone aglycones ++ ++ +++ - +

Coumarins + ++ - - -

Lignans + - - ++ -

Mono, sesqui diterpenes ++ ++ - - -

Naftoquinones - - - - -

Phenolic compounds +++ - - + +++

Saponins + - - - ++

Tannins Condensed + - - - -

Hydrolysable Trace - ++ ++ +

Triterpenes and steroids + +++ ++ - -

Xanthines - - - - - The tested samples were CME: crude methanolic extract; fractions: (H: hexane; DCM: dichloromethane; EA: ethyl acetate; Et: ethanol); Scale of the class of compounds: (-) absent, (+) present at low levels, (++) abundant, (+++) very abundant.

7.3.2 Cytotoxicity of L. tridentata leaves extract and fractions on cancer cell lines

The cytotoxic effects of the CME and fractions obtained from L. tridentata leaves,

as well as of standard compounds, namely, NDGA, quercetin and kaempferol, against

HT29 (human colon carcinoma cells), NCI-H292 (human lung cancer cells) and HEp-2

(human laryngeal carcinoma cells) are depicted in Table 7.2. DCM and EA fractions

demonstrated a remarkable antiproliferative effect against all the three cancer cell lines,

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inhibiting between 81.4 and 93.7% the cell proliferation at 50 µg/mL. Significant

antiproliferative effect of the CME against HT29 and HEp-2 cell lines was also

observed, with inhibition percentages of 86.1 and 78%, respectively. Hexane fraction

showed a moderate cytotoxic activity against NCI-H292 cancer cells line, and a strong

effect against HEp-2 cell line. None interesting result was found for the cytotoxic

activity of the Et fraction against any of the cell lines studied. Overall, fractions obtained

with polar aprotic solvents, in particular dichloromethane and ethyl acetate, showed

remarkable results concerning their cytotoxic activity.

Some studies have demonstrated the potential of NDGA to inhibit the proliferation

of human cancer cell lines. Youngren et al. (2005) related anti-breast cancer properties of

NDGA to the direct inhibition of two important receptor tyrosine kinases, the insulin-like

growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu), which have a

crucial role in regulating cancer cell growth and survival. Another interesting study

developed by Seufferlein et al. (2002) demonstrated that NDGA potently inhibits

anchorage-independent growth of human pancreatic and cervical cancer cells in soft

agar, and delays growth of pancreatic and cervical tumors established in athymic mice,

inducing apoptosis of these cancer cells in vitro and in vivo. However, in the present

study, NDGA showed a strong antiproliferative effect only for the HEp-2 human

carcinoma cell line, and a moderate effect for the remaining two cell lines (Table 7.2).

Similar results were observed for the standard compound quercetin, while kaempferol

showed low cytotoxic activity against HT29 and HEp-2 cell lines, and no cytotoxic

activity against NCI-H92 cell line. In fact, the efficiency of most natural drugs might be

explained by the synergistic or additive effects of several components rather than arising

from a single compound. Different bioactive compounds in a mixture interact each other

providing a combined effect that can be similar to the sum of the effects of the individual

components (additive), or greater than the sum of the individual components

(synergistic) (Ginsburg and Deharo, 2011).

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Table 7.2 Cytotoxic activity screening (inhibition of cell viability, in %) of the crude

methanolic extract and fractions obtained from L. tridentata leaves on three cancer cell

lines measured by the MTT assay.

Tested samplesCell lines

a

HT29

b

NCI-H292 HEp-2

CME 86.1 ± 0.7 64.4 ± 1.0 78.0 ± 2.9

H 48.2 ± 2.6 62.4 ± 4.4 90.2 ± 0.7

DCM 89.1 ± 0.1 86.3 ± 3.8 81.4 ± 1.5

EA 93.7 ± 2.8 85.6 ± 4.2 90.5 ± 1.9

Et 30.1 ± 6.6 NI 44.4 ±3.6

NDGA 49.8 ± 5.6 54.7 ± 4.8 75.5 ± 11.5

Quercetin 43.0 ± 0.3 37.5 ± 2.8 77.9 ± 3.2

Kaempferol 29.4 ± 14.0 NI 37.2 ± 1.5 a The tested samples were CME: crude methanolic extract; fractions: (H: hexane; DCM: dichloromethane; EA: ethyl acetate; Et: ethanol); NDGA, quercetin, kaempferol: reference compounds used as controls.

b

NI: no inhibition of cell growth.

Results are expressed as percentage growth inhibition ± standard deviation; HT29 (human colon carcinoma cells), NCI-H292 (human lung cancer cells) and HEp-2 (human laryngeal carcinoma cells).

In order to reach a more clear understanding of the latter statement, HPLC analyses

of the fractions obtained from L. tridentata leaves were carried out. These analyses

showed the presence of NDGA, quercetin, and kaempferol at different concentrations on

the DCM, EA, and Et fractions, but not in the H fraction (Table 7.3). Despite the lack of

antiproliferative effect of NDGA and quercetin against most of the cell lines, and

kaempferol against all the cell lines evaluated, combination among them and/or

combination of them with other specific compounds not identified in the present study,

might explain the antiproliferative potential of the DCM and EA fractions. On the other

hand, the absence or presence at low concentrations of these three bioactive compounds

in the H and Et fractions, respectively, could also be responsible for the poorer

antiproliferative activity of these fractions against the human cancer cell lines evaluated,

mainly HT29 and NCI-H292 cell lines.

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Table 7.3 Quantification of quercetin, NDGA and kaempferol (in mg/g of plant material)

in crude methanolic extract and fractions from L. tridentata leaves.

Tested samples Quercetin a Kaempferol NDGA

CME 8.7 21.5 35.8

H - - -

DCM 0.3 6.9 3.2

EA 8.5 11.9 16.5

Et 0.4 0.4 0.2 a

The tested samples were CME: crude methanolic extract; fractions: (H: hexane; DCM: dichloromethane; EA: ethyl acetate; Et: ethanol).

Complete dose-response curves were then developed, and IC50 values were

calculated for the CME, DCM and EA fractions against the same three cell lines (HT29

(human colon carcinoma cells), NCI-H292 (human lung cancer cells), and HEp-2

(human laryngeal carcinoma cells)). IC50 values of 12.51 and 12.79 μg/ml were obtained

against HEp-2, with the DCM fraction and CME, respectively. Additionally, the DCM

fraction provided the best results for antiproliferation of HT29 and NCI-H292, with IC50

values of 24.94 and 24.18 μg/ml, respectively. The capacity of several phytocompounds

such as anthraquinones, triterpenes and anthocyanins, among others, on the inhibition of

human cancer cell proliferation has been well documented (Chiang et al., 2005; Kamiya

et al., 2010; Zhang et al., 2005), and DCM and EA fractions have, among others

components, these bioactive compounds. Another interesting aspect to be mentioned are

the higher concentrations of quercetin, kaempferol and NDGA in the EA fraction

compared to the DCM fraction (Table 7.3); however, more interesting antiproliferative

results were observed for the DCM fraction. Once again, this occurrence could be

explained by the presence of specific bioactive compounds not quantified in the present

study and their synergy on the DCM fraction. Therefore, further studies would be useful

to identify such compounds and also to determine their mechanisms of action, providing

more detailed information for the development of an anticancer agent.

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Taking into account the criteria of the American National Cancer Institute, which

recognizes an active extract based at an IC50 lower than 30 µg/mL (Suffness and

Pezzuto, 1990) further studies were developed using the CME and DCM fraction against

a different human colon carcinoma cell line. NDGA was also tested as a reference

compound. The effect of DCM fraction, CME and NDGA on cell viability/proliferation

and apoptosis in HCT116 cells was established by the MTT and nuclear condensation

assays, respectively. As shown in Fig. 7.1, NDGA was slightly more effective in

decreasing cell viability/proliferation in HCT116 cells after 48 h treatment than CME

and DCM fraction. Dose-response curves characterized by a nonlinear relationship

between the effect on HCT116 cells viability/proliferation and different plant extract

concentrations were plotted and IC50 was determined. The estimated IC50

values for the

CME, DCM fraction and NDGA were 18.7, 15.5 and 14.1 µg/mL, respectively (Fig.

7.2). These results show that NDGA and DCM fraction presented a stronger cytotoxic

activity against HCT116 cell line compared to CME.

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Fig. 7.1 Effect on cell viability/ proliferation of different concentrations of (A) crude

methanolic extract, (B) dichloromethane fraction (DCM), and (C) pure

nordihydroguaiaretic acid (NDGA), after 48 h of treatment, in HCT116 colon carcinoma

cells, using MTT assays. Results are presented as mean ± standard deviation of at least 3

independent experiments. *p ≤0.05, *** p≤0.01, and *** p≤0.001.

A

B

C

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Fig. 7.2 Dose-response curves for IC50 determination for the (A) crude methanolic

extract (CME), (B) dichloromethane fraction (DCM), and (C) pure nordihydroguaiaretic

acid (NDGA), after 48 h of treatment, in HCT116 colon carcinoma cells, using MTT

assays. Results are presented as mean ± standard deviation of at least 3 independent

experiments. *p≤0.05, *** p≤0.01, and *** p≤0.001.

A

B

C

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To further study the induction of apoptosis in HCT116 cells by CME, DCM

fraction and NDGA, apoptotic-related nuclear condensation was monitored. Quercetin,

known for its capacity to induce apoptosis (Xavier et al., 2009), was used as a reference

compound. Several studies have reported the apoptosis inducing effects of NDGA in

human cancer cells through several targets of actions, including arachidonic acid

pathways, protein kinase C pathways and the PDGF receptor system (Domin et al., 1994;

Tang et al., 1996; Seufferlein et al., 2005; Zavodovskaya et al., 2008). In the present

study, this compound demonstrated less apoptotic induction effect by nuclear

condensation in HCT116 cells compared to the CME and DCM fraction (Fig. 7.3). DCM

fraction showed the strongest effect in apoptosis induction. More detailed studies are

needed to elucidate the apoptotic effects of the tested compounds and extracts, which

could be analyzed by western blot, such as by evaluating the expression of the positive

mediators of apopotosis (p53 and Bax), as well as the negative regulator (Bcl-2) as well

as the cleavage of caspases.

Fig. 7.3 Effect on nuclear condensation of different concentrations of crude methanolic

extract, dichloromethane fraction (DCM), and pure nordihydroguaiaretic acid (NDGA),

after 48 h of treatment, in HCT116 colon carcinoma cells. The control used consisted of

dimethyl sulfoxide (DMSO), and quercetin was used as a reference compound. Results

are presented as mean ± standard deviation of at least 3 independent experiments.

*p≤0.05, *** p≤0.01, and *** p≤0.001.

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7.4 Conclusion

Ethnopharmacological knowledge is crucial in guiding which plants may have

potential for the development of anticancer products. The present findings provide

important information about L. tridentata suggesting that the compounds present in the

dichloromethane fraction extracted from the leaves of this plant possess anticancer

activity against colorectal carcinoma cells. This effect is due to cell growth inhibition

and induction of cell death by apoptosis, which can be due, at least in part, to the effects

of NDGA. In a next stage, detailed pharmacological and in vivo studies would be useful

in order to perform more extensive biological evaluations.

7.5 References

Argueta V. (1994) Atlas of the Traditional Mexican Medicinal Plants, vol. II. National Indigenous

Institute, Mexico (in Spanish).

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131

CHAPTER 8

General Conclusions

This chapter presents the major conclusions of this thesis and

recommendations/suggestions for further research in this field.

132

CHAPTER 8

GENERAL CONCLUSIONS

133

8.1 Conclusions

The main objective of this thesis was the recovery of bioactive compounds from

L. tridentata leaves by using different techniques, and the evaluation of the biological

activities of the produced extracts. In order to cover successfully the thesis aims, several

subjects were studied and strategies were implemented. In particular, the evaluation of

alternative extraction techniques for the recovery of bioactive compounds, such as

microwave-assisted extraction and solid-state fermentation using a fungal strain; and the

evaluation of the antibacterial and cytotoxic activities of the crude methanolic extract

and fractions obtained from L. tridentata leaves.

The main contributions of this thesis were the following:

- Microwave-assisted extraction (MAE) was proved to be a faster and more efficient

method for extraction of the nordihydroguaiaretic acid (NDGA) from Larrea tridentata

leaves when compared to the conventional heat-reflux extraction (HRE). The best results

of NDGA extraction by MAE might be explained by a greater extent of cell rupture of

the plant material during the extraction process;

- Methanol in a concentration of 90% (v/v) was an efficient organic solvent to recover

bioactive compounds (NDGA, kaempferol and quercetin) from L. tridentata leaves by

solid-liquid extraction. Additionally, the produced extracts showed higher antioxidant

capacity and contents of total flavonoids and protein than extracts produced with other

solvents (ethanol, acetone and distilled water). The extracts produced using 90% (v/v)

methanol showed as well significantly higher antioxidant potential and NDGA content

when compared to the extracts obtained by MAE.

- Submitting L. tridentata leaves to solid-state fermentation (SSF) with the fungus

Phanerochaete chrysosporium caused a major disorganization of the material structure.

However, this occurrence did not promote significant liberation nor an improvement of

chemical extraction of NDGA, Q and K from the plant. Some increase of the total

phenolic, flavonoids and protein contents in the extracts were obtained after the plant

fermentation, but no effect on the total antioxidant activity of the extracts was observed.

CHAPTER 8

GENERAL CONCLUSIONS

134

- The ethyl acetate fraction resulting from the fractioning of the crude methanolic extract

obtained from L. tridentata leaves was efficient to inhibit the growth of the bacterial

strain methicillin-resistant S. aureus, which represents an important step for the search

and development of a new antibacterial agent. This fraction also presented elevated

concentrations of nordihydroguaiaretic acid, kaempferol and quercetin.

- Dichloromethane fraction resulting from the fractioning of the crude methanolic extract

obtained from L. tridentata leaves show cell antiproliferative effect against human

cancer cell lines HT29 (colon carcinoma cells), NCI-H292 (lung cancer cells), and HEp-

2 (laryngeal carcinoma cells). This fraction also possess anticancer activity against the

HCT116 colorectal carcinoma cells line by inhibiting cell growth and inducing cell death

by apoptosis. These studies might represent an important step for the search and

development of a new anticancer agent.

CHAPTER 8

GENERAL CONCLUSIONS

135

8.2 Recommendations

Despite the main objectives have been achieved, some work still stays to be done

in order to develop an efficient and environmentally friendly procedure for the extraction

of bioactive compounds able to provide extracts with both high quality and biological

activity, while precluding any toxicity associated to the use of solvents. Thus, some

recommendations and guidelines for future works in this field could be the following:

- Evaluating the utilization of different fungal strains for the recovery of bioactive

compounds from L. tridentata leaves by solid-state fermentation process, and

determining the biological activity of the produced extracts;

- Studying further toxicological and pharmacological effects in order to confirm the

hypothesis of using phytochemicals from L. tridentata leaves as antibacterial agents.

- Developing more detailed studies related to the potential of dichloromethane fraction

from L. tridentata leaves as an anticancer agent.

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