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UNIVERSITY OF SÃO PAULO
SCHOOL OF PHARMACEUTICAL SCIENCES OF RIBEIRÃO PRETO
DANIEL PECORARO DEMARQUE
Chemical and biological studies of tannins and anthraquinones
acting on the gastrointestinal tract
Estudos químico e biológico de taninos e antraquinonas com
atuação no sistema gastrointestinal
Doctoral thesis presented to the Graduate Program of School of Pharmaceutical Sciences of Ribeirão Preto/USP for the degree of Doctor in Sciences.
Concentration area: Natural and synthetic products
PhD student: Daniel Pecoraro Demarque Supervisor: Prof. Dr. Norberto Peporine Lopes Co-supervisor: Dr. Christian Merten
Versão corrigida da Tese de Doutorado apresentada ao Programa de Pós- Graduação em Ciências Farmacêuticas em 27/11/2017. A versão original encontra- se disponível na Faculdade de Ciências Farmacêuticas de Ribeirão Preto/USP.
Ribeirão Preto 2017
FICHA CATALOGRÁFICA
AUTORIZO A REPRODUÇÃO E DIVULGAÇÃO TOTAL OU PARCIAL DESTE TRABALHO, POR QUALQUER MEIO CONVENCIONAL OU ELETRÔNICO, PARA FINS DE ESTUDO E PESQUISA, DESDE QUE CITADA A FONTE.
Demarque, Daniel Pecoraro Chemical and biological studies of tannins and anthraquinones acting on the gastrointestinal tract. Ribeirão Preto, 20 17. 164 p.: il. ; 30cm.
Tese de Doutorado, apresentada à Faculdade de Ciências Farmacêuticas de Ribeirão Preto/USP – Área de concentração: Produtos Naturais e Sintéticos. Orientador: Lopes, Norberto Peporine. Co-orientador: Merten, Christian
- Antraquinonas. 2. Taninos. 3. Farmacognosia.
ACKNOWLEDGEMENTS
I would like to thank for grant 2014/18052-0 to São Paulo Research Foundation
(FAPESP). Any opinions, findings, and conclusions or recommendations expressed
in this material are those of the author(s) and do not necessarily reflect the views of
FAPESP.
I would like to thank my supervisor, prof. Norberto P. Lopes for all the support
and encouragement during this period. Thank you for the patient guidance, advice,
and especially for teaching me much more than just chemistry. I would like also to
thank my co-supervisor Christian Merten for the help in VCD spectroscopy part.
I would like to thank all members of Núcleo de Pesquisa em Produtos
Naturais e Síntéticos , NPPNS, for all the years of working, sharing, complaining,
discussing and living together. Thanks for the help, advice, and friendship. People
like you made this process much more pleasant.
I would like to thanks my friends in Germany. It was a real privilege to work
with everybody from Merten research group in Germany.
I would like to thank José Carlos Tomáz, Izabel Cristina Casanova Turatti
and Jacqueline Nakau Mendonça for technical support. As well as for analyses,
advice, and friendship. Additionally, I would like to thank Amanda Boaretto and Dr.
Flávio Macedo Alves for collecting and identifying barbatimão.
It would have been impossible to develop in vivo studies without the help and
assistance of Daniel Roberto Callejon. Also, I would like to thank Danielle Rocha
Pinho for help with cascarosides isolation and Gibson G. de Oliveira for the
assistance and advice in histological cuts.
I would like to thank Prof. Dr. Denise B. da Silva for all support, advice and
guidance in this project. Also, I would like to thank my collaborators Rui M. P. da
Silva, Prof. Dr. Enilza Spreafico, Leando F. Santos and Prof. Dr. Andréia M.
Leopoldino for in vitro tests. In addition, I am very grateful for all the people who
directly or indirectly contributed to this work that I did not mention here.
I must express my gratitude to RESOLV from Ruhr Universität Bochum for
supporting my stay in Germany.
I would also like to mention my gratitude to all employees and professors
from Faculdade de Ciências Farmacêuticas de Ribeirão Preto and all Universidade
de São Paulo for supporting the development of this project.
Finally, I thank my amazing family and friends for their love and support. In
particular, I would like to thank my parents, brothers, sisters, and in-laws for always
encouraging and helping during these years. Thank you Fernanda for making my life
happier, making me smile, and inspiring me. I would not have made it this far without
you.
i
RESUMO
DEMARQUE, D. P. Estudos químico e biológico de taninos e antraquinonas que atuam no sistema gastrointestinal. 2017. 164f. Tese (Doutorado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo, Ribeirão Preto, 2017.
Diversas plantas com atuação no trato gastrointestinal – ricas em taninos e antraquinonas – ainda deixam muitas dúvidas quanto a sua caracterização estrutural, o seu mecanismo de ação e toxicidade. O presente trabalho objetivou o estudo químico e biológico de duas plantas utilizadas para doenças relacionadas ao sistema gastrointestinal: barbatimão ( Stryphnodendron rotundifolium ), rico em procianidinas e taninos (utilizada para combater gastrite); e cáscara sagrada ( Rhamnus purshiana ), rica em antraquinonas (utilizada para constipação). Inicialmente foi realizado um estudo químico com as plantas envolvendo isolamento e caracterização de componentes do extrato por RMN (^1 H, 13 C, DEPT e NOESY), espectrometria de massas (MS), infravermelho e dicroísmo circular vibracional (VCD). Essas substâncias foram utilizadas para a padronização dos extratos utilizados em estudo biológico. O estudo biológico envolveu testes in vitro para avaliar a toxicidade de diferentes componentes do extrato de cáscara sagrada e teste in vivo com barbatimão, visando, através do tratamento de animais e remoção do estômago para estudo em microscopia confocal, entender o mecanismo de proteção gástrica. Com o desenvolvimento do trabalho foi possível desenvolver novas metodologias para a identificação de antraquinonas utilizando espectrometria de massas e provar a veracidade de regras empíricas utilizadas para a determinação da configuração absoluta de moléculas dessa classe. Os testes in vitro com antraquinonas isoladas e padrões comerciais indicam a possibilidade de aprimoramento dos perfis químicos utilizados para a produção de medicamentos a base de extratos de cáscara sagrada, visando à redução de toxicidade. Quanto ao barbatimão, o estudo químico possibilitou a aplicação da técnica de VCD para a diferenciação de procianidinas diasteroisomericas. Os estudos in vivo confirmaram a eficácia do uso de taninos para combater gastrite através da formação de um revestimento protetor.
Palavras-chave: taninos, antraquinonas, gastrite, laxativo, farmacognosia.
ii
ABSTRACT
DEMARQUE, D. P. Chemical and biological studies of tannins and anthraquinones acting on the gastrointestinal tract. 2017. 164 f. Thesis (Doctoral). Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo, Ribeirão Preto, 2017.
Several plants with action in the gastrointestinal tract - rich in tannins and anthraquinones - cause many doubts concerning their chemical characterization, mechanism of action and toxicity. The present work aimed to develop a chemical and biological study of two plants used for gastrointestinal system related diseases: barbatimão ( Stryphnodendron rotundifolium ), rich in procyanidins and tannins (used to treat gastritis); and cascara sagrada ( Rhamnus purshiana ), rich in anthraquinones (used for constipation). Initially, a chemical study with plants was performed comprising isolation and characterization of components of the extract through NMR (^1 H, 13 C, DEPT and NOESY), mass spectrometry (MS), and infrared and vibrational circular dichroism (VCD). These substances were used for extract standardization used in biological studies. The in vitro tests were performed in order to evaluate the toxicity of different components of the cascara extract. The in vivo tests with barbatimão aimed to better understand the mechanism of gastric protection, by treating animals with the extract, removing their stomachs, and analyzing them with fluorescence microscopy. Through this work we were able to develop new methods for identifying anthraquinones by mass spectrometry and to prove empirical NMR rules used to determine the absolute configuration of molecules of this class. The tests with isolated anthraquinones indicated the possibility of improving chemical profile to further reduce cascara sagrada toxicity. The barbatimão chemical study allowed the application of VCD technique for the differentiation of diasteroisomeric procyanidins, and confirmed the effectiveness of using tannins for gastritis treatment through the formation of a protective coating.
Keywords: tannins, anthraquinones, laxative, gastritis, pharmacognosy.
iv
Figure 18: (A) Results of percentage of cell viability in different concentrations for extracts and isolated compounds. (B) Western blot result of caspase assay. (-) indicates absence of compound; (+) indicates presence of compound. PARP: poly(ADP-ribose) polymerase; RIP: Receptor-interacting protein (RIP); Casp: caspase. .................................................................................................................... 57
Figure 19: Chemical structures of gallocatechin, epigallocatechin and robinetinidol isolated from barbatimão. .......................................................................................... 61
Figure 20: HPLC-MS analysis of isolated compounds. Top: chromatogram of gallocatechin (RT 3.9 min), robinetinidol (RT 5.6 min) and epigallocatechin (RT 5. min). Bottom: MS spectra of correspondent compounds. .......................................... 61
Figure 21: VCD spectra of BTMPrep03 (gallocatechol), BTMPrep07 (robinetinidol) and BTMPrep09 (epigallocatechin). .......................................................................... 62
Figure 22: VCD spectra overlap of all gallocatechin conformers. .............................. 63
Figure 23: Vibrational modes of four main bands in 1600 and 1300 cm-1.................. 63
Figure 24: Comparison of epigallocatechin (black) and gallocatechol (red) VCD (above) and IR (bottom) spectra. Calculated spectra are shown in the left and experimental in the right. ........................................................................................... 64
Figure 25: Comparison between BTM03 (gallocatechin) and BTM (epigallocatechin) subtracted spectrum with calculated trans-cis subtracted spectrum and cis-trans subtracted spectrum. ........................................................................... 65
Figure 26: MS spectra of BTMFr16-31 obtained by MALDI. ...................................... 66
Figure 27: Fluorescence confocal microscopy images of stomach cross sections. First column (A1-D1): visible light image; second column (A2-D2): overlap of visible and fluorescence light images; third column (A3-D3): fluorescence light image. Line A: Stomachs tissues from untreated animals. Line B: Stomach tissues from untreated animals impregnated with S. rotundifolium crude extract. Line C: Stomach tissues from treated animals with S. rotundifolium crude extract (400 mg/mL); non-washed tissues (euthanized after 0.5 h). Line D: Stomach tissues from treated animals with S. rotundifolium crude extract (400 mg/mL); PBS-washed tissues (euthanized after 0. h). .............................................................................................................................. 69
v
TABLES
Table 1: MS parameters optimized for cascarosides quantification (QT – quantification; Conf – confirmation). .......................................................................... 29
Table 2: Mass spectrometer conditions (DP: declustering potential, EP: entrance potential, CE: collision energy, CXP: collision cell exit potential, DT: Dwell time) to quantification of Catechin (CAT), Gallocatechin (GCT), Gallic Acid (GC) using 10- hydroxycascaroside A (10Casc-A) as internal standard. ........................................... 34
Table 3: NMR data (^1 H, 13 C and Dept-135; D 2 O) of 10-hydroxycascaroside B (Comparison with Cascaroside B (MeOD; 300MHz)(MANITTO, PAOLO et al., 1993a) .................................................................................................................................. 42
Table 4: NMR data (^1 H, 13 C and Dept-135; D 2 O) of 10-hydroxycascaroside B (Comparison with Cascaroside B (MeOD; 300MHz)(MANITTO, PAOLO et al., 1993a). ...................................................................................................................... 44
Table 5: Cascarosides content in CasEtAcPh and CasCrEx. .................................... 56
Table 6: NMR data (^1 H and 13 C) of gallocatechin, robinetinidol and epigallocatechin (MeOD-d4; 400 MHz). ............................................................................................... 62
Table 7: Tannin series identified in BTMFr16-31. PCY: Procyanidin, PDE: Prodelphinidin............................................................................................................ 67
vii
HPLC High Pressure Liquid Chromatography
HR-MS High Resolution Mass Spectrometry
IR Infrared
IS Ionization Voltage
LDH Lactate Dehydrogenase
LDL Low Density Lipoprotein
LC-MS Liquid Chromatography coupled with Mass Spectrometry
LC-MS/MS Liquid Chromatography coupled with Mass Spectrometry in tandem
MD Molecular Dynamics
MS Mass spectrometry
NOE Nuclear Overhauser Effect
NOESY Nuclear Overhauser Effect Spectroscopy
ORD
PARP
Optical Rotatory Dispersion Poly(ADP-ribose) polymerase
PBS Phosphate Buffered Saline
PCY Procyanidin
PDE Prodelphinidin
PNASP Polyphenols not Absorbed on Skin Powder
QT Quantification
RDA
RIP
Retro-Diels Alder Receptor-interacting protein
RT Retention Time
SGPT/ALT Glutamate-pyruvate Transaminase/Alanine Transaminase
SS Standard Solution
SUS Sistema Único de Saúde (Brazilian Public Health System)
TFA Trifluoroacetic acid
THP Tradicional Herbal Products
TLC Thin Layer Chromatography
TNF Tumor Necrosis Factor
TOF Time of Flight analyser
TP Total Phenolics
UV Ultraviolet
VCD Vibrational Circular Dichroism
VLDL Very Low Density Lipoprotein
viii
TABLE OF CONTENTS
RESUMO …………….....….……..……………….………………………….................… i ABSTRACT …………………………………………………….…………................….… ii FIGURES ................................................................................................................... iii TABLES ..................................................................................................................... v
INTRODUCTION
Introduction _____________________________________________________________ 3
Daniel P. Demarque
1. INTRODUCTION
Medicinal plants are the oldest disease treatments known to man and are still
important and useful therapeutic tools. According to the Brazilian Association of Companies
from the Phytotherapeutic Sector (ABIFISA, in Portuguese, Associação Brasileira das
Empresas do Setor Fitoterápico ) herbal medicines are responsible for about US$ 20 billion in
profits, globally (GUIA DA FARMÁCIA, 2016). For the Brazilian market the continuous
growth of the herbal medicines – 6.1% in 2013 and 8.0% in 2014 compared to the previous
years – is an outlier in the industry’s general growth trend.
Due to the importance of this sector for the Brazilian and global market and the
effectiveness of adherence to therapeutic treatments using herbal medicines, the Brazilian
Public Health System (SUS, in Portuguese, Sistema Único de Saúde ) has supported them as
alternatives to classic treatments. Furthermore, SUS also recommends increasing the research
development of herbal medicines for the most commonly used plants (BRASIL, 2006).
The resumption of herbal medicine treatments in the last 10 years, especially by SUS
makes this treatment official, mobilized the Brazilian Health Regulatory Agency (ANVISA,
in Portuguese, Agência Nacional de Vigilância Sanitária ) to create two different product
categories in 2014: Traditional Herbal Products (THP) and Herbal Medicines (HM), where
vegetable drugs have to undergo the same scientific tests as common medicines to be
classified as HM (BRASIL, 2014). Consequently, the efficacy, safety, and mechanism of
action of the HM should be proved.
In light of these regulatory modifications, the variability of secondary metabolism,
the chemical complexity and the deficiency of scientific evidence about the pharmacological
and toxicological mechanism of some widely used plants still compromises the vegetable
drug’s efficacy and safety to become an HM. This can affect even commonly used and known
species, such as those used for constipation and gastritis treatment. Species that act in the
gastrointestinal tract (GIT) have a chemical complexity which includes a huge range of
compounds with wide stereochemical variations. In addition, the toxicological aspects of
some plants that are used as laxatives are still not completely elucidated, nor the mechanism
of action of plants used for gastritis treatment.
The changes in Brazilian regulatory system point toward the requirement of scientific
studies for HM development. In order to produce scientific information to include THP in
HM classification, important chemical, pharmacological and toxicological issues that have
been neglected for plants used as laxatives and to treat gastritis need to be solved. Therefore,
4 _____________________________________________________________ Introduction_
Daniel P. Demarque
this thesis focuses on the chemical and biological problems concerning some plants with GIT
action.
1.1. Plants acting on the gastrointestinal tract (GIT)
The GIT is a collection of organs that are responsible for converting food into
energy. The dysfunctions which medicines act on include: dyspepsia, gastroesophageal reflux,
irritable bowel syndrome, inflammatory conditions of the upper tract (ulcers, esophagitis,
gastritis), chronic gastrointestinal infections, dysbiosis and constipation (BONE; MILLS,
2013). Between them, dyspepsia and constipation are very common disturbances treated with
medicinal plants, since most cases are not particularly severe.
The non-severity of some gastrointestinal problems, along with the popular common
sense of non-toxic herbs contributes to drug abuse and, consequently, side effects emerge. For
instance, even a natural tea to treat constipation may cause severe toxic effects neglected by
lay people.
This drug abuse is related to several reasons. Individuals who abuse laxatives can be
divided into the following groups: individuals with eating disorders (ROERIG et al., 2010;
TOZZI et al., 2006), individuals who use laxatives when constipated and continue to overuse
them to the point that their intestine becomes refractory (BAKER; SANDLE, 1996), and
individuals who use them to lose weight or for sports reasons (MARQUART; SOBAL, 1994).
Regarding the drugs used for gastric dysfunctions, such as dyspepsia, the main
concern is an empirical explanation for their action. The tissue binding property of some
chemicals is the reasonable explanation how some phenol-rich species improve gastric
problems. Considering the necessity to convert THP into HM, the science of natural products
should better understand how gastric problems are improved by these medicines, how this
process happens, and which characteristics of the chemicals are necessary to them became
part of an HM to treat gastric problems. Furthermore, this same concern applies to the
toxicological aspects of laxative plants, where understanding the role of each compound can
help to develop safer medicines.
