Chemical study and antilithiatic activity of Caesalpinia ...

INSTITUTE OF PHARMACY AND FOOD

UNIVERSITY OF HAVANA

CUBA

DEPARTMENT PHARMACEUTICAL SCIENCES

UNIVERSITY OF ANTWERP

BELGIUM

Chemical study and antilithiatic activity

of Caesalpinia bahamensis Lam. (brasilete)

Thesis submitted in fulfillment of the requirements for the degree of

Doctor in Pharmaceutical Sciences

Alejandro Felipe González

Antwerp, 2020

INSTITUTE OF PHARMACY AND FOOD

UNIVERSITY OF HAVANA

CUBA

DEPARTMENT PHARMACEUTICAL SCIENCES

UNIVERSITY OF ANTWERP

BELGIUM

Chemical study and antilithiatic activity

of Caesalpinia bahamensis Lam. (brasilete)

Chemisch onderzoek en anti-lithiasis activiteit

van Caesalpinia bahamensis Lam. (brasilete)

Proefschrift voorgelegd tot het behalen van de graad van Doctor in de

Farmaceutische Wetenschappen aan de Universiteit Antwerpen

te verdedigen door

Alejandro FELIPE GONZÁLEZ

Promotoren

Prof. Dr. L. Pieters

Dr. Yamilet I. Gutiérrez Gaitén

Dr. René Delgado Hernández

Antwerpen, 2020

AKNOWLEDGEMENTS

AKNOWLEDGEMENTS

This dissertation is the result of, not only of my own personal efforts, but also the support of family,

friends, and colleagues who have helped me during this academic path,

• To my mother Daisy González Osorio, a very brave woman who had to work alone and hard to facilitate

my personal and professional successes.

• To my brother Jorge Felipe González who has been an example to follow. I thank him for the impulse

he has given me to continue with my studies.

• To my best friend Joelmir Estrada Folch for always being present in good and bad times.

• To my first professional mentor Adrián Sánchez Machado for introducing me to the world of science

and motivating me to follow it.

• To my other mentor Ramón Scull Lizama for his timely advices and disposition to assist me in difficult

moments.

• To my sons Fernando Felipe Gómez and Lian José Felipe Díaz for being patient during all the time I

had to spend to conclude this dissertation while they continuously asked “Dad, when are you ending?”

• To my colleague Yamilet Irene Gutiérrez Gaitén for trusting in my work and patience.

• To my colleague René Delgado Hernández for expanding my knowledge of science and fighting for

me to exceed my goals and open my horizons.

• To my mentor Luc Pieters for accepting me as his PhD student and spending his precious time on

lengthy revisions and corrections.

• To Wim Vanden Berghe and Claudina Pérez Novo for helping during my stays in Belgium, making

my life happier despite the distance.

• To Kenn Foubert for his teaching and his patience during my research.

• To the members of the NatuRa group; specially, Stefaniya Velichkova, Andrés Rivera, Tania Naessens

and Mamadou Aliou Baldé; for your friendship and your help in my research, thanks

• To VLIR-UOS projects for the support for the development of this research.

• To Carlos Rafael Núñez Cairo for your disposition to assist me in my biological research.

• To Ana Cristina Noa Rodríguez for joining my life at the right time and moving ahead together.

To all them and to other peoples that I did not mention; thanks!

TABLE OF CONTENT

LIST OF ABBREVIATION 1

SUMMARY 2

SÍNTESIS 3

SAMENVATTING 4

CHAPTER 1. Introduction

1.1. Renal lithiasis 5

1.2. Pharmacological models for the study of oxalate renal stones 8

1.2.1. Oxalate calcium crystallization (in vitro) 8

1.2.2. Oxalate-induced cell injury (in vitro) 9

1.2.3. Ethylene glycol-induced urolithiasis in rats (in vivo) 9

1.2.4. Implantation of a foreign corps through surgery (in vivo) 10

1.3. Traditional medicine in renal lithiasis: A therapeutic solution? 10

1.4. Cuban herbal medicine and renal lithiasis 13

1.5. The genus Caesalpinia: Chemical and biological studies 16

1.5.1. Homoisoflavonoids as potential chemical markers of the genus Caesalpinia 17

1.6. Caesalpinia bahamensis 20

1.7. Aim of this work 21

References 22

CHAPTER 2. Pharmacognostic study

2.1. Introduction 31

2.2. Material and Methods 32

2.2.1. Plant material 32

2.2.2. Morphological study 32

2.2.3. Physic-chemical parameters of the drug 32

2.2.4. Preparations of the extracts 32

2.2.5. Physic-chemical parameters of the extracts 33

2.2.6. Phytochemical characterization of the extracts 33

2.2.7. Total phenols and flavonoids content 33

2.2.8. Statistical analysis 34

2.3. Results 34

2.3.1. Morphological analysis of the drug 34

TABLE OF CONTENT

2.3.2. Physic-chemical parameters of the drug 35

2.3.3. Physic-chemical parameters of the extracts 36

2.3.4. Phytochemical characterization of the extracts 37

2.3.5. Total phenols and flavonoids content 38

2.4. Discussion 38

2.5. Conclusion 40

References 40

CHAPTER 3. Fatty acids, terpenoids and phytosterols



3.2.1. Plant material 43

3.2.2. Preparation and fractionation of the extract 44

3.2.3. Identification of compounds by GC/MS 44

3.3. Results and Discussion

44

3.4. Conclusion 48

References 48

CHAPTER 4. Homoisoflavonoids and antilithiatic activity



4.2.1. Plant material and preparation of the extracts 51

4.2.2. Isolation of homoisoflavonoids 52

4.2.3. UPLC-UV-MS analysis 55

4.2.4. Antilithiatic activity 55

4.2.4.1. Animals 55

4.2.4.2. Ethylene glycol-induced urolithiasis 56

4.2.4.3. Biochemical parameters 56


4.3. Results 57

4.3.1. Phytochemical analysis 57

4.3.2. Biological evaluations 60

4.4. Discussion 62

TABLE OF CONTENT

4.5. Conclusion 65

References 65

CHAPTER 5. Diuretic activity and acute oral toxicity

5..1. Introduction 69

5.2. Material and methods 70

5.2.1. Plant material and preparation of the extracts 70

5.2.2. Animals 70

5.2.3. Diuretic activity 71

5.2.4. Acute oral toxicity 71


5.3. Results 72

5.3.1. Diuretic activity 72

5.3.2. Acute oral toxicity 74

5.4. Discussion 74

5.5. Conclusion 76

References 76

GENERAL CONSIDERATION AND FUTURE PERSPECTIVES 79

CONCLUSIONS 87

RECOMMENDATIONS 88

CURRICULUM VITAE 89

LIST OF ABBREVIATIONS

1

LIST OF ABBREVIATIONS

δ: Chemical shift

13C-RMN: 13 Carbon Nuclear Magnetic Resonance

1H- RMN: Protonic Nuclear Magnetic Resonance

BSTFA + 1% TMCS: N,O-bis(trimethylsilyl)-trifluoroacetamide with 1% of trimethylsilyl chloride

CG/MS: Gas Chromatography tandem Mass Spectrometry

COSY: Correlated Spectroscopy

DEPT: Distortionless Enhancement by Polarization Tranfer

ELSD: Evaporating light scattering detector

HMBC: Heteronuclear Multiple Bond Correlation

HPLC: High Performance Liquid Chromatography

HSQC: Heteronuclear Simple Quantum Correlation

J: Coupling constant

LC/MS: Liquid Chromatography tandem Mass Spectrometry

NMR: Nuclear Magnetic Resonance

Rf: Retention factor

SD: Standard deviation

TLC: Thin Layer Chromatography

TMS: Tetrametilsilane

UPLC: Ultra Performance Liquid Chromatography

UV: Ultraviolet

SUMMARY

2

SUMMARY

Renal lithiasis is a pathology with high incidence and prevalence around the world. Its treatment is

based on minimal invasive surgery. For this reason, the scientific community has focused its efforts on

the search for new drugs for the treatment of renal lithiasis. Caesalpinia bahamensis Lam. is a medicinal

plant used by the Cuban population for the treatment of renal stones. There is only little scientific

information about this plant species.

Macro- and micro-morphological characteristics of the stem of this plant were identified and the

physicochemical parameters of the drug were determined through the analysis of its quality parameters.

In addition, the chemical characteristics were established by chromatographic and ultraviolet profiles

and the total content of phenols and flavonoids was quantified. All these results were shown for the first

time for this species.

An hydroalcoholic extract was prepared from the stem, followed by liquid / liquid partition and further

chromatographic separations, resulting in the isolation and structure elucidation of six homo-

isoflavonoids, which were identified by Nuclear Magnetic Resonance (NMR) spectroscopy and Mass

Spectrometry (MS) as: 3-(2-hydroxy-4-methoxybenzyl)chromane-4,7-diol (metasappanin) (1), 10-

methyl-protosappanin B (2), (iso)-10-methyl-protosappanin B (3), brazilin (4), protosappanin B (5) and

(iso)-protosappanin B (6). In addition, seventy-four compounds were identified by Gas Chromatography

/ Mass Spectometry (GC/MS), mainly fatty acids, sesquiterpenoids and phytosterols. These compounds

were reported for first time for this species, while compound 1 was reported for first time as a natural

product.

The diuretic and antilithiathic activity of the aqueous and hydroalcoholic extracts was demonstrated in

animal models and their traditional use was corroborated. The antilithiathic activity of the extracts was

reported for the first time for the genus Caesalpinia.

SÍNTESIS

3

SÍNTESIS

La litiasis renal es una patología con alta incidencia y prevalencia mundial, cuyo tratamiento definitivo

implica el uso de técnicas quirúrgicas de mínimo acceso, por lo cual, varios investigadores han centrado

sus esfuerzos en la búsqueda de nuevas drogas para el tratamiento de esta patología. Caesalpinia

bahamensis Lam es una planta utilizada en la medicina tradicional cubana para el tratamiento de los

cálculos renales sobre la que existe poca información científica.

Mediante el análisis de los parámetros de calidad de la droga se identificaron las características macro y

micro morfológicas del tallo de la planta y se determinaron sus parámetros físico-químicos, los cuales

estaban en correspondencia con los parámetros establecidos para drogas vegetales. Se realizó la

caracterización química mediante los perfiles cromatográficos y ultravioleta de los extractos acuoso e

hidroalcohólico y se cuantificaron los fenoles y flavonoides totales de los extractos. Todos estos

resultados se muestran por primera vez para la especie.

El desarrollo de una secuencia analítica, con la combinación de métodos de extracción convencionales y

métodos cromatográficos, permitió aislar seis homoisoflavonoides del extracto hidroalcohólico de la

planta, que fueron identificados con la ayuda de técnicas de Resonancia Magnética Nuclear como: 3-(6’-

hidroxi-4’-metoxibenzil)cromano-4,7-diol (metasapanina) (1), 10-metil-protosapanina B (2), (Iso) 10-

metil-protosapanina B (3), brasilina (4), protosapanina B (5) e (Iso) protosapanina B (6). Se

identificaron otros setenta y cuatro compuestos mediante CG/EM, con un predominio de ácidos grasos,

terpenoides y fitosteroles. Los ochenta compuestos identificados se reportaron por primera vez para esta

especie, de ellos, el compuesto 1 se reportó por primera vez en el campo de los productos naturales.

Además, se demostró el efecto antilitiásico y diurético de los extractos en modelos animales, lo cual

permitió corroborar su uso tradicional. La actividad antilitiásica fue reportada por primera vez en el

género.

SAMENVATTING

4

SAMENVATTING

Renal lithiasis is een pathologie met hoge incidentie en prevalentie wereldwijd. De behandeling is

gebaseerd op minimale invasieve chirurgie. Daarom worden ook veel inspanningen gedaan om nieuwe

geneesmiddelen te ontwikkelen tegen deze aandoening. Caesalpinia bahamensis Lam. is een medicinale

plant, die door de Cubaanse bevolking gebruikt wordt voor de behandeling van nierstenen. Er is slechts

zeer weinig wetenschappelijk informatie beschikbaar over deze plantensoort.

Macro-en micro-morfologische karakteristieken van de stam van deze plant warden geïdentificeerd en

de fysicochemische parameters van het geneesmiddel bepaald door de analyse van de

kwaliteits¬parameters. Daarenboven werden chemische karaktaristieken vastgelegd onder de vorm van

chromatografische en ultraviolet-profielen, en werd het total gehalte fenolen en flavonoiden bepaald. Al

deze resultaten werden voor de eerste keer bekomen voor deze plantensoort.

Een hydro-alcoholisch extract van de stam werd bereid, gevolgd door vloeistof / vloeistof verdeling en

verdere chromatografische opzuivering, wat resulteerde in de isolatie en structuuropheldering van zes

homo¬isoflavonoiden, met behulp van Nucleaire Magnetische Resonantie (NMR) spectroscopie en

Massa Spectrometrie (MS) als: 3-(2-hydroxy-4-methoxybenzyl)chromane-4,7-diol (metasappanin) (1),

10-methyl-protosappanin B (2), (iso)-10-methyl-protosappanin B (3), brazilin (4), protosappanin B (5)

en (iso)-protosappanin B (6). Daarenboven werden 74 producten geïdentificeerd met Gas

Chromatografie / Massa Spectrometrie (GC/MS), voornamelijk vetzuren, sesquiterpenen en fytosterolen.

Deze producten werden voor de eerste keer beschreven voor deze soort, en product 1 werd voor de

eerste keer beschreven als natuurproduct.

De diuretische en anti-lithiasis activiteit van de waterige en hydroalcoholische extracten werd

aangetoond in diermodellen, en het traditioneel gebruik bevestigd. De anti-lithiase activiteit van deze

extracten werd voor de eerste keer gerapporteerd voor het genus Caesalpinia.

CHAPTER 1:

Introduction

Part of this chapter was published as:

Felipe A, Pieters L, Delgado R. Effectiveness of Herbal

Medicine in Renal Lithiasis: A review. Siriraj Medical

Journal 2020; 72(2): 188-194.

http://dx.doi.org/10.33192/Smj.2020.25


INTRODUCTION

5

1.1. Renal lithiasis

Renal lithiasis can be defined as the deposition of stones in the urinary tract due to an alteration of the

normal crystallization conditions of urine [Grasses et al., 2006]. It is currently the third most frequent

urological disease after urinary tract infections and prostate problems [Cook et al., 2016]. It has a

prevalence that ranges between 4-15% of the world population [Mittal et al., 2016] and a high

recurrence rate, that is, the probability of repeating a renal lithiasis episode is 40% after 5 years of the

first calculi and 60% after 10 years [Cano et al., 2015]. It is a health problem with greater incidence in

people between 30-60 years of age and more common in men than in women [Nalini et al., 2016].

The difference in prevalence between countries is associated with the combination of genetic and

environmental factors, including dietary habits, climatic conditions and socio-economic status [Cook et

al., 2016]. For example, several studies show that the incidence is higher in populations of warm

countries compared to populations in cold countries. It has also been found that high consumption of

salt, animal protein, calcium, fatty acids and sugar are risk factors for the development of kidney stones

[Cook et al., 2016; Nalini et al., 2016]. Finally, renal lithiasis has been associated with a family history

of kidney stones and with some diseases such as diabetes, hypertension, hyperthyroidism, obesity,

metabolic syndrome, gout and urinary tract infections [Cook et al., 2016; Sarroca & Arada, 2015].

The stones may be composed of calcium phosphate, uric acid, struvite, cystine and calcium oxalate.

Oxalate stones are the most frequent, being present in more than 80% of the uroliths [Sarroca & Arada,

2015]. For this reason, this study will focus on oxalate stones.

The mechanisms involved in the formation of calcific stones are not fully understood [Mittal et al.,

2016]. Renal stone formation is a biological process that involves physicochemical changes and

supersaturation of urine [Alelign & Petros, 2018]. It is explained through of the loss of equilibrium

INTRODUCTION

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between promoters and inhibitors of crystallization [Aggarwal et al., 2013], urine composition and renal

morphoanatomy [Torres et al., 2013].

Recently, promoters and inhibitors of crystallization have been referred to as modulators, which can be

small molecules of low-molecular weight that modify supersaturation of the urine by serving as

chelators of calcium and oxalate by forming soluble complexes. The formation of this complex depends

on numerous physicochemical factors such as the concentration of individual (competing) chemical

species, the relative magnitude of the formation constants of the complexes themselves, the pH and ionic

strength of the urine in which the process occurs [Rodgers, 2017]. As a result of supersaturation, solutes

precipitation in urine leads to nucleation and then crystal concretions are formed [Alelign & Petros,

2018].

The first step in the formation of kidney stones is the formation of a nucleus (termed as nidus), normally

apatite (calcium phosphate) due to the fact that heterogeneous nucleation is easier than homogeneous

nucleation in the physiological conditions of urine [Grasses et al., 1988]. It is the transformation from a

liquid to a solid phase in a supersaturated solution [Tsujihata, 2008]. A widely held theory is that of

Randall’s plaques, which proposes that subepithelial interstitial calcium-based deposits act as nuclei for

stone formation. These plaques originate adjacent to the thin limbs of loops of Henle as spherical

particles, which could be related to the high local ion concentrations at this site, and can extend to the

interstitium [Johri et al., 2010; Green & Ratan, 2013; Sethmann et al., 2017]. Recent studies have

investigated the role of oxalate-degrading bacteria. These form apatite structures that serve as a

crystallization center for the formation of stones and could be a pharmacological target to avoid the

nucleation process. Also, existing epithelial cells, urinary casts, RBCs, and other crystals in urine too

can act as nucleating centers in the process of nuclei formation termed as heterogeneous nucleation

[Alelign & Petros, 2018].

INTRODUCTION

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The growth process is very important because small stones are expulsed by urine, but the big stones

require medical treatment. This process can be favored by the retention of microcrystals in the

urothelium and controlled by lithogenesis inhibitors [Torres et al., 2013; Estévez et al., 2013]. Recent

studies have demonstrated that oxalate produces damage to renal cells by the generation of reactive

oxygen species [Vaitheeswari et al., 2015], which increase the surface expression of phosphatidylserine,

sialic acid, hyaluronan, osteopontin, or the glycoprotein receptor CD44, resulting in more crystal

adhesion and formation of the nidus for the formation of stones [Mittal et al. 2016]. On the other hand,

renal cavities with low urodynamic efficacy retain urine for long periods of time in the upper urinary

tract, which favors the formation of stones [Grases & Costa, 1999]. Finally, there are substances in the

urine acting as inhibitors of lithogenesis; also, there are substances which have complexing properties

with some ions involved in the precipitation process decreasing their concentration [Grasses et al.,

1988]. Several inhibitors have been found in urine, such as citrate, phytate, magnesium and

pyrophosphate ions [Torres et al., 2013] and other molecules such as uropontin, osteopontin, bikunin,

and Tamm-Horsfall protein [Green & Ratan, 2013]; but citrate, magnesium and phytate have been the

most studied [Grases & Costa, 1999; González, 2013].

The treatment of the calculi will depend of their size and site, and of any symptoms and signs,

particularly of obstruction. When the stones are less than 10 mm, they can be expulsed with

pharmacologic treatment. When the calculi are larger than 10 mm or when they cannot be expulsed with

pharmacologic treatment, it is necessary to use minimally invasive surgery [Sarroca & Arada, 2015;

Johri et al., 2010], such as extracorporeal shockwave lithotripsy (ESWL), percutaneous nephrolithotomy

(PCNL), or ureteroscopy (URS), which has revolutionized acute and complex stone management [Mittal

et al., 2016]. The problem is that these techniques don’t prevent the likelihood of new stone formation

INTRODUCTION

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because they often result in incomplete stone clearance [Bashir & Gilani, 2011; Mittal et al., 2016;

Vaitheeswari et al., 2015].

Then, to reduce the rate of stone recurrence, interventions such as lifestyle advice and some forms of

medical therapy are necessary [Mittal et al., 2016]. For example, the increase of liquid, potassium,

magnesium, calcium, vegetable and fruit intake and the decrease of sodium, animal protein and fat

consumption have been associated with reduction of renal stones [Grasses et al., 2006; Nalini et al.,

2016; Johri et al., 2010; Semins & Matlaga, 2010; Arrabal et al., 2013]. On the other hand, many drugs

have been used to prevent recidives, for example, thiazide diuretics, potassium citrate, allopurinol,

NSAIDs, calcium antagonists, pyridoxine and alpha-blockers [Sarroca & Arada, 2015; Johri et al., 2010;

Semins & Matlaga, 2010; Arrabal et al., 2013]. However, scientific evidence about the efficacy of

pharmacological therapy is less convincing [Bashir & Gilani, 2011; Estévez et al., 2013; Rathod et al.,

2012]. All these facts indicate the need for new therapeutic approaches for the treatment of renal stones

[Vaitheeswari et al., 2015], therefore, new alternatives are being tested using herbal medicine or

phytotherapy [Estévez et al., 2013].

1.2. Pharmacological models for the study of oxalate renal stones

1.2.1. Oxalate calcium crystallization (in vitro)

In vitro oxalate calcium crystallization is a method widely used because it is easy and economic. It

imitates the nucleation, aggregation and growth of crystals, but does not comprise the action of

endogenous promoters and inhibitors and the biological processes involved in stone formation [Pérez et

al., 2016]. In this method, calcium oxalate crystals are obtained by a chemical reaction between calcium

chloride and sodium oxalate, generally in water with Tris-HCl buffer and in the presence of NaCl

[Aggarwal et al., 2013; Mittal et al., 2016; Vaitheeswari et al., 2015; Godínez & Volpato, 2008]; also, it

is possible to perform the assay in urine [Pérez et al., 2016; Touhami et al., 2007]. The design of the

INTRODUCTION

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method differs qualitatively and quantitatively among researchers [Pérez et al., 2016]. In general,

sodium citrate is used as positive control and spectrophotometric methods are used to evaluate the

antiurolithic effect [Bashir & Gilani, 2011; Vaitheeswari et al., 2015; Pérez et al., 2015]

1.2.2. Oxalate-induced cell injury (in vitro)

This method is used to demonstrate the protective effect of the drugs on damage produced by oxalate in

renal cells, which favors crystal adhesion and formation of the nidus for the development of the calculi

[Mittal et al., 2016; Touhami et al., 2007; Vaitheeswari et al., 2015]. MDCK and NRK 52E cells are

used in this model. The cells are incubated with sodium oxalate in the presence of the test samples. Cell

injury is evaluated by measuring cell viability through monitoring lactate dehydrogenase (LDH) leakage

into the medium [Aggarwal et al., 2013; Tayal et al., 2012; Aggarwal et al., 2010].

1.2.3. Ethylene glycol-induced urolithiasis in rats (in vivo)

In the in vivo model of chemically-induced urolithiasis, ethylene glycol is the substance most used. A

mix of 0.75% ethylene glycol with 1-2% ammonium chloride is administered to the rats to promote

hyperoxaluria and calcium oxalate deposition in the kidneys. Evaluation includes histological

examination and determination of biochemical markets in serum and ions in the urine. In general, the

markers most evaluated are: calcium, magnesium, phosphorus, urea and creatinine. Also, pH and weight

are measured [Bashir & Gilani, 2011; Touhami et al., 2007; Novaes et al., 2014; Divakar et al., 2010].

Other chemical substances have been used to induce renal lithiasis, for example, vitamin D3, calcium

gluconate, ammonium oxalate, gentamicin sulfate and L-hydroxyproline. Chemical induction causes

renal damage, and for this reason it does not represent the clinical evolution of the disease [Pérez et al.,

2016].

INTRODUCTION

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1.2.4. Implantation of a foreign corps through surgery (in vivo)

This method consists of the implantation of a foreign corps in the urinary bladder through a small

incision [Pérez & Vargas, 2006; Morales et al., 2015]. The implanted corps simulates the nuclei for the

formation of renal stones, and therefore this model is more representative of the etiology of the

formation of calculi in humans. Zinc and magnesium corpses are most used in the scientific community

[Pérez et al., 2016]. The results are evaluated by the difference of the weight of the corps before and

after the experiment [Pérez & Vargas, 2006; Morales et al., 2015].

1.3. Traditional medicine in renal lithiasis: A therapeutic solution?

The use of medicinal plants dates from the beginnings of humanity, when people had no other effective

therapeutic resources to treat their diseases. This knowledge was transmitted through legends,

pictographs and various monographs until our days [Rodríguez et al., 2015]. The oldest written evidence

of medicinal plants’ usage for preparation of drugs has been found on a Sumerian clay slab from

Nagpur, approximately 5000 years old. Other ancient references were shown in "The Chinese book on

roots and grasses" written by Emperor Shen Nung around 2500 BC, "The Indian holy books Vedas" and

“The Ebers Papyrus”, written about 1550 BC [Petrovska, 2012].

According to data from the World Health Organization (WHO), 80% of the world’s population uses

plants as a remedy to cure their diseases [Escalona et al., 2015]. On the other hand, it is known that

around 20% -30% of the medicines available on the market are derived from natural products [Majouli

et al., 2017].

Recently, the use and commercialization of medicinal herbs has increased in developed and developing

countries, linking several multinational companies, which have obtained benefits of up to $ 7.00 billion

in Europe, $ 3.2 billion in the United States and $ 2.3 billion in Asia [Table 1]. The reasons for this

increase are the preference of consumers for natural therapies; concerns regarding undesirable side

INTRODUCTION

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effects of modern medicines and the belief that herbal drugs are free from side effects, since millions of

people all over the world have been using herbal medicines for thousands of years; great interest in

alternative medicines; preference of populations for preventive medicine due to increasing population

age; the belief that herbal medicines might be of effective benefit in the treatment of certain diseases

where conventional therapies and medicines have proven to be inadequate; a tendency towards self-

medication; improvement in quality, proof of efficacy and safety of herbal medicines and high cost of

synthetic medicines [Calixto, 2000].

The good acceptance of the population of herbal medicine, the wide traditional knowledge about

medicinal plants, the few scientific studies that support the therapeutic properties of these and the

interest of the pharmaceutical industry in the development of phytopharmaceuticals constitute

opportunities in the research of herbal medicines as therapeutic alternatives for several diseases,

especially for those in which conventional medicine has not been very effective.

Table 1. Revenues obtained from sales of herbal medicines in some countries

Country Income [USD]

Germany $3.5 billion

United States of America $3.2 billion

Japan $2.1 billion

France $1.8 billion

Italy $700.0 million

United Kingdom $400.0 million

Spain $300.0 million

Netherlands $100.0 million

On the other hand, herbal medicines usually contain a range of pharmacologically active compounds.

This could be an advantageous characteristic for the therapeutic application of herbal medicines, since

INTRODUCTION

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sometimes beneficial synergisms are established in the treatment of some diseases, being able to be

more effective than synthetic drugs.

As mentioned earlier, several researchers have focused their attention on herbal medicine for the

treatment of renal lithiasis because currently, this pathology is treated by minimal access surgery and

because pharmacological therapy is less convincing.

The traditional knowledge about medicinal plants is the first clinical evidence on efficacy of herbal

medicines; however, scientific studies are necessary to corroborate the ethnobotanical information

[Calixto, 2000]. Recently, a list of antilithiatic plants used by the population of different countries of the

world was published, in which already 500 species belonging to 106 families were identified. The most

representated families in this study were the Asteraceae (87), Fabaceae (71), Lamiaceae (58), Rubiaceae

(17), Solanaceae (12), Phyllanthaceae (9), Zingiberaceae (9), Rutaceae (9), Polygonaceae (8) and

Urticaceae (8) [Ahmed et al., 2017; Ahmed & Hasan, 2017; Ahmed & Hasan, 2017a]. Other reported

families were the Rosaceae (41), Poaceae (24), Malvaceae (23), Brassicaceae (20) and Boraginaceae

(13) [Ahmed et al., 2017a; Ahmed & Hasan, 2017b]. The wide traditional knowledge of antilithiatic

plants favors the study of herbal medicine in this pathology because it increases the chances of finding

an effective therapeutic treatment.

Despite the widespread use of plants in traditional medicine, their therapeutic application is limited due

to the lack of scientific studies that support their therapeutic properties, especially clinical studies

[Calixto, 2000]. However, a study done by Newman & Cragg (2016) shows that 50% of the drugs

approved in the period 1981 to 2014 originated from natural products. This evidence shows that plants

are an effective resource for the treatment of diseases; however, studies that support their use in

therapeutics are required, which is one of the weaknesses in the clinical application of herbal medicine.

INTRODUCTION

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Several in vitro, in vivo and clinical studies have been developed in plants traditionally used for the

treatment of renal lithiasis with promising results; however, phytochemical studies of these plant species

have been insufficient. Consequently, the validity of the studies is limited because without

phytochemical characterization, quality control is difficult and reproducibility of results questionable.

The available information shows that some possible mechanisms of action of plant extracts include an

increased excretion of urinary citrate, decreased excretion of urinary calcium and oxalate, and the ability

to inhibit the crystallization process of calcium oxalate. The activity could also be attributable to

diuretic, antioxidant or antibacterial effects [Butterweck & Khan, 2009].

In summary, the good acceptance of natural products by the world’s population, the interest of the

pharmaceutical industry in the development of them, the traditional knowledge of a great variety of

plants for the treatment of lithiasis and the promising results of the scientific studies carried out are

elements that support the theory that plants could be an effective therapeutic resource for the treatment

of renal lithiasis; however, phytochemical and biological studies that support this theory are still highly

needed.

1.4. Cuban herbal medicine and renal lithiasis

In Cuba, the first evidence about the use of medicinal plants was found in the primitive community,

where “el behíque” was the second most important person in the community. Among its functions was

the treatment of patients through remedies made with medicinal plants [Callejas et al., 2010]. On the

other hand, in the wars of independence (XIX century), “los mambises” found in the Cuban flora a

solution to the cure of their wounds and diseases. For example, José Martí mentioned in his Campaign

Diary the benefits of “hijereta”, “cilantro” honey and other remedies used in camps [Martí, 2003]. In

1945, the Cuban scientist Juan Tomás Roig published his book "Plantas Medicinales y Aromáticas de

Cuba" where he described more than five thousand medicinal species used by the Cuban population

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[Roig, 2012]. Recently, the Ministry of Public Health of Cuba created the National Program of Natural

and Traditional Medicine and elaborated the National Therapeutic Guide of Phytodrugs and bee-derived

products for their application in health institutions.

The great variety of medicinal plants in the Cuban flora, together with the wide ethnobotanical

knowledge, increases the opportunities for the search of new therapies in the treatment of various

diseases, of which renal affections have been one of the most treated. However, the phytotherapeutic

potential of the island is still virgin. For example, in ethnobotanical studies made in Cuba, one hundred

seventy-nine species have been used in the renal system, of which only 9% have been evaluated

pharmacologically [Boffil, 2008; Scull et al., 1998]. In Table 2 a compilation is listed of some plants

used for the treatment of kidney stones according to ethnobotanical studies carried out in different areas

of the country.

Also, several in vitro and in vivo studies have demonstrated the antilithiatic effect of other medicinal

plants used by the population, for example Costus spiralis (Jacq.) Roscoe, Phyllanthus niruri L.,

Bergenia ligulate Engl., Origanum vulgare L., Hibiscus sabdariffa L., Zea mays L. [Bashir & Gilani,

2011; Pérez et al., 2016], Selaginella lepidophylla (Hook. & Grev.) Spring [Estévez et al., 2013],

Berberis trifoliata Hartw. ex Lindl. [Pérez et al., 2015], Punica granatum L. [Rathod et al., 2012] and

Terminalia arjuna (Roxb. ex DC.) Wight & Arn. [Mittal et al., 2016]. Generally, this effect has been

attributed to polyphenols [Grases et al., 2015] and flavonoids [Touhami et al., 2007]. On the other hand,

berberine is an isoquinoline alkaloid that has antiurolithic effects against calcium oxalate stones

mediated through a combination of antioxidant, diuretic, urinary alkalinizing and hypocalciuric effects

[Bashir & Gilani, 2011].

15

Table 2. Medicinal plants traditionally used in Cuba to treat renal lithiasis

Plant species Family Part(s) Preparation Reference

Blechum pyramidatum Lam. Acanthaceae Aerial Decoction [Beyra et al.,2004]

Caesalpinia bahamensis Lam. Caesalpinaceae Stem Decoction [Godínez & Volpato, 2008]

Chiococca alba (L.) Hitchc. Rubiaceae Root Decoction [Hernández & Volpato 2004]

Cymbopogon citratus (DC.) Stapf. Poaceae Stem Decoction [Godínez & Volpato, 2008]

Cyperus rotundus L. Cyperaceae Root Decoction [Hernández & Volpato 2004]

Erythroxylum havanense Jacq. Erythroxylaceae Root Decoction [Hernández & Volpato 2004]

Guazuma ulmifolia Lam. Sterculiaceae Bark Decoction [Hernández & Volpato 2004]

Heliotropium angiospermum Murray Boraginaceae Leaves Infusion [Godínez & Volpato, 2008]

Lepidium virginicum L. Brassicaceae Leaves Decoction [Volpato et al., 2009]

Lonchocarpus pentaphyllus (Poir.) DC. Fabaceae Stem, Root Decoction [Beyra et al.,2004]

Momordica charantia L. Cucurbitaceae Leaves Infusion [Godínez & Volpato, 2008]

Peperomia pellucida (L.) Kunth Piperaceae Aerial Decoction [Hernández & Volpato 2004]

Polypodium polypodiodes (L.) Watt Polypodiaceae Leaves Decoction [Beyra et al.,2004]

Roystonea regia (Kunth) O.F. Cook Arecaceae Root Decoction [Godínez & Volpato, 2008]

Boldoa purpurascens Cav. ex Lag. Bignoniaceae Aerial Decoction [Godínez & Volpato, 2008]

Trichilia glabra L. Meliaceae Leaves Infusion [Godínez & Volpato, 2008]

Urera baccifera (L) Gaudich ex Wedd Urticaceae Root Decoction [Godínez & Volpato, 2008]

Xanthium strumarium L. Asteraceae Root Decoction [Godínez & Volpato, 2008]

Xiphidium caerelum Aubl. Haemodoraceae Leaves Infusion [Riverón et al., 2015]

Zea mays L. Poaceae Hair Infusion [Godínez & Volpato, 2008]

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1.5. The genus Caesalpinia: Chemical and biological studies

The genus Caesalpinia (Fabaceae) is represented by more than 500 species distributed in tropical and

subtropical regions [Zanin et al., 2012]. From a phytochemical point of view, flavonoids, steroids,

terpenoids, galactomannans and gallotannins have been isolated from the genus [Zanin et al., 2012;

Cerqueira et al., 2009; Castañeda et al., 2012]. These structures have been associated with the

pharmacological properties demonstrated in the genus; such as, analgesic, anti-inflammatory, antiulcer,

antimicrobial, hypoglycemic, antitumoral and antioxidant [Zanin et al., 2012].

For example, the gallotannins isolated from C. spinosa were related with the capacity of the drug to

increase the sensitivity to doxorubicin in a leukemia cell line [Castañeda et al., 2012]. The oleanolic acid

isolated from the methanolic extract of the seed of C. paraguarensis showed moderated activity against

B. subtilis and S. aureus [Woldmichael et al., 2003]. Similar results were reported for the methanolic

extract of the leaves and flowers of C. pulcherrima, possibly associated with the presence of

isovouacapenol [Ragasa et al., 2002]. Other antimicrobial constituents have been isolated from the

genus Caesalpinia, such as, α-amyrin, β-amyrin, lupeol acetate, lupeol, deoxycaesaldekarine C,

bentaminine 1 and bentaminine 2 [Zanin et al., 2012]. On the other hand, Hsu et al. (2012) isolated

thirteen polyphenols related to gallic acid with antioxidant properties. Figure 1 shows some of the

structures previously mentioned.

Besides these compounds, species from the genus Caesalpinia interestingly produce unusual compounds

such as uncommon biflavonoids and a rare subclass of flavonoids, named homoisoflavonoids. The first

group is more distributed within plants, while homoisoflavonoids are restricted only to some vegetal

species from the Fabaceae and Asparagaceae [Baldim et al., 2017].

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Figure 1. Terpenoids isolated from the genus Caesalpinia

1.5.1. Homoisoflavonoids as potential chemical markers of the Caesalpinia spp.

Homoisoflavonoids have a general structure of 16 carbons containing two phenyl rings and one

heterocyclic ring. Homoisoflavonoids are biosynthesized from cinnamic acid derivatives along with

malonyl‐CoA subunits. The resulting compound, an aromatic polyketide, is the precursor of chalcones.

In the following step, the aromatic polyketide undergoes Claisen and enolization reactions, which lead to

the formation of the chalcone backbone. An additional carbon is added to the chalcone, provided by S‐

methyl moiety from methionine, creating the homoisoflavonoid skeleton containing 16 carbons. Thus,

there is the formation of 3′‐hydroxy‐chalcone as a precursor, which is transformed to 3‐benzylchroman‐

4‐one (homoisoflavonoids sappanin-type). Subsequently, different cyclization lead to the formation of

other types of homoisoflavonoids as protosappanins, caesalpins, brazilins and scillascillins (Figure 2)

[Baldim et al., 2017].

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Figure 2. Biosynthetic pathway of homoisoflavonoids

In some Caesalpinia spp. rare dimers of homoisoflavonoids (biflavonoids) have been isolated. For

example, the dimerization of a protosappanin with brazilin leads to the structures of protosappanin E-1,

protosappanin E-2 and neosappanone A [Nagai & Nagumo, 1990; Nguyen et al., 2005]. Similarly, the

dimerization of two units of brazilin leads to neoprotosappanin [Nguyen et al., 2005] and

caesalpinioflavone originates from the fusion of a flavone with sappanchalcone [Zanin et al., 2015].

Several biological properties of the genus Caesalpinia have been related to the occurrence of

homoisoflavonoids. For example, the homoisoflavonoids sappanchalcone, 3’-deoxy-4-O-methyl-

episappanol, brazilin, brazilein and sappanol, isolated from the stems of C. sappan, showed antioxidant

activity due to their capacity to scavenge free radicals and to avoid lipid peroxidation [Lee et al., 2010;

Liang et al., 2013, Mezbah et al., 2015].

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Among the homoisoflavonoids, brazilin has been widely studied. It showed anticoagulant activity acting

as receptor agonist in human platelets [Chang et al., 2013]; antimicrobial activity by inhibiting the

synthesis of bacterial proteins and DNA; anti-inflammatory activity in macrophages and chondrocytes,

reducing mRNA expression and the secretion of the proinflammatory cytokines IL-6 and TNF-α;

hypoglycemic activity, increasing the action of the 6-phosphofructo-2-kinase (PEK-2) and pyruvate

kinase, and vasorelaxation by activating Ca2+/calmodulin dependent NO synthetase. In addition,

hepatoprotective activity has been demonstrated as well [Nirmal et al., 2015; Mueller et al., 2016]. A

similar compound, brazilein, showed anthelmintic activity and cytotoxicity activity on tumoral skin cells

[Liang et al., 2013], while caesalpinioflavone showed this activity in human cancer cell lines, such as

HepG2 and Hep3B (liver), MDA-MB-231 and MCF-7 (breast), A549 (pulmonary), and CA9-22

(gingival) [Zanin et al., 2015]. Besides sappanin, other compounds have shown anti-inflammatory

activity, such as sappanol, episappanol, protosappanin A, protosappanin B, protosappanin C,

protosappanin E-2, neoprotosappanin, sappanone B, 3-deoxy-sappanone B and sappanchalcone [Nguyen

et al., 2005; Mueller et al., 2016].

According to the European Medicines Agency (EMA), chemical markers are “chemically defined

constituents or groups of constituents of an herbal substance, an herbal preparation or an herbal

medicinal product which serve for quality control purposes, independent of whether they have any

therapeutic activity”. EMA describes two different categories of markers. The constituents of an herbal

medicine responsible of its therapeutic activity or active markers; and the constituents that are

characteristics of its taxon or analytical markers [Rivera et al., 2017].

In this sense, homoisoflavonoids can be used as analytical markers for Caesalpinia spp. because they are

metabolites characteristic of the genus Caesalpinia. In addition, some biological properties

demonstrated for the genus have been associated with the antilithiatic activity, such as, antioxidant,

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20

antimicrobial and anti-inflammatory activities. For example, during the formation of renal stones,

oxalate crystals generate free radicals, producing renal cell damage and increasing the expression of pro-

lithiatic substances that participate in the adhesion step [Mittal et al., 2016; Vaitheeswari et al., 2015].

Recently, the presence of oxalate-degrading nanobacteria that form apatite structures serving as a

crystallization center for stone formation has been reported [Alelign y Petros, 2018]. Then, the

homoisoflavonoids should be considered as bioactive compounds in renal lithiasis; however, other

studies more in depth are required to demonstrate this hypothesis.

1.6. Caesalpinia bahamensis

Caesalpinia bahamensis Lam. (Fabaceae) is a medicinal plant used by the Cuban population to treat

renal and hepatic diseases, diabetes and peptic ulcers [Roig, 2012; Setzer et al., 2015]. It is commonly

named as bois rouge de la Jamaique, brasilete de Jamaica, leño de Jamaica (Antillas francesas), brasileto

wood of Jamaique (Antillas inglesas) [Roig, 2012], brasilete colorado [Fuentes et al., 2000] and brasilete

(Cuba) [MINSAP, 1994]. In Cuba, it is located in Pinar del Río (Las Martinas, Península de

Guahanacabibes and Sierra de los Órganos) [Dominicis et al., 1995; Fuentes et al., 2000], Holguín

(Moa) [MINSAP, 1994], Camagüey and Isla de la Juventud [Roig, 2012].

The diuretic effect of the aqueous extract has been evaluated in rodents with similar results as

hydrochlorothiazide, a well-established diuretic drug [Felipe et al., 2011; Pérez et al., 2011]. In in vitro

studies, the dichloromethane extract of the bark of C. bahamensis showed cytotoxic effects against SK-

Mel-28 (human melanoma), MDA-MB-231 (human mammary adenocarcinoma) and 5637 (human

bladder carcinoma) cells [Setzer et al., 2015], the acetone extract showed poor antimicrobial activity

against E. coli, S. aureus and C. albicans [Abreu et al., 2017] and three fractions of the stem showed

antioxidant activity [Felipe et al., 2019]. Chemically, the presence of saponins and absence of flavonoids

has been determined by phytochemical screening [Dominicis et al., 1995] and t-muurolol, α-bisabolol,

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nerolidol, palmitic acid, linoleic acid, oleic acid, stearic acid, octacosanol, β-sitosterol and

monooeloylglycerol have been identified by GC/MS [Felipe et al., 2019] Apart from this, the scientific

information about this species is limited.

The wide traditional use of the plant and the lack of scientific information are the main reasons to justify

this research.

1.6. Aim of this work

This thesis aims to identify the constituents responsible for the antilithiatic activity of the stem extract of

Caesalpinia bahamensis Lam. in order to support the traditional use of this plant species in Cuba.

The research envisaged in this thesis comprised five stages:

1. To determine the morphological characteristics and quality parameters of the stem of Caesalpinia

bahamensis Lam.

2. To characterize the physicochemical parameters and preliminary chemical composition of the

aqueous and hydroalcoholic extracts from the stem of Caesalpinia bahamensis Lam.

3. To quantify flavonoids and phenols in the aqueous and hydroalcoholic extracts from the stem of

Caesalpinia bahamensis Lam.

4. To isolate and to identify the main constituents of the hydroalcoholic extract from the stem of


5. To evaluate the antilithiatic activity of the aqueous and hydroalcoholic extracts from the stem of


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Petrovska BB. Historical review of medicinal plants’ usage. Pharmacognosy Reviews 2012; 6(11): 1–5.

Ragasa CY, Hofileña JY, Rideout JA. New Furanoid Diterpenes from Caesalpinia pulcherrima. Journal

of Natural Product 2002; 65(8): 1107-1110.

Rathod NR, Biswasa D, Chitmeb HR, Ratnac S, Muchandia IS, Chandrad R. Anti-urolithiatic effects of

Punica granatum in male rats. Journal of Ethnopharmacology 2012; 140: 234-238.

Rivera A, Ortíz OO, Bijttebier S, Vlietinck A, Apers S, Pieters L, Catherina C. Selection of chemical

markers for the quality control of medicinal plants of the genus Cecropia. Pharmaceutical Biology

2017; 55(1): 1500-1512.

Riverón FB, Hernández Y, García A, Escalona RY. 2015. La colección de plantas medicinales del Jardín

Botánico de Holguín, Cuba: su importancia social y científica. Revista del Jardín Botánico Nacional

2015; 36: 219-222.

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Cuba, 2012. Págs.: 228-229.

Sarroca M & Arada A. Litiasis renal. AMF 2015; 11(6): 314-323.

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INTRODUCTION

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Soc. 2015; 26(4): 804-809.

CHAPTER 2:

Pharmacognostic study

This chapter was published as:

Felipe A, Gutiérrez YI, Scull R, Noa AC, Beverly D,

Foubert K, Pieters L, Delgado R. Pharmacognostic

study of the stem of Caesalpinia bahamensis and

characterization of its aqueous and hydroalcoholic

extracts. Journal of Pharmacognosy and

Phytochemistry 2019; 8(3): 3079-3083.

PHARMACOGNOSTIC STUDY

31

2.1. Introduction

The use of medicinal plants dates from the beginnings of humanity, when people had no other effective

therapeutic resources for the treatment of their diseases. This knowledge was transmitted through

legends, pictographs and various monographs until our days [Rodríguez et al., 2015]. According to data

from the World Health Organization (WHO), 80% of the world’s population uses plants as a remedy to

cure their diseases [Escalona et al., 2015]. On the other hand, it is known that around 20% - 30% of the

medicines available in the market are derived from natural products [Majouli et al., 2017]. Due to the

wide use of natural products, pharmacognostic studies are required to ensure the quality of the plants

used in the production of phytotherapeutics, thus guaranteeing their safety and efficacy [Chanda, 2014].

Caesalpinia bahamensis Lam. (Leguminosae) is a medicinal plant used by the Cuban population to treat

renal and hepatic diseases, diabetes and peptic ulcers [Roig, 2012]. The diuretic effect of the aqueous

extract has been evaluated in rodents with similar results to furosemide, a well-established diuretic drug

[Felipe et al., 2011]. In in vitro studies, the dichloromethane extract of the bark of C. bahamensis

showed cytotoxic effects against SK-Mel-28 (human melanoma), MDA-MB-231 (human mammary

adenocarcinoma) and 5637 (human bladder carcinoma) cells [Setzer et al., 2015] and the acetone extract

showed poor antimicrobial activity against E. coli, S. aureus and C. albicans [Abreu et al., 2017]. In

addition, the chloroform, ethyl acetate and methanol fractions of the stem showed antioxidant activity in

the FRAP and DPPH assays [Felipe et al., 2019]. The non-polar fraction of a methanolic extract has

been analyzed by gas chromatography - mass spectrometry (GC-MS), and a series of fatty acids,

terpenoids and phytosterols has been identified [Felipe et al., 2017], but apart from this, the scientific

information about this species is limited.

In the present study, the morphological characteristics and the quality parameters of the stem of C.

bahamensis and its hydroalcoholic and aqueous extracts are reported for the first time. Also, the


32

preliminary chemical composition of both extracts was determined and flavonoids and phenol were

quantified, as the first step for the development of a future herbal formulation.

2.2. Material and Methods

2.2.1. Plant material

Stems of C. bahamensis were collected in March 2017 at Cañada Arroyón, Artemisa, Cuba. It was

identified in the National Botanical Garden of Cuba, where a voucher specimen (No. 85369) was

deposited. The materiel was dried in an oven at 40 °C during seven days and milled until the size of the

particles was less than 2 mm.

2.2.2. Morphological study

The macro-morphological characteristics were determined by visual inspection. To identify the micro-

morphological characteristics, the milled drug was decolorized with 10% sodium hypochlorite and

colorized with 1% safranin in water. Observations were done with an optical microscope Novel with a

coupled camera.

2.2.3. Physic-chemical parameters of the drug

Residual humidity, total ashes, soluble ashes in water, non-soluble ashes in 10% hydrochloric acid and

extractive substances in water, 30% ethanol, 50% ethanol and 80% ethanol were determined. The

residual humidity was determined by the azeotropic method. All assays were done according to well-

established official methods and procedures [WHO, 1992].

2.2.4. Preparation of the extracts

One kg of dried and powdered stem material was macerated with 15 L of distillated water for seven days

at room temperature in the dark, filtered and in this way the aqueous extract was obtained. The ethanol

80% extract was obtained using the same procedure.


33

2.2.5. Physic-chemical parameters of the extracts

Organoleptic properties, total solids, refraction index and relative density were determined according to

the methods described by Miranda & Cuéllar (2000).

2.2.6. Phytochemical characterization of the extracts

Phytochemical characterization of the extracts was done through phytochemical screening and their Thin

Layer Chromatographic (TLC) and Ultraviolet (UV) profiles.

The phytochemical screening was carried out through the assays of Dragendorff [alkaloids], Wagner

[alkaloids], Baljet [lactones and coumarins], Liebermann-Burchard [triterpenes and steroids], Fehling

[reducing sugars], foaming [saponins], ferric trichloride [polyphenols], ninhydrin [amino acids],

Bornträger [anthraquinones], and Shinoda [flavonoids]. Color changes of the extracts by applying the

mentioned reagents were observed [Miranda & Cuéllar, 2000].

The TLC profile was established on silica gel F254 plates (Merck) using chloroform / methanol (9:2) as

mobile phase. The plate was observed at visible light, under UV light at 254 and 366 nm; and after

spraying with vanillin 2% in sulphuric acid followed by applying heat.

Ultraviolet spectra of the extracts were recorded from 200 nm to 400 nm on a X Lambda 35 UV-Vis

spectrophotometer.

2.2.7. Total phenols and flavonoids content

The total amount of phenolic compounds was determined by the Folin–Ciocalteu reagent [Hsu et al.,

2017]. A total of 200 µL of extract was dissolved with 10 mL of Folin–Ciocalteu solution and 8 mL of

saturated sodium carbonate solution. After 90 min, the absorbance was recorded at 765 nm with an UV–

Vis spectrophotometer. Gallic acid was used as standard.

The total amount of flavonoids was determined using the aluminum trichloride (AlCl3) reagent [Hsu et

al., 2017]. A volume of 1.5 mL (1 mg/mL) of extract was added to an equal volume of a 2% AlCl3


34

solution. The mixture was vigorously shaken, and the absorbance was recorded at 367 nm after 10 min

of incubation with an UV–Vis spectrophotometer. Quercetin was used as standard.

2.2.8. Statistical analysis

For the physicochemical parameters, the mean and standard deviation were determined. The Student t-

test was used to compare the content of phenols and flavonoids of the extracts.

2.3. Results

2.3.1. Morphological analysis of the drug

The stem of C. bahamensis has a compact and cylindrical form. The internal surface is stringy and

orange, and the external surface is wrinkled and gray (Figure 3).

Figure 3. Macroscopic characteristics of the stems of Caesalpinia bahamensis

A: Internal surface; B: External surface

By microscopic inspection, it is possible to observe acute and elongated fibers composed of

sclerenchyma cells and highly lignified secondary walls (Figure 4A), surrounding to xylemic vessels

(Figure 4B-E), typical of fibrous and compact stems. Filiform sclerids and suber were observed as well

(Figure 4F-G). In histochemical analysis, the presence of flavonoids was observed by the apparition of a

yellow color after tinction with potassium hydroxide 10% in ethanol (Figure 5A). Also, the presence of

phenols was evidenced by the apparition of a reddish-brown color after tinction with trichloride ferric 10

% in water (Figure 5B) [Cuéllar et al., 2000].


35

Figure 4. Microscopic characteristic of the stem of Caesalpinia bahamensis

F: Fibers; VX: Xylemic vessels; E: Sclerids; EF: Filiform sclerids; S: Suber

Figure 5. Histochemical analysis on the stem of Caesalpinia bahamensis

A: Flavonoids; B: Phenols

2.3.2. Physico-chemical parameters of the drug

Some quality parameters of the plant material are listed in Table 3. Total ashes constitute a base to

determine the purity and identity of drugs. The highest value established by WHO for this parameter is

5% [WHO, 1992]. On the other hand, insoluble ashes in hydrochloric acid are indicative of the presence

of heavy metals in the drug [Woisky & Salatino]. The value established in the Chinese Pharmacopeia for

this parameter is below 1.5% [Commission CP, 2015]. Our results suggest that the plant material does

not contain heavy metals, or only a little quantity. The residual humidity below 10% does not permit the

growth of micro-organisms and prevents degradation of the drug [Miranda & Cuéllar, 2000]. Finally, the


36

results for the soluble constituents suggest that the highest extraction rate of secondary metabolites from

this drug is in ethanol 50% and 80%; for this reason, ethanol 80% was used as extraction solvent and

compared with the aqueous extract in relation to its traditional use.

Table 3. Physico-chemical parameters of the stem of Caesalpinia bahamensis

Parameter Values (%)

Total ashes 1.33 ± 0.81

Water-soluble ashes 0.04 ± 0.00

Acid-insoluble ashes [in HCl 10%] 0.01 ± 0.00

Moisture content 8.01 ± 1.04

Water soluble constituents 4.60 ± 0.33

Ethanol (30%) soluble constituents 6.16 ± 1.32



* The values are expressed as meean ± standard deviation

2.3.3. Physico-chemical parameters of the extracts

The parameters pH, total solids, relative density were higher in the aqueous extract, while the parameter

refractive index was higher in the hydroalcoholic extract. Both extracts showed a slightly acidic pH,

indicative of the presence of phenolic acids in the samples (Table 4).

Table 4. Physic-chemical parameters of the aqueous and hydroalcoholic extract

Parameters Aqueous

extract

Hydroalcoholic

extract

pH 5.37 ± 0.01 a 5.27 ± 0.01 b

Total solids [%] 0.63 ± 0.13 c 0.35 ± 0.002 d

Refraction index 1.3283 ± 0.0004 e 1.3567 ± 0.0001 f

Relativity density [g/mL] 1.0052 ± 0.0035 g 0.8724 ± 0.0001 h

* The values are expressed as medium ± standard deviation. Different letters indicate significant

differences (p<0.05) among extracts


37

2.3.4. Phytochemical characterization of the extracts

The presence of lactones, coumarins, triterpenes, steroids, reducing sugars, phenolic compounds,

quinones, saponins and flavonoids was evidenced for both extracts by phytochemical screening. The

results of the Dragendorff, Mayer and Wagner assays were negative, indicating the absence of alkaloids

in the extracts.

In the TLC profile, three spots were observed in visible light: two of them near to the solvent front and

the other near to the application point. They showed fluorescence under UV light at 254 nm, indicative

of chromophores. Also, fluorescence at 366 nm was observed, indicating the presence of conjugated

groups. When the spots were revealed with vanillin, they developed pink and brown colors. In general,

this comportment is associated with phenolic compounds and flavonoids. The presence of flavonoids

was evidenced through the UV profile by the presence of two bands at 282 and 445 nm in the extracts,

associated with these metabolites (Figure 6).

Figure 6. Ultraviolet (A) and TLC (B) profiles of the extracts of the stems of Caesalpinia

bahamensis

Aq: Aqueous extract; H: Hydroalcoholic extract


38

2.3.5. Total phenols and flavonoids content

Phenols and flavonoids were quantified (Table 5). The hydroalcoholic extract contained a higher

quantity of flavonoids than the aqueous extract. In contrast, the content of total phenols compounds was

highest for the aqueous extract.

Table 5. Content of phenols and flavonoids of the extracts

Parameters Aqueous

extract

Hydroalcoholic

extract

Content of phenols [mg/mL] 148.43 ± 8.01 a 7.07 ± 0.12 b

Content of flavonoids [mg/mL] 0.24 ± 0.03 c 0.570 ± 0.007 d

*The values are expressed as medium ± standard deviation. Different letters are indicative of significant

differences (p<0.05)

2.4. Discussion

Pharmacognosy is the science that studies the physical, chemical, biochemical and biological properties

of drugs, drug substances, or potential drugs of natural origin, and it also includes the search for new

drugs from natural sources [Biswas, 2015].

The establishment of the quality parameters of drugs guarantees the safety and efficacy of the finished

herbal medicine [Govindaraghavan & Sucherb, 2015], and avoids the adulteration or substitution of the

plants used as raw materials [Chanda, 2014].

Caesalpinia bahamensis Lam. (Leguminosae) is a medicinal plant used by the Cuban population to treat

renal and hepatic diseases, diabetes and peptic ulcers [Roig, 2012]. In earlier studies, this species

showed diuretic, antimicrobial, antioxidant and cytotoxic activity [Felipe et al., 2011; Setzer et al., 2015;

Abreu et al., 2017; Felipe et al., 2019]. Despite the fact that studies carried out on this species are

scarce, the preliminary results are encouraging, converting it into a potential source for the development

of herbal medicines, which requires knowledge of its quality parameters and phytochemical

composition.


39

The quality parameters of the stem of C. bahamensis and its aqueous and hydroalcoholic extracts were

evaluated for the first time in this study. The macromorphological and micromorphological

characteristic of the stem permitted to observe the presence of fibers and scarified or woody xylem

vessels, which corresponds to the woody and compact structure of the drug. The values of the evaluated

physico-chemical parameters were within the established general quality limits for medicinal plants.

In this study, the aqueous and hydroalcoholic extract of the stem of C. bahamensis were obtained. The

aqueous extract was chosen in correspondence with the traditional use of the plant while the

hydroalcoholic extract was selected because it had the highest content of extractables. The quality

parameters of both extracts were determined and compared.

The highest percentage of soluble constituents was observed in the 50% and 80% ethanol extracts.

However, the level of total solids in the aqueous extract was higher than in the hydroalcoholic extract. In

this study, a predominance of polar compounds in the extracts was observed through its TLC profiles.

This comportment can explain the higher quantity of total solids obtained from the aqueous extract. On

the other hand, the determination of soluble constituents requires a stir system increasing the yield of

extraction, which can explain an increase in the content of soluble constituents in hydroalcoholic

mixtures. The influence of these two factors can explain these differences [Sultana et al., 2019;

Nantitanon et al., 2010].

The presence of flavonoids and phenols in the extracts was tested by the Shinoda and aluminum

trichloride assays. Also, the TLC profile showed spots related to flavonoids and phenolic compounds.

The UV profile showed bands at 282 and 445 nm, corroborating the presence of flavonoids in the

extracts [Tsimogiannis et al., 2007; Anouar et al., 2012]. This suggests that flavonoids and phenols can

be used as markers in the quality parameters of the extracts.


40

Based on these results the amount of phenols and flavonoids in the extracts was determined. The

quantity of flavonoids was higher in the hydroalcoholic extract, but this was not the case for the phenols.

The hydroalcoholic mixtures have been used to obtain flavonoid-rich fractions. On the other hand, a

higher content of phenols in the aqueous extract suggest the presence of glycosylated phenols, which are

more soluble in water than non-glycosylated phenols [Vázquez et al., 2008; Do et al., 2014].

2.5. Conclusion

The quality parameters of the stem of Caesalpinia bahamensis and its aqueous and hydroalcoholic

extracts were evaluated, being within the general limits established for medicinal plants. The content of

flavonoids was higher in the hydroalcoholic extract while in the aqueous the phenolic compounds

predominated. The results presented in this study have not been previously reported for this species.

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dependent density functional theory study. Food Chemistry 2012; 131: 79-89.

Biswas D. Role of reverse pharmacognosy in finding lead molecules from nature. Indian Journal of

Research in Pharmacy and Biotechnology 2015; 3(4): 320-323.

Chanda S. Importance of pharmacognostic study of medicinal plants: An overview. Journal of


Commission CP. Pharmacopoeia of the People's Republic of China. (Chinese Medical Science and

Technology Press: Peking), 2015. Pags. 337-345.


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Do KD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, Ju YH. Effect of

extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of

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Felipe A, Marrero D, Scull R, Cuéllar A, Gutiérrez YI. Composición química de una fracción apolar del

extracto metanólico de la madera de Caesalpinia bahamensis Lam (brasilete). Revista Cubana de

Ciencias Farmacéuticas y Alimentarias 2017; 3(2): 1-8


antioxidant capacity of three fractions from the stem of Caesalpinia bahamensis Lam. Journal of

Pharmacy & Pharmacognosy Research 2019; 7(1): 12-20.

Govindaraghavan S & Sucherb NJ. Quality assessment of medicinal herbs and their extracts: Criteria

and prerequisites for consistent safety and efficacy of herbal medicines. Epilepsy & Behavior 2015;

52: 363-371.

Hsu BY, Lin SW, Stephen B, Chen BH. Simultaneous determination of phenolic acids and flavonoids in

Chenopodium formosanum Koidz. (djulis) by HPLC- DAD-ESI–MS/MS. Journal of Pharmaceutical

and Biomedical Analysis 2017; 132: 109-116.




42

Miranda MM, Cuéllar AC. Manual de prácticas de laboratorio: Farmacognosia y productos naturales.

Félix Varela: La Habana, 2000.

Nantitanon W, Yotsawimonwat S, Okonogi S. Factors influencing antioxidant activities and total

phenolic content of guava leaf extract. LWT - Food Science and Technology 2010; 43: 1095-1103.


medicinales en terapéutica. Acta Farmacéutica Portuguesa 2015; 4(1): 42- 52.


Cuba, 2012. Págs.: 228-229.

Setzer MC, Schmidt J, Moriarity DM, Setzer WM. A phytopharmaceutical survey of Abaco Island,

Bahamas. American Journal of Essential Oils and Natural Products 2015; 3(1): 10-17.

Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of

selected medicinal plant extracts. Molecules 2009; 14: 2167-2180.

Tsimogiannis D, Samiotaki M, Panayotou G, Oreopoulou V. Characterization of flavonoid subgroups

and hydroxy substitution by HPLC-MS/MS. Molecules 2007; 12: 593- 606.

Vázquez G, Fontenla E, Santos J, Freire MS, González- Álvarez J. Antorrena G. Antioxidant activity

and phenolic content of chestnut (Castanea sativa) shell and eucalyptus (Eucalyptus globulus) bark

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CHAPTER 3:

Fatty acids, terpenoids

and phytosterols


Felipe A, Marrero D, Scull R, Cuéllar A, Gutiérrez YI.

Composición química de una fracción apolar del

extracto etanólico de la madera de Caesalpinia

bahamensis Lam (brasilete). Revista de Ciencias

Farmacéuticas y Alimentarias 2017; 3(2):1-8.

FATTY ACIDS, TERPENOIDS AND PHYTOSTEROLS

43

3.1. Introduction

Recently the use and commercialization of medicinal herbs has increased in developed and developing

countries. The reasons for this increase are due to the preference of consumers for natural therapies;

concerns regarding undesirable side effects of modern medicines and the belief that herbal drugs are free

from side effects [Calixto, 2000].

The great variety of medicinal plants in the Cuban flora, together with the wide ethnobotanical

knowledge, increases the opportunities for the search of new therapies in the treatment of various

diseases, of which the renal affections have been one of the most treated in ethnobotany. However, the

phytotherapeutic potential of the island is still virgin [Felipe et al., 2020].

Caesalpinia bahamensis Lam. is a medicinal plant traditionally used in Cuba to treat renal and hepatic

diseases, diabetes and peptic ulcers [Roig, 2012]. In pharmacological studies the diuretic, cytotoxicity,

antimicrobial and antioxidant properties have been demonstrated [Felipe et al., 2011; Setzer et al., 2015;

Abreu et al., 2017; Felipe et al., 2019]. In contrast, there are no scientific reports about its chemical

composition.

The present research aims to identify the non-polar components of the hydroalcoholic extract of the

stems of Caesalpinia bahamensis by Gas Chromatography / Mass Spectrometry (GC/MS)


3.2.1. Plant material

Stems of C. bahamensis were collected in March 2017 at Cañada Arroyón, Artemisa, Cuba. It was

identified in the National Botanical Garden of Cuba, where a voucher specimen (No. 85369) was

deposited. The materiel was dried in an oven at 40 0C during seven days and milled until the size of the

particles was less than 2 mm.


44

3.2.2. Preparation and fractionation of the extract

One kilogram of the stems of Caesalpinia bahamensis was extracted with five liters of ethanol 80% by

maceration for seven days at room temperature and in absence of light. Two-hundred milliliters of the

extract was concentrated under reduced pressure and extracted with petroleum ether by liquid/liquid

extraction. The petroleum ether fraction was concentrated until the apparition of a yellow oil.

3.3.3. Gas Chromatography / Mass Spectrometry (GC/MS)

The petroleum ether fraction was analyzed by GC/MS on a Trace 2000 instrument (TRACE 2000 GC,

Interscience Thermo Quest, Belgium). Prior to analysis, 300 μL of the test sample was derivatized by

adding 100 μL of N,O-bis(trimethylsilyl)-trifluoroacetamide with 1% of trimethylsilyl chloride (BSTFA

+ 1% TMCS) reagent and 100 μL of chloroform. The mix was stirred and maintained at 30 °C for 30

min. An HP-5Ms (30 m, 0.25 mm ID x 1.0 μm) column (Hichrom Limites, UK) was used. The inlet and

detector temperatures were 280 °C and 250 °C, respectively. Helium gas was used as the mobile phase.

The identification of compounds was done using the NIST 2000 data base.

2.3. Results and Discussion

Gas Chromatography / Mass Spectrometry (GC/MS) is a potent method for the analysis of natural

products because it is a simple, fast and economic. In addition, mass spectrometry also provides

structural information, but GC/MS can only be used for volatile compounds, being the main

disadvantage of the method [Kitson et al., 2002].

In this research, seventy-four compounds were identified in the petroleum ether fraction of the

hydroalcoholic extract of the stems of C. bahamensis (Table 6). Compounds were grouped as

butanediol, fatty acids, high molecular weight (HMW) alcohols, hydrocarbons, phytosterols, terpenoids

and others. Terpenoids (26.64 %), fatty acids (26.45 %) and phytosterols (14.61 %) were found to be the

major metabolites (Figure 7).


45

Table 6. Compounds identified in the non-polar fraction of the hydroalcoholic extract

RT (min) Compounds RA (%)

7.08 2,3-butanediol 5.82

7.20 2,3-butanediol (isomer) 7.95

9.07 benzoic acid 0.13

9.30 glycerol 0.14

9.43 tridecane 0.13

10.10 2-tetradecene 0.34

10.15 tetradecane 0.10

10.79 γ- murolene 0.47

10.93 α- murolene 1.21

11.04 γ -cadinene 0.56

11.08 δ-cadinene 1.32

11.23 α-calacorene 0.33

11.39 hexadecene 0.64

11.43 hexadecane 0.06

11.80 cubenol 0.50

11.91 δ-cadinol (α-murolol) 4.94

12.02 α-cadinol 5.62

12.17 α-bisabolol 8.02

12.75 1-octadecene 0.87

13.15 tetradecanoic acid 0.22

13.58 methyl ester of hexadecenoic acid 0.13

13.77 methyl ester of palmitic acid 1.39

13.93 pentadecanoic acid 0.25

14.57 hexadecenoic acid 0.44

14.72 7,11,15-trimethyl-3-methylen-hexadeca-1,6,11,14-tetraen 3.62

14.80 palmitic acid 4.49

15.22 methyl ester of linoleic acid 0.84

15.26 methyl ester of oleic acid 0.70

15.40 heptadecenoic acid 0.09

15.46 methyl ester of stearic acid 0.57

15.62 heptadecanoic acid 0.27

15.71 1-octadecanol 0.09

15.79 ethyl ester of linoleic acid 0.05

15.83 ethyl ester of oleic acid 0.08

16.05 1-docosen 0.15

16.29 linoleic acid 4.14

16.34 oleic acid 3.35

*RT: Retention Time; RA: Relative abundance


46

Table 6. Compounds identified in the non-polar fraction of the hydroalcoholic extract (Cont.)

RT (min) Compounds RA (%)

16.54 stearic acid 2.34

17.26 methyl ester of eicosanoic acid 0.08

17.40 nonadecanoic acid (C19:0) 0.06

17.49 1-eicosanol 0.04

18.08 eicosenoic acid 0.05

18.30 eicosanoic acid (C20:0) 0.31

19.19 heneicosanoic acid 0.07

19.33 bis(2-ethylhexyl) phtalate 0.55

19.72 monopalmitin 0.11

19.98 methyl ester of tricosanoic acid 0.02

20.08 docosanoic acid 0.30

20.57 heptacosane 0.03

20.86 methyl ester of tetracosanoic acid 0.08

20.94 triosanoic acid 0.26

21.00 1-tetracosanol 0.05

21.41 monoestearin 0.13

21.76 squalene 1.11

21.80 tetracosanoic acid 1.00

22.55 methyl ester of hexacosanoic acid 0.12

22.60 pentacosanoic acid 0.12

22.67 1-hexacosanol 0.56

23.42 hexacosanoic acid 1.23

23.46 1-heptacosanol 0.33

24.10 stigmastan-3,5-dien 0.19

24.17 methyl ester of octacosanoic acid (C28:0 Me) 0.44

24.28 1-octacosanol 2.89

24.54 cholesterol 0.68

25.01 octacosanoic acid 2.72

25.39 campesterol 2.02

25.64 stigmasterol 3.81

25.73 1-triacontanol 0.41

26.10 β-sitosterol 7.20

26.46 9,19-cyclolanost-24-en-3-ol 0.90

26.53 9,19-cyclolanost-24-en-3-ol (isomer) 1.34

26.91 stigmast-4-en-3-ona 0.71

27.31 lup-20[29]-en-3-ol-acetate 0.32

27.58 1-tetratiacontanol 1.07

*RT: Retention Time; RA: Relative abundance


47

Figure 7. Distribution of the compounds identified in the ether petroleum fraction of the

hydroalcoholic extract of the stems of Caesalpinia bahamensis

Fatty acids were represented by thirty-one structures. Of them, oleic (3.35%), linoleic (4.14%), palmitic

(4.49%) and stearic (2.34%) acids were most prominent. Anti-inflammatory and oxidant activities, as

well as the capacity to reduce Prostatic Benign Hyperplasia (PBH) have been reported for these

compounds [Pérez et al., 2011; Molina et al., 2011].

Another representative group of the sample were the phytosterols, mainly including β-sitosterol (7.20%),

stigmasterol (3.81%) and campesterol (2.02%). β-Sitosterol and stigmasterol have been previously

reported for the genus Caesalpinia [Jagdale et al., 2019]; however, the presence of stigmasterol in the

genus has not been reported yet. β-Sitosterol shows a wide spectrum of biological properties; among

them, treatment of diabetes mellitus type II [Pandey et al., 2013], inhibition of cancer cells growth in

vitro, increasing of the urinary flow in patients with PBH and anti-inflammatory, lipid-lowering and

antiulcer activities [Jagdale et al., 2019].

In this research, sesquiterpenoids were the major compounds, more specifically α-bisabolol (8.02%), α-

cadinol (5.62%) and δ-cadinol (4.94%). However, only the presence of spatulenol in C. pulcherrima has

been previously reported [Matiz et al., 2011].


48

The therapeutic activity of plant extracts is usually related to synergistic and simultaneous action of

several phytochemicals. Then, searching for new drug candidates from natural products is often

hampered by the complexity of the mixtures [Thomford et al., 2018]. In this sense, the present research

constitutes the first chemical study of C. bahamensis. Also, campesterol was reported for first time in

the genus Caesalpinia.

2.4. Conclusion

Seventy-four compounds were identified in the stems of Caesalpinia bahamensis for first time,

including α-bisabolol (8.02%), 2,3-butanediol (7.95%), β-sitosterol (7.20%), α-cadinol (5.62%) and δ-

cadinol (4.94%), palmitic acid (4.49%), linoleic acid (4.14%) and campesterol (2.02%) as the major

compounds.

References




(phytotherapeutic agents). Brazilian Journal of Medical and Biological Research 2000; 33: 179-189.

Contreras JL, Amor D, Garcia A, Perez MC, Bratoeff EA, Labastida C. A chromatographic study of

flavonoids and fatty acids of four Caesalpinia species. PHYTON 1995; 57(1): 31-35.



2011; 3(2): 300- 308.





49

Felipe A, Pieters L, Delgado R. Effectiveness of Herbal Medicine in Renal Lithiasis: A Review. Siriraj

Med J. 2020; 72: 188-194.

Jagdale RA, Somkuwar AP, Bhoye SK, Sarode KG, Limsay RP. In vivo anti-inflamatory activity and

GC-MS analysis of hydroethanolic extract of Caesalpinia bonducella seeds. Journal of


Kitson FG, Larsen BS, McEwen CN. Gas Chromatography and Mass Spectrometry. Academic Press,

2002. Pag. 3-23.

Matiz GE, Franco LA, Rincón J. Actividad antiinflmatoria de flores y hojas de Caesalpinia pulcherrima

L. (Swarts). Salud UIS 2011; 43(3): 281-287.

Molina V, Ravelo Y, Mas R, Carbajal D, Arruzazabala ML. Anti- inflammatory and gastric effects of D-

002, aspirin and naproxene and their combined therapy in rats with cotton pellet-induced granuloma.

Lat J Pharm 2011; 30(9): 1709-1713.

Pandey AK, Gupta PP, Lal VK. Preclinical evaluation of hypoglycemic activity of Ipomoea digitata

tuber in streptozotocin-induced diabetic rats. J. Basic. Clin. Physiol. Pharmacol. 2013; 24(1): 35-39.

Pérez Y, Molina V, Oyarzábal A, Mas R. Tratamiento farmacológico en la Hiperplasia Prostática

Benigna. Revista Cubana de Farmacia 2011; 45(1): 109-126.


Cuba, 2012. Págs.: 228-229.



Thomford NE, Senthebane DA, Rowe A, Munro D, Seele P, Maroyi A et al. Natural Products for Drug

Discovery in the 21st Century: Innovations for Novel Drug Discovery. Int. J. Mol. Sci. 2018; 19:

1578-1600.

CHAPTER 4:

Homoisoflavonoids and

antilithiatic activity

This chapter was submitted as:

Felipe A, Gutiérrez YI, Scull R, Foubert K, Pieters L,

Delgado R. A new sappanin isolated from the

hydroalcoholic extract of the stem of Caesalpinia

bahamensis Lam (brasilete). [Submitted to Brazilian

Journal of Pharmacognosy].

Felipe A, Núñez CR, Gutiérrez YI, Scull R, Zumata MC,

Iglesias E, Pazo N, Foubert K, Pieters L, Delgado R.

Phytochemical characterization and antilithiatic

activity of Caesalpinia bahamensis Lam. (brasilete).

[Submitted to Journal of Pharmacy and

Pharmacognosy].

HOMOISOFLAVONOIDS AND ANTILITHIATIC ACTIVITY

50

4.1. Introduction

Renal lithiasis can be defined as the deposition of stones in the urinary tract due to an alteration of the

normal crystallization conditions in urine (Grasses et al., 2006). Renal lithiasis is a health problem of

high prevalence and recurrence rates around the world (Cano et al., 2015). Currently, it is the third most

frequent urological disease after urinary tract infections and prostate problems (Cook et al., 2016). It has

a prevalence that ranges between 4-15% of the world population (Mittal et al., 2016) and it has a high

recurrence rate of 40% after 5 years and 60% after 10 years (Cano et al., 2015). For the treatment of

renal stones minimally invasive surgery is used; it is effective to break the calculi, but it does not reduce

recurrence rates. On the other hand, many drugs have been used, such as thiazide diuretics, potassium

citrate and non-steroidal anti-inflammatory drugs (NSAIDs); but they are only used for preventing or

treating the symptoms (Alelign & Petros, 2018). For these reasons, many studies have been focused on

understanding the mechanism involved in renal lithiasis, and the development of a herbal medicine as a

new drug for the treatment and prevention of this pathology and its recurrences is a promising approach.

In Cuba, several plant species have been recognized in ethnobotanical studies for the treatment of renal

problems. However, their phytotherapeutic potential is still virgin. In ethnobotanical studies made in

Cuba, 179 species have been used in relation to the renal system, of which only 9% have been evaluated

pharmacologically, including Caesalpinia bahamensis Lam. (Leguminosae), a medicinal plant widely

distributed in Cuba and the Caribean, and known as “brasilete” (Felipe et al., 2020). The aqueous

maceration of this plant is used by the Cuban population to treat renal and hepatic diseases, diabetes, and

peptic ulcers (Roig, 2012). However, experimental studies to support the traditional knowledge are

lacking (Felipe et al., 2020). In pharmacological studies, the diuretic effect of an aqueous extract of

stems has been evaluated in rodents with similar results as furosemide, a well-established diuretic drug

(Felipe et al., 2011). The dichloromethane extract of the bark of C. bahamensis showed in vitro


51

cytotoxic effects against SK-Mel-28 (human melanoma), MDA-MB-231 (human mammary

adenocarcinoma) and 5637 (human bladder carcinoma) cells (Setzer et al., 2015) and the acetone extract

showed weak antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida

albicans (Abreu et al., 2017). In addition, the chloroform, ethyl acetate and methanol fractions of the

stems showed antioxidant activity in the FRAP and DPPH assays (Felipe et al., 2019a). Chemically, 74

compounds have been identified in the non-polar fraction of a methanolic extract of the species, using

gas chromatography - mass spectrometry (GC-MS). In this study, fatty acids, terpenoids, and

phytosterols were reported as the major compounds of this fraction (Felipe et al., 2017). A comparative

pharmacognostic study of the aqueous and hydroalcoholic extracts demonstrated the presence of

flavonoids and phenolic compounds as the major metabolites. In addition, the chemical composition of

both extracts was similar according to HPLC analysis. However, the total yield and quantity of the

flavonoids was higher for the hydroalcoholic extract (Felipe et al., 2019b). Apart from this, the scientific

information about this species is limited. This study reports the isolation and identification of the main

constituents of stems of Caesalpinia bahamensis, and the evaluation of the in vivo antilithiatic activity in

a rat model of an aqueous and hydroalcoholic extract of stems of Caesalpinia bahamensis, according to

its traditional use in Cuba.

4.2. Materials and methods

4.2.1. Plant material and preparation of the extracts

Stems of Caesalpinia bahamensis Lam. (Leguminosae) were collected in March 2017 at Cañada

Arroyón, Artemisa, Cuba (22°46'45.7"N 83°04'18.6"W). The material was identified in the National

Botanical Garden of Cuba, where a voucher specimen (No. 85369) was deposited. The material was

dried in an oven (AI-SET-DNE 600, Shanghai, China) at 40 °C for seven days and milled (Manesti,

Italy) until the size of the particles was less than 2 mm. Aqueous and hydroalcoholic extracts were


52

obtained by maceration at room temperature in the dark, during 24 h. Five milliliters of solvent was used

for each gram of dry plant material. After that, the extracts were dried under reduced pressure in a rotary

evaporator (IKA).

4.2.2. Isolation of homoisoflavonoids

Thirty grams of the dried hydroalcoholic extract were suspended in acid water (pH<3), and liquid-liquid

partition was performed with dichloromethane, yielding the following fractions: dichloromethane (5.79

g), interphase (3.90 g) and acid water (17.92 g). The dichloromethane fraction was dried, dissolved in

methanol 90%, and fractionated with petroleum ether by liquid/liquid partition. The subsequent

methanol fraction was separated by flash chromatographic (Reveleris1 iES, Grace), consisting of a

binary pump with four solvent selections, an ultraviolet (UV) and evaporating light scattering detector

(ELSD) and a fraction collector, using the Reveleris NavigatorTM software. A packed Flash Grace

Reveleris silica cartridge (40 g, 40µm) was used. A gradient of increasing polarity of dichloromethane,

ethyl acetate and methanol was applied, yielding the semi-pure fractions F1a-02/02 (25.4 mg) and F1a-

16/01 (177.3 mg). This fraction was further purified by semipreparative liquid chromatography with a

sample manager, injector and collector (2767), a quaternary gradient module (2545), a System Fluidics

Organizer, an HPLC pump (515), a photodiode array detector (2998), and a Micromass Quattro mass

spectrometer with triple quad detection (TQD) (Waters, Milford, MA, USA), running MassLynx

software version 4.1. A RP-18 column was used for the separation. Formic acid 0.1% in ultrapure water

and formic acid 0.1% in acetonitrile were used as mobile phase. In this way compounds 1 (3.6 mg) and 2

(13.8 mg) were obtained.

The acid water fraction was submitted to 200 g of MCI gel column chromatography using a gradient

from 5% to 100% of methanol in ultrapure water, yielding a semi-pure fraction (3.97 g). This fraction

was further separated by flash chromatography on a column packed with silica gel 40 g using a gradient


53

of increasing polarity of dichloromethane, ethyl acetate and methanol to obtain compound 3 (466.8 mg)

and compound 4 (7.3 mg).

NMR spectra of the isolated constituents were recorded on a DRX-400 (Bruker, Germany) instrument,

operating at 400 MHz for 1H and 100 MHz for 13C, and 1D and 2D NMR spectra were recorded using

standard Bruker experiments.

Structures were confirmed by high resolution mass spectrometric analysis using a Xevo G2_XS QTOF

(Quadrupole – Time of Flight) instrument (Waters, Milford, MA, USA) equipped with MassLynx

software version 4.1. Data was recorded in negative ESI (Electro Spray Ionisaton) mode from m/z 50 to

1500 and then analysed in sensitivity mode (approximate resolution: 22,000 FWHM). Leucine

Enkephalin was used as lock mass.

Metasappanin [3-(2-hydroxy-4-methoxybenzyl)chromane-4,7-diol] (1): 1H-NMR (400 MHz,

MeOD) δ 7.14 (1H, d, J = 8.3 Hz, H-5), 6.93 (1H, d, J = 8.4 Hz, H-2’), 6.43 (1H, dd, J = 8.4, 2.6 Hz, H-

3’), 6.38 (1H, dd, J = 8.3, 2.4 Hz, H-6), 6.32 (1H, d, J = 2.5 Hz, H-5’), 6.24 (1H, d, J = 2.4 Hz, H-8),

4.10 (1H, ddd, J = 10.7, 4.0, 1.1 Hz, H-2), 3.97 (1H, t, J = 10.9 Hz, H-4), 3.69 (3H, s, OMe-4’), 3.09

(1H, dd, J = 16.9, 7.1 Hz, H-9), 2.56 (1H, m, H-9), 2.49 (1H, m, H-3). 13C-NMR (100 MHz, MeOD) δ

160.66 (C-4’), 160.24 (C-7), 156.73 (C-6’), 154.63 (C-8a), 132.43 (C-2’), 131.03 (C-5), 114.41 (C-4a),

113.02 (C-1’), 109.31(C-6), 108.64 (C-3’), 103.59 (C-8), 102.55 (C-5’), 70.99 (C-4), 66.47 (C-2), 55.67

(OMe-4’), 31.39 (C-3), 25.51 (C-9). ESI-MS m/z 301.1073 [M-H]- (calculated m/z 301.1076 [M-H]-)

10-methyl-protosappanin B (2a): 1H-NMR (CD3OD, 400 MHz), δ: 2.59 (2H, s, H-8), 3.47 (1H, d, J =

11.3 Hz, H-13a), 3.55 (1H, d, J = 3.5 Hz, H-13b), 3.86 (3H, s, OCH3-4), 3.87 (1H, s, H-6a), 4.13 (1H, d,

J = 12.2 Hz, H-6b), 6.44 (1H, d, J = 2.3 Hz, H-4), 6.53 (1H, dd, J = 8.4, 2.3 Hz, H-2), 6.74 (1H, s, H-

12), 6.83 (1H, s, H-9), 6.98 (1H, d, J = 8.4 Hz, H-1); 13C-NMR (CD3OD, 100 MHz) δ: 40.2 (C-8), 56.5

(OCH3), 68.5 (C-13), 73.1 (C-7), 76.5 (C-6), 108.2 (C-4), 111.6 (C-2), 116.5 (C-9), 117.3 (C-12), 123.8


54

(C-1a), 127.5 (C-8a), 133.3 (C-1), 133.6 (C-12a), 146.2 (C-11), 147.6 (C-10), 159.2 (C-3), 159.4 (C-4a).

MS m/z 317.1017 [M-H]- (calculated: 317.1025).

Iso-10-methyl-protosappanin B (2b): 1H-NMR (CD3OD, 400 MHz), δ: 2.69 (1H, d, J = 13.4 Hz, H-

8a), 2.80 (1H, d, J = 13.4 Hz, H-8b), 3.37 (1H, d, J = 11.2 Hz, H-13a), 3.58 (1H, d, J = 4.1 Hz, H-13b),

3.86 (3H, s, OCH3), 3.87 (1H, s, H-6a), 4.34 (1H, d, J = 11.8 Hz, H-6b), 6.51 (1H, d, J = 2.8 Hz, H-4),

6.57 (1H, dd, J = 8.3, 2.2 Hz, H-2), 6.68 (1H, s, H-12), 6.89 (1H, s, H-9), 6.98 (1H, d, J = 8.4 Hz, H-1);

13C-NMR (CD3OD, 100 MHz) δ: 42.7 (C-8), 56.5 (OCH3), 65.8 (C-13), 73.4 (C-7), 77.2 (C-6), 108.9

(C-4), 112.2 (C-2), 115.7 (C-9), 117.7 (C-12), 125.1 (C-1a), 128.2 (C-8a), 132.7 (C-1), 133.6 (C-12a),

146.2 (C-11), 147.6 (C-10), 159.5 (C-3), 160.6 (C-4a). MS m/z 317.1017 [M-H]- (calculated: 317.1025).

Protosappanin B (3a): 1H-NMR (CD3OD, 400 MHz), δ: 2.67 (2H, s, H-8), 3.46 (1H, d, J = 11.3 Hz, H-

13a), 3.55 (1H, d, J = 4.1 Hz, H-13b), 3.85 (1H, d, J = 12.2 Hz, H-6a), 4.14 (1H, d, J = 12.2 Hz, H-6b),

6.43 (1H, d, J = 2.2 Hz, H-4), 6.54 (1H, dd, J = 9.0, 2.2 Hz, H-2), 6.72 (1H, s, H-12), 6.70 (1H, s, H-9),

6.97 (1H, d, J = 8.4 Hz, H-1); 13C-NMR (CD3OD, 100 MHz) δ: 40.0 (C-8), 68.3 (C-13), 73.0 (C-7), 76.7

(C-6), 108.2 (C-4), 111.5 (C-2), 120.0 (C-9), 117.5 (C-12), 124.1 (C-1a), 127.5 (C-8a), 133.2 (C-1),

132.5 (C-12a), 144.7 (C-11), 144.9 (C-10), 158.9 (C-3), 159.3 (C-4a). MS m/z 303.0857 [M-

H]- (calculated: 303.0869).

Isoprotosappanin B (3b): 1H-NMR (CD3OD, 400 MHz), δ: 2.49 (1H, d, J = 13.8 Hz, H-8a), 2.56 (1H,

d, J = 13.7 Hz, H-8b), 3.37 (1H, br, H-13a), 3.52 (1H, d, J = 4.5 Hz, H-13b), 3.85 (1H, d, J = 12.2 Hz,

H-6a), 4.38 (1H, d, J = 11.8 Hz, H-6b), 6.51 (1H, br, H-4), 6.56 (1H, dd, J = 8.3, 2.2 Hz, H-2), 6.66 (1H,

s, H-12), 6.73 (1H, s, H-9), 6.97 (1H, d, J = 8.4 Hz, H-1); 13C-NMR (CD3OD, 100 MHz) δ: 42.5 (C-8),

65.7 (C-13), 73.3 (C-7), 76.9 (C-6), 108.7 (C-4), 112.1 (C-2), 119.0 (C-9), 117.8 (C-12), 125.2 (C-1a),

128.1 (C-8a), 132.7 (C-1), 132.0 (C-12a), 144.7 (C-11), 144.9 (C-10), 159.0 (C-3), 160.4 (C-4a). MS

m/z 303.0857 [M-H]- (calculated: 303.0869).


55

Brazilin (4): 1H-NMR (CD3OD, 400 MHz), δ: 2.76 (1H, d, J = 15.7 Hz, H-7a), 3.01 (1H, d, J = 15.7 Hz,

H-7b), 3.67 (1H, d, J = 4.3 Hz, H-6a), 3.92 (1H, br, H-6b), 3.95 (1H, s, H-12), 6.28 (1H, d, J = 2.5 Hz,

H-4), 6.46 (1H, dd, J = 8.3, 2.5 Hz, H-2), 6.59 (1H, s, H-11), 6.69 (1H, s, H-8), 7.17 (1H, d, J = 8.4 Hz,

H-1); 13C-NMR (CD3OD, 100 MHz) δ: 42.5 (C-7), 51.0 (C-12), 70.8 (C-6), 78.1 (C-6a), 104.3 (C-4),

109.9 (C-2), 112.4 (C-8), 112.8 (C-11), 115.5 (C-1a), 131.3 (C-1), 132.2 (C-7a), 137.4 (C-11a), 145.3

(C-9), 145.6 (C-10), 155.7 (C-3), 157.8 (C-4a). measured mass m/z 285.0754 [M-H]-(calculated:

285.0763).

4.2.3. UPLC-UV-MS analysis

The UPLC (Ultraperformance Liquid Chromatography)-UV profile was established on an Acquity LC-

UV system equipped with a Xevo G2-XS QTOF spectrometer (Waters, Milford, MA, USA), running

MassLynx software version 4.1. For analysis, 10 µL sample at 0.1 mg/mL was injected on a BEH-

Shield-RP18 column (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA, USA) which was kept at 40 °C.

The mobile phase consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B),

and the gradient was set as follows (min/B%): 0/2, 1/2, 14/26, 24/65, 26/100, 29/100, 31/98, 36/98. The

flow rate was 0.4 mL/min. UV data was recorded at 280 nm. Mass spectrometric data was recorded in

ESI (-) mode from m/z 50 to 1500 and the analyzer was set in sensitivity mode (approximate resolution:

22,000 FWHM). The spray voltage was set at -0.8 kV; cone gas flow and desolvation gas flow at 50.0

L/h and 1000.0 L/h, respectively; and source temperature and desolvation temperature at 120 °C and 550

°C, respectively. Leucine Enkephalin was used as lock mass.

4.2.4. Antilithiatic activity

4.2.4.1. Animals

Wistar rats (200 – 250 g body weight) were obtained from the animalarium of CENPALAB (Havana,

Cuba) and kept in collective cages at 22 °C under a 12-h light/dark cycle (lights on at 07:00 h) with free


56

access to laboratory food and tap water. This investigation followed the international regulations for

animal care and was approved by Ethical Committee of the Institute of Basic and Preclinical Sciences

“Victoria de Girón”, Cuba (Agreement 25-2019; November 2019).

4.2.4.2. Ethylene glycol-induced urolithiasis

The antilithiatic activity of the hydroalcoholic and aqueous extracts was evaluated according to the

procedure described by Bashir & Gilani (2011) with slight modifications. Thirty-two female Wistar rats

were divided into four treatment groups of equal size as follows: healthy control group (1), lithiasis

group (2), aqueous extract of C. bahamensis group (3) and hydroalcoholic extract of C. bahamensis

group (4). A preliminary dose-response study using doses of 200, 100 and 10 mg/kg of each extract was

performed. All the main studies reported were carried out at the dose of 200 mg/kg, according to the

ethnomedical uses that reflect the use of the extracts in traditional herbal medicine. The healthy control

group received common water during the complete experiment. The lithiasis group and all test groups

received a solution of ethylene-glycol 0.75% and ammonium chloride 0.5% during 21 days. After that,

the lithiasis group was treated with common water and the test groups with the aqueous or

hydroalcoholic extract of the stems of C. bahamensis at a dosage of 200 mg/kg during seven days. At

the end of the experiment, the rats were transferred into metabolic cages, and urine was collected. Blood

was collected through cardiac puncture from animals under ether anesthesia to obtain the plasma.

4.2.4.3. Biochemical parameters

The urine volume was quantified during 6 h and the urinary flow was calculated. Concentrations of

calcium (urine and plasma) and oxalate (urine) were determined (Radiometer ABL 800 FLEX), as well

as plasmatic creatinine (HITACHI 2020).

Histopathological analysis


57

Animals were sacrificed at the end of the experiment, and the right kidney was excised, washed with

physiological saline solution and fixed in 10% neutral buffered formalin. It was processed and stained

with Hematoxylin and Eosin (H & E) and according to the Von Koss method, which selectively stains

calcium oxalate (CaOx) for microscopic examination (Conn, 1962). The slides were observed under a

microscope (Motic BA 310) and polarised light at 40x.


All statistical comparisons between the groups are made by means of One-Way Analysis of Variance

(ANOVA) with the post hoc Student-Newman-Keuls test. A P-value less than 0.05 was regarded as

significant.

4.3. Results

4.3.1. Phytochemical analysis

Four homoisoflavonoids were isolated and identified by 1D and 2D NMR spectroscopy and mass

spectrometry according to published data. This class of compounds has a complicated stereochemistry;

they exist as an inseparable mixture of 2 conformers, which explains the doubling of signals as observed

in the NMR spectra (Zhao et al., 2016).

Compound 1 was isolated for first time in the field of natural products (Figure 8A). In the 1H-NMR and

COSY-spectrum, two sets of ABX-type aromatic coupling patterns were observed, the first one at δ

7.14 (1H, d, J = 8.3 Hz), δ 6.38 (1H, dd, J = 8.3, 2.4 Hz) and δ 6.24 (1H, d, J =2.4 Hz), and the second

one at δ 6.93 (1H, d, J =8.4 Hz), δ 6.43 (1H, dd, J = 8.4, 2.6 Hz) and δ 6.32 (1H, d, J = 2.6 Hz). This

was confirmed by the presence of 12 aromatic signals in the 13C-NMR spectrum, corresponding to 6

quaternary signals and 6 CH signals, as evident from the DEPT-spectra. The CH-signals were correlated

with their respective H-signals in 1H-NMR through an HSQC experiment. The chemical shift of 4 of the

quaternary carbons, i.e. at δ 160.66, 160.24, 156.73 and 154.63 was indicative for O-substitution,


58

including one aromatic methoxy group (δH 3.69, δC 55.67), which showed an HMBC correlation with

the quaternary carbon at δC 160.66. The position of the methoxy group at C-4’ was evidenced by the

HMBC correlation between this carbon and the 1H-NMR signal at δH 6.43. Other signals in the aliphatic

part of the 1H- and 13C-NMR spectra included 2 CH-signals (δH 3.97, δC 70.97 and δH 2.49, δC 31.39)

and 2 CH2-signals (δH 4.10, δC 66.47 and δH 3.09, δC 25.51), which was in agreement with the

heterocyclic ring of a homoisoflavonoid-like structure. Detailed analysis of the 2D NMR spectra and the

correlations listed in Table 7 allowed to elucidate the structure of compound 1 as 3-(2-hydroxy-4-

methoxybenzyl)chromane-4,7-diol (Figure 8A).

Table 7. NMR data of metasappanin isolated from the stems of C. bahamensis.

C No δH (multiplicity, J, integration)

δC

(ppm) HMBC

2 4.10 (ddd, J = 10.7, 4.0, 1.1 Hz, 1H) 66.5 25.5, 31.4, 71.0, 154.6

3 2.49 (m, 1H) 31.4 25.5, 66.5, 113.0, 114.4, 131.0

4 3.97 (t, J = 10.9 Hz, 1H) 71.0 25.5, 31.4, 66.5, 71.0, 154.6, 160.2

4a 114.4 5 7.14 (d, J = 8.3 Hz, 1H) 131.0 71.0, 103.0, 131.0, 154.6, 160.2

6 6.38 (dd, J = 8.3, 2.4 Hz, 1H) 109.3 103.0, 114.4, 160.2

7 160.2 8 6.24 (d, J = 2.4 Hz, 1H) 103.0 109.3, 114.4, 160.2

8a 154.6 9 3.09 (dd, J = 16.9, 7.1 Hz, 1H), 2.56 (m, 1H) 25.5 31.4, 66.5, 71.0, 114.4, 132.4,156.7

1' 113.0 2' 6.93 (d, J = 8.4 Hz, 1H) 132.4 25.5, 102.6, 132.4, 156.7, 160.6

3' 6.43 (dd, J = 8.4, 2.6 Hz, 1H) 108.6 102.6, 113.0, 160.6

4' 160.6 5' 6.32 (d, J = 2.6 Hz, 1H) 102.6 108.6, 113.0, 156.7, 160.6

6' 156.7 4'-OCH3 3.69 (s, 3H) 55.6 160.6

Compound 2 was identified as a mixture of the enantiomers 10-methyl-protosappanin B (1a) (Figure

8B) and iso-10-methyl-protosappanin B (1b) in agreement with previously published data (Zhao et al.,

2016). Compound 3 was identified as a mixture of the enantiomers protosappanin B (2a) (Figure 8C)

and isoprotosappanin B (2b) according to previously published NMR assignments (Zhao et al., 2016).


59

Compound 4 was identified as brazilin (Figure 8D) according to previously published NMR data

(Nirmal et al., 2015).

Figure 8. Homoisoflavonoids isolated from the hydroalcoholic extract of the stems of C.

bahamensis

A: metasappanin; B: 10-methylprotosappanin B; C: Protosappanin B; D: Brazilin

The hydroalcoholic extract was analyzed by UPLC-UV-MS. In the obtained chromatogram (Figure 9)

the major peaks could be attributed to the isolated compounds brazilin (7.23 min) and (iso)

protosappanin B (7.45 min), while (iso-)10-methyl-protosappanin B (9.50 min) and metasappanin () are

a minoritarian constituents of the extract. Mass spectrometric analysis of the peak with retention time of

8.20 min revealed the presence of 2 compounds with an m/z [M-H]- value of 303.0865 and 333.0971.

The pseudomolecular ion of the peak at Rt 8.65 min also showed a value of m/z 333.0971 [M-H]-.


60

Tentative identification of these compounds revealed the presence of homoisoflavonoids with an

elemental composition of C16H16O6 and of C17H18O5.

Figure 9. UPLC-UV-MS profile of the hydroalcoholic extract of the stem of C. bahamensis

The presence of homoisoflavonoids in C. bahamensis was reported for first time in this study; however,

protosappanin B, (iso) protosappanin B and brazilin have been isolated before in other species of the

genus Caesalpinia. (Baldim et al., 2017). In contrast, metasappanin was reported for first time in the

field of natural products. Considering that brazilin and protosappanin B are the major compounds of the

hydroalcoholic extract, they can be used as chemical marker for quality control.

4.3.2. Biological evaluation

The increase of the calcium and oxalate concentration in urine of the lithiasis group compared to the

healthy control group demonstrated the effect of the ethylene-glycol and ammonium chloride solution

on the formation of renal stones. In contrast, after administration of aqueous and hydroalcoholic extracts

of the stems of C. bahamensis at dosage of 200 mg/kg, a reduction of these parameters was observed,

demonstrating their antilithiatic activity (Figure 10). Other doses were tested in a preliminary study (data


61

not shown), at dose of 100 and 10 mg/kg with a significant anti-lithiatic effect at the dose of 100 mg/kg

at dose of 10 mg /kg and below, no antilithiatic effect was obtained.

Figure 10. Urinary concentration of calcium (A) and oxalate (B) in rats

Different labels indicate significative differences (p<0.05)

In addition, the absence of red spots in the renal tubules of the kidney sections stained with Von Koss

(Figure 11A) and the absence of white spots under polarized light microscopy (Figure 11B) in rats of the

groups treated with the extracts corroborated these results.

Figure 11. Kidney section of the rats observed at microscopy

A: Von Koss tinction; B: Under polarized light


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On the other hand, the lithiasis and the treated groups showed a higher plasmatic creatinine level

compared to the control group, indicating renal damage (Figure 12A). On the kidney sections stained

with hematoxylin/eosin signs of renal damage were observed, such as loss of the morphology of the

Bowman Capsule and renal tubules (Figure 12B).

Figure 12. Evidences of renal damage in Wistar rats

A: Urinary concentration of plasmatic creatinine; B: Kidney section (hematoxylin-eosin tinction).

Different labels indicate significative differences (p<0.05)

In summary, in our experimental conditions, the extracts of the stems of C. bahamensis showed

antilithiathic activity, but they did not show effect on renal damage produced by renal stones.

4.4. Discussion

The pharmacological efficacy of herbal medicines depends on their chemical composition (Ernst, 2005).

Medicinal plants are composed of a wide variety of chemical compounds related to external factors such

as environmental conditions and age of the plant. For this reason, it is necessary to identify chemical

markers in order to guarantee their quality, efficacy and safety (Butt et al., 2018). According to the


63

European Medicines Agency (EMA), chemical markers are “chemically defined constituents or groups

of constituents of an herbal substance, an herbal preparation or an herbal medicinal product which serve

for quality control purposes, independent of whether they have any therapeutic activity”. EMA describes

two different categories of markers. The constituents of an herbal medicine responsible of its therapeutic

activity or active markers; and the constituents that are characteristics of its taxon or analytical markers

(Rivera, 2017). In the present study, four homoisoflavonoids were isolated from the hydroalcoholic

extract of the stems of Caesalpinia bahamensis for the first time.

Of them, metasappanin was reported for first time in the field of natural products. In related

homoisoflavonoids (sappanins), the benzyl ring usually is monosubstituted (in the paraposition at C-4’)

or disubstituted (2 substituents in ortho-position at C-3’ and C-4’), associated with chemical shifts

between δC 140 to 150 (Abegaz & Knife, 2019; He et al., 2016; Namikoshi et al., 1987; Zhao et al.,

2014) but in compound 1, these shifts were not observed. A meta-substitution (at C-4’ and C-6’) is

unusual. Therefore, since this compound has not been reported before, the name “metasappanin” was

adopted. Still, the configuration at C-3 and C-4 needed to be established. The large coupling constant of

10.9 Hz between H-3 and H-4 suggested an axial-axial relationship (Pérez & Ortíz, 2015), as shown in

Fig. 1. This implies an equatorial position for the hydroxy- and benzyl-substituent, which indeed, is the

most stable configuration. The structure was confirmed by accurate mass spectrometry, establishing a

molecular formula of C17H18O5. Similar structures have been isolated in the Caesalpinia genus, such as,

sappanol, episappanol, caesalpinianone and 3′‐ deoxy‐ 4‐ O‐ methylsappanol. In fact, the sappanins are

the most common homoisoflavonoids in this genus (Baldim et at., 2017). Most compounds have a 3,4-

dihydroxy substitution, or a carbonyl at C-4.

Brazilin and protosappanin B were identified as the major compounds. In biological studies,

protosappanin B has been studied as anti-inflammatory (Mueller, 2016) and anti-tumoral drug (Yang et


64

al., 2016; Yang et al., 2019). For brazilin various activities have been demonstrated, such as

anticoagulant, antimicrobial, antioxidant, antitumoral, hypoglycemic and hepatoprotective (Nirmal et

al., 2015). Homoisoflavonoids are a rare subclass of flavonoids restricted to some families (Baldim et

al., 2017. They have been previously isolated from several species of the genus Caesalpinia, such as C.

sappan, C. crysta, C. bonduc, C. pyramidalys and C. pulcherrima (Zanin et al., 2012). Therefore,

protosappanin B and brazilin were proposed as a chemical marker for the quality control of the stems of

C. bahamensis and its preparations.

In the present study, the antilithiatic activity of the aqueous and hydroalcoholic extracts of the stems of

C. bahamensis was evaluated for first time. We only showed the pharmacological efficacy obtained at

the maximum dose used according to the ethnomedical experience for this plant, previously analyzed.

Despite both extracts showed similar antilithiatic activity, the effect on the elimination of calcium

oxalate of the hydroalcoholic extract was significantly higher than the aqueous extract. A previous

comparative analysis of these extracts demonstrated that flavonoids are present in a larger amount in the

hydroalcoholic extract compared to aqueous extract (Felipe et al., 2019b). Recently, Zheng et al. (2018)

described the role of flavonoids in the treatment of renal lithiasis. The authors explained the reduction of

oxalate in urine by the capacity of some flavonoids to inhibit the synthesis of oxalate, which also may

have been the case in our experiment. On the other hand, the extracts did not show an effect on renal

damage produced by renal stones. In previous studies on natural products using the ethylene glycol-

induced urolithiasis method, extracts have been administered longer than 28 days (Kumar et al., 2016;

Patel & Shah, 2017). In the present study, the rats were treated only for seven days, which may explain

the absence of these effects.


65

4.5. Conclusion

Four homoisoflavonoids were isolated for first time from Caesalpinia bahamensis; two of them,

protosappanin B and brazilin, were proposed as a chemical marker for the quality control of the plant

material and its extracts. The aqueous and hydroalcoholic extracts of the stems of Caesalpinia

bahamensis showed antilithiatic activity in a lithiasis model in Wistar rats.

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He DY, Li YP, Tang HB, Luo L, Ma RJ, Wang JH, Wang LQ. Phenolic compounds from the twigs and

leaves of Tara (Caesalpinia spinosa). Journal of Asian Natural Products Research 2016; 18(4): 334-

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Kumar D, Niranjan P, Alok S, Kulshreshtha S, Dongray A, Dwivedi S. A brief review on medicinal

plant and screening method of antilithiatic activity. International Journal of Pharmacognosy 2016;

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Mittal A, Tandon S, Singla SK, Tandon C. In vitro inhibition of calcium oxalate crystallization and

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Namikoshi M, Nakata H, Nuno M, Ozawa T, Saitoh T. Homoisoflavonoids and Related Compounds.

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Patel KM, Shah SK. Evaluation of antiurolithiatic activity of Lawsonia inermis Linn. in rats. Int. J.

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Natural Product Research 2014; 28(2):102-105.

APPENDIX A: NMR SPECTRUMS OF HOMOISOFLAVONOIDS ISOLATED

Figure A-1. 1H-NMR spectrum of compound 1 (metasappanin)


Figure A-2. 13C-NMR, DEPT 90 and DEPT 135 spectrums of the compound 1 (metasappanin)


Figure A-3: COSY spectrum of compound 1 (metasappanin)


Figure A-4: HSQC spectrum of compound 1 (metasappanin)


Figure A-5: HMBC spectrum of compound 1 (metasappanin)


Figure A-6. 1H-NMR spectrum of compound 2 (10-methyl protosappanin B and (iso) 10-methyl protosappanin B)


Figure A-7. 13C-NMR, DEPT 90 and DEPT 135 spectrums of compound 2 (10-methyl protosappanin B and (iso) 10-methyl

protosappanin B)


Figure A-8. COSY spectrum of compound 2 (10-methyl protosappanin B and (iso) 10-methyl protosappanin B)


Figure A-9. HSQC spectrum of compound 2 (10-methyl protosappanin B and (iso) 10-methyl protosappanin B)


Figure A-10. HMBC spectrum of compound 2 (10-methyl protosappanin B and (iso) 10-methyl protosappanin B)


Figure A-11. 1H-NMR spectrum of compound 3 (brazilin)


Figure A-12. 13C-NMR, DEPT 90 and DEPT 135 spectrums of compound 3 (brazilin)


Figure A-13. HSQC spectrum of compound 3 (brazilin)


Figure A-14. Comparative 1H-NMR spectrum of compound 2 and 4 (protosappanin B and (iso) protosappanin B)

The signal at δ 3.87 ppm observed in the compound 2 was not evidenced in the spectrum of compound 4, demonstrating the absence of the

methoxy group in protosappanin B (compound 4)

Compound 2

Compound 4


Figure A-15. Comparative 1H-NMR spectrum of compound 2 and 4 (protosappanin B and (iso) protosappanin B)

The signal at δ 56.49 ppm observed in the compound 2 was not evidenced in the spectrum of compound 4, demonstrating the absence of the

methoxy group in protosappanin B (compound 4)

Compound 4

Compound 2

CHAPTER 5:

Diuretic activity and

acute oral toxicity


Felipe A, Núñez CR, Gutiérrez YI, Scull R, Noa AC,

Foubert K, Pieters L, Delgado R. Diuretic Activity and

Acute Oral Toxicity of Caesalpinia bahamensis Lam.

extracts (brasilete). International Journal of

Pharmaceutical and Phytopharmacological Research

2020; 10(3): 65-69.

DIURETIC ACTIVITY AND ACUTE ORAL TOXICITY

69

5.1. Introduction

The use of plants for medicinal purposes dates back to the very origins of human history, when people

had no other effective therapeutic resources to treat their diseases. This knowledge was transmitted

through legends, pictographs and various monographs until our days (Rodríguez et al., 2015). According

to data from the World Health Organization (WHO), 80% of the world’s population uses plants as a

remedy to cure their diseases (Escalona et al., 2015). On the other hand, it is known that around 20% -

30% of the medicines available on the market are derived from natural products (Majouli et al., 2017).


medicine; however, scientific studies are necessary to corroborate the ethnobotanical information

(Calixto, 2000).

The Cuban population has a wide ethnobotanical knowledge about medicinal plants for the treatment of

various diseases, and renal affections are among the most treated in ethnobotany. However, the

phytotherapeutic potential of the island is still virgin. For example, in ethnobotanical studies made in

Cuba, 179 species have been used against diseases of the renal system, of which only 9% have been

evaluated pharmacologically (Felipe et al., 2020).

Caesalpinia bahamensis Lam. is a medicinal plant used by the Cuban population to treat renal and

hepatic diseases, diabetes and peptic ulcers (Roig, 2012). The diuretic (Felipe et al., 2011), antioxidant

(Felipe et al.,2019a), cytotoxic (Setzer et al., 2015) and poor antimicrobial activities (Abreu et al., 2017)

have previously been reported. A total of 74 compounds have been identified in the non-polar fraction of

a methanolic extract of the species, using gas chromatography - mass spectrometry (GC-MS). In this

study, fatty acids, terpenoids and phytosterols were reported as the major compounds of this fraction

(Felipe et al., 2017). A comparative pharmacognostic study of the aqueous and hydroalcoholic extracts

demonstrated the presence of flavonoids and phenolic compounds as the major metabolites. In addition,


70

the phytochemical composition of both extracts was similar according to HPLC analysis. However, the

total yield and quantity of the flavonoids was higher for the hydroalcoholic extract (Felipe et al., 2019b).

Apart from this, the scientific information about this species until now is very limited.

This study aimed to evaluate the diuretic activity and acute oral toxicity of the aqueous and

hydroalcoholic extracts of the stems of C. bahamensis as a continuation of the studies to evaluate its

efficacy and safety.


5.2.1. Plant material and preparation of the extracts

Stems of Caesalpinia bahamensis Lam. (Leguminosae) were collected in March 2017 at Cañada

Arroyón, Artemisa, Cuba (22°46'45.7"N 83°04'18.6"W). The material was identified in the National

Botanical Garden of Cuba, where a voucher specimen (No. 85369) was deposited. The material was

dried in an oven (AI-SET-DNE 600, Shanghai, China) at 40 °C for seven days and milled (Manesti,

Italy) until the size of the particles was less than 2 mm. Aqueous and hydroalcoholic extracts were

obtained by maceration at room temperature in the dark, during 24 h. Five milliliters of solvent was used

for each gram of dry plant material. After that, the extracts were dried under reduced pressure in a rotary

evaporator (IKA). The plant material and the extracts were previously characterized (Felipe et al.,

2019b).

5.2.2. Animals

Wistar rats (200 – 250 g body weight) were obtained from the animalarium of CENPALAB (Havana,

Cuba) and kept in collective cages at 22 °C under a 12-h light/dark cycle (lights on at 07:00 h) with free

access to laboratory food and tap water. This investigation followed Cuban laws for this type of

preclinical studies in agreement with the international regulations for animal care, and was approved for


71

the Ethical Committee of the Institute of Basic and Preclinical Sciences “Victoria de Girón”, Medical

University of Havana, Cuba (Agreement 28-2019; December 2019).

5.2.3. Diuretic activity

Diuretic activity was evaluated in Wistar rats using metabolic cages. Fifty-four male Wistar rats were

divided into five groups of equal size (n=6) as follow:

Group 1: Physiological solution of sodium chloride 0.9% (negative control)

Group 2: Furosemide in CMC (20 mg/kg) (positive control)

Group 3: Hydrochlorothiazide in CMC (10 mg/kg) (positive control)

Group 4: Dried aqueous extract of C. bahamensis in CMC (10 mg/kg)



Group 7: Dried hydroalcoholic extract of C. bahamensis in CMC (10 mg/Kg)



All treatments were suspended in Carboximethyl cellulose (CMC) and administered by intragastric

gavage using a volume of 3 mL per rat. After that, the rats were placed in metabolic cages and the

excreted urine volume was measured every hour during four hours. The urinary flow was calculated and

the urinary concentration of sodium and potassium was determined by flame photometry (Corning 400,

USA).

5.2.4. Acute oral toxicity

The in vivo toxicological properties of the aqueous and hydroalcoholic extracts for the stems of C.

bahamensis were investigated according to OECD Guideline 423, consisting of a single-dose 14-day

acute oral toxicity study. The Globally Harmonized System (GHS) was used for the classification


72

(Jonsson et al., 2013). Six male and six female Wistar rats were randomly assigned to four independent

blocks of experiments (3 rats of each sex per block; n = 3). According to the OECD Guideline, three

animals of each sex were treated with the extracts at a dose of 2000 mg/kg body weight as a starting

dose to study acute toxicity. After treatment, signs of toxicity, including changes in skin and fur, eyes,

respiration and behavioral pattern, and/or mortality in all animals were observed periodically during the

first 24 h (0.5, 1, 6, and 24 h), and then once daily for 14 days. Post-treatment, animals were weighed

weekly. At day 15, all animals were anesthetized by petroleum ether inhalation, sacrificed by cervical

dislocation, and their internal organs were observed (Worasuttayangkurn et al., 2019).


All statistical comparisons between the groups were made by means of One-Way Analysis of Variance

(ANOVA) with the post hoc Student-Newman-Keuls test. A p-value less than 0.05 was regarded as

significant.

5.3. Results

5.3.1. Diuretic activity

The diuretic activity of the aqueous and hydroalcoholic extracts was evaluated and compared with

furosemide and hydrochlorothiazide, well established diuretic drugs. The rats receiving the extracts

showed a significant increase (p < 0.05) of the urinary flow and the levels of Na+ and K+ compared to

the negative control group; similar values were obtained as for the hydrochlorothiazide group.

Furosemide showed the highest values for these parameters [Table 8]. The groups of animals that

received doses of the studied extracts of 100 mg/kg also produced statistically significant results, which

demonstrated diuretic pharmacological efficacy at this dose. The 10 mg/kg dose did not produce

statistically significant results for both extracts, demonstrating a dose-dependent effect.


73

The highest diuresis was observed two hours after administration of the extracts, similar to

hydrochlorothiazide. In contrast, furosemide showed the highest diuresis after one hour [Figure 13].

Table 8: Effect of the extracts of C. bahamensis on the urinary flow and urinary concentration of

sodium and potassium in Wistar rats (n=6)

Group Urinary flow C(Na) C(K)

(ml/min) (mEq) (mEq)

G1. Sodium chloride 0.9% 0.011 ± 0.005 a 47.67 ± 4.50 a 26.67 ± 3.04 a

G2. Furosemide 0.043 ± 0.007 b 85.67 ± 2.53 b 40.67 ± 2.65 b

G3. Hydrochlorothiazide 0.032 ± 0.004 c 76.33 ± 9.18 c 33.33 ± 2.16 c

G4. Aqueous extract 10 mg/Kg 0.013 ± 0.008 a 45.23 ± 3.17 a 23.85 ± 2.83 a

G5. Aqueous extract 100 mg/Kg 0.021 ± 0.007 d 59.00 ± 4.13 d 28.57 ± 1.18 d

G6. Aqueous extract 200 mg/Kg 0.028 ± 0.008 c 73.00 ± 2.24 c 35.28 ± 3.07 c

G7. Hydroalcoholic extract 10 mg/Kg 0.015 ± 0.004 a 44.72 ± 5.18 a 22.49 ± 2.56 a

G8. Hydroalcoholic extract 100 mg/Kg 0.019 ± 0.003 d 61.06 ± 4.19 d 27.28 ± 1.03 d

G9. Hydroalcoholic extract 200 mg/Kg 0.030 ± 0.005 c 77.15 ± 3.23 c 34.15 ± 1.28 c

Values are expressed as mean ± SD (Standard Deviation). Different superscripts indicate significant

differences (p <0.05) between groups.

Figure 13. Kinetics of urinary volume excreted for each group


74

In summary, the extracts of the stems of C. bahamensis showed a diuretic activity similar to

hydrochlorothiazide in Wistar rats at a dosage of 200 mg/kg.

5.3.2. Acute oral toxicity

The results of the study showed that 14 days after administration of a high dose of the aqueous and

hydroalcoholic extracts, no signs of toxicity and mortality were observed. Body weight increased in the

time, a parameter indicative of absence of or low toxicity [data not shown]. Since vital organs, including

liver, kidneys, heart lungs, and spleen, are functionally crucial organs often impaired by toxic

substances, gross examinations of these internal organs were performed to identify potential signs of

organ-targeted toxicity. No lesions were found upon macroscopic examination of the internal organs of

any animals treated with the extracts. According to the Globally Harmonized System (GHS), the extracts

of C. bahamensis were classified as drugs of category 4. These results suggest that extracts of C.

bahamensis are not acutely toxic after oral administration.

5.4. Discussion

Diuretics are defined as any substance that increases urine flow and thereby water excretion. The

majority acts by reducing sodium chloride reabsorption at different sites in the nephron, thereby

increasing urinary sodium, and consequently, water loss (Wile, 2012). They are classified as loop

diuretics, distal convoluted tubule diuretics or thiazides, potassium-sparing diuretics, carbonic anhydrase

inhibitors and osmotic diuretics. This classification is based on their predominant site of action along the

nephron and by the mechanism by which they inhibit transport (Ellison, 2019). They are among the most

commonly used drugs for disease conditions such as congestive heart failure, nephritic syndrome,

cirrhosis, renal failure, hypertension, and pregnancy toxaemia. However, many diuretic drugs derived

from chemical sources are associated with adverse effects on quality of life including impotence,


75

hyperglycaemia, ototoxicity, gout and fatigue (Huda & Debnath, 2017). Therefore, it is necessary to

search for alternative fewer toxic treatments.

In this research, the diuretic activity of the aqueous and hydroalcoholic extracts of C. bahamensis was

evaluated and compared with furosemide and hydrochlorothiazide. They are a well-stablished drug and

their mechanism of action can be explained according to the differences in the urinary flow, time of

action and ion concentrations in urine (Sarafidis et al., 2010). For example, furosemide has the highest

excretion of water and sodium in the urine but a short duration of action; for this reason, it is used to

treat edemas and acute diseases. In contrast, hydrochlorothiazide excretes less water and sodium with

respect to furosemide, but it has a long duration of action and it is used mainly for chronic diseases (Min

& White, 2009).

The rats that received the extracts of C. bahamensis showed similar values of urinary flow and

concentration of sodium and potassium as the rats of the hydrochlorothiazide group. This suggests that

C. bahamensis acts with a similar mechanism as hydrochlorothiazide at the distal convoluted tubule,

where ~ 5 – 10% of filtered NaCl is reabsorbed. However, other studies are necessary to corroborate this

hypothesis.

In a previous study, the diuretic activity of the aqueous extract of the stems of C. bahamensis was

evaluated (Felipe et al., 2011). In general, aqueous extracts are used in traditional medicine as tea or

decoction, but these formulations are not stable for a long time and the dosage is not exact. Therefore,

the use of hydroalcoholic extracts is more common in the pharmaceutical industry for the development

of herbal medicines. For this reason, it was necessary to evaluate also the diuretic effect of the

hydroalcoholic extract of the stems of C. bahamensis. Also, the hydroalcoholic extract was found to be

richer in flavonoids than the aqueous extract (Jonsson et al., 2013). The presence of flavonoids can be

associated with the diuretic activity of the drug, because flavonoids increase the activity of prostacyclin


76

synthase, leading to the release of renal prostaglandin that has been implicated in diuresis (Yakubu et

al., 2019).

On the other hand, the acute oral toxicity of the extracts was evaluated in order to start safety studies of

this herbal preparation. In this sense, both extracts did not show any acute toxicity in the experimental

conditions. Therefore, C. bahamensis can be a safe candidate drug to be used as a diuretic; however, it is

necessary to evaluate the effects after chronic administration taking into account the pharmacological

similarity with hydrochlorothiazide, a drug used in chronic diseases.

5.5. Conclusion

The aqueous and hydroalcoholic extracts of the stems of Caesalpinia bahamensis showed a diuretic

activity similar to hydrochlorothiazide and they did not show acute oral toxicity. These studies

contribute to endorse the popular use of this plant species for diuretic purposes, and are part of the set of

studies that may contribute to its future development as a herbal formulation for the treatment of kidney

diseases.

References


medicinales en terapéutica. Acta Farmacéutica Portuguesa 2015; 4(1): 42- 52.



Medicinales 2015; 20(4): 429-439.




(phytotherapeutic agents). Braz. J. Med. Biol. Res. 2000; 33(2): 179-189.


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Felipe A, Pieters L, Delgado R. Effectiveness of Herbal Medicine in Renal Lithiasis: A review. Siriraj

Medical Journal 2020; 72(2): 188-194.

Roig JT. Plantas medicinales, aromáticas o venenosas de Cuba, 2nd edn., Ciencia y Técnica: La Habana,

2012.



2011; 3(2): 300- 308.



Pharmacy & Pharmacognosy Research 2019a; 7(1): 12-20.






extracto metanólico de la madera de Caesalpinia bahamensis Lam (brasilete). Revista Cubana de

Ciencias Farmacéuticas y Alimentarias 2017; 3(2): 1-8

Felipe A, Gutiérrez YI, Scull R, Noa AC, Beverly D, Foubert K, Pieters L, Delgado R. Pharmacognostic

study of the stem of Caesalpinia bahamensis and characterization of its aqueous and hydroalcoholic

extracts. Journal of Pharmacognosy and Phytochemistry 2019b; 8(3): 3079-3083.

Jonsson M, Jestoi M, Nathanail AV, Kokkonen U, Anttila M, Koivisto P et al. Application of OECD

Guideline 423 in assessing the acute oral toxicity of moniliformin. Food and Chemical Toxicology.

2013; 53:27-32.


78

Worasuttayangkurn L, Nakareangrit W, Kwangjai J, Sritangos P, Pholphana N, Watcharasit P et al.

Acute oral toxicity evaluation of Andrographis paniculata-standardized first true leaf ethanolic

extract. Toxicology Reports. 2019; 6:426–430.

Wile D. Diuretics: a review. Ann Clin Biochem 2012; 49: 419–431.

Ellison DH. Clinical Pharmacology in Diuretic Use. CJASN 2019; 14: 1248–1257.

Huda EA, Debnath J. Evaluation of diuretic activity of aqueous extract of leaves of Centella asiatica.

World Journal of Pharmaceutical Research 2017; 6(10): 494-500.

Sarafidis PA, Georgianos PI, Lasaridis AN. Diuretics in clinical practice. Part I: Mechanisms of action,

pharmacological effects and clinical indications of diuretic compounds. Expert Opin. Drug Saf. 2010;

9(2): 243-257.

Min B, White CM. A Review of Critical Differences Among Loop, Thiazide, and Thiazide-Like

Diuretics. Hosp. Pharm. 2009; 44(2): 129–149.

Yakubu MT, Oyagoke AM, Quadri LA, Agboola AO, Oloyede HOB. Diuretic activity of ethanol extract

of Mirabilis jalapa (Linn.) leaf in normal male Wistar rats. Journal of Medicinal Plants for Economic

Development 2019; 3(1): 64-70.

GENERAL DISCUSSION

AND FUTURE

PERSPECTIVES

GENERAL DISCUSSION AND FUTURE PERSPECTIVES

79


The use of plants for medicinal purposes dates back to the very origins of human history, when people

had no other effective therapeutic resources to treat their diseases. This knowledge was transmitted

through legends, pictographs and various monographs until our days (Rodríguez et al., 2015). According

to data from the World Health Organization (WHO), 80% of the world’s population uses plants as a

remedy to cure their diseases (Escalona et al., 2015). On the other hand, it is known that around 20% -

30% of the medicines available on the market are derived from natural products (Majouli et al., 2017).


medicine; however, scientific studies are necessary to corroborate the ethnobotanical information

(Calixto, 2000).

In Cuba, the great variety of medicinal plants, together with the wide ethnobotanical knowledge,

increases the opportunities for the search of new therapies in the treatment of various diseases, of which

renal affections have been one of the most treated. However, the phytotherapeutic potential of the island

is still virgin (Felipe et al., 2020).

Caesalpinia bahamensis Lam. is a medicinal plant used by the Cuban population to treat renal and

hepatic diseases, diabetes and peptic ulcers (Roig, 2012). The diuretic (Felipe et al., 2011), antioxidant

(Felipe et al.,2019), cytotoxic (Setzer et al., 2015) and poor antimicrobial activities (Abreu et al., 2017)

have previously been reported. Apart from this, the scientific information about this species until now is

very limited.

Pharmacognosy is the science that studies the physical, chemical, biochemical and biological properties

of drugs, drug substances, or potential drugs of natural origin, and it also includes the search for new

drugs from natural sources (Biswas, 2015).


80

In the present research, the pharmacognostic study of the stems of Caesalpinia bahamensis and their

aqueous and hydroalcoholic extract was carried out. In general, the parameters evaluated are in

correspondence with the general parameters stablished for natural products (Comission CP, 2015). Also,

the better solvent for the extraction was ethanol 30 and 80%. During preliminary phytochemical

characterization, flavonoids and phenols were identified as the main metabolites; then, these metabolites

were quantified in both extracts. The hydroalcoholic extract showed the highest content of flavonoids,

while the aqueous extract showed the highest content of total phenols.

The establishment of the quality parameters of drugs guarantees the safety and efficacy of the finished

herbal medicine (Govindaraghavan & Sucherb, 2015), and avoids the adulteration or substitution of the

plants used as raw materials (Chanda, 2014). The pharmacognostic study permitted to establish the

quality parameters of the drug and the extracts, as well as to choose flavonoids and total phenols as

general chemical markers.

In the genus Caesalpinia, several homoisoflavonoids have been isolated (Zanin et al., 2012).

Homoisoflavonoids are chemical structures restricted only to some vegetal species from the Fabaceae

and Asparagaceae (Baldim et al., 2017). Antioxidant, antimicrobial, anti-inflammatory, cytotoxic and

hypoglycemic activities have been demonstrated for these metabolites (Mezbah et al., 2015; Nirmal et

al., 2015; Mueller et al., 2016; Liang et al., 2013; Zanin et al., 2015). According to the European

Medicines Agency (EMA), chemical markers are “chemically defined constituents or groups of

constituents of an herbal substance, an herbal preparation or an herbal medicinal product which serve for

quality control purposes, independent of whether they have any therapeutic activity” (Rivera et al.,

2017).

The next step of the present research aimed to isolate and to identify specific chemical markers for

Caesalpinia bahamensis. In this sense, four homoisoflavonoids were isolated and identified from the


81

hydroalcoholic extract of the stems of Caesalpinia bahamensis for first time in the species. One of them

(metasappanin) was reported for first time in the field of natural products and two others were chosen as

chemical markers (brazilin and protosappanin B). In addition, another seventy-four compounds were

identified by GC/MS in the petroleum ether fraction obtained from the hydroalcoholic extract.

As mentioned earlier, Caesalpinia bahamensis has been traditionally used as antilithiatic. According to

the ethnobotanical knowledge, extracts from the stems are prepared by aqueous maceration. However, in

the pharmacognostic analysis, the hydroalcoholic extract showed the highest percentage of soluble

constituents and content of flavonoids. Previous reports suggest that flavonoids and phenols are related

with the antilithiatic activity in herbal medicines (Grases et al., 2015; Touhami et al., 2007; Zeng et al.,

2018)

For this reason, a comparative biological study of the aqueous and hydroalcoholic extract was done,

including antilithiatic and diuretic activities and acute oral toxicity assays. Both extracts showed

antilithiatic and diuretic activities in Wistar rats using the ethylene glycol-induced urolithiasis and

individual metabolic cages methods, respectively. Despite both extracts showed similar antilithiatic

activity, the effect on the elimination of calcium oxalate of the hydroalcoholic extract was significantly

higher than the aqueous extract, possibly associated with the higher content of flavonoids determined in

the hydroalcoholic extract. On the other hand, the diuretic activity of both extracts was similar to

hydrochlorothiazide used as a positive control. In addition, no acute oral toxicity was observed for the

extracts.

During time plant NPs have been used as traditional medicines, remedies, potions and oils without any

knowledge about the bioactive compounds contained inside but just considering results of hundreds of

centuries of empirical knowledge (Bernardini et al., 2018). It has been estimated that approximately

over half of the pharmaceuticals in clinical use today are derived from natural products. Some natural


82

product-derived drugs that are a hallmark of modern pharmaceutical care include quinine, theophylline,

penicillin G, morphine, paclitaxel, digoxin, vincristine, doxorubicin, cyclosporine and vitamin A among

many other examples (Mubanga et al., 2017).

Renal lithiasis is a pathology of high incidence, prevalence and recurrence rate around the world (Cano

et al., 2015). For the treatment of renal stones minimally invasive surgery is used; it is effective to break

the calculi, but it does not reduce recurrence rates. On the other hand, many drugs have been used, such

as thiazide diuretics, potassium citrate and non-steroidal anti-inflammatory drugs (NSAIDs); but they

are only used for preventing or treating the symptoms (Alelign & Petros, 2018). For these reasons, many

studies have been focused on understanding the mechanisms involved in renal lithiasis, and the

development of an herbal medicine as a new drug for the treatment and prevention of this pathology and

its recurrences is a promising approach (Felipe et al., 2020).

Current drug discovery strategies and modern medicine discard the use of whole plant extracts and are

driven by single compound-based medicine. Taking the whole plant or extracts with no isolation of

components as practiced in traditional medicine, produces a better therapeutic effect than individual

compounds. This is important as most of the plant metabolites likely work in a synergistic fashion or

concurrently to give the plant extract its therapeutic effect. However, searching for new drug candidates

from natural products is often made difficult by the complexity of the molecular mixtures (Thomford et

al., 2018).

The present research provides novel results for Caesalpinia bahamensis, contributing to the

development of a potential new drug for the treatment of renal lithiasis. The quality parameters of the

drug and the extracts were stablished, including chromatographic and UV profiles. The contents of

flavonoids, total phenols, brazilin and protosappanin B were proposed as chemical markers; however,

further studies will be focused on the development of a quantification method for brazilin and


83

protosappanin B and to standardize the quality parameters of the drug and the extracts. In addition, the

effectiveness of the aqueous and hydroalcoholic extracts of the stems of Caesalpinia bahamensis in

renal lithiasis has been demonstrated in Wistar rats, using the ethylene glycol-induced urolithiasis

method; however, other in vivo and in vitro methods should be used in order to understand the

mechanism of action and to determine the dose-response curve. Also, sub-chronic and chronic

toxicological studies are necessary in the future to demonstrate the safety of the extracts.

References




2018: 1-12.



From biosynthesis to human health. Geneva: IntechOpen, 2017.

Bernardini S, Tiezzi A, Masci VL, Ovidi E. Natural products for human health: an historical overview of

the drug discovery approaches. Natural Product Research 2018; 32(16): 1926-1950.

Biswas D. Role of reverse pharmacognosy in finding lead molecules from nature. Indian Journal of

Research in Pharmacy and Biotechnology 2015; 3(4): 320-323.


(phytotherapeutic agents). Brazilian Journal of Medical and Biological Research 2000; 33: 179-189.




84

Chanda S. Importance of pharmacognostic study of medicinal plants: An overview. Journal of


Commission CP. Pharmacopoeia of the People's Republic of China. (Chinese Medical Science and

Technology Press: Peking), 2015. Pags. 337-345.



Medicinales 2015; 20(4): 429-439.



2011; 3(2): 300- 308.





Med J. 2020; 72: 188-194.

Govindaraghavan S & Sucherb NJ. Quality assessment of medicinal herbs and their extracts: Criteria

and prerequisites for consistent safety and efficacy of herbal medicines. Epilepsy & Behavior 2015;

52: 363-371.

Grases F, Prieto RM, Fernandez-Cabot RA, Costa-Bauzá A, Tur F, Torres JJ. Effects of polyphenols

from grape seeds on renal lithiasis. Oxidative Medicine and Cellular Longevity 2015; 2015: 1-6.

Liang CH, Chan LP, Chou TH, Chiang FY, Yen CM, Chen PJ, et al. Brazilein from Caesalpinia sappan

L. antioxidant inhibits adipocyte differentiation and induces apoptosis through caspase-3 activity and


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anthelmintic activities against hymenolepis nana and anisakis simplex. Evidence-Based

Complementary and Alternative Medicine 2013; 2013: 1-14.



Mezbah G, Young C, Chung D, Kim KA, Hoon S. One-step isolation of sappanol and brazilin from

Caesalpinia sappan and their effects on oxidative stress-induced retinal death. BMB Rep. 2015;

48(5): 289-294.

Mubanga P, Mayoka G, Mutai P, Chibale K. The Role of Natural Products in Drug Discovery and

Development against Neglected Tropical Diseases. Molecules 2017; 22: 58-99.



2016; 7: 1671–1679.



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2017; 55(1): 1500-1512.


medicinales en terapéutica. Acta Farmacéutica Portuguesa 2015; 4(1): 42-52.


Cuba, 2012. Págs.: 228-229.


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Thomford NE, Senthebane DA, Rowe A, Munro D, Seele P, Maroyi A et al. Natural Products for Drug

Discovery in the 21st Century: Innovations for Novel Drug Discovery. Int. J. Mol. Sci. 2018; 19:

1578-1600.

Touhami M, Laroubi A, Elhabazi K, Loubna F, Zrara I, Eljahiri Y, et al. Lemon juice has protective

activity in a rat urolithiasis model. BMC Urology 2007; 7:18.

Zanin JLB, Carvalho BA, Martineli PS, Santos MH, Lago JHG, Sartorelli P, Viegas C, Soares MG. The

genus Caesalpinia L. (Caesalpiniaceae): Phytochemical and pharmacological characteristics.

Molecules 2012, 17: 7887-7902.

Zanin JLB, Massoni M, Santos MH, Freitas GC, Niero ELO, Schefer RR, et al. Caesalpinioflavone, a

new cytotoxic biflavonoid isolated from Caesalpinia pluviosa var. peltophoroides. J. Braz. Chem.

Soc. 2015; 26(4): 804-809.

Zeng X, Xi Y, Jiang W. Protective role of flavonoids and flavonoid-rich plant extracts against

urolithiasis: a review. Food Sciences and Nutrition 2018; 59(7): 1-44.

CONCLUSIONS AND

RECOMMENDATIONS

CONCLUSIONS

87

CONCLUSIONS

1. The quality parameters of the stems of Caesalpinia bahamensis are in correspondence with the

general parameters established for herbal medicines according to the Chinese Pharmacopoeia.

2. Qualitative and quantitative analysis of the aqueous and hydroalcoholic extracts was carried out.

Total phenol, flavonoids, brazilin and protosappanin B were proposed as chemical markers for further

quality control analysis.

3. Seventy-four compounds were identified in the non-polar fraction of the hydroalcoholic extract by

Gas Chromatography / Mass Spectrometry, including fatty acids, terpenoids and phytosterols as the

major compounds.

4. The homoisoflavonoids metasappanin, protosappanin B, (iso)-protosappanin B and brazilin were

isolated and characterized in the hydroalcoholic extract. Of them, metasappanin was reported for first

time as a natural product.

5. Both extracts showed diuretic and antilithiatic activities and absence of acute oral toxicity in Wistar

rats.

RECOMMENDATIONS

88

RECOMMENDATIONS

1. To validate the quantification methods for brazilin and protosappanin B in the extracts of Caesalpinia

bahamensis.

2. To standardize the quality parameters of the drug and extracts of Caesalpinia bahamensis.

3. To evaluate the antilithiatic activity of the extracts of Caesalpinia bahamensis in other biological

models in order to understand the possible mechanism of action.

4. To evaluate the sub-chronic and chronic toxicity of the extracts of Caesalpinia bahamensis in order

to guarantee the safety of the extract.

5. To develop a phyto-drug based on the hydroalcoholic extract of the stems of Caesalpinia

bahamensis.

CURRICULUM VITAE

CURRICULUM VITAE

89

PERSONAL

Name: Alejandro Felipe González

Date of birth: 26 January 1986

Place of birth: Havana, Cuba

E-mail: [email protected]

EDUCATION

2005-2010 Bachelor in Pharmaceutical Sciences, great distinction

Institute of Pharmacy and Food. University of Havana

Havana, Cuba

2012-2014 Master in Chemical Pharmaceutical


Havana, Cuba

2016–2020 PhD student in Pharmaceutical Sciences


Havana, Cuba

2016–2020 PhD student in Pharmaceutical Sciences

Natural Products & Food Research and Analysis (NatuRA)

University of Antwerp

Wilrijk, Belgium

mailto:[email protected]

CURRICULUM VITAE

90

WORKING EXPERIENCE

2010-2014: Specialist A in Drug Control. Minster of Interior. Havana, Cuba.

2015: Technician C in University Management

2016-2020: Doctoral training and work at Natural Products & Food Research and Analysis (NatuRA)

lab at University of Antwerp, Belgium as part of the VLIR-UOS project “Implementation

of a state-of-the-art reference center for pharmaceutical and pharmacological studies in

Cuba, to support the use of natural product formulations composed of indigenous

phytomedicinal and nutraceutical molecules, in the National System of Health (NSH) in

Cuba”.

2016-Present: Research staff at Pharmacognosy lab, Institute of Pharmacy and Food, University of

Havana, Cuba.

SCIENTIFIC EXPERIENCE

PROJECTS

2015-2020: Standardization of raw materials of natural origin for the development of

phytopharmaceutical drugs. Registered in: Institute of Pharmacy and Food. University of

Havana (Cuba).

2015-2020: Chemical and biological studies of antilithiatic plants. Registered in: Minster of Public

Health (Cuba).

2016-2020: Implementation of a state-of-the-art reference center for pharmaceutical and

pharmacological studies in Cuba, to support the use of natural product formulations

composed of indigenous phytomedicinal and nutraceutical molecules, in the National

System of Health (NSH) in Cuba. Registered in: University of Havana (Cuba) and

University of Antwerp (Belgium). Supporting by: VLIR-UOS Projects.

CURRICULUM VITAE

91

PUBLICATIONS

1. Felipe A, García G, Scull R, Herrera Y, Fernández Y. Efecto diurético de los extractos acuosos y

secos de Caesalpinia bahamensis Lam (brasilete) en ratas Wistar. Revista Colombiana de Ciencia

Animal 2011; 3(2): 300-308.

2. Felipe A, Marrero D, Scull R, Cuéllar A, Gutiérrez YI. Composición química de una fracción apolar

del extracto metanólico de la madera de Caesalpinia bahamensis Lam (brasilete). Revista de Ciencias

Farmacéuticas y Alimentarias 2017; 3(2): 1-8.

3. Gutiérrez YI, González J, Scull R, Ocanto Z, Felipe A, Mayoral JL, Monan M. Pharmacognostical,

phytochemical and antioxidant evaluations of Guettarda calyptrata A. Rich. Open Access Library

Journal 2018; 5: 1-11. https://doi.org/10.4236/oalib.1104921

4. García C, Felipe A, Naessens T, Foubert K, Delgado R, Mora C, Martínez V. Validation of an

analytical method by HPLC applicable to the cuban Mangiferin. Ars Pharmaceutica 2018; 59(4): 1-7.

http://dx.doi.org/10.30827/ars.v59i4.7430

5. Felipe A, Hernández I, Gutiérrez YI, Scull R, Carmenate LM, Pieters L, Rodeiro I, Delgado R.

Phytochemical study and antioxidant capacity of three fractions from the stem of Caesalpinia

bahamensis Lam. Journal of Pharmacy & Pharmacognosy Research 2019; 7(1): 12-20.

6. Felipe A, Gutiérrez YI, Scull R, Noa AC, Beverly D, Foubert K, Pieters L, Delgado R.

Pharmacognostic study of the stem of Caesalpinia bahamensis and characterization of its aqueous and

hydroalcoholic extracts. Journal of Pharmacognosy and Phytochemistry 2019; 8(3): 3079-3083.

7. Felipe A, Pieters L, Delgado R. Effectiveness of Herbal Medicine in Renal Lithiasis: A review.

Siriraj Medical Journal 2020; 72(2): 188-194. http://dx.doi.org/10.33192/Smj.2020.25

https://doi.org/10.4236/oalib.1104921

http://dx.doi.org/10.30827/ars.v59i4.7430


CURRICULUM VITAE

92

8. Felipe A, Núñez CR, Gutiérrez YI, Scull R, Noa AC, Foubert K, Pieters L, Delgado R. Diuretic

Activity and Acute Oral Toxicity of Caesalpinia bahamensis Lam. extracts (brasilete). International

Journal of Pharmaceutical and Phytopharmacological Research 2020; 10(3): 65-69.

CONGRESS, WORKSHOPS AND MEETINGS

Oral presentations

2015: 3rd Meeting of Pharmaceutical and Food Sciences (Havana, Cuba). Chemical and biological study

of Caesalpinia bahamensis Lam (brasilete).

2016: 1st International Congress of Esthetic, Cosmetology and Esthetic Medicine (Havana, Cuba).

Caesalpinia bahamensis: A new potential source in phytocosmetic.

2016: 3rd Workshop of Advances in Bionanomaterials, Biomolecules and Pharmaceutical Technologies

(Havana, Cuba). Pharmacognostic study, identification of metabolites and biological effects of


2017: 22nd Conference of Chemic (Santiago de Cuba, Cuba). Chemical study and diuretic activity of


2018: 5th International Congress of Pharmacology and Natural Products (Topes de Collantes, Cuba).

Pharmacological activities and chemical composition of Caesalpinia bahamensis Lam.

2018: 12nd National Congress of Pharmacology and Therapeutic (Camagüey, Cuba). Phytochemical

study and antioxidant activity of three fractions of the stems of Caesalpinia bahamensis Lam.

2018: 4th International Congress of Research-Development and Technological Innovation in the

Biopharmaceutical Industry (Havana, Cuba). Extracts obtained from Caesalpinia bahamensis

Lam; comparative pharmacognostic studies and pharmacological potentialities.

2019: 28th International Congres of the Society Italo-Latinoamerican of Ethnomedicine (Havana, Cuba).

Chemical study and antilithiatic effect of Caesalpinia bahamensis Lam.

CURRICULUM VITAE

93

2019: 6th International Congress on Pharmacology and Therapeutics (Havana, Cuba). Identification of

compounds and antilithiatic activity of Urera baccifera.

Posters

2018: 18th World Congress of Pharmacology (Kyoto, Japan). 1) Mangiferin, a naturally occurring

glucosylxanthone obtained by spray drying with increased solubility using HPMC, as an active

pharmaceutical ingredient for new pharmacotherapeutic applications. 2) Pharmacognostic,

chemical and pharmacological studies of Caesalpinia bahamensis Lam (brasilete).

Chemical study and antilithiatic activity of Caesalpinia ...

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