PHYTOCHEMICAL AND INHIBITION STUDIES OF GARCINIA KOLA HECKEL (GUTTIFERAE) SEED EXTRACTS ON SOME KEY ENZYMES INVOLVED WITH DIABETES

ABSTRACT

Garcinia kola is an Angiosperm belonging to the family Guttiferae. It is known in commerce as bitter kola. The plant seeds have been used in the treatment of a wide range of diseases including diabetes, and its importance in folkloric medicine as a purgative, mastcatory, aphrodisiac etc. is eminent. Diabetes mellitus is a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia leading to severe complications such as neuropathy, nephropathy, retinopathy and foot ulcer. n-Hexane, ethyl acetate and methanol extracts were prepared successively in a soxhlet apparatus at 50ºC. Qualitative phytochemical screening was carried out. Column chromatographic analysis was carried out on the ethyl acetate extract and the structure of the isolated compound was elucidated via Gas Chromatography-Mass Spectrophotometry and Fourier Transformed-Infra Red spectroscopy. Pancreatic α-amylase and intestinal α-glucosidase were extracted from porcine pancrease and rat small intestine under specified conditions. Steroids/triterpenes, phenolics, flavonoids, cardiac glycosides, alkaloids, coumarins and phlobatannins were detected. Methanol, ethyl acetate and n-hexane extracts inhibited α-amylase with IC₅₀ = 0.78 ± 0.32 mg/ml, 3.44 ± 3.46 mg/ml, 4.89 ± 4.62 and α-glucosidase IC₅₀ = 2.67 ± 0.74 mg/ml, 1.68 ± 1.27 mg/ml, 10.29 ± 4.08 mg/ml respectively. The compound ZAAK was isolated from ethyl acetate extract. Fourier Transformed-Infrared spectra revealed the presence of carboxylic acid and an ester in ZAAK. Total ion chromatogram of ZAAK revealed three major peaks corresponding to ZAAK₁ ZAAK₂ and ZAAK₃. The mass spectra identified ZAAK₁ ZAAK₂ and ZAAK₃ as 1-pentadecanecarboxylic acid, (Z)-11-Octadecenoic acid and octadecanoic acid, 2-(2-hydroxyethoxy) ethyl ester respectively.

CHAPTER ONE

1.0 INTRODUCTION

1.1

Medicinal Plants

Since   ancient   times,   people   have   been   exploring   nature   particularly   plants    in search  of  new  drugs.  This  has  resulted  in  the  use  of  large  number  of  medicinal  plants

with  curative  properties  to  treat				various  diseases  (Verpoorte,  1998).  Nearly  80%  of
the  world’s	population  relies		on	traditional	medicines  for  primary  health  care,  most
of  which  involve  the  use		of	plant  extracts		(Sandhya,  et  al.,  2006).  Medicinal  plants
are  the  richest  bio-resources  of				drugs  for  traditional  medicinal  systems,  modern
medicines,	nutraceuticals,	food		supplement,   pharmaceuticals   and   precursors   for
synthetic drugs (Hammer et al., 1999).

Secondary metabolites are responsible for the medicinal activity of medicinal plants, hence, due to their large biological activities. These metabolites have been used for centuries in traditional medicine. These secondary metabolites can be classified into three chemically distinct groups viz: alkaloids, terpenoids, and phenolics (Mazid et al., 2011).

1.1.1 Alkaloids

Alkaloids are naturally occurring chemical compounds containing one or more nitrogen atoms (usually in a heterocyclic ring) and are basic in nature (Evans, 2009). Many alkaloids are toxic and often have a pharmacological effect, which makes them to be used as medications and recreational drugs (Guillermo and Victor, 1999).

1.1.2 Phenolics

Plants have limitless ability to synthesize aromatic secondary metabolites, most of which are phenols or their oxygen-substituted derivatives (Geissman, 1963). Important subclasses in this group of compounds include phenols, phenolic acids, quinones, flavonoids, tannins and coumarins. These groups of compounds show antimicrobial effect and serves as plant defense mechanisms against pathogenic microorganisms. They are synthesized by plants in response to microbial infection (Dixon et al., 1983) and are often found effective in vitro as antimicrobial substance against a wide array of microorganisms (Bennet and Wallsgrove, 1994). They also show anti-allergic, anti-inflammatory and anticancer activity (Spencer and Jeremy, 2008).

1.1.3 Terpenoids

The terpenoids form a large and structurally diverse family of natural products. They are derived from five-carbon isoprene units, and according to the number of isoprene molecules incorporated, they can be classified into hemiterpenes (C₅), monoterpenes (C₁₀), sesquiterpenes (C₁₅), diterpenes (C₂₀), triterpenes (C₃₀), tetraterpenes (C₄₀), and polyterpenes such as rubber (Dewick, 2002). They occur widely in the leaves and fruits of higher plants, conifers, citrus and eucalyptus (Breitmaier, 2008). Vast majority of the different terpenes structures produced by plants as secondary metabolites are presumed to be involved in defense as toxins and feeding deterrents to a large number of plant feeding insects and mammals (Gershenzon and Croteau, 1991

1.2   Plants in Traditional Medicine

Plants have formed the basis of sophisticated traditional medicine (TM) practices that have been used for thousands of years by people in China, India, and many other countries (Sneader, 2005). They are important source of medicines, especially in developing countries that still use plant-based TM for their healthcare (Salim et al., 2008). It has been extensively documented that plants still form the bases of traditional medicine system and that plant based system continue to play an essential role in healthcare for over 80% of the world population (WHO, 2002).

1.3   Plants in Modern Medicine

Modern medicine has benefited enormously from plants used in traditional medicine as a source of natural products (Kinghorn, 1992). An estimate showed that over 50% of the best selling pharmaceuticals in use today are derived from or mimics of natural products (Newman and Cragg, 2007). In more recent history, the use of plants as medicines has involved the isolation of active compounds, beginning with the isolation of morphine 1 from opium poppy (Papaver somniferum) in the early 19th century (Samuelsson, 2004). Other early drugs from medicinal plants were also isolated due to drug discovery research of which some are still in use (Butler, 2004). Crude morphine (a pain reliever) was found to be readily converted to codeine 2 (painkiller) when boiled in acetic anhydride (Marderosian and Beutler, 2002). Digitoxin 3, a cardiotonic glycoside isolated from Digitalis purpurea L. (foxglove) enhances cardiac conduction (Marderosian and Beutler, 2002). The anti-malarial drug quinine 4 isolated from the bark of Cinchona succirubra has been used for centuries for the treatment of malaria fever (Marderosian and Beutler, 2002).

Fig 1.1 Chemical structure of earliest drugs developed from plant

Other drugs that were introduced into western medicine include an L-histidine-derived alkaloid, Pilocarpine 5 isolated from Pilocarpus jaborandi (Rutaceae) is used in the treatment of chronic open-angle glaucoma and acute angle-closure glaucoma (Aniszewski, 2007). Also, Vinblastine 6 and vincristine 7 both anti-neoplastic agents isolated from Catharanthus roseus (L.) G. Don (Apocynaceae) (formerly Vinca rosea L.) (Van Der Heijden et al., 2004) and artemisinin 8 isolated from Artemisia annua L. (Asteraceae)which is now a good remedy against the multidrug resistant strains of Plasmodium parasites (Namdeo et al., 2006). Galantamine 9, a natural product isolated from Galanthus woronowii Losinsk., (Amaryllidaceae) is now used in the treatment of Alzheimer’s disease (Pirttila et al., 2004). Medicinal plant derived natural products play a dominant role in drug discovery for the treatment of human diseases in modern medicine (Koehn and Carter, 2005).

6 R=CH3

7 R=CHO

Fig 1.2 Chemical structure of some medicinal natural products of plant origin

8                                                                                              9

Fig 1.2 Continued

Several plant natural products have made significant impact in cosmetics, perfumes and spices market. Few examples include arbutin 10 from Arctostaphylos uva-ursi Spreng for skin whitening and melanin-inhibiting, rutin 11 from Afrormosia laxiflora Harms for antioxidant and as emollient. Plant derived compounds of the terpenoid and phenolic types have also demonstrated their potentials as commercial sweetener (Kinghorn and Soejarto, 2002). Few examples include Hernandulcin [6-(1,5-dimethyl-1-hydroxy-hex-enyl)-3-methylcyclohex-2-enone] 12 from leaves and flowers of Lipia dulci Trev

(Verbanaceae), periandrin V (3β-O-(β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl)-25-al-olean-18(19)-en-30-oic acid) 13 from the rhizomes of Periandra dulci L. (Leguminosae) (Brazilian Licorice) are much sweeter than sucrose (Hashimoto et al., 1983). Other uses of plant extracts and plant products include pest control for agricultural purposes (Tewary et al., 2005; Asawalam et al., 2007).

Fig 1.3 Chemical structure of some natural products of plant origin in use as sweeteners and cosmetics

1.4 Impact of Technological Advancement in Medicinal Plant Research

A number of advances in capability and technology are fostering a renaissance in natural product research and directly or indirectly reducing the historical impediments to development of natural products (Schuster, 2001; McChestney et al., 2007). The advantage in methodologies for separation technologies such as High Performance Liquid Chromatography (HPLC) and countercurrent partition chromatography have further expanded the capacity for separation of plant chemical constituents (Pauli, 2006). Structure elucidation technology has improved especially with the development of high field NMR (Korfmacher, 2005; Phillipson 2007) allowsing rapid and straight forward structure elucidation. Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) are now being coupled with detectors in what is known as hyphenated techniques (GC-MS, LC-MS, LC-NMR, LC-MS-NMR etc.) this enable direct (online) identification of plants constituent prior to their isolation (Hostettman and Wolfende, 2004). Although the active principle isolated from plant may not necessary replace the plant extract (Phillipson, 2001), drugs are now discovered as a result of chemical studies directed at the isolation of the active substances from plants used in traditional medicine. Where the drugs do not possess the optimal properties for their use in human or animal medicine, they would be subjected to structure modification in order to improve their biological properties (Guthikonda et al., 1987).

1.5 Challenges in Medicinal Plant Research

Despite the great contribution of the plant kingdom, it has only been haphazardly investigated. A few plants have been exhaustively studied while many have not been studied at all. It has been estimated that only 5-15% of the approximately 350,000 species of higher plants have been systematically investigated for the presence of bioactive compounds (Cragg, et al., 1997). It is well known that some of these important sources of drugs are fast getting extinct due to many factors which include agricultural development, indiscriminate destruction of flora and so on (Rukangira, 2009). This is a result or growing trade demands for cheaper healthcare products and new plant-based therapeutic markets in preference to more expensive target-specific drugs and biopharmaceuticals. This habitat loss is the greatest immediate threat to biodiversity and as such it has stimulated positive legal as well as research interest in order to document the potential of these plants before they disappear completely or results in lose genetic diversity. Folk healers and their orally transmitted traditions are more vulnerable than medicinal plants themselves because many healers/indigenous knowledge holders aged and are dying or being killed in ethnic, religious or political crises with their knowledge left unrecorded. Traditional medicine is well known to be associated with secrecy (Sofowora, 2008). Families with this knowledge would like to keep their information and knowledge to themselves for fear of being marginalized in the race to exploit the commercial values of their medicine. Nowadays, younger generation show less interest in acquiring knowledge of plants from parents due to expansion of modern education and to some extent modern medicine (Weldegerima, 2009). This is another great challenge to drug discovery and development. Despite the long history of success in the application of natural products as drugs during the past couple of decades, research into natural products has experienced a steady global decline. This decline could be partly attributed to the introduction of high-throughput synthesis (HTS) and combinatorial chemistry (Eldridge et al., 2002)

1.6 Statement of Research Problem

Diabetes mellitus is a chronic metabolic disease which now afflicts 3% of the world population. Around 95 % of diabetic patients are diagnosed with type 2, diabetes (Attele et al., 2002). The disease is characterized by hyperglycemia. Prevalence of hyperglycemia has been on the increase, not only because of genetic factor/reason but also due to individual lifestyle, thus becoming one of the major causes of death especially in developing countries (Akinloye et al., 2013). It has been reported that long standing hyperglycemia with diabetes mellitus leads to the formation of advanced glycosylated end-products which are involved in the generation of reactive oxygen species, leading to oxidative damage, particularly to heart and kidney (Rolo and Palmeira 2006). The disease has also been reported to be associated with disturbances in learning, memory, and cognitive skills in the diabetic patients (Akram, 2013). Estimates have shown that at least 150 million people world-wide have diabetes, of which two-thirds live in developing countries (Salisu et al., 2009) such as Nigeria. Based on World Health Organization (WHO) report, the number of diabetic patients is expected to increase from 171 million in year 2000 to 366 million or more by the year 2030 (Wild, et al., 2004). It appears that available treatments, including attempts at lifestyle alterations and drug therapies including insulin, are insufficient to stem the tide.

1.7 Justification

Current oral anti-diabetic agents such as insulin releasers, insulin, sensitizers and α-glucosidase inhibitors, have modest efficacy and limited of modes of action. In addition, current anti-diabetic drugs usually have adverse side effects, decreased efficacy over time, ineffectiveness against some long-term diabetic complications and low cost-effectiveness (Grover et al., 2002). One   therapeutic   approach    for   treating   diabetes   is    to    decrease   the     postprandial

hyperglyceamia		(Kwon	et  al.,	2007)  which		can  be		achieved  by		retarding  the
absorption	of	glucose	through	the	inhibition	of	the	carbohydrate		hydrolyzing
enzymes  such  as  α-amylase  and				α-glucosidase  in  the			digestive  tract,		bringing		about
delay  in	carbohydrate		digestion	and	prolongs	overall	carbohydrate		digestion		time,

causing a reduction in the rate of glucose absorption and consequently blunting the postprandial plasma glucose rise (Ranilla et al., 2010). Varieties of therapeutic drugs such as Acarbose, Voglibose and Miglitol, all synthetic inhibitors of microbial origin are available for the management of postprandial hyperglycaemia but are not cost

 

effective

and

produce  adverse

side

effects  such

as

abdominal

distention,

flatulence

and  diarrhea  resulting  from  excessive  inhibition

of

pancreatic

α-amylase.

Although,

food  grade  α-amylase  inhibitors  from  dietary  plant  extracts

are

potentially

safer  and

may  be

a  preferred

alternative

for

modulation  of

carbohydrate

digestion

and

control

of  glycaemic  index  (McCue  et  al.,  2005).  Hence,  a  good

strategy

to

managing

diabetes

with

lesser

side  effects

is

to  identify

the

natural

inhibitors

from

diatery

 

sources having mild inhibitory effect on α-amylase and strong inhibitory activity α-glucosidase.

 

11

 

Literature   search    have    shown    that    G.   kola     seeds    have    anti-diabetic    property.

 

However,  there  is   dearth  of  information   concerning   the  biochemical   pathway  and    the

 

bioactive  constituents  that		maybe  responsible  for  this  activity.  Therefore,  identifying
the   possible	biochemical	pathway   and   the	bioactive	constituents	that	maybe
responsible  for	the  reported	anti-diabetic  property	of  G.  kola	seeds  could	be  of	value

 

in the control of the disease with safe and effective drug of natural product origin

1.8   Aim and Objectives of the Research

1.8.1 Aim of research

To characterize the bioactive constituents from Garcinia kola seeds and evaluate its inhibitory activity on some key enzymes related to diabetes.

1.8.2 Objectives of research

1. To extract and isolate bioactive constituent(s) from Garcinia kola
2. To identify the constituents via spectroscopic means

• To evaluate the inhibitory effects of methanol, ethyl acetate and n-hexane extracts on glycosidase (α-amylase and α-glucosidase) enzymes

1.9 Hypothesis

Garcinia kola seeds contain bioactive constituents with inhibitory activity on glycosidase (α-amylase and α-glucosidase) enzymes related to diabetes

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