ABSTRACT
Dried and pulverised leaves of A. boonei De Wild (Apocynacea) were extracted using methanol for 48 h. The methanol extract obtained was defatted using n-hexane and fractionated using ethylacetate. The ethyl acetate fraction of the extract was subjected to vacuum liquid chromatography (VLC) in silica gel using gradients of hexane-ethyl acetate. The VLC fractions were further separated on Sephadex LH-20. A total of 10 compounds were successfully isolated and purified using the reverse phase semi preparative HPLC (L-7100, Merck/Hitachi). The
structures of these compounds were determined using UV, HPLC-MS, one-dimensional: 1H, 13C
and DEPT NMR, and two-dimensional: 1H1H COSY, HMQC, and HMBC NMR. The antioxidant properties were assessed using the 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test. The isolated compounds were also subjected to anti-microbial studies using Agar well diffusion technique against the organisms: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Based on the spectral data, the isolated compounds were identified as: quercetin-3-O- [α-L-rhamnopyranosyl(1→6) β-D- glucopyranoside] (1); quercetin-3-O- [α-L-rhamnopyranosyl(1→6) β-D-galactopyranoside] (2); kaempferol-3-O-[α-L-rhamnopyranosyl(1→6) β-D-glucopyranoside] (3); kaempferol-3-O-[α -L- rhamnopyranosyl(1→6) β-D-galactopyranoside] (4); quercetin-3-O-[α -L- rhamnopyranosyl(1→4) β-D-glucoctopyranoside] (5); kaempferol-3-O-[α -L- rhamnopyranosyl(1→4) β-D-glucopyranoside] (6); quercetin-3-O-[α -L-rhamnopyranosyl(1→2) β-D-glucopyranoside] (7); quercetin-3-O-[α -L-rhamnopyranosyl(1→2) β-D-galactopyranoside] (8); chlorogenic acid (9) and 4,5-dicaffeoylcinnamic acid (10). Compounds 1, 2, 5, 7,
8 (derivatives of quercetin) and 9, a caffeic acid derivative, showed a dose dependent antioxidant activity on DPPH free radical scavenging model with IC50 values of 52, 48, 36, 66, 56 and 22 μg/mL respectively. The three kaempferol derivatives (3, 4 and 6) showed poor anti-oxidant activity (IC50 >100 μg/mL). This suggests that the presence of at least two ortho coupled OH groups in RING B of the flavonoid nucleus is necessary for a good antioxidant activity. Compounds 7 and 8 were active against Escherichia coli with MIC values of 1.77 μg/mL and
1.92 μg/mL respectively. The profound antioxidant activity of the isolated quercetin derivatives and chlorogenic acid may explain the ethnomedicinal use of the leaf extract in the management of inflammatory disorders. These groups of compounds isolated could be very useful for SAR studies as well as other studies on the effects of glycosyl substitution patterns on chemical shifts of the ring carbons of flavonoid nuclei.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Medicinal Plants
Over the centuries humans have relied on plants for basic needs such as food, clothing, and shelter, all produced or manufactured from plant matrices (leaves, woods, fibers) and storage parts (fruits, tubers). Plants have also been utilized for additional purposes, namely: arrow and dart poisons for hunting, poisons for murder, hallucinogens used for ritualistic purposes, stimulants for endurance, and hunger suppression, as well as inebriants and medicines (Salim et al., 2008).They have also formed the basis of sophisticated Traditional Medicine (TM) systems that have been in existence for thousands of years and continue to provide mankind with new remedies (Barboza et al., 2009). They provide a large bank of rich, complex and highly varied structures which are unlikely to be synthesized in laboratories (Newman, 2008).
Some of the oldest known medicinal systems of the world such as Ayurveda of the Indus civilization, Arabian medicine of Mesopotamia, Chinese and Tibetan medicine of the Yellow River civilization of China and Kempo of the Japanese are based mostly on plants. These ancient cultures are known for their systematic collection of information on herbs and their rich and well-defined herbal pharmacopoeias. Although some of the therapeutic properties attributed to plants have proven to be erroneous, medicinal plant therapy is based on the empirical findings of hundreds and thousands of years (Gurib Fakim, 2006).
A medicinal plant is therefore defined as (1) any plant used in order to relieve, prevent or cure a disease or to alter physiological and pathological process, or (2) any plant employed as a source of drugs or their precursors (Arias, 1999). A phytopharmaceutical preparation or herbal medicine is any manufactured medicine obtained exclusively from
plants (aerial and non-aerial parts, juices, resins and oil), either in the crude state or as a pharmaceutical formulation (Rates, 2001). Phytotherapy is referred to as the study of the use of plant extracts from natural origin as medicines or health-promoting agents. The main difference between phytotherapy medicines and the medicines containing the herbal elements lies in the methods of plants processing. The preparation of medicines containing herbal elements involves the extraction of the chemically clean active substances, while in the case of phytotherapy medicines all complex active substances of plant are incorporated in the crude natural form. Phytotherapeutic medicines do not include drugs from medicinal plants made for homeopathy, anthroposophic medicine, as well as non-standardized mixture of plant and synthetic bioactive substances or isolated in a pure form from natural bioactive substances.
There is ample archaeological evidence indicating that medicinal plants were regularly employed by people in prehistoric times. In several ancient cultures botanical products were ingested for biomedically curative and psychotherapeutic purposes (Halsberstein, 2005). However, medicinal plants are constrained by procedures such as classification, identification, and characterization. Nearly 50,000 species of higher plants have been used for medicinal purposes. In systems of traditional healing, major pharmaceutical drugs have been either derived from or patterned after compounds from biological diversity (Bisset, 1994).
Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend themselves against attack from predators such as insects, fungi and herbivorous mammals. Many of these phytochemicals have beneficial effects on long-term health when consumed by humans,
and can be used to effectively treat human diseases. The beneficial medicinal effects of these plant materials typically result from the combinations of secondary products present in the plant making the medicinal actions of plants unique to particular plant species or groups. As a result, the combinations of secondary products in a particular plant are often taxonomically distinct (Kaufman et al., 1999).
Phytochemicals are actually divided into (1) primary metabolites/products such as sugars and fats, which are found in all plants; and (2) secondary metabolites/products – compounds which are found in a smaller range of plants, serving a more specific function (Meskin and Mark, 2002). For example, some secondary metabolites are toxins used to deter predation and others are pheromones used to attract insects for pollination. It is these secondary metabolites and pigments that can have therapeutic actions in humans and which can be refined to produce drugs – examples are inulin, a naturally occurring form of fruit sugar, extracted from dahlia root tubers (Williams, 1895), quinine from the cinchona, morphine and codeine from the poppy, and digoxin from the foxglove (Meskin and Mark, 2002). Chemical compounds in plants mediate their effects on the human body through processes identical to those already well understood for the chemical compounds in conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects (Briskin,
2000; Lai and Roy 2004; Tapsell et al. 2006). In contrast to synthetic pharmaceuticals based upon single chemicals, many phytomedicines exert their beneficial effects through the additive or synergistic action of several chemical compounds acting at single or multiple target sites associated with a physiological process. This synergistic or additive
pharmacological effect can be beneficial by eliminating the problematic side effects associated with the predominance of a single xenobiotic compound in the body (Tyler, 1999).
The development of ethnomedicine is usually based on information transferred from generation to generation among peoples in rural societies. This knowledge is often acquired through trial and error methods, and is majorly based on speculation and superstition. For instance, it is common knowledge that plants are more likely to survive if they contain potent compounds which deter animals from eating them. This helps the plants to survive in the midst of an adverse environmental condition, stress and competition or to overcome a prevalent infection destroying other plants around. The pharmaceutical and biotechnological industries are much interested in using this knowledge for the discovery, development and application, within biodiversity, of new active products on health and new genes with properties for food improvement (Heinrich & Gibbons, 2001). Chemical analyses and biological assays have begun to play an important role in ethnobotanical studies and there are now numerous examples where scientific analyses have provided objective evidence to validate traditional plant use, for example Homalanthus nutans (G. Forst.) Guill. (Euphorbiaceae), used by Samoan healers against the viral disease yellow fever; extracts have been found to exhibit potent antiviral activity, particularly against the human immunodeficiency virus HIV-1 (Balick & Cox, 1996). This however does not rule out that a lot of claims in ethnomedicine are baseless. Tyler listed some common fallacies including claims that there is a conspiracy to suppress safe and effective herbs, herbs cannot cause harm, that whole herbs are more effective than molecules isolated from the plants, herbs are superior to drugs, the doctrine of signatures (the belief that the shape of the plant indicates its function) is valid, dilution of substances increases their potency (a doctrine of the pseudoscience of homeopathy), astrological alignments are significant, animal testing is not
appropriate to indicate human effects, and that anecdotal evidence is an effective means of proving a substance works. Tyler believes that none of these beliefs have any basis (Tyler and Robbers, 1999; Tyler, 2012).
As new uses of medicinal plants are discovered and popularized, the concern for sustainability is being increasingly addressed; concern over the growth in biopiracy also combines with the critical need for the conservation of species and their habitat (Science Reference Services, 2008). A 2008 report from the Botanic Gardens Conservation International (BGCI) (representing botanic gardens in 120 countries) warned that “cures for things such as cancer and HIV may become ‘extinct before they are ever found’.” They identified 400 medicinal plants at risk of extinction from over-collection and deforestation, threatening the discovery of future cures for disease. This means that complementary medicinal plant/tree-planting exercises of these endangered plant species are necessary in order to preserve them.
The search for new molecules, nowadays, has taken a slightly different route where the science of ethnobotany and ethnopharmacognosy are being used as guide to lead the chemist towards different sources and classes of compounds (Gurib-Fakim, 2006). Plant derived natural products hold great promise for discovery and development of new pharmaceuticals (McChesney et al., 2007). Since ancient times, medicinal plants have been harvested from the wild (Mshigeni et al., 1991; Balick & Cox, 1996; Sheldon et al., 1997; Dhillion & Ampornpan, 2000; Singh & Padmalatha, 2004). In many rural communities, traditional medicine (TM) is still viewed as the mainstay of primary health care systems (Bannerman et al., 1983; Manandhar, 1994; Svarstad & Dhillion, 2000; Manandhar, 2002) due to its
effectiveness, cultural preference or absence of modern alternatives (Plotkin & Famolare,
1992; Taylor et al., 1995; Balick et al., 1996; Tabuti et al., 2003).
1.2 Phytochemistry in Drug Developement
Despite the recent interest in drug discovery by molecular modelling, combinatorial chemistry, and other synthetic chemistry methods, natural-product-derived compounds are still proving to be an invaluable source of medicines for humans (Salim et al., 2008). Plants remain rich sources of lead compounds (e.g. alkaloids such as, morphine, cocaine, digitalis, quinine, tubocurarine, nicotine, and muscarine). Many of these lead compounds are useful drugs in themselves (e.g. the alkaloids, morphine and quinine), and others have been the basis for synthetic drugs (e.g. local anaesthetics developed from cocaine). In 1805, morphine became the first pharmacologically active compound to be isolated in pure form from a plant, although its structure was not elucidated until 1923 (Sneader , 2005). The 19th century marked the isolation of numerous alkaloids from plants used as drugs, for instance, atropine (Atropa belladonna), caffeine (Coffea arabica), cocaine (Erythroxylum coca), ephedrine (Ephedra species), morphine and codeine (Papaver somniferum), pilocarpine (Pilocarpus jaborandi Holmes), physostigmine (Physostigma venenosum), quinine (Cinchona cordifolia Mutis ex Humb.), salicin (Salix species), theobromine (Theobroma cacao), theophylline (Camellia sinensis), and (+)-tubocurarine (Chondodendron tomentosum Ruiz & Pav.) (Sneader, 2005). Following these discoveries, bioactive secondary metabolites from plants were later utilized more widely as medicines, both in their original and modified forms (Sneader, 1996; Samuelsson, 2004).
Although relatively few plant-derived drugs have been launched into the market over the last few years, many plant-derived compounds are currently undergoing clinical trials for the
potential treatment of various diseases. The majority of such drugs under clinical development are in the oncological area, including new analogs of known anticancer drugs based on the camptothecin-, taxane-, podophyllotoxin-, or vinblastine-type skeletons (Butler,
2005).
Plants as sources or starting points of drugs is associated with some advantages, for instance, the selection of a candidate species for investigations can be done on the basis of a long-term use by humans (ethnomedicine). This approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use. On the other hand, more often than not, drug discovery and eventual commercialization would pressurize the resource substantially and might lead to undesirable environmental concerns. While synthesis of active molecule could be an option, not every molecule is amenable for complete synthesis. Hence, certain degree of dependence on the lead resource would continue. For instance, anticancer molecules like etoposide, paclitaxel, docetaxel, topotecan, and irinotecan continue to depend upon highly vulnerable plant resources for obtaining the starting material since a complete synthesis is not possible (Katiyar et al., 2012). One major challenge in drug development from plants remains that it is time consuming and very costly. The process of identifying the structures of active compounds from an extract could take weeks, months, or even years, depending on the complexity of the problem. Nowadays, the speed of bioassay-guided fractionation has been improved significantly by improvements in instrumentation such as high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS)/MS (liquid chromatography, LC-MS), higher magnetic field-strength nuclear magnetic resonance (NMR) instruments, and robotics to automate high-throughput bioassays (Salim et al., 2008). Screening of plant extract libraries can be problematic due to the presence of compounds that may either
autofluoresce or have UV absorptions that interfere with the screen readout, but fractionation of extracts can be used to alleviate some of these types of problems. Also, most high- throughput screening assay methods have been developed with computational filtering methods to identify and remove potentially problematic compounds that can give false- positive results (Walters and Namchuk, 2003).
1.3 Folkloric Uses of Alstonia boonei
Fresh leaves, stem bark and root bark extracts of Alstonia boonei de Wild have been used for centuries now in various parts of the northern tropical Africa and some parts of Asia for the treatment of various ailments ranging from malaria to inflammatory diseases as well as hypertension. Plants with such a wide range of validated folkloric use are expected to contain compounds which show good antioxidant properties. Alstonia boonei , especially the leaves has, however not been fully chemically investigated in order to identify the bioactive compounds.
1.4 Statement of Problem
Plants have provided humans with many of their essential needs, including life-saving pharmaceutical agents. In the last few years, some new plant-derived drugs have been launched onto the market, and many more are currently undergoing clinical trials. As a vast proportion of the available higher plant species have not yet been screened for biologically active compounds, drug discovery from plants should remain an essential component in the search for new medicines, particularly with the development of highly sensitive and versatile analytical methods (Salim et al., 2008).
1.5 Aims and Objectives of the Study
The aim of the study is to isolate and characterise the principles/constituents of the ethyl acetate fraction of Alstonia boonei de Wild leaves and trace the principles to some known pharmacological effects especially those of folkloric origin. This will enhance further studies and drug development on them. To achieve this, the following specific objectives will be pursued:
I. Extraction and fractionation to obtain the ethyl acetate fraction of the leaves of
Alstonia boonei de Wild.
II. Isolation of the constituents by vacuum column chromatography, separation and purification using HPLC.
III. Structure elucidation of the isolated constituents by a combination of UV, HPLC- EIMS, 1D 1H-NMR, COSY, HMBC, HMQC, 13C-NMR, and DEPT analyses.
IV. Screening of the isolated constituents for antimicrobial and antioxidant effects using established in vitro models.
This material content is developed to serve as a GUIDE for students to conduct academic research
CHARACTERISATION OF THE COMPOUNDS OF THE ETHYLACETATE EXTRACT OF THE LEAVES OF ALSTONIA BOONEI DE WILD AND THEIR ANTIOXIDANT AND ANTIMICROBIAL POTENTIALS>
PROJECTOPICS.com Support Team Are Always (24/7) Online To Help You With Your Project
Chat Us on WhatsApp » 07035244445
DO YOU NEED CLARIFICATION? CALL OUR HELP DESK:
07035244445 (Country Code: +234)YOU CAN REACH OUR SUPPORT TEAM VIA MAIL: [email protected]