CHARACTERISATION OF THE COMPOUNDS OF THE ETHYLACETATE EXTRACT OF THE LEAVES OF ALSTONIA BOONEI DE WILD AND THEIR ANTIOXIDANT AND ANTIMICROBIAL POTENTIALS

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.

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