PHYTOCHEMICAL AND ANTIBACTERIAL STUDIES OF Ensete gilletii ROOT EXTRACTS

ABSTRACT

Ensete gilletii commonly called wild banana, is ethno-medicinally used for the treatment of diarrhea, dysentery, typhoid, stomach pain, kidney stone and liver disorders by herbal practitioners. The dried pulverized roots of the plant were subjected to exhaustive, successive soxhlet extraction using petroleum ether, chloroform and methanol. The result revealed that a higher percentage yield was obtained from the methanol extract (MEG). The GC-MS analysis of the petroleum ether crude (PEG) extract revealed about eighty compounds which are mainly terpenes and hydrocarbons, while the chloroform crude (CEG) extract showed twenty one compounds. The phytochemical screening of the root (PEG, CEG and MEG) extracts revealed the presence of flavonoids, phenols, saponins, tannins, terpenoids, glycosides and sterols. TLC chromatograms show that sub-fraction PEG3 have a better resolved profile with the solvent system; Hex:EtOAc; 5:1 (Rf 0.45, 0.7). A compound was isolated from sub-fraction PEG3c; the isolated compound  (PEG3c1)  was  characterized  using  physical,  chemical  and  spectroscopic techniques. The antibacterial activities of the extracts were evaluated by the disc diffusion method against selected organisms, in comparison with standard reference (ampicillin). The extracts were active against Salmonella typhii, Escherichia coli, Klebsilla pneumoniae, Shigella dysentriae and Staphylococcus aureus with zones of inhibition ranging from 16 mm to 28 mm when compared to a standard drug. MIC and MBC values were 40 mg/cm3 and 80 mg/cm3 respectively. The results confirmed the ethno-medicinal claims on the plant and could be used as a lead to the discovery of potent medicaments.

CHAPTER ONE

1.0  INTRODUCTION

1.1 Background to the Study

1.1.1 Importance of plants

Plants are living organisms of the kind exemplified by trees, shrubs, grasses, vines, ferns and mosses, usually absorbing water, and inorganic nutrients through its roots (Merriam-Webster,

2009). They are either wild plant species or domesticated wild species that grow spontaneously in self-maintaining populations, natural or semi-natural ecosystems and could exist independently of constant human activity. Domesticated plants species are those that spring up through human actions such as selection or breeding and depend on management for their occurrence (Calixto, 2000). Green plants obtain most of their energy and food from sunlight through the process of photosynthesis by primary chloroplasts that are derived from endosymbiosis with cyanobacteria. The leaves of plants vary in shapes, colour and size (Ahn, 2017). Green plants provide a significant proportion of the world’s molecular oxygen and are the basis of most of earth’s ecosystems, particularly on land.

Plants yield grains, tubers, fruits and vegetables, form essential foods for both humans and animals and have been made a part of domestic use over the years. Plants have many cultural uses and are also used as ornaments, building materials, writing materials and in great variety, they have been the source of medicines and drugs (Samuelsson, 2004). They produce oxygen in exchange of carbon (IV) oxide, and aid in air purification and also prevent direct ultra-violet rays from reaching the earth surface. They are the primary habitat for thousands of animals and microorganisms. Plants help moderate the temperature and generate shelter from sun, rain, storm and wind. Many species of plants are explored locally as a source of traditional herbs and in certain spiritual activities (Summer, 2000). They are also important in synthesis of modern drugs.

1.1.2 Medicinal uses of plants

A medicinal plant is a plant which one or more of its organs, contain substances that can be used for therapeutic purposes or which are precursors for chemo-pharmaceutical semi-synthesis (WHO, 2005). For centuries, plants have been used for medicinal purposes (Samuelsson, 2004). In recent years, the use of plants as a source of medicine has become an increasingly incorporated into scientifically based system of healing. The increasing demand on natural products has improved studies into the scientific basis and therapeutic abilities of plants, allowing herbal practitioners gain acceptance among the medical sector (Karunamoorth et al., 2012). The medicinal uses of plants were formulated through observations of animals and by trial, error, failure and eventually successful (RamGopal, 2006). Medicinal plants are regarded as rich sources of components which can be used in drug manufacturing. In recent time, plants are found in diverse medicinal applications ranging from; the production of medicines to cosmetics, herbal drinks and food supplements and other items in daily uses. Herbs are found to have significant role particularly in modern time industries. The system is based on the belief that plants have the vast ability for curing and improving health when used as medicine and as herbal food products (Goyal, 2008; Rajakumar, 2014). The therapeutic properties of plants could be based on their anti-oxidant, anti- microbial, anti-pyretic, anti-inflammatory and anti-diabetic effects of the phytochemical constituents present in them (Adesokan et al., 2008).

Hasler (2005), had earlier stated that herbs which have supporting, enhancing or curative potentials should be added to food products in order to improve their medicinal effects. Scientific value of plants is on the rise, resulting from microbes such as; virus, bacteria and fungus originated diseases which are widespread and most organisms have developed resistance against synthetic drugs, thereby increasing the need for a more effective, reliable and natural remedy (Gurib-Fakim, 2006; WHO, 2018). According to World Health Organization, medicinal plants would be the greatest source for obtaining an array of drugs. Therefore, potentially active plants should be studied to better understanding of their properties, safety practices in addition to usefulness (Nascimento et al., 2013).

Most plants are regarded as important source of nutrition and soothing effect, as a result, they are recommended for their therapeutic values. Some of these plants include; ginger, green tea, garlic, cinnamon, walnut, aloe vera and turmeric to mention a few. Some plants and their derivatives are considered as important source for active ingredients which are used in aspirin, toothpaste, mouth- wash, body-wash, soap and in other herbal products formulation (Tarbuti et al., 2003). According to several documented reports, many drugs listed as conventional medications were originally obtained from plants. Medicinal plant therapy is based on the empirical findings of hundreds of years of use. Staniszewska et al. (2003) reported that Cupressus sempervirens (cypress), Glycyrrhiza  glabra  (licorice),  Commiphora  species  and  Papaver  somniferum  (poppy juice), among others are used for the treatment of ailments ranging from coughs and colds to parasitic infections and inflammation. The interest in nature as a source of potential chemotherapeutic agents by both local and foreign researchers is on the increase. Cragg and Newmann (2005) had earlier recorded that 50% of drugs in clinical use were produced from natural products and their derivatives.

Plants synthesize hundreds of chemical compounds for several functions, including; defense against insects, fungi, diseases and herbivorous mammals (Tapsell et al., 2006). Several phytochemical constituents with potentials or confirmed biological activities have been identified over the years, mainly through ethno-botanical studies. Medicinal plants are widely used in non- industrialized societies and rural communities, mainly because they are readily available and cheaper with few side effects compared to modern synthetic medicines. In Africa, the practice of herbal medicine is well known and established; as a result, most of the plants that are used for medicinal and other purposes come from rural areas due to the existing knowledge of herbalist. Majority of the claims by local practitioners have been proven to be true, while; some are still under review.  The annual global export value of 50,000 to 70,000 types of plants suspected to contain  medicinal properties was estimated to be 2.2 billion US$  in 2012, while in 2017, the potential global market for botanical extracts and medicines was estimated at several hundred billion dollars (Ahn, 2017; Medicinal and Aromatic, 2017).

1.1.3 Plant constituents

Plants produce a vast number of chemical compounds that are known as secondary metabolites

/natural products/phytochemicals/phytoconstituents/bioactive compounds. Secondary metabolites are the chemicals that are not required for the direct survival of plant, but are synthesized to increase adaptation of the plant by protecting it from pathogens, herbivores, insects and other environmental factors (Kennedy and Wightman, 2015; Shalini and Shampathkumar, 2012). Phytochemicals accumulate in different parts of plant, such as; the root, stem, bark, leaves, fruits and seeds. Plant natural products belong to various classes of compounds, including; isoprenoids, phenylpropanoids, triterpenoids, alkaloids, glycosides, tannins, flavonoids, saponins, essential oils and other related constituents which exert physiological actions in addition to the carbohydrates, proteins and lipids that are often found as conjugated products of compounds derived from different biosynthetic pathways (Gurib-Fakim, 2006). Most of plant secondary metabolites are therapeutically active, while others are inactive. In recent time, the active constituents responsible for medicinal actions of plants have been screened, studied, observed, isolated and characterized. Phytochemicals (from the Greek word phyto, meaning plant) are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those ascribed to macronutrients and micronutrients (Azene et al., 2016). Several plants have been reported to contain phytochemicals which are responsible for their medicinal values. Salicylic acid, a precursor of aspirin, was originally derived from white willow bark and the meadowsweet plant. Cinchona bark is the source of malaria-fighting quinine (Manuchair, 2002). It was recorded that most of the major anticancer drugs are natural products, either from plants or micro-organisms, such as; Bleomycin, Doxorubicin, Vincristine, Vinblastine paclitaxel (Taxol), Ironotecan (a camptothecin derivative), Etoposide and Tenoposide (Manuchair, 2002; Gurib-Fakim, 2006). Herbal researchers believe that plant medicinal effects in general are the sum of their constituents and cannot be reduced to just a specific compound alone. Researches has proven that medicinal plants contain chemicals that have been sub-divided into fourteen main groups; alkaloids, anthocyanins, anthraquinones, cardiac glycosides, coumarins, cynogenic glycosides, flavonoids, glucosilinates, minerals,  phenols, saponins, tannins, vitamins and volatile oils (Taiz and Zeiger, 2006).  The active plant constituents are usually classified by their chemical structure rather than their effect.

Even though compounds found in plants are of many kinds but most are in four major bio- chemical classes; alkaloids, glycosides, polyphenols and terpenes (Kennedy and Wightman, 2015).

1.1.4 Preliminary extraction of phytochemicals using different methods

Extraction is the separation of medicinally active constituents of plant tissues using selective solvents through standard procedures. The extracts obtained from plants are relatively complex mixtures of metabolites which are usually in liquid, semisolid, solid state or in dry powder form, after evaporation of the solvent (Prashant et al., 2011). An important factor that controls the choice of solvents used in an extraction is the type of phytochemical constituents that are to be extracted (Handa et al., 2008). Several methods adopted for plant extraction include; maceration, infusion, percolation, digestion, decoction, hot continuous extraction (soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, and phytonic extraction (with hydrofluorocarbon solvents). For aromatic plants, hydrodistillation techniques (water distillation, steam distillation, water and steam distillation), hydrolytic maceration followed by distillation, expression and enfleurage (cold fat extraction, fragrance extraction) may be employed. Some of the recent extraction methods for aromatic plants include headspace trapping, solid phase micro- extraction, protoplast extraction, microdistillation, thermomicrodistillation and molecular distillation (Bimakr, 2010; Handa et al., 2008; Prashant et al., 2011).   Most extraction methods can be direct, which involves bringing the plant material in contact with the solvent for a period of time; or serial which involves the use of many solvents of varying polarities successively on the same material. Soxhlet extractor can be very useful for the exhaustive and successive extraction of plant constituents with various solvents, but this cannot be used for thermolabile or degradable compounds. This challenge may be overcome by extracting under reduced pressure and temperature (Ncube et al., 2008; Nikhal et al., 2010). Several researchers have used various organic and inorganic solvents while extracting constituents from plant samples.

1.2 Statement of the Research Problem

The spread of diseases caused by microorganisms is a major concern in the pharmaceutical world as thousands of people suffer from persistent microbes originated ailments, owing to the fast increase of bacteria resistance to the common antibiotics, which has raised the need for alternative active compounds from plants in order to treat the bacterial infections (Akinpelu et al., 2008; WHO, 2018). Resistance to medications are emerging and spreading globally, threatening the use of synthetic drugs in the treatment of common bacterial infectious diseases, resulting into prolonged illness, disability and sometimes death. The use of synthetic drugs, though effective is often associated with side effects which cannot be ignored as there is a continuous increase in reported relapse resulting from the synthesized components. Synthetic drugs can be relatively expensive, rare in local communities in comparison with natural products; such as plants (Karunamoorth et al., 2012).

1.3 Justification of the Study

Studies have shown that natural products are sources of potent antibacterial drugs and many researchers are compelled to further investigate the biological activities of various medicinal plants. The use of herbal medicines for the treatment of infections is popular, especially in rural communities due to their availability, effectiveness, little or no side effects.

The ever increasing need for the discovery of new and effective means for the control and cure of various ailments has become a global concern, so that the search for natural cure and control of diseases have increased. The roots of Ensete gilletii is traditionally used for the treatment of bacterial infections, such as; diarrhea, dysentery, stomach ache and infertility. Extensive literature search have revealed no much information on the isolation and characterisation of phytochemical constituents as well as investigation of the ethno-medicinal antibacterial claim on roots of Ensete gilletii. Therefore, this study was undertaken.

1.4 Boundaries of the Study

The present study focuses on sequential extraction of phytochemicals of Ensete gilletii roots using three different organic solvents; petroleum ether, chloroform and methanol. The phytochemical constituents of the three extracts will be screened, for their antibacterial potentials.

1.5 Aim and Objectives of the Study

Aim

This research work is focused on the phytochemical, characterisation of chemical constituents and antibacterial studies of Ensete gilletii root extracts.

Objectives

In order to achieve the aim of this study, the following objectives were undertaken:

i.   Quantitative screening of the pulverized sample using standard procedures.

ii.  Successive extraction of phytochemicals from the dried roots of Ensete gilletii using petroleum ether, chloroform and methanol by the continuous extraction soxhlet) method.

iii. Preliminary antibacterial screening of the crude extracts using standard methods. iv. Qualitative screening of the crude extracts using standard methods.

v.   Isolation and purification of one or more phytochemical(s) from the extract with promising spots using chromatographic techniques.

vi. Structural  elucidation  of  the  isolated  compound(s)  using  physical,  chemical  and spectroscopic techniques.

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