THE ANTIMICROBIAL ACTIVITY OF THE CRUDE METHANOL EXTRACT AND FRACTIONS OF ALBIZIAZYGIA STEM- BARK.

CHAPTER ONE

1.0 INTRODUCTION

Plants had been used for medicinal purposes long before recorded history. Ancient Chinese and Egyptian papyrus writings describe medicinal uses for plants as early as 3000BC. Indigenous cultures(such as African and Native Americans) used herbs in their healing rituals, while others developed traditional medical systems in which herbal therapies were used. Researchers found that people in different parts of the world tended to use the same or similar plants for the same purposes.Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses (Pretty,2011).

Herbal medicines include herbs, herbal materials, herbal preparations, and finished herbal products that contain parts of plants or other plant materials as active ingredients. (W.H.O., 2008).Recently, the World Health Organization estimated that 80% of people worldwide rely on herbal medicines for some part of their primary health care. The remaining 20% of individuals living in industrialized countries uses, in at least 25% of cases, pharmaceuticals which have been directly derived from plant products. The use of medicinal plants in complementary and alternative medicine has seen a great increase in recent years especially in Asia and Europe. One of the main reasons for the increasing use of traditional medicine is a growing trend for patients to take a more proactive approach to their own health and to seek out different forms of self-care. In the process, many consumers have turned to natural traditional medicinal products and practices, under the assumption that “natural means safe”(W.H.O, 2004). A vast family of plant kingdom has been explored from time immemorial for their medicinal values. These include: Euphorbiaceae, Fabaceae, Rubiaceae, Apocynaceae, Solanaceae, Sterculiaceae and many others. Many of these plants used as medicine were derived from ethnomedicinal plant sources.Plants have diverse pharmacological roles they play in the health of people, these pharmacological roles have depended on the occurrence of varieties of chemical components in the plants (Duke and Wain, 1981). Medicinal plants are sources of a number of novel chemical compounds(Boret al., 1998). The metabolites that have effects on human health are known as phytochemicals. Phytochemicals are compounds derived from plants which possess vast biological properties. They are divided into two major types: primary and secondary plant metabolites. Examples of primary plant metabolites include: carbohydrates, amino acids, fatty acids, lipids, steroids and phenolics.Secondary metabolites are responsible for the medicinal effects of plants (Deyu, 2007).Examples of such chemicals include alkaloids, flavonoids, saponins, phenol compounds, steroids and proteins (Negemet al., 1980)

Significance of Medicinal Plants to Man

Many of medicines today are produced indirectly from medicinal plants. For example, reserpine from Rauwolfiavormitoria, Digoxin from Digitalislanata.

Plants are directly used as medicine by a majority of cultures around the world. For example, Chinese medicine and Indian medicine.

Many food crops have medicinal value, hence are sold to generate income. For example, cocoa.

Medicinal plants are sources for new drugs. It is estimated that there are more than 250,000 flowering plant species of important medicinal value.

Studying medicinal plants helps to understand plant toxicity and protect human and animals from natural poison.

Cultivation and preservation of medicinal plants protect biological diversity, for example metabolic engineering of plants.

The study of medicinal plants has created an essential area for further studies and research, and the discovery of new lead compounds for drug development (Verpoorte, 2009).

1.1 FAMILY: FABACAEAE

The Fabaceae or Leguminosae commonly known as the legume, pea, or bean family, are a large and economically important family of flowering plants. It includes trees, shrubs and herbaceous plants perennials or annuals, which are easily recognized by their fruits (legume) and their compound, stipulated leaves. The group is widely distributed and is the third-largest land plant family in terms of number of species, behind only the Orchidaceae and Asteraceae, with 730 genera and over 19,400 species (Judd et al., 2002). The largest genera are Astragalus(over 2,400 species), Acacia (over 950 species), Indigofera(around 700 species), Crotalaria (around 700 species), and Mimosa (around 500 species), which contain around 9.4% of all flowering plant species (Magalion and Sanderson, 2001). The Fabaceae is the most common family found in tropical rainforests and in dry forests in the Americans and Africa (Burhametal., 2004).

1.2 GENUS: AlbiziaAlbizia is a genus of about 150 species of mostly fast-growing subtropical and tropicaltrees and shrubs in the subfamily Mimosoideae of the familyFabaceae. The genus is pantropical, occurring in Asia, Africa, Madagascar, America and Australia, but mostly in the Old World tropics. In some locations, some species are considered weeds. They are commonly called silk plants, silk trees, or sirises. Some species are commonly called mimosa, which more accurately refers to plants of genus Mimosa. Species from southeast Asia used for timber are sometime termed East Indian walnut. The leaves are pinnately or bipinnately compound. The small flowers are in bundles, with stamens much longer than the petals. The stamens are usually showy, although in some species such as A. canescens the flowers are inconspicuous. Unlike those of Mimosa, Albizia flowers have many more than 10 stamens. Albizia can also be told apart from another large related genus, Acacia, by its stamens, which are joined at the bases instead of separate. Albizias are important forage, timber, and medicinal plants, and many are cultivated as ornamentals for their attractive flowers ( Lowry et al., 1994)

1.3 SPECIE: ALBIZIA ZYGIA

BotanicalClassificationKingdom PlantaeSubkingdom Tracheobionta– Vascular plants

Super division Spermatophyta – Seed plants

Division Magnoliophyta– Flowering plants

Class Magnoliopsidia– DicotyledonsSubclass RosidaeOrder FabalesFamily Fabaceae– mimosoidae –Pea family

Genus AlbiziaSpecies Albiziazygia (DC.) Macbr– albizia (USDA, plants)

CommonNamesEnglish: West African albizia, West African walnut 

Efik: AyinretaIgbo:NyieavuYoruba:Ayin-rela(Orwaet al., 2009)

1.3.1 Geographic distribution

Albiziazygia is widespread in tropical Africa, occurring from Senegal in the west to Kenya in the east and northern Angola and Tanzania in the south (Apertorgbor, 2007). Geographical area include: Benin, Cameroun, Central African Republic, Democratic Republic of Congo, Gabon, Gambia, Ghana, Guinea Bissau, Guinea, Ivory Coast, Kenya, Liberia, Mali, Mozambique, Nigeria, Senegal, Sudan, Tanzania, Uganda.(USDA, 2014)

Botanical description

Albiziazygia is a deciduous tree 9-30 m tall with a spreading crown and a graceful architectural form. Bark is grey and rough. Young branchlets are densely to very sparsely clothed with minute crisped puberulence, usually soon disappearing but sometimes persistent. Leaves pinnate, pinnae in 2-3 pairs and broadening towards the apex, obliquely rhombic or obovate with the distal pair largest, apex obtuse, 29-72 by 16-43 mm, leaves are glabrous or nearly so. Flowers subsessile, pedicels and calyx puberulous, white or pink; stamina tube exserted for 10-18 mm beyond corolla, Fruit pod oblong, flat or somewhat transversely plicate, reddish-brown in colour, the seeds of A. zygia are smaller (7.5-10 mm long and 6.5 to 8.5 mmwide) and flatter than either of the other Albizia species, but have the characteristic

round shape, with a slightly swollen center (Orwaet al 2009).

Ecology

Albiziazygia occurs in lowland semi-deciduous and evergreen forest, in East Africa up to 1400 m altitude. In West Africa it is most common in secondary forest in the semi-deciduous foresst zone, but can also be found in lower numbers in both forested savanna and evergreen forest. It is locally common and shows no preference for wet or dry sites. It is tolerant to acid soils and shows some drought tolerance(Apertorgbor, 2007).

1.4 EthnomedicinalUsesBark: 

In traditional medicine, bark sap is instilled in the eyes to treat ophthalmia. A bark decoction is administered to treat bronchial diseases, fever (including malaria) and female sterility, and as a purgative, stomachic, antidote, vermifuge and aphrodisiac. Pounded or rasped bark is applied externally to treat yaws, sores, wounds and toothache(Apertorgbor, 2007).

Root: 

Ground roots are added to food to treat cough and as an expectorant (Apertorgbor, 2007). It is also used as an antidote, cutaneous and sub-cutaneous parasitic infection (Burkill, 1985).

The roots have been reported to be used in the treatment of tuberculosis in Lake Victoria region in Kenya (Abereetal., 2014).

Leaf:

Leaf decoctions are used to treat fever and diarrhea, dysentery, insanity and pain killers (Apertorgbor, 2007; Burkill, 1985).

1.5 ChemicalCompositionAlbiziazygiahas been found to contain of bioactive xanthones, benzophenones and flavonoids in the stem bark of Albiziazygia (Oyewoet al., 2004)

The methanol extract of the stem bark contain alkaloid, saponins, glycosides, steroids, resins and reducing sugars, while flavonoid and cardio active glycosides were only observed with hexane extract (Oloyodeet al., 2013). 

In addition to lupeol, chondrillasterol and p-hydroxybenzoic acid, three flavones were isolated for the first time from the bark of Albiziazygia. The latter compounds were identified as 3’,4’,7-trihydroxyflavone (1), 3-Omethoxyfisetin (2), and 3’,7-dihydroxyflavonone (3).Their structures were determined by Nuclear magnetic resonance and Mass spectrometry techniques and confirmed by comparison with the literature (Ndjakouet al., 2007).



BenzophenoneXanthone

LupeolFigure 1.0 Structures of some isolated compounds of Albizia zygia1.6 Reported Pharmacological Activities.

Antiprotozoal Activity

The methanol extract of the stem bark exhibited antiprotozoal activity (IC50 1.0 µg/ml) against Plasmodium falciparum strain K1, the protozoa responsible for malaria, and Trypanosomabrucei and rhodesiense (IC50 0.2 µg/ml), which causes African trypanosomiasis (HYPERLINK "http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746526/" \l "R12"Ndjakou et al., 2007).

Analgesic Activity

Analgesic and toxicological activity of methanol extract of Albiziazygia. The analgesic activity was evaluated using acetic acid-induced writhing response and hot plate model in Swiss albino mice (Abereetal., 2014).

Binding and Coating Agent

Albiziazygia gum has been used as a binding agent in tablet formulations. Experiment have demonstrated its good potential for use as compression coating for drugs targeting the colon, being capable of protecting the core tablet in the physiological environment of the stomach and small intestine, but susceptible to degradation by the colonic bacterial enzymes leading to release of the drug (Oyewoet al., 2004).



Figure 1.2: Photograph of Albiziazygia stem-bark



Figure 1.3: Photograph ofAlbiziazygia leaves



Figure 1.4 Photographof freshly collected Albiziazygia stem bark

1.7Antioxidants

Free radicals are highly reactive chemicals that have the potential to harm cells. They are created when an atom or a molecule (a chemical that has two or more atoms) either gains or loses an electron (a small negatively charged particle found in atoms). Free radicals are formed naturally in the body and play an important role in many normal cellular processes (Diplocket al., 1998). At high concentrations, however, free radicals can be hazardous to the body and damage all major components of cells, including DNA, proteins, and cell membranes. The damage to cells caused by free radicals, especially the damage to DNA, may play a role in the development of cancer and other health conditions (Valkoet al., 2007).

Abnormally high concentrations of free radicals in the body can be caused by exposure to ionizing radiation and other environmental toxins. When ionizing radiation hits an atom or a molecule in a cell, an electron may be lost, leading to the formation of a free radical. The production of abnormally high levels of free radicals is the mechanism by which ionizing radiation kills cells. Moreover, some environmental toxins, such as cigarette smoke, some metals, and high-oxygen atmospheres, may contain large amounts of free radicals or stimulate the body’s cells to produce more free radicals. Free radicals that contain the element oxygen are the most common type of free radicals produced in living tissue. Another name for them is “reactive oxygen species,” or “ROS (Diplocket al., 1998; Valkoet al., 2007).

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols (Fushuaet al., 2007).

Antioxidants in other words are chemicals that interact with and neutralize free radicals, thus preventing them from causing damage. Antioxidants are also known as “free radical scavengers.” The body makes some of the antioxidants it uses to neutralize free radicals. These antioxidants are called endogenous antioxidants. However, the body relies on external (exogenous) sources, primarily the diet, to obtain the rest of the antioxidants it needs. These exogenous antioxidants are commonly called dietary antioxidants. Fruits, vegetables, and grains are rich sources of dietary antioxidants. Some dietary antioxidants are also available as dietary supplements(Valkoet al., 2007). Examples of dietary antioxidants include beta-carotene, lycopene, and vitamins A, C, and E (alpha-tocopherol). The mineral element selenium is often thought to be a dietary antioxidant, but the antioxidant effects of selenium are most likely due to the antioxidant activity of proteins that have this element as an essential component (i.e., selenium-containing proteins), and not to selenium itself (Davis et al.,2012).

FlavonoidsThese are polyphenolic compounds made of 15 carbon atoms, with 2 aromatic rings connected by a three carbon bridge. According to IUPAC nomenclature, they can be grouped into three viz; Flavones, Isoflavonoids, Neoflavonoids (Ghasemzadeh, 2011).

Flavonoids and phenolics acids are the most important groups of secondary metabolites and bioactive compounds in plants (Kim et al., 2003). They are also a kind of natural product and 

antioxidant substance capable of scavenging free superoxide radicals, anti-aging and reducing the risk of cancer(Bodeker, 2000).Flavonoids are widely distributed in plants and they are the most important plant pigment for flower colouration. In higher plants they are involved in UV filtration, symbiotic nitrogen fixation and pigmentation of flora. They are physiological regulators (chemical messenger) in some cases and also serve as cell cycle inhibitors (Sneader, 2000).Flavonoids constitute a wide range of substances that play important role in protecting biological systems against the harmful effects of oxidative processes on macromolecules, such as carbohydrates, proteins, lipids and DNA (Halliwell and Gutteridge, 1989).

Flavonoids occur in the form of quercetin and found in citrus fruits. They are also present in teas, wine and dark chocolate. They are popularly known in research for their in vitro antioxidant activity. At high experimental concentrations, in vitro antioxidant activities of flavonoids may be stronger than those of vitamin C and E.Flavonoids are able to inhibit aldose reductase enzyme (that converts sugars to sugar alcohols) and is implicated with diabetic complications, such as neuropathy, heart disease and retinopathy (Thorne Research, Inc., 2001). Antidiabetic activity of flavonoids and phenolic acids has been reported by several studies (Weissmanet al., 2004).



Flavone backbone



Neoflavonoid

IsoflavanFigure 1.5Structures of the major classes of flavonoid1.9 Classification of Antimicrobial Agent (Hugo and Russel, 1996).

Antimicrobial agents are defined as chemical compounds which are capable of inhibiting the life process of micro-organisms in small concentrations. They may be derived from micro-organisms or from other sources as plants. Agents can be classified into the following categories.

Disinfectants

These are agents used to reduce microbial population to a level which is harmful neither to health, pharmaceutical product nor to perishable goods.

Antiseptics

These are agents that kill or inhibit micro-organisms on living tissue or in contact with the body.

Preservations

There are chemicals or physical agents used to prevent microbial proliferation in pharmaceuticals, cosmetics, foods, drinks and industrial products.

ChemotherapeuticagentsThese are agents used for oral or systemic treatment of infections in man and animals.

Evaluation of antimicrobial agents can be divided into those that determine the bacteriostatic and those that determine bactericidal activities of the agents. Bacteriostatic agents are agents that merely inhibit the growth of micro-organisms without killing than while bactericidal agents actually kill the micro-organisms

Methods used to determine bacteriostatic activity (Hugo and Russel, 1996).

a. Serial dilution method

In this method, the antimicrobial agent is serially diluted and added to the molten agar which is then allowed to set and the organisms are then inoculated on the surfaces of the agar and incubated at 370C for 24-48 hours. The usefulness of this method is that several organisms can be tested on one plate and hence can be used to determine the spectrum of activity of a compound.

b. Agar diffusion method 

This method depends on the ability of the test agent to diffuse through the agar. It is a qualitative test rather than a quatitative test although it has been adapted to provide a fully qualitative one for the assay of antibiotics and other agents.

This method is subdivided into

Ditch-plate method

In this method, molten agar is poured into a plate and allowed to set. A spatula is inflamed and used to make a ditch in the plate and the agent is poured into the ditch.

The organisms are then streaked across the agar right-angle to the ditch and incubated. The result is then read as a clear region around the ditch.

Ii. Seeded plate method

In this method the nutrient agar is melted and the temperature allowed to fall to below 40-450C. The innoculum is mixed with the molten agar and poured into a sterile Petridishes. In this condition the nutrient agar is said to be seeded. Holes are bored in the set nutrient agar using sterile cork borers. The antimicrobial agent is then introduced into the wells. The plates are then incubated for 24-48hours at 370C

Method used for Bactericidal Activity (Hugo and Russel, 1996).

Bactericidal activity means actual killing of the organisms by the antimicrobial agent for specified or variable period of time, after which the surviving organisms are recovered. The method used include;

- Viable count method

- Phenol coefficient test



JUSTIFICATION OF STUDY

Albiziazygia stem bark is currently used by traditional healers in the management of infectious diseases amongst other disease conditions in Africa. Evaluation of the antimicrobial activity will reveal the place of Albizia zygia in the management of some of these disease conditions.

Also, free radicals and micro-organisms seems to be more and more ubiquitous in our day to day life as exemplified by the misuse of drugs, pollution, and smoking. This however poses a challenge to the body's protective or defence mechanisms and can overwhelm it to cause diseases and infections including atherosclerosis, Alzheimer's disease, cancer, and dysentery. Antioxidants and antimicrobial agents serves to salvage the situation and hence Albizia zygia stem bark is being investigated for its antioxidant and antimicrobial activities.

OBJECTIVES OF STUDY

To determine the secondary plant metabolites present in Albiziazygia stem-bark.

To investigate the antioxidant activity of the crude methanol extract and fractions of Albiziazygia stem- bark.

To investigate the antimicrobial activity of the crude methanol extract and fractions of Albiziazygia stem- bark.