Phytochemical and Antimicrobial Studies of the Root Bark Extracts of Acacia Ataxacantha Dc (Fabaceae)
Chapter One
AIM AND OBJECTIVES OF THE RESEARCH
Aim of the Research
The research aims to screen the root-bark of Acacia ataxacantha of the family Fabaceae for bioactive constituents, together with the structural elucidation of such constituents.
Objectives of the Research
- Collection, proper botanical identification, drying and pulverizing of the roots of the plant.
- Extraction of the pulverized plant material using different solvents based on the eluotropic series i.e. from non-polar (petroleum ether 60-80oC) to polar(methanol).
- Phytochemical screening for bio-active compounds using the crude
- Antibacterial and Antifungal screening of the
- Analytical separations involving several consecutive steps of chromatographic techniques and
- Verification of the purity of the isolated compounds. Structural elucidation and characterization of the isolated compounds using available spectral techniques such as FTIR, 1H NMR, 13C NMR and DEPT.
CHAPTER TWO
LITERATURE REVIEW
GENERAL BOTANICAL FEATURES OF THE FABACEAE FAMILY
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 plantsperennials or annuals, which are easily recognized by their fruit (legume) and their compoundstipulated leaves. This family is widely distributed and is the third-largest land plant family in terms of number of species, behind only the Orchidaceae and Asteraceae, with 630 genera and over 18,860 species (Stevens, 2001). The five largest of the 630 legume genera are Astragalus (over 2,000 species), Acacia (over 1000 species), Indigofera (around 700 species), Crotalaria (around 700 species) and Mimosa (around 500 species), which constitute about a quarter of all legume species. About 18,000 legume species are known, amounting to about 7% of flowering plant species. Fabaceae is the most common family found in tropical rain forest and in dry forests in the Americas and Africa (Magallon and Sanderson, 2001).
The Fabaceae have a wide variety of growth forms including trees, shrubs or herbaceous plants or even vines or lianas. The herbaceous plants can be annuals, biennials or perennials, without basal or terminal leaf aggregations. They are upright plants, epiphytes or vines. The latter support themselves by means of shoots that twist around a support or through cauline or foliar tendrils. Plants can be heliophytes, mesophytes or xerophytes (Watson and Dallwitz, 2007).
Taxonomic Position of Family Fabaceae/ Leguminosae:
Division: Angiospermae Class: Dicotyledonae Sub Class: Polypetalae Series: Calyciflorae
Order: Rosales
Family: Fabaceae (Leguminosae)
Characteristic Features of Fabaceae Family (Common to all Sub- Families)
Habit: Legumes are trees (Acacia, Laburnum), shrubs (gorse, furze) and (majority) herbs (clovers, medicago). They may be runner, climbers or with twining stems.
Habitat: Contains about 690 genera and over 18,000 species. Found in varying habitats based on their sub family characteristics.
Roots: Roots contain nodules. Nitrogen fixing bacteria harbour at these roots in a symbiotic manner.
Leaves: compound, alternate, simple and pulvinate (swollen leaf base)
Inflorescence: Predominantly racemose type (that is, a cluster of stalked flowers are borne on a main stalk in an upward and arranged fashion; older flowers below and younger above)
Flowers: zygomorphic (irregular arrangement), hypogynous (ovary is above other floral parts) , heterochlamydeous (distinct calyx and corolla present), and pentamerous (floral members arrangement in fives)
Androecium: Stamens ten in number; monadelphous (stamens fused fully or partially into a tube), diadelphous (stamens are fused in two groups) or free
Gynoecium: Monocarpellary (single carpel), unilocular (single cavity) with marginal placentation (ovule placed at the side of the ovary).
Fruit: Legume or lomentum (Mittra, 2010).
CHAPTER THREE
MATERIALS AND METHODS
APPARATUS, INSTRUMENTS AND REAGENTS
Apparatus and Instruments
- Rotary Evaporator
- Oven
- Autoclave
- Pre-coated Chromatographic Plates (20 × 20 cm) andTank
- Silica gel (60 – 120mesh)
- Chromatographic Column
- Separating Funnel
- Incubator
- Ultraviolet lamp (254 – 366nm)
- Perkin Elmier Spectrum 8400S FT-IR Spectrometer
- Bruker – Avance 400 MHz FT – NMR Spectrometer
Solvents
- Methanol
- Ethylacetate
- Chloroform
- Petroleum ether (60 – 80 0C)
All solvents and reagents used were of high analytical grade supplied by Sigma- Aldrich. The solvents were distilled before use.
CHAPTER FOUR
RESULTS
EXTRACTION
Extraction of the pulverised root of Acacia ataxacantha(1 kg) and fractionation gave various weights as shown in Table 4.1
RESULTS OF PHYTOCHEMICAL SCREENING EXPERIMENTS
The crude plant extracts were screened using the methods of Harborne (1984), Sofowora (1993), and Trease and Evans (1989)and the results obtained are recorded in Table 4.2
CHAPTER FIVE
DISCUSSION
PHYTOCHEMICAL SCREENING
The antimicrobial activities of plant extracts have been linked to the presence of some bioactive compounds. These secondary metabolites also serve to protect the plants themselves against bacterial, fungal and viral infections (El-Mahmood and Amey, 2007). These bioactive compounds are known to work synergistically to produce various effects on the human and animal subjects (Amagase, 2006).
The screening of crude extracts of Acacia ataxacantha for phytochemical constituents has revealed the presence of flavonoids, steroids/ triterpenes, glycosides, tannins, saponins and alkaloids. Carbohydrate is present in all extracts except petroleum ether; while anthraquinones were absent in all the tested extracts. Steroids are important drugs used as cardiac depressants, hypotensive, sedatives and anti- dysenteric agents (Ghani, 1990).
Tannins have been reported to have various physiological effects like anti-irritant, antisecretolytic, antiphlogistic, antimicrobial and antiparasitic effects (Trease and Evans, 2002). Alkaloids act as anti-malarial, anti-amoebic agents and astringents (Ghani, 1990). Flavonoids and tannins possess antimicrobial activity, the antimicrobial activity of flavonoids is due to their ability to complex with extracellular and soluble protein and to complex with bacterial cell wall while that of tannins may be related to their ability to inactivate microbial adhesions, enzymes and cell envelop proteins (Cowan, 1999). This result is similar to the phytochemical screening results of the stem bark of Acacia nilotica as reported by Banso (2009).
CHAPTER SIX
SUMMARY, CONCLUSION AND RECOMENDATION
SUMMARY
The root of Acacia ataxacantha DC was collected fromEdumoga, Benue state Nigeria and was properly identified at the Herbarium of Biological Sciences A.B.U. Zaria with voucher number 1707. It was air-dried, pulverised and extracted. The crude extracts were subjected to phytochemical and antimicrobial screening. The Phytochemical screening gave positive results for flavonoids, glycosides, saponins, steroids/ triterpenes, tannins and alkaloids. The screening of the methanol, ethyl acetate, chloroform and petroleum ether extracts showed that the plant root could inhibit the growths of Bacillus subtilis, Streptococcus pneumonia, Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Escherichia coli, Candida albicans and Candida krusei in varying degrees; but were not active on Corynebacterium ulcerans, Streptococcus faecalis, Proteus mirabilis and Candida tropicalis. The minimum inhibitory concentration (MIC) of the extracts were determined. Methanol and chloroform fractions had MIC of 5 mg/ml and petroleum ether had 10 mg/ml for all the test organisms, while ethyl acetate was the most active with 2.5 mg/ml for Bacillus subtilis, Escherichia coli, Salmonella typhi and Klebsiella pneumonia. The Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) showed that ethyl acetate extract could kill Bacillus subtilis, Escherichia coli and Klebsiella pneumonae at a concentration of 5 mg/ml. Ethyl acetate extract was the most active, thus was subjected to chromatographic separations that led to the isolation of compounds ABA and ABA 1. The compounds were analysed spectroscopically (FTIR, 1H NMR, 13C NMR and DEPT) and found to be α-amyrenol ((3β)-Urs-12-en-3-ol) and lupeol ((3β)-lup-20(29)-en-3-ol) respectively. The antimicrobial activity of ABA was determined with the same organisms. The MIC and MBC/MFC of ABA was found to be 12.5 and 25 µg/ml respectively against B.subtilis, E.coli and S.typhi.
CONCLUSION
The phytochemical analysis of the root bark of Acacia ataxacantha was found to contain some components which are of medicinal value. They include alkaloids, glycosides, flavonoids, tannins and steroids/triterpenes. Antimicrobial screening of root extracts from the plant showed that the extracts were able to inhibit the growth of some bacteria and fungi such as Bacillus subtilis, Streptococcus pneumonia, Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella
typhi and Escherichia coli; Candida albicans and Candida krusei. In conclusion, the findings in this research have justified the use of this plant in ethnomedical treatment of pneumonia, excessive cough, yellow fever, respiratory diseases, dysentery, and wounds which are caused by some of the organisms used in this study andother infections in which sparfloxacin and cefuroximeare used fortreatment.
RECOMMENDATION
Further work can be carried out to isolate more bioactive compounds from the plant. There is need for biological and pharmacological studies of the isolated compound to determine their mode of action for possible use as chemotherapeutic drugs by humans.
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