STUDIES ON THE CHEMICAL CONSTITUENTS OF THE LEAVES AND SEEDS OF HYPTIS SPICIGERA LAM

STUDIES ON THE CHEMICAL CONSTITUENTS OF THE LEAVES AND SEEDS OF HYPTIS SPICIGERA LAM

ABSTRACT

The extracts of the leaves of H.spicigera were screened for the presence of secondary metabolites: alkaloids, glycosides, flavonoids, carbohydrates, tannins, sterols, terpenes and resins. The results of the phytochemical screening showed all the extracts with the exception of hexane extract to contain the secondary metabolites analysed in high and moderate amounts. Antimicrobial activities of the crude extracts of H.spicigera against a broad spectrum of microorganisms namely: Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Corynebacterium ulcerans, Salmonella typhi, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae and Candida albicans were carried out. All the extracts showed bactericidal activity against the entire antibiotic resistant microorganisms tested with some degree of variations against standard drug, penicillin. The methanolic extract of H.spicigera exhibited significant bactericidal activity at low concentration of 2.5mg/ml while S.aureus, N.gonorrhoeae and C.albicans were resistant at MBC of 5.0mg/ml. The insecticidal properties of H.spicigera leaf extracts (hexane, ethyl acetate and methanol) tested against Callosobruchus maculatus on cowpea was carried out. Azadirachtin was used as standard check along with the extracts tested. Ethyl acetate extract showed the highest percent mortality of 98% each at 72hrs, while 6% and 10% were recorded for the emergence of the C.maculatus. Ethyl acetate extract and azadirachtin standard check

showed oviposition deterrent at 4.16% and 2.78% respectively. Hexane extract showed the least percent (2%) seed damage while the control gave the highest percent (88%) of seed damage at 16 weeks. Hyptis spicigera seed oil was characterized using GC-MS and UV-VIS spectrometry for its fatty acid profile, tocopherol and physicochemical properties. The oil content was 21% while unsaturated fatty acids were linoleic acid (71.85 %) and palmitic acid (16.06%) as predominant fatty acids. Tocopherol content was 186.15mg/ml while Vitamin A was absent. The study showed potentials of Hyptis spicigera seed oil to have high oxidative stability which could be suitable for food and beverage as well as other industrial applications while the tocopherol content could improve human health. Purification of the ethyl acetate fraction by High Performance Liquid Chromatography (HPLC) yielded 3-buten-2-enol on the basis of GC-MS. The best separation using the analytical High Performance Liquid Chromatography (HPLC) was achieved at 0.8ml/min. The hydrodistillation of the fresh leaves of H.spicigera gave a colourless volatile oil with yield of 0.65%.The volatile oil gave forty compounds on the basis of GC-MS with low composition of cineole (4.11%) and caryophyllene (2.61%) while α-pinene (12.16%) β-pinene (9.47%) and α-phellandrene (10.19%) were predominant compounds. Crude ethyl acetate extract of H.spicigera leaves was fractionated through solvent separation followed by thin layer and column chromatography. Two relatively pure constituents were obtained and characterized through spectral studies. The structure elucidation of the constituents was established as ursolic acid and its derivative (3β-hydroxy-urs-12-en-28-oic acid) and 3β-hydroxy-20-isopropyl-urs-12-an-28-oic using spectroscopic techniques including 1D and 2D NMR such as 1H NMR, 13C NMR, DEPT, HMQC, HMBC, COSY, NOESY as well as MS, and IR. Toxicity studies on the dichloromethane and methanolic extracts of Hyptis spicigera were carried out on mice intraperitoneally. The LD50 was calculated using the method of Karber (LD50, 2534mg/kg) which showed the plant to be moderately toxic based on the WHO toxicity rating. The biological activity of one of the new compounds (ursolic acid) was determined against a trypanosome (T.brucei brucei).The compound was evaluated in vitro for activity against Trypanosoma brucei brucei (Tbb) and was found to possess antitrypanosomal activity in vitro in a dose dependent pattern at 0.4μg/ml in 3 minutes.

Background to the study

Natural products can either be of prebiotic origin or originate from microbes, plants, or animal sources (Nakanishi, 1999). As chemicals, natural products include such classes of compounds as terpenoids, polyketides, amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and so forth. Natural products do not just occur by accident or products of convenience of nature, but more than likely are a natural expression of the increase in complexity of organisms (Jarvis, 2000). Interest in natural sources to provide treatments for pain palliatives, or curatives for a variety of maladies or recreational use reaches back to the earliest points of history. Nature has provided many things for humankind over the years, including the tools for the first attempts at therapeutic intervention (Nakanishi, 1999). The Nei Ching is one of the earliest health science anthologies ever produced and dates back to the thirtieth century B.C. (Nakanishi, 1999). Some of the first records on the use of natural products in medicine were written in cuneiform in Mesopotamia on clay tablets and date to approximately 2600 B.C. (Cragg and Newman, 2001; Nakanishi, 1999). Indeed, many of these agents continue to exist in one form or the other to this day and are used for the treatment of inflammation, influenza, cough, and parasitic infestation among others. Chinese herb guides document the use of herbaceous plants as far back in time as 2000 B.C (Holt and Chandra, 2002). For a variety of different reasons, the interest in natural products continues to this very day (Barron and Vanscoy, 1993; Bhattaram et al., 2002; Chan, 1995; Holt and Chandra, 2002; Koh and Woo, 2000; Marriott, 2001). The first commercial pure natural product introduced for therapeutic use is generally considered to be the narcotic morphine, marketed by Merck in 1826 (Newman et al., 2000). The first semi-synthetic pure drug based on a natural product, aspirin, was introduced by Bayer in 1899.