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African Journal of Biomedical Research, Vol. 9, No. 3, Sept, 2006, pp. 205-211 Full Length Research Article In-vitro Anti-Microbial and Brine-Shrimp Lethality Potential of the Leaves and Stem of Calotropis procera (Ait). *1Oladimeji, H.O, 2Nia, R and 1Essien, E. E. 1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo,
Uyo Received: February
2006 Code Number: md06033 ABSTRACT Calotropis procera (Family, Asclepiadeae) is implicated in a variety of ethno-medicinal therapies ranging form oral, skin, gastro-intestinal to respiratory problems. The effects of this plant on the sensitivity of micro-organisms need verification and its potential for cytotoxicity needs to be investigated. Thus, the anti-microbial and brine–shrimp lethality studies on the leaves and stem were carried out. The crude ethanolic extract of the stem and its ethylacetate fraction elicited good antibacterial activity against clinical strains of Bacillus subtilis, Staphylococcus aureus but gave minimal activity against Escherichia coli, Klebsiella pneumoniae and Salmonella typhi but none against the fungal isolate; Candida albicans. The brine-shrimp lethality assay analyzed using the Finney probit method showed that the crude ethanolic extracts of the leaves and stem displayed LD50 values at 192 ppm and 182 ppm respectively. These findings indicate the potential of the plant as panacea for infectious diseases and also reveal a novel potential in the fight against tumors in man. Keywords: Anti-microbial, extracts, fractions, brine-shrimp lethality, Calotropis procera INTRODUCTION The genus, Calotropis belongs to the Family, Asclepiadeae. The species found in this genus include C.busseana,C.inflexa,C.syrica and C.procera.Calotropis procera(Ait)is a drought-resistant, salt-tolerant weed found along degraded roadsides, lagoon edges and in overgrazed pastures. It is native to Nigeria and many other countries in tropical Africa, Asia and Latin America where the plant is of high socio-economic value (F.A.O.1986; Abbas et al,1992). The bark is used traditionally in the treatment of coughs, dermatitis, dysentery, elephantiasis, jaundice, leprosy, sore-gums swellings and ulcers(von Maydell,1986). It is also used in dehairing hides, tanning, brewing, curdling milk and as an arrow and spear poison (Leeuwenberg,1987). The latex is used on conjunctiva, epiphora, in local anesthesia, to treat ringworm and other skin diseases. It also serves as a source of renewable energy, hydrocarbons (Arora,1982), green manure, sulphur dioxide emission and an indication of exhaust soil (Leeuwenberg,1987). The flower is used as a digestive, tonic for asthma and catarrh while the sap serves as a rubefacient and purgative. The floss of the seeds is used as substitute cotton wool in surgical operations (von Maydell, 1986). The leaves are eaten by goats, occasionally sheep in times of need but rarely by cattle and other livestock because they are slightly toxic. The stem is used in native roofing of huts and also as source of charcoal (Taylor,2004). Compounds such as asclepsin and mudarin reportedly isolated from this plant have been found to posses emeto-cathartic, digitalic, bactericidal and vermicidal properties while calotropin is cardio-toxic (Taylor,2004). Consequently, this study was embarked upon to confirm or otherwise, the sensitivity of microbes to extracts of the plant and also to investigate its potential for cytotoxic activity MATERIALS AND METHODS Plant collection, identification and authentication. The fresh leaves and stem of Calotropis procera (Ait) were collected throughout the month of November, 2004 at a location inside the University of Uyo (Main campus) Uyo, Akwa Ibom State. These plant parts were identified and authenticated by the taxonomist of Faculty of Pharmacy where voucher specimens (NoH43) and (NoH44) were deposited. Chemicals, media, micro-organisms and other materials. Chemical reagents: The reagents used in this study were, Butanol, Chloroform, Ethanol and Methanol; all of AnalaR grade(East Anglia Chemicals Limited, England), Silica gel (254GF), Streptomycin sulphate and Nystatin(Unique Pharmaceuticals, Lagos, Nigeria). Culture media: The media used were Mueller Hinton II Agar (Biotec Oxiod Limited, England, No13904) and Sabouraud Dextrose Agar (International Diagnostic Group, England, No049041). Micro-organisms:The micro-organisms(Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi and Candida albicans) clinically isolated from human specimens; urine, faeces, wounds and vaginal swabs originally obtained from the University of Uyo Teaching Hospital, Uyo were collected in sterile bottles and refrigerated at 0-5°C. They were then subjected to laid down convectional biochemical methods (Gibson and Khoury,1986; Murray et al,1985) for identification and preservation. Other materials: Sea water was collected from Kuramo Beach, Lagos, brine- shrimp eggs; Artemia salina (Leach) (San Francisco Bay Brand Inc. Newark, CA 94560, U.S.A.) and plastic soap case. Extraction and processing The plant parts i.e. the leaves and stem of C. procera were air –dried and powdered respectively on an electric mill. The resultant ground powders were then extracted with 50% aqueous ethanol at room temperature(25°C) for 72h. The filtrates were evaporated to dryness using a rotary evaporator (Buchi CH-9230, Laboratorium-Technic,Flawk/SG, Switzerland). The dried crude ethanolic extracts were then investigated for plant metabolites (alkaloids, saponins, tannins, cardiac glycosides, terpenes, anthraquinones, phlobotannins and flavonoids) as described in phytochemical methods (Stahl et al,1965;Brain and Turner,1975;Harbone,1984; Akerele,1984;Trease and Evans,1989). The dried crude ethanolic extracts were dissolved in water and then chromatographed respectively on silica gel (254GF) column with chloroform: ethylacetate: butanol(1:1:1) mixture in a gradient elution. Eluates showing similar (T.L.C) profiles were pooled and bulked separately resulting in chloroform, ethylacetate and butanol fractions which were evaporated to dryness and then subjected to anti-microbial screening. Anti-microbial sensitivity test: The media (Mueller Hinton II Agar and Sabouraud Dextrose Agar) were prepared according to Manufacturers’ instructions, poured into large sterile petri-dishes (diameter,13.5cm) and allowed to set. The Agar-cup diffusion method was employed for the anti-microbial susceptibility testing. 20ml of the media agar seeded with 0.1ml of 10-2 dilution of the organisms were introduced into the petri-dishes. After solidification, uniform and equidistant wells of 6mm diameter were cut in the agar by using a sterile cork- borer(Washington,1995). Concentrations of 20mg/ml and 40mg/ml of the dried crude ethanolic extracts and the fractions at 5mg/ml dissolved in de-ionized water were separately introduced into wells. Also, concentrations of 1mg/ml of Streptomycin sulphate, 1mg/ml of Nystatin and de-ionized water were introduced into separate wells as positive and negative controls respectively. The experiments were carried out in triplicates. The plates were left at room temperature (25-30°C) for 2h to allow for diffusion and then incubated at 37°C for 24h. Zones of growth inhibition were measured in millimeters (mm). Thereafter, the minimum inhibitory concentration (M.I.C.);the lowest concentration of a sample that inhibits the growth of a micro-organism was determined by the standard serial dilution technique (Hugo and Russel, 1994; Washington,1995) using the Mueller Hinton II Agar and Sabouraud Dextrose Agar as media for the bacteria and fungus respectively. Brine-shrimp lethality assay Hatching of shrimp eggs Some sea water was placed in a small plastic with perforated dividing dam which was fabricated from a plastic soap case. Some shrimp eggs were added to one side of the divided dam tank. This side was darkened by covering it with a plastic lid while the other compartment was exposed. The set- up was left for 48h for the shrimp eggs to hatch and mature as nauplii. Mature nauplii usually swim to the exposed compartment. Preparation of vials for testing A stock solution of sample was prepared by dissolving 20mg of the sample in 2ml methanol/de-ionized water (1:1v/v). To obtain the desired final concentrations such as 1000mg/ml, 100mg/ml and 10mg/ml; 0.5ml, 0.05ml and 0.005ml of the stock were transferred into the three vials respectively. The solvent was then evaporated by leaving the vials in a vacuum desiccator for 24h. Ten shrimp nauplii were counted into each vial(i.e.30 nauplii per dilution).The total volume of solution in each vial was adjusted to 5ml by adding the sea water (5ml/vial). The control (methanol/de-ionized water (1:1v/v) was prepared in the same way except that the sample was omitted. The vials were maintained in the laboratory with normal fluorescent illumination and the set-up left for 24h.The number of survivors usually swimming was counted with the aid of a magnifying lens for each of the vials at the end of 24h.Thus, the number of the dead was computed; hence the LD50 in p.p.m. (parts per million) was determined using the Finney probit analysis software (McLauglin,1988; McLauglin et al,1991). RESULTS AND DISCUSSION The phytochemical screening of the crude ethanolic extracts of leaves and stem of Calotropis procera showed that they both contain alkaloids, saponins, tannins, cardiac glycosides, terpenes, flavonoids and phlobotannins (Table 1).This confirms previous studies as reported in Taylor (2004). However, anthraquinones were absent in both extracts. From the anti-microbial results presented on Table 2, it could be inferred that the extracts elicited concentration-dependent activities. The crude ethanolic extract of the stem gave higher antibacterial activity against B.subtilis and S.aureus than the extract of the leaves. However, the activity given by both extracts against E.coli, K.pneumoniae and S.typhi were comparably less.(Table 2).This is not surprising because both extracts tested positive for tannins which had been implicated in previous studies to be anti-microbial (Adesina, et al, 2000;Burapadaja and Bunchoo,1995).It is very probable that these bioactive compounds might have played a significantly similar role in the observed activity. Furthermore, these results have partly justified some of the uses of the plant in ethno-medicine (von Maydell,1986). The activity recorded by these extracts then necessitated their purification by chromatography into fractions. Table 1. The phytochemical screening of the crude ethanolic extracts of leaves and stem of Calotropis procera(Ait).
Key: A - Crude ethanolic extract of the leaves; B - Crude ethanolic extract of the stem. + - Trace; ++ - Moderate; +++ - Abundant; - - Absent Table 2. The antimicrobial sensitivity of the crude ethanolic extracts and fractions of leaves and stem of Calotropis procera in de-ionized water.
Key: *The diameter of
zone of inhibition is zone of inhibition and cup-size (6mm) Table 3. The minimum inhibitory concentration (M.I.C.) of the crude ethanolic extracts and the ethylacetate fractions of leaves and stem of Calotropis procera on test microbes
ND-Not detected. Table 4. The brine-shrimp lethality assay of the crude ethanolic extracts of leaves and stem of Calotropis procera
Expectedly, the fractions afforded greater antibacterial activity against the test organisms than the activity given by their respective mother extracts (Table 2).This could be attributed to level of purity inherent in the fractions. It was observed that among the fractions, the ethylacetate fractions gave the highest antibacterial activity while the chloroform fractions recorded the least. This was so because the extracts were largely insoluble in chloroform, thereby affording insoluble fractions which gave comparably smaller antibacterial activity.(Table 2). However, the crude ethanolic extracts and the fractions demonstrated poor anti-fungal activity against C.albicans. This could be due to the nature of the fungus; its structure differing from the cell-wall of bacteria and resembling those of higher plants, hence limiting the permeation of substances into it. The results of the M.I.C. determinations as presented on Table 3 generally show that higher concentrations of the extracts were required it inhibit the growth of the microbes than those required of the fractions. The same reason of level of purity applies. The brine-shrimp assay determines the lethalities of materials toward brine-shrimp larvae (nauplii) and in doing so predicts the ability to kill cancer cells in cell-cultures, kill various pests and exert a wide range of pharmacologic effects. The shrimp nauplii have been used for a number of bioassay systems in which natural products extracts, fractions or pure isolates are tested at concentration of 1000mg/ml,100mg/ml and 10mg/ml in vials containing 5ml of brine and ten nauplii in each of the three replicates (Meyer et al,1982).The LD50 values in p.p.m. are estimated with 95% confidence using the appropriate mathematical estimates; the Finney probit analysis program being the model routinely employed. The LD50 values of the crude ethanolic extracts of leaves and stem of C.procera and other plants in literature (Kupahan et al, 1969; Ma et al 1989; Ma et al 1990; McLauglin et al,1991) are presented on Table 4.The results show that both extracts(leaves and stem) C. procera displayed LD50 values of 192ppm and 182ppm. These values compare favorably with those of Myrsine Africana (114ppm), Pogonopus speciosus (50ppm) and Persia major (2.6ppm) whose LD50 values below the 200ppm are generally considered as ‘significant’((Kupahan et al,1969; Ma et al,1989;Ma et al,1990;McLauglin et al,1991; Oladimeji et al, 2005).This is to be expected because the crude ethanolic of the leaves and stem tested positive for saponins (Table 1).This class of metabolites had been implicated in previous studies to be cytotoxic (Kupahan et al,1969). These particular results obtained in this study have revealed a novel potential of the plant in the fight against tumors in man. REFERENCES
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