Received on: 16-05-2014 Accepted on: 24-05-2014 Published on: 06-06-2014
Owolarafe Tajudeen Alowonle
Department of Biochemistry and Forensic Science, Nigeria Police Academy, Wudil, Kano. P.M.B. 3474, kano.
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Phytochemical investigation and brine shrimp lethality
assay of extracts of picralima nitida (apoceanacea )staph.
Seeds
1Owolarafe T. A., 2 Dosunmu S. O., 4Yakubu M. T., 1Lawal A. T., 3Akolade J. O., 1Muhammed M. B., 1Ononamadu C. J.
1Department of Biochemistry and Forensic Science, Nigeria Police Academy, Wudil, Kano. P.M.B.
3474 Kano;
2Department of Chemistry, University of Ilorin, P.M.B. 1515 Ilorin, Nigeria. 3Biotechnology Advanced Laboratory, SHEDA Science and Technology complex Sheda. P.M.B.
186, Abuja.
4Department of Biochemistry, University of Ilorin, P.M.B. 1515 Ilorin, Nigeria.
ABSTRACT
Phytochemical study was undertaken on the seed of picralima nitida staph. Phytochemical analysis based on test of colouration and precipitate formation was carried out on four different extracts of P. nitida seed , extraction was based solvents polarities and are aqueous, methanol, ethylacetate and hexane. Also brine shrimp Lethality Assay (BSLA) of the extracts was carried out to detect possible cytotoxicity effects and possible pharmacological activities of the plant seed. Cytotoxicity was evaluated in terms of LC50 (lethality concentration) were carriedout in three replicates of the concentration of the seed extracts and survining brine shrimp were recorded after 24 hours. The tests carried out shows the presence of saponin, tannins, flavonoids, steroids, alkaloids, glycoside and anthraquinones. However no reducing sugar was observed with alkaloids, glycoside and steroids found/observed in all the extracts. The extracts were potent against
the bri e shri p ith LC5 alues of 7, 7, a d 9 pp (μg/ L), respectively.This indicates the presence of bioactive components in these seed extracts which could account for the reported pharmacological effects of the plant. Thus, the results support the uses of these plant seeds in traditional medicine. Keywords: phytochemical, picralima nitida seed, alkaloid, glycoside and steroids, brine shrimp, Lethality Assay , LC50 .
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Introduction
Plant derived medicines have made a large contribution to human health and still the future of pharmaceutical drug discovery. According to Newman and Cragg, 2007 approximately one-half of all licensed drug that were registered worldwide in the 25 years periods prior to 2007 were natural products or their synthetic derivatives. Nearly, all cultures and civilizations from ancient times to the present day have depended fully or partially on herbal medicine because of their effectiveness, affordability, availability, low toxicity and acceptability (Akharaiyi and Bobye, 2010). The general acceptability of herb products have been limited by lack of dose regimen, adequate toxicity data (Pousset, 1988) and large information about the bioactive content of these plants.
Picralima nitida staph (Apocynaceae family) is a medicinal plant with diverse end-use (Keay, 1989) and widely distributed in the tropical rain forest of Africa, when fully grown, it is about 20 metres high with white flower and large paired fruit (keay et al., 1964). In
Nigeria it is popularly referred to as Osi-igwe by the
igbos and Abere by the yorubas. In Benin Republic it is known as Gbe-fon dangne , in Ghana as Adangme, Ivory Coast as Abure Ebissi, Sierra Leone: susu Balunyi (burkill, 1985). The fruit is broadly obovoid, smooth and glabrous measuring about 15 cm long and 10 cm in diameter. Each fruit contains flattened seeds embedded in pulp (Aguwa et al., 2001).
Extracts from its seeds, fruit rind and stem bark demonstrated antimalarial activity (Iwu and Klayman, 1992), Antimicrobial effect (Fakeye et al., 2002), anti – inflamatory and analgesic action (Duwiejuna, 2002) and others uses in various herbal preparations. Nevertheless, there is no sufficient information regarding the phytochemical study of P. nitida such as screening for it phytochemical constituents to detect all the secondary metabolites in the seed which is majorly used in various herbal preparations.
The shrimp lethality assay was proposed by Michael and his team in 1956 and later developed by Vanhaecke and his team in 1981. It is based on the ability to kill laboratory-cultured Artemia nauplii brine shrimp. The assay is considered a useful tool for preliminary assessment of toxicity (Solís et al, 1993) and it has been used for the detection of fungal toxins ( Harwig and scott, 1971) plant extract toxicity (McLauglin et al., 1991), and cyanobacteria toxins (Jaki et al., 1999 ).
Materials and methods Plant Material
The seeds of picralima nitida used in this study were obtained from the local herbal market in Ilorin, Nigeria and authenticated at the herbarium unit of the Forest
Research Institute of Nigeria (FRIN) Ibadan, where voucher specimen was deposited with voucher Number FHI108794.
Preparation of Extracts
Aqueous Extract: the protocol of Afolayan and Yakubu, 2009 was followed; the dried seeds were pulverized (milled) with electric milling machine after drying to constant weight. 500 grams of the powder was extracted in 3000ml of distilled water and left to stand for 72 hours at room temperature with stirring at intervals. The extracts were filtered with wathman (Maidstone, UK) No. 42 (125mm) filter paper, and the resulting filtrate was freeze dried (Vir Tis bench top K, Vir Tis Co. Gardinar, NY) and the dry sample obtained was stored for further use.
Methanol Extract: the procedure of Inya-Agha et al., 2006 was followed, the dried pulverized seeds were macerated with methanol for 24 hours with intermittent shaking. The material was filtered and concentrated in vacuo using rotary evaporator.
The same above procedure was used. Ethylacetate and hexane extracts were obtained and labelled with others appropriately.
Phytochemical Screening: The phytochemical analysis was carried out respectively on the four obtained extracts, aqueous extract, methanol extract, ethylacetate extract and Hexane extract using standard procedures to identify the constituents as described by Edeoga et al., 2005, harbourne 1978 and Sofowora 1993.
Test for tannins: About 0.5 g of the dried powdered samples was boiled in 20 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black colouration.
Test for saponin: About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and filtered. 10ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth.
13 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow colouration was observed indicating a positive test for flavonoids.
Test for steriods: Two ml of acetic anhydride was added to 0.5 g methanolic extract of each sample with 2 ml H2S04. The colour changed from violet to blue or green in some samples indicating the presence of steroids.
Test for cardiac glycosides (Keller-Killani test): 5ml of each extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was under layed with 1 ml of concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.
Alkaloid determination using Harborne (1973) method: 5ml of each extracts was treated with 2 ml of
. % hydrochloric acid and Mayer’s reagent was added
drop wise to the extract until the precipitation was
Brine Shrimp Lethality Assay (BSLA): The protocol of Miller and Tainter was adopted as described by partition for dark (covered) and light areas. Shrimp eggs were added into the dark side of the chamber while the lamp above the other side (light) will attract the hatched shrimp. Two days were allowed for the shrimp to hatch and mature as nauplii (larva). After two days, when the shrimp larvae are ready, 4 mL of the seawater was added to each test tube and 10 brine shrimps were introduced into each tube. Thus, there were a total of 30 shrimps per dilution. Then the volume was adjusted with seawater up to 5 mL per test tube. The test tubes were left uncovered under the lamp. The number of surviving shrimps were counted and recorded after 24 hours. Using probit analysis, the lethality concentration (LC50) was assessed at 95% confidence intervals. LC50
value of less than μg/mL is toxic while LC5 value of greater than μg/mL is non-toxic. The percentage mortality (%M) was also calculated by dividing the
In the present investigation, the phytochemical screening has been done in the various extracts of picralima nitida staph. Seeds and the results are presented in Table 1. The hexane extract exhibited the presence of alkaloids, saponins, glycoside, steroid and Anthraquinones. The ethylacetate extract showed the presence of alkaloids, flavonoid, glycoside and steroid. The methanol extract contained alkaloids, tannins, glycoside and steroid. Aqueous extract contained alkaloids, tannins, saponins, glycoside and steroid. The various extract shows the presence alkaloid, glycoside and steroids with the exception of while only hexane extract that shows the presence of anthraquinones. These differences can be attributed to the solubility of the bioactives in different solvent of degree of polarity (Ekpo and Etim, 2009). The results of phytochemical analysis has comprehensively validate the various therapeutically importance place on picralima nitida seeds by the folk medicine and ethnobotanical data. Herbal extracts contain different phytochemicals with biological activity that can be valuable therapeutical index (kanchana et al., 2012). Various secondary metabolites have been implicated to exhibit a wide range of biological effect and protection against different diseases (Augusti and Cherian, 2008). Alkaloids exhibit marked physiological effects such as antibacterial (veronika et al., 2006), analgesic effect (Stray, 1998). Saponin and glycoside exhibit cytotoxicity effect (Tsuyosh, et al., 1997). Favonoids and Tannins are phenolics compounds and plants phenolics are a major group of compounds that act as primary antioxidants
Table 1: The analysis of phytochemicals in the various extracts of
picralima nitida staph. Seeds
Key: + = presence; ++ = presence in high concentration; - = absence
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Table 2: The number of shrimp nauplii that survived after treatment with different extract of p. nitida seed, the percentage mortality and
LC50 Value
T= number of trials for each concentration.
They observed that the crude extracts of p nitida as having antimicrobial activity against E. coli and salmonella spp. This maybe due to the ability of the components of the extract to bind to the cell wall of the bacterial thereby (has) inhibiting its synthesis probably because flavonoids and tannin present in the plant extract (Lilybeth and Olga, 2013). Aqueous-ethanollic extracts is also observed by Darko, 1994 to exhibit anti-inflammatory activity, he suggested that due to the presence of alkaloids which has been implicated for it analgesic activity (Stray, 1998). Also antioxidant activity of methanolic extract of p. nitida was observed by Osa and Abiodun, 2012. Despite the fact that Brine Shrimp Lethality Assay (BSLA) is inadequate to determine the mechanism of action of various bioactive component present in different extracts of p. nitida seed, it is very useful in providing preliminary screening for researcher and this can be verified with bioassay of different purified isolated of the plant.(extracts)
Conclusion
The phytochemical screening of picralima nitida seed exhibit the presence of alkaloids, saponins, glycoside, flavonoid, steroid and Anthraquinones and brine shrimp lethality assay shows LC50 of Aqueous, Methanol, Ethylacetate and Hexane extracts were 317ppm, 317ppm, 110ppm and 29ppm respectively, from this result, it is evident that the seed picralima nitida staph. may have curative properties against several human pathogens and suggest its importance in traditional medicine of tropical region of Africa. For further studies, effort should be devoted to characterising the bioactive components through activity-specific assays.
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