Topclass Journal of Herbal Medicine Vol. 3(3) pp. 14-16, 26 Sept., 2014
Available online at http://www.topclassglobaljournals.org
ISSN 2315-8840 ©2014 Topclass Global Journals
Submitted 3/5/14 Accepted 23/8/14
Short Communication
Phytochemistry and cytotoxicity of
Prosopis spicigera
using Brine Shrimp Lethality assay
Wasim Ahmed*
1, Mushtaq Ahmad
1, A. Rauf
2, Sania Feroz
2, Zakir Khan
3, Zia-Ul-Haq
31
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, Kpk, Pakistan
2
Institute of Chemical Sciences, University of Peshawar, K.P.K Peshawar-25120, Pakistan 3
Institute of Chemical Sciences, Gomal University D.I.Khan, Kpk, Pakistan
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Abstract
Phytochemical assessment is imperative for the corroboration of a variety of bioactive compounds in plants. This study was aimed to screen the methanolic crude extract of Prosopis Spicigera for its phytochemistry and cytotoxicity potential. Our results showed the presence of alkaloids, saponins, steroids, reducing sugar, flavonoids, tennins and terpenoids. The results also showed 100% shrimps deaths at 1000 µgm/ml. This confirmed the presence of anti tumorous compounds in the plant which are needed to be isolated for future use in order to synthesize potential drugs.
Key Words: Prosopis spicigera, Phytochemistry, Cytotoxicity, Brine shrimp
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INTRODUCTION
Medicinal plants are of great significance to the health of individuals and communities. The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds (Hill, 1952). Being a source of remedial agents, plants always played an important role in the human history. About 80% of the world’s populations still depend on plants for primary health care. Even in present day Western medication, about 25% of prescribed medicines are still derived either directly or indirectly from plants. In Pakistan, researchers are incessantly working towards the search of new and useful drugs from higher plants. Every country of the world is virtually active in this search to a limited degree. (Farnsworth, 1980; Duke, 1985; Tyler, 1987).
Bioactive compounds are often toxic to Artemia salina (leach) shrimp larvae. In many pet shops, the eggs of the brine shrimp A. salina are readily available as fish food. (Kivack et al., 2001; Carballo et al., 2002). When placed in artificial seawater, the eggs hatch within 48 h, producing large numbers of larvae. This is a speedy, reasonably priced, general bioassay, which has been developed for screening, fractionation and monitoring of physiologically active natural products (Carballo et al., 2002). In the present research investigation, methanolic crude extract of P. Spicigera was screened for its phytochemical and cytotoxic potential.
MATERIALS AND METHOD consecutive weeks. It was then mechanically grinded into powder using a local grinder machine.
Extraction
100 gm powder of P. Spicigera was extracted in 1 L commercial grade methanol (Merck Lab) and randomly shaken for 3 h on a shaker machine. It was then kept for 5 days at room temperature. After this time period, the extract was filtered by using wattman filter paper No 1 and the filtrate was further concentrated under reduced pressure on rotavap at 380C. The concentrated methanolic crude extract was then applied to lyophilizer and converted into very fine powder form which was stored at 40C in a Falcon tube
PREPARATION OF SAMPLE
Sample was prepared by dissolving 5 mg of crude extract powder in 5 ml methanol. From this stock solution; different sub-solutions were prepared i.e. 100 µgm/ml, 250 µgm/ml, 500 µgm/ml and 1000 µgm/ml for cytoxic assay.
Phytochemical Profiling
The crude extract was evaluated to spot bioactive compounds by using standard phytochemical screening tests (Uddin et al., 2014). This was done by adding H2SO4 to crude extract and also adding some dragendroff’s reagent, alkaloids were confirmed upon appearance of orange red precipitates. Tannins were determined by adding few drops of ferric chlorides to aqueous solution of crude which were confirmed by the emergence of dark green solution. Diluted NaOH was added to 0.2 g of crude. After that, HCl was added. A yellow solution that turns colorless is the indication for the presence of flavonoids.
1 g of crude was mixed in 3 ml of water and heated until boiling. Appearance of frothing showed the presence of saponins. Small quantity of crude extract was shaken solution. Red precipitate shows the presence of
Ahmed et al., 2014..Topcls. J. Herb. Med. 3(3)15
phlobatanins. To 1 g of crude extract, few drops of acetic acid were added. It was gently warmed, cooled and then a drop of H2SO4 was added.
The color changed to green which shows the presence of steroids. 0.2 g of crude extract was mixed with 2 ml of chloroform and concentrated H2SO4 (3 ml) was carefully added to form a layer. The formation of a reddish brown coloration at the interface is the positive results for the yellow color indicates the presence of betacyanin.
Cytotoxic Brine Shrimp Assay
A cytotoxic activity of methanolic crude extract of P. Spicigera was conceded according to the standard procedure of (Meyer-Albert et al., 1992). Sample was prepared by dissolving 5 mg of crude plant extract in respective solvent (methanol) to form stock solution of 5 mg/ml in methanol and further diluted into 100, 500, and 1000 μg/ml. 2.8 g commercial sea salt (Sigma) was dissolved in 100 ml of dH2O with constant stirring for 2 h. Brine shrimp eggs were hatched in a shallow rectangular tray. A plastic divider with several 2 mm holes were clamped in the tray to make 2 uneven compartments.
The eggs were speckled into the smaller compartment and were covered with aluminum foil while the larger compartment was illuminated. After 24 h, larvae were observed in the lightened compartment. 1 ml of all sub-solutions were put into cleaned test tubes and allowed to evaporate the methanol. Residues were re-dissolved in 3 ml saline. A positive control was maintained by pouring 3 ml saline in a test tube. 8 brine shrimp were transferred to all the tubes and the volume increased up to 5 ml. After 24 h, survivors were counted with the help of 3x magnifying glass and calculation was done using Abbot’s formula;The results are shown in Figure 1.
Percentage of death = (Sample-control/control) × 100
RESULTS
Ahmed et al., 2014..Topcls. J. Herb. Med. 3(3)16
Figure 1. Results showing %survivial of brine shrimps with varying concentrations of extract
Table 1. Phytochemical screening of Prosopis Spicigera methanolic crude extract
Type of Test Results Alkaloids + Tannins + Flavonoids + Saponins + Reducing sugars + Phlobatanins - Steroids + Anthocyanin - Betacyanin - Terpenoids + Emodins -
anti tumorous compounds within it.
DISCUSSION
Phytochemical screening test play important role in the isolation of rare, new, novel compounds and the discovery of drugs. Plants are like the factory for bringing about natural products in a most selective and precise way. The chemical analysis of P. Spicigera revealed the presence of various secondary metabolites such as alkaloids, saponins, steroids, terpenoids and emodins. (Table 1)
Results showed that the brine shrimp survival is inversely proportional to the concentration of the extract used. Kanegusuku et al., (2001) reported organic fraction of Rubus imperialis (C.) which showed more cytotoxicity. Zaidi et al., (2006) studied that methanolic fraction of Arceuthobium oxycedri possessed 100% lethality for brine shrimps at high dose which are in accordance with
results obtained. The results of present study suggest that methanolic fraction possess some bioactive constituents having anticancer activities that can be the point of interest for new drugs possessing anticancer and protective role against different pathogens.
CONCLUSION
Results showed good cytotoxicity against Brine shrimps which means P. spicigera can be used as a cytotoxic agent due to the presence of certain bioactive compounds which are needed to be isolated and characterized for further use.
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