Phytochemical studies and thin layer chromatography of leaves and flower extracts of S enna siamea lam for possible biomedical applications

Senna siamea is a medium-size, evergreen plant which has been utilized as a source of food, medicine and other agricultural purposes in different communities. However, there is dearth of information in regard to its possible biomedicinal uses, especially in Nigeria. Thus the preliminary phytochemical analysis and thin layer chromatography (TLC) separation was done using methanol, n-hexane and ethyl acetate (1:3:1) as solvent system while iodine vapour as spotting agent. The phytochemical screening of methanol extracts of leaves revealed the presence of cardiac glycoside, flavonoid, saponin, alkaloid and tannins while chloroform extracts of leaves revealed saponin only. Ethyl acetate and petroleum ether extracts revealed absence of all these phytochemicals. The chloroform, ethyl acetate and petroleum ether extracts of flower revealed absence of saponin, flavonoids, tannins and alkaloids but with traces of saponin and anthraquinones. TLC separation showed nine (9) spots each of chloroform and ethyl acetates, six (6) spots of methanol, three (3) spots of petroleum ether from leaves extracts. While, three (3) spots each of ethyl acetate and methanol, six (6) spots of chloroform were identified for flower extracts. No water spot separated from both leaves and flower extracts. From our findings, it can be concluded that S. siamea lam contains some significant phytochemicals that can exhibit desired therapeutic activities such as hypoglycemia, anti-arrthymia and antimicrobial. However, there is the need to conduct further pharmaceutical analyses on test extracts in order to establish these biomedical applications.


INTRODUCTION
Plants have been found to be the source of energy for the animal kingdom.In addition, plant can synthesize a large variety of chemical substances that are of physiological significance (Kretovich, 2005).The active phytochemical principles produced by plants include, alkaloids, phenolic, anthraquinones, flavonoids, phenols, saponins, steroid, tannins, terpenes etc (Namukobea et al., 2011).
Medicinal plants are those that contains one or more of its phytochemicals that can be used for the synthesis of useful therapeutic agents (Sofowora, 2000).The wide range of medicinal plant parts like flowers, leaves, barks, stems, fruits, roots extracts are used as powerful raw drug possessing a variety of pharmacological activities (Momin et al., 2012).In the last two centuries, there have been serious investigations into the chemical and biological activities of plants and these have yielded compounds for the development of synthetic organic chemistry and the emergence of medicinal chemistry as a route for the discovery of more effective therapeutic agents (Roja and Rao, 2000).
Senna siamea is native to Southeast Asia from India, Sri Lanka, and Thailand to Indonesia, Burma, and Malaysia and forms part of the warm and wet tropical forests.The species has been introduced in Africa and America.S. siamea is effective in managing constipation association with a number of causes including surgery, childbirth and the use of narcotic pain relievers (Hill, 1992).It is used locally as antimalarial drugs especially when decocted (the leaves and bark) (Lose et al., 2000).In traditional medicine, the fruit is used to charm away intestinal worms and to prevent convulsion in children.The young fruits and leaves are also eaten as vegetables in Thailand.The flowers and young fruits are used as curries (Kiepe, 2001) and as an antimalarial (Otimenyin et al., 2010).The stem bark extract was reported to have analgesic and anti-inflammatory effects (Ntandu et al., 2010).Isolated compounds, emodu and lupeol from the ethyl acetate fraction of the stem bark of S. siamea were reported to be the active principles responsible for the antiplasmodial property with IC 50 values of 5 µg/ml, respectively (Ajaiyeoba et al., 2008).Sub-chronic studies of the aqueous stem bark extract of the plant in rats did not show significant toxic effect after seven weeks of administration (Mohammed et al., 2012) This study was designed to determine the phytochemical compositions as well as to perform thin layer chromatography separation of the leaves and flowers extracts of S. siamea in order to create awareness of its possible medicinal and nutritional values.

MATERIALS AND METHODS
These include the test plant (the fresh leaf and flower of S. siamea), beaker, conical flask, measuring cylinder (large and small), glass funnel, glass stirrer, cotton wool, spatula, bunsen burner, top mettler weighing balance, test tubes, stainless steel tray, thermostat water bath, oven, syringe and needle, aluminum foil paper, hand gloves, mortar and pestle, analytical weighing balance, test-tube holder, refrigerator, meter rule, sieves (No. 5), bottles, UV fluorescence analysis cabinet tripod stand, wire gauze, capillary tubes, retort stand, thin layer chromatography (TLC) paper, TLC tank, test tube rack, tiles and filter paper.

Sterilization
All work surfaces were comprehensively disinfected with cotton wool soaked in antiseptic fluid to minimize contamination during work process.

Dry heat sterilization
An hot air oven was used to sterilize the conical flasks, forceps, office punch, wire loop and filter paper discs (wrapped in foil paper) and beaker at 160°C for 45 min.

Moist heat sterilization
All materials used in the course of this research project that are not sensitive to moist heat sterilization were adequately sterilized using autoclave and detergents.Materials such as glass wares, beakers and conical flasks etc. were properly washed with detergent and water so as to remove dirt and contaminants and were allowed to dry prior to usage.These materials were then sterilized in a portable laboratory autoclave at 121°C for 15 min.

Collection, authentication and processing of plant materials
The fresh leaves and flowers of S. siamea were collected from the botanical garden of University of Maiduguri.Plant materials were identified and authenticated by a taxonomist, Professor S. S. Sanusi of the Department of Biological Sciences, University of Maiduguri, Nigeria in respect with the description in published literatures (Dalziel, 1958;Keay et al., 1989).The plant materials were dried under shade at our Pharmaceutical Chemistry Laboratory for about four weeks and then made into powdered form, using mortar and pestle and then sieved.

Extraction
The method of extraction in this experiment was by maceration.The general process on a small scale, consist of placing the powdered plant material (250 g) of leave was soaked in 500 ml methanol while that of flower was soaked with different solvents that is water, petroleum ether, methanol, chloroform and ethyl acetate (in order of decreasing polarity) in 1 L capacity conical flasks stopper and kept for 48 h with intermittent shaking.The cold extracts thus obtained were filtered with Whatman No. 1 filter paper into different conical flask and allowed to dry at room temperature under normal atmospheric pressure.50 g of the powdered leaves were soaked in 100 ml distilled water and the extract was obtained using the aforementioned method.

Phytochemical analysis
Phytochemical analysis for the qualitative detection of alkaloids, anthraquinone, carbohydrates, flavonoids, tannins and saponins was carried out on the extracts as described by Trease and Evans (2010), Sofowora (1993) and Harbone (1973).

Thin layer chromatography (TLC)
Commercially available standard TLC plate was used with standard particle size range to improve reproducibility.The absorbent silica gel coated on an aluminum foil of 22 cm length, 11.5 cm breadth and 0.3 cm thick plate for leaves while 22 cm length, 11.9 cm breadth and 0.3 cm thick plate for flower.Small spot of the solution containing the sample was applied on the plate 1.5 cm from the bottom marked by a line ruled using a pin.For a multiple spotted plate, the spots are applied 1 cm apart to avoid cross contamination and interference as they move up the plate.

Spotting and development
The sample spotted on the plate was allowed to dry before the plate was placed into the chromatographic tank which was covered immediately after which its atmosphere is completely saturated with solvent (mobile phase).The reaction was then monitored as the solvent moved up the plate (elutes the sample) using mobile phase solvent ratio 1:3:1 of methanol, n-hexane and ethyl acetate, respectively.When the solvent reaches the top of the plate, it is removed, marked and dried.

Visualization
Following separation of the solvent, the plate was removed and dried; the spots detected using various techniques and reagents.This includes visualization in daylight; viewing under UV at 254 and 366 nm i.e. short and long wavelengths and spraying with spotting reagent, using iodine vapor tank.

Findings
The phytochemical screening of methanol extracts of leaves revealed the presence of cardiac glycoside, flavonoid, saponin, alkaloid and tannins while chloroform extracts of leaves revealed saponin only.Ethyl acetate and petroleum ether extracts revealed absence of all these phytochemicals.The chloroform, ethyl acetate and petroleum ether extracts of flower revealed absence of saponin, flavonoids, tannins and alkaloids but with traces of saponin.Anthraquinones glycosides was absent in all the extracts.

Extraction process for leaves and flowers
This is seen in Tables 1 and 2.

Phytochemical screening results
This is seen in Tables 3 to 9.

Thin layer chromatography (TLC)
Extracts of leaves and flowers were individually applied on the origin, they dissolved and moved with the solvent, each extract separated into bio constituents and moved to different locations.After all the spots became clear.UV fluorescence lamp at 254 nm was used to visualize and identify all the various spots.However, at 366 nm and daylight, spots were not clearly visualized.On exposure to iodine vapour, spots of various extracts became darker.TLC separation showed nine (9) spots each of chloroform and ethyl acetates, six (6) spots of methanol, three (3) spots of petroleum ether from leaves extracts.While, three (3) spots each of ethyl acetate and methanol, six (6) spots of chloroform were identified for flower extracts.No water spot separated from both leaves and flower extracts.

Leaves TLC
Length of the plate = 22 cm.Breath of the plate =11.5 cm.Thickness of the plate = 0.1 cm.Solvent front of the plate = 18.3 cm.R f value = distance move by the solute ÷ distance move by the solvent.
Tables 10 to 13 shows the TLC results of leaves.

Flowers TLC
Length of the plate = 22 cm.Breath of the plate = 11.9 cm.Thickness of the plate = 0.1 cm.Solvent front of the plate = 17.9 cm.R f value = distance move by the solute ÷ distance move by the solvent.
Tables 14 to 16 shows TLC results of flowers.

DISCUSSION
S. siamea plants grow virtually everywhere in Nigeria and Maiduguri in particular.The plant has been used in this region for the treatment of typhoid fever and fever related conditions.Traditionally, it has also been used for treatment of jaundice, abdominal pain, menstrual pain, and hypoglycemic agent among diabetics.Ethno medicinally, S. siamea is used as laxative, blood cleaning agent, cure for digestive system and genitourinary  disorders, herpes and rhinitis (Aliyu, 2006).When decocted, S. siamea leaves are locally used as antimalaria drug (Lose et al., 2000).Previous studies on S. siamea extracts have confirmed some of the traditional uses: antiplasmodial activity (Gbeassor et al., 1990;Nsonde-Ntandou et al., 2005;Mbatchi et al., 2006).Antibacterial activities of the extract were tested against thirteen pathogenic bacteria and were compared with the standard antibiotic, kanamycin by measuring the zone of inhibition diameter and expressed in millimeter (mm) (Hailu et al., 2005;Dahiru et al., 2013).Phytochemical screening reveals that methanolic extract contains carbohydrate, cardiac glycosides, saponins, flavonoids, tannins and alkaloids.The extracting solvent used are decreasing order of polarity in which each of them extract a number of solvent to their own polarity depending on the active metabolites the plant contained.Based on this experiment the alkaloid, tannins and saponins content of this can be responsible for its antibacterial activity (Dahiru et al., 2013) Preliminary phytochemical analysis showed that leaf extracts of S. siamae possesses alkaloids, saponins, tannins and glycosides which is in support with studies done by Momin et al. (2012), Edeoga et al. (2005) and Bukar et al. (2009).Phytoconstituents such as saponins, phenolic compounds and glycosides when present in S.  siamea have been reported to inhibit bacterial growth and to be protective to plants against bacterial and fungal infections (Gonzalel and Mather, 1982;Okwute, 1992).Cardiac glycosides have also been found useful in

Table 3 .
Test for carbohydrate.