INTRODUCTION:
India is rich in wide variety of medicinal plants. Plants are used for the treatment of various ailments from ancient times. Strychnos is one of the largest genuses that comprises of approximately 200 known species and among that 44 species are endemic to Asia1. Strychnos colubrina Linn belongs to the family Logainacea is widely distributed in Kerala. The plant is been used as a traditional medicine from ancient times. It is one of the important medicinal plants of tribes. The plant is mainly used for the treatment of Snake bite, dyspepsia, malaria, intermittent fevers, swellings in chicken pox, joint pain, Diarrhoea, tumor, Febrifuge, Intermittent fever, dyspepsia, malarial cachexia2 etc. The bioactive components of the plant are not properly exploited. So the present study aims on the extraction, quantitative and qualitative phytochemical analysis of Strychnos colubrina Linn. The GC-Ms study reveals the various phyto constituents present in the extracts.
MATERIAL AND METHODS:
Plant Material:
The ariel parts of Strychnos colubrina Linn was collected from village of Idukki in the month of October 2014. The plant material was identified and authenticated by the Botany department at Nirmala college of Arts and Science, Muvattupuzha, Kerala, India. The ariel parts of the plant was shade dried at ambient temperature and the dried materials were crushed into fine powder using an electric blender. The powder was stored in polythene bags before use.
Preparation of Extracts:
The powdered sample material was extracted with 4 four solvents (pet. ether, chloroform, methanol and water) by successive extractions. About 200g of powdered plant material was weighed and defatted with petroleum ether (1L) using soxhlet apparatus at 40°C for 48hrs. Then the residue was extracted with chloroform, methanol and water (750mL) successively as like pet. ether extraction. The extracts were concentrated in a rotary evaporator to yield a dark brown mass (5 g).
Preliminary Phytochemical Studies:
The preliminary Phytochemical studies were performed by using petroleum ether , chloroform, methanol and aqueous extracts of dried ariel parts of Strychnos colubrina Linn to screen the presence of various secondary metabolites according to the standard procedure described by Harborne3.
Quantitative estimation of Active constituents:
i). Alkaloids:
The extract of plant sample was treated with 0.1N HCl and aqueous acidified layer thus obtained was partitioned with chloroform in a separating funnel. The chloroform layer is rejected. The aqueous layer was basified with ammonium hydroxide and then partitioned with chloroform. The chloroform layer was concentrated and tested for alkaloids with alkaloid testing reagents 4.
ii). Tannins
Dissolve of 1g of sample in 100ml water. Pipette out10ml into a 250ml conical flask and add 25ml of indigo sulphonic acid solution. Makeup the volume to100ml with distilled water. Titrate, with constant stirring, against 0.1 N potassium permanganate solution. End point is a golden yellow colour. Conduct a blank test by titrating 25ml of indigo sulphonic acid in 75 ml of water and calculate the content of total tannins. Each ml of 0.1 N potassium permanganate solution is equivalent to 0.004157g of tannin compound calculated as tannic acid 5, 6, 7.
Tannins % = (Blank-sample) x Normality of KMnO4 x0.4147x10
 |
0.1 x Weight of sample
iii). Sterols:
Extract 5g of sample with 25ml methanol, filter and evaporate to 25ml. The standard is prepared by dissolving 1g in1L methanol and 5 concentration of standard β-sitosterol (1g/L) (5μL,10μL, 15 μL, and 20 μL) are prepared. These standards along with sample were spotted onto 6.0 x 10.0 cm HPTLC plates silica gel 60 F 254 (Merck, Germany)coated aluminum plates. Chromatogram tanks (Twin Trough Chamber 20x10cm) were preconditioned for 3min at 80°C in an oven using diethyl ether: pet ether (1:1) as solvents. The solvent front position is 80mm and volume is 10mL. After developing the spots, it is derivatized by treating with 20mlof LB reagent and dried at 100°C for 10min in an oven and scan at 254nm and 366nm using CAMAG TLC Scanner. Calculate the concentration of sterols from calibrated graph8, 9, 10.
iv). Saponins:
About 5 gm of plant powder was put into a conical flask and then 50 ml of 20% aqueous ethanol was added. The sample was heated with continuous stirring at 550c over a hot water bath for 4 hours. This mixture was filtered and the remaining residue re-extracted with another 100 ml 20% ethanol. Both the extract combined and reduced up to 40 ml over water bath at 900c. The concentrate obtained was transferred into a 250 ml separating funnel and 10 ml of diethyl ether was added and shaken vigorously. In separating funnel, two separate layers were observed out of which aqueous layer was recovered and the ether layer was discarded. The process of purification was repeated. To the aqueous extract 30 ml of n- butanol was added. A combined n- butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the sample obtained were dried in oven to the constant weight and the saponin percentage was calculated 11
v). Flavanoids:
10 g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight 12
GC-MS Analysis.
The GC-MS analysis of the C. carandas was performed using a Clarus 680 Perkin Elmer gas chromatography equipped with an Elite-5 capillary column (5% diphenyl, 95% dimethyl polysiloxane) (30.0m × 0.25mmID× 250 𝜇m) and mass detector turbo mass of the company which was operated in EI mode. Helium was the carries gas used at a flow rate of 1 mL/min. The injector was operated at 200°C and the oven temperature was programmed as follows:60°C for 2min and 10°C/min until 300°C. Interpretation of GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight, and structure of the components of the test materials were ascertained 13.
In-Vitro Anti-Inflammatory Activity by HRBC membrane Stabilization method:
i). Preparation of Human Red Blood Cells (HRBC) Suspension
Fresh whole human blood (from healthy volunteers who had not taken NSAIDs for 2weeks) was collected and mixed with equal volume of sterilized Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in water). The blood was centrifuged at 3000 rpm for 10 min and packed cells were washed three times with isosaline(0.85%, pH 7.2). The volume of the blood was measured and reconstituted as 10% v/v suspension with isosaline.
ii). Hypo tonicity Induced Hemolysis
The principle involved here is stabilization of human red blood cell membrane by hypo tonicity induced membrane lysis. The assay mixture contains 1ml phosphate buffer [pH 7.4, 0.15 M], 2 ml hyposaline [0.36 %], 0.5 ml HRBC suspension [10 % v/v] with 0.5 ml of plant extracts and standard drug diclofenac sodium of various concentrations (50, 100, 250, 500, 1000, 2000 μg/ml) and control (distilled water instead of hypo saline to produce 100 % hemolysis) were incubated at 37°C for 30 min and centrifuged respectively. The hemoglobin content in the suspension was estimated using spectrophotometer at 560 nm. Diclofenac (100 and 200 g/ml) was used as reference standard and a control was prepared by omitting the extracts. The percentage of hemolysis of HRBC membrane can be calculated as follows 14,15,16,17: % Hemolysis = (Optical density of Test sample / Optical density of Control) X 100 The percentage of HRBC membrane stabilization can be calculated as follows:
100- Optical density of drug treated sample
% Protection = X
100
Optical density of control
RESULTS AND DISCUSSION:
Extraction:
The successive extraction of powdered plant sample was carried out with four solvents and the results are shown in Table-1. The extraction process was carried out by hot percolation method. The extraction process was carried out at a temperature of 40-60°Cand the colour of obtained extracts were green and maximum yield was obtained for aqueous extract
Preliminary phytochemical analysis:
The phytochemical screening of Strychnos colubrina Linn whole plant extracts was done with pet. ether, chloroform, methanol and water. The components present in various extracts were found to be alkaloids, saponins, glycosides, proteins and amino acids, sterols, flavonoids, carbohydrates, phenols and tannins. The chloroform extract had shown the presence of alkaloids, glycosides, sterols, flavonoids and proteins. The methanolic extract contains alkaloids, flavonoids, tannins, sterols and carbohydrates. The aqueous extract contains only saponins
Figure 1:.Arieal parts of Strychnos colubrina Linn