Biochemical characterization and cell immobilization of costus pictus D. Don with special reference to antidiabetic property

Abstract

Kerala, a state known for its indigenous knowledge and traditional healing practices, is endowed with thousands of medicinal plants. More than 1200 species of plants are being used in the indigenous system of medicine in the state. The use of synthetic chemicals in modern medicine has been causing several side effects. Hence, more and more scientific and commercial activities are now directed towards plant-based medicines. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2004-2006. The objective of the study was the biochemical characterization and cell immobilization of Costus pictus with special reference to antidiabetic property.The hypoglycemic properties of the plant has been reported both in streptozotocin and alloxan induced diabetes in animals. The toxicity study of crude extract of the plant was also studied (Benny, 2004a; Benny et al., 2004a & b) and reported a positive sign of non-toxicity. Based on the above facts the biochemical characterisation and cell immobilization of Costus pictus was carried out and this was compared with antidiabetic plants like ginger, gymnema, adhatoda, neem and tulsi. Molisch's test, Iodine test, fehling's test, benedict's test and seliwanoff's test confirmed the presence of carbohydrates in Costus pictus and in all selected plants with varying intensities. Carbohydrate tests indicated the presence of monosaccharides of ketohexoses origin in all selected plants including Costus pictus. xanthoproteic reaction, glyoxylic reaction for tryptophan and modified million’s test confirmed the presence of proteins having aromatic amino acids in Costus pictus and in all selected crops. TLC also confirmed the presence of - amino acids by giving pink colour with ninhydrin. The best separation of phenolic spots occurred on acid hydrolysis. The phenols appeared as dark absorbing spots in 253 nm and fluorescent spots in 366 nm. Phenol spots appeared as blue with folins reagent. Four compounds were isolated in TLC from Costus pictus. Ethanol was used as the solvent for the extraction of flavonoids from leaf samples. There was only one spot (Rf value 70) at 253nm in Costus pictus. The red colour production observed in neem and tulsi leaf samples confirmed the presence of condensed tannins. The presence of four hydrolysable tannin in Costus pictus was confirmed. The TLC for terpenoids, Costus pictus and ginger expressed same compound at Rfx100 value 71.4. Gymnema, adhatoda and neem had different spots. In TLC the carotenoids expresed different coloured spots such as green-chlorophyll b, blue green-chlorophyll a, yellow green-xanthphyll and yellow-β-carotene in ascending order under visible light. TLC for plant acids, Costus pictus only gave spot only similar to that of oxalic acid. Mayer’s test, dragendorff’s test and wagner’s test showed positive result for alkaloid in all the plants studied. All the above antidiabetic plants for comparison were subjected to electrophoresis. Protein extraction was carried out using Tris-HCl, Tris-HCl + ingredients and Phosphate buffers. The Tris-HCl +ingredients buffer was found to be more appropriate on account of discrete and distinct banding pattern. Protein-banding pattern in SDS denatured gel conditions and isozyme (peroxidase) banding in non denaturing gels were also studied. The 10% gel gave good separation of bands. The bands were clear in coomassive brilliant blue stain than in the silver staining. Costus pictus exhibited four bands. Isozyme analysis was carried out using peroxidase enzyme system. Costus pictus exhibited four bands in which three are similar to that of ginger. The cells of Costus pictus were immobilized using sodium alginate. Immobilized cells kept for secondary metabolite production in ½ MS liquid media showed the presence of phenolics, terpenoids different Rf values and plant acids, indicating the utility of the technique of producing secondary metabolites from Costus pictus in vitro. Considering the presence of similar hexoketoses, free amino acids and / or soluble protein and phenolic compounds in gymnema and Costus pictus cannot be ruled out for the medicinal use of Costus pictus as an antidiabetic plant. Detailed study on Costus pictus with respect to protein and oxalic acid are needed to obtain more information of the antidiabetic property and toxicity to biological system.