CHAPTER XI STUDY OF STEROIDAL SAPOGENINS 1
STUDY OF STEROIDAL SAPOGENINS The present investigation describes the isolation and identification of Steroidal Sapogenins from roots, shoots and fruits of Abutilon indicum, Datura Stramonium, Withania somnifera and Tribulus terristris collected from kuchaman city area of Nagaur district. Materials and Methods The dried and powdered plant parts (roots, shoots and fruits) were used for extraction of sapogenins. Extraction Procedure Each of the dried samples were hydrolysed with 30% hydrochloric acid. The hydrolysed test samples were washed separately with distilled water till ph 7. Test samples thus obtained were dried and Soxhlet extracted in benzene (Nag et al., 1979). Each of the benzene extracts of the various test samples were separately dried in vacuo and taken up in chloroform for further analysis of its steroidal sapogenins. Qualitative Estimation Thin Layer Chromatography Each of the crude extracts along with the reference sapogenins (diosgenin, gitogenin, hecogenin, kryptogenin, smilagenin, tigogenin and yamogenin) were dissolved in chloroform and applied separately on silica gel G coated and activated glass plates. These glass plates were developed in an organic solvent mixture of benzene: ethyl acetate 85:15 (Heble et al., 1968). The developed glass plates were air dried and visualized under UV light showed 2 fluorescent spots in some test samples which on spraying with 50% sulphuric acid and subsequent heating at 100º C for 10 minutes showed two spots conciding with that of their standard reference compounds as hecogenin and diosgenin (Rf 0.23 and 0.42) respectively in some of the test samples ; hecogenin (dark yellow Rf 0.23) and diosgenin (brownish green Rf 0.42). A few other solvent systems (hexane : acetone, 8:2; acetone : benzene, 1:2; hexane : ethyl acetate, 3:1 ; benzene : ethyl acetate, 3:1) were also tried, but benzene : ethyl acetate (85:15) gave excellent results in the present investigation. 2
Preparative Thin Layer Chromatography (PLC) Each of the extracts along with the standard reference steroids were applied separately on thickly (0.3 mm to 0.4 mm ) silica gel G coated and activated glass plates. The plates were developed in an organic solvent mixture of benzene : ethyl acetate (85:15). The developed plates were air dried and visualized under UV light. Two fluorescent spots (Rf 0.23 and 0.42) corresponding with those of the standard reference samples of hecogenin and diosgenin in test samples were separately marked and collected along with silica gel from unsprayed plates, eluted with chloroform, dried, crystallized separately with methanol and chloroform. Each of the crystallized isolates were subjected to colorimetery (for quantitative estimation ), and melting point (Melting Point apparatus, Toshniwal, India) studies along with their respective standard reference steroids. Quantitative Estimation of Sapogenins Steroidal sapogenins were estimated following the Spectro- photometric method of Sanchez et al. (1972). Standard stock solution of diosgenin and hecogenin were separately prepared from 10 ug to 120 ug in chloroform and applied separately on silica gel coated and activated glass plates along with a parallel run of blank. These glass plates were rum in solvent mixture of benzene and ethyl acetate (85:15), air dried and kept in a chamber saturated with iodine vapours. The resulting colour spots were marked and the plates were kept in an oven at 100ºC for 15 minutes so as to evaporate the excess of iodine. Spots of diogenin, hecogenin and blank zones from the parallel run were separately scrapped along with the absorbent eluted with 5 ml of methanol and than centrifuged. From each of the samples 4 ml of aliquots was taken up and evaporated to dryness on a water bath. To each of the resulting residue, 4 ml of 80 % methanolic sulphuric acid, was added and kept for 2 hrs. Absorbences from each of the known samples were measured on a spectronic- 20- colorimeter (Bausch and Lamb) set at 405 nm against a blank (8% methanolic sulphuric acid ) and a regression curve of various concentrations against optical densities was computed which followed the beer s law. Absorbences from each of the unknown samples were also taken in a similar manner and their concentrations were determined (mg/g.d.w.) by comparing with those of the samples were examined and the mean values taken. 3
Results and Discussion Presence of diosgenin and hecogenin from all the selected samples were confirmed by co-chromatography (diosgenin, Rf 0.42 and hecogenin, Rf 0.23) mp (207-208ºC and 264-266ºC) respectively. In present study the sapogenins (diosgenin and hecogenin) were not detected in any parts of Abutilon indicum. In the plant parts of Withania somnifera such as roots, shoots and fruits have only diosgenin (0.04 mg/g.d.w.,0.05 mg/g.d.w., and 0.08 mg/g.d.w.) respectively (Table 11.1). Maximum amount of diosgenin was present in shoots of Datura Stramonium (0.10 mg/g.d.w.) while, minimum in roots of Withania somnifera (0.04 mg/g.d.w.). (Table 11.1) Maximum amount of hecogenin was present in shoots of Datura Stramonium (0.12 mg/g.d.w.) while, Minimum in roots of Datura Stramonium (0.05mg/ g.d.w.). (Table 11.1) Maximum (0.22 mg/g.d.w) amount of total sapogenin contents were present in shoots of Datura Stramonium while, minimum (0.04 mg/g.d.w) in roots of Withania somnifera. (Table 11.1) A number of plant species were investigated for their sapogenin contents in vivo and in vitro.diosgenin is principally obtained from the under ground portion of Dioscorea spp. (Khanna et al., 1978b: Staba, 1977; Singh et al., 1965 Mandal and Chatterjee, 1985 and Ravishankar and Grewal, 1986). It has also been reported from a few species of Solanum (Madeva and stephanova, 1965; Mookherjee and Methew, 1969; Varshney and Dube, 1970; Doepke et al., 1976; Khanna et al., 1978b; Carle et al., 1977; Kaul and Atal, 1978 and Khanna, 1984) and from other plant species such as Asparagus species (Kar and Sen, 1984; Kar, 1985) Fagonia cretica and Lycium barbarum (Harsh, 1982), Kalistromia puescens (Chakravarti et al., 1976; Nag et al., 1979) Trigonella corniculata (Khanna, 1984). Hecogenin, tigogenin and diosgenin reported from various plant parts (roots, shoots and fruits ) and tissue culture of Zygophyllum simples (Mathur, 1988). Singh (1989) reported the presence of diosgenin, hecogenin, kryptogenin and tigogenin from intact plant parts of Ocimum americanum and Abutilon pannosum. 4
Kapoor, B.B.S. (1991) reported presence of diosgenin and kryptogenin in Fagonia cretica. Diasgenin has also been reported from tissue cultures of Lycium barbarum (Grover, 1984; Nag,1995), Tribulus alatus (Jit, 1985; Nag, 1995). Seetzenia orientalis (Sethia, 1988; Nag, 1995) and Zygophyllum simplex (Mathur, 1988; Nag, 1995). Savikin Fodulovic Katarina et al. (1998) observed the dioesgenin in five callus lines of Dioscarea balcanica. This investigation presents evidence that sapogenins can be isolated from intact plant parts of growing in kuchaman city area of nagaur district. These plants have sufficient amount of sapogenins and could be a good source of production of steroidal sapogenins. 5
GRAPH 11.1 TABLE-11.1 Steroidal Sapogenins in various plant parts of selected plant species (mg/g.d.w.) Name of sterols Datura stramonium Withania somnifera Tribulus terristris Root Shoot Fruit Root Shoot Fruit Root Shoot Fruit Diosgein 0.09 0.10 0.07 0.04 0.05 0.08 0.05 0.08 0.06 Hecogenin 0.05 0.12 0.09 - - - 0.06 0.10 0.09 Total Contents 0.14 0.22 0.16 0.04 0.05 0.08 0.11 0.18 0.15 6
7