133 gel 'G' by using EtOAc : MeOH in various proportions. The details of column chromatography are given below. Column Chromatography Length of the Column : 150. cm Diameter of the Column : 5.0.cm Weight of the crude extract: 5.5gm Weight of the Sigel'G : 190 gm Table - 6 Column Chromatography Fraction No. | Eluants Spoton TLC | Remarks lo 1-5 EtOAc - MeOH Nil Sticky mass 2. 6-12 BOR seen Nil No solid mass 3, 1325 | etoxs-meon | one | compound 06) 4. 26-32 FOR Seon Nil Gummy mass. —_—18:4) STUDY OF FRACTIONS (13-25) The eluates collected from fractions 13-25 have same Ri values, therefore mixed together. On removal of the solvent it yielded an yellow needles of compound DG (2.81 g), which gave single spot on TLC examination.134 STUDY OF GLYCOSIDE (DG) It was analysed for molecular formula C2zsH3201s, mp 280- 281°C (Mj* 608 (EIMS). It gave positive response to Molisch test for glycosidic nature and the following colour reactions for flavonoidal nature. (i) Pink colour with Mg-HCl (i) | Green Colour with FeCl (ii) Bright yellow colour with Boric acid in the presence of citric acid. ELEMENTAL ANALYSIS Found Calculated for CasH32Ois C= 55.26% C=55.24% H= 5.26% H= 5.27% Molecular weight 608 (By EIMS) ACETYLATION OF THE GLYCOSIDE (DG) The acetylation of the glycoside (DG) was carried in similar method as described for glycoside (K!) on page No. 54 of the thesis. The acetylated derivative of the glycoside was crystallised from MeOH as white crystals and was analysed for molecular formula CusHsoO24,mp 183-184%C and (M]* 986 (EIMS).ELEMENTAL ANALYSIS Found Calculated for CasHsoOz« C= 55.98% C= 55.98% H= 5.08% H= 507% % Acetyl group = 39.23 % Acetyl group = 39.24 Molecular weight 986 (By EIMS) ACID HYDROLYSIS OF THE GLYCOSIDE (DG) The glycoside (60 mg) was taken in a 150 mi round bottomed flask and refluxed with 10% HCI on a water bath for 6 hr., at 100°c. On cooling and removal of the solvent yielded yellow amorphous product of the aglycone, which was separated by filtration and the filtrate was examined separately for the identification of sugar moieties. STUDY OF AGLYCONE (DG-A) The aglycone (DG-A) was crystallised from EtOH as yellow needles, and was analysed for molecular for CisHizOs, mp 287°¢ and [MJ* 300 (EIMS). It gave single spot on TLC examination by using CHCh - MeOH- HzO (10:5:2) and !2 vapours as visualising agent. The aglycone (DG-A) gave the following colour reactions for its flavonoidal nature.136 {i) Pink colour with Mg/HCI (i) Green colour with FeCls (iii) Red colour with Zn/ HCI {iv) Bright yellow colour with Boric acid in the presence of citric acid. Elemental Analysis Found Calculated for CisHi2Os C=6401% C= 640% H= 402% H= 40% Molecular weight 300 (By EIMS) ACETYLATION OF THE AGLYCONE (DG-A) The acetylation of the aglycone (DG-A) was caried out as similar manner as described on page no. 54 of the thesis. The acelyl derivative of the aglycone was crystallised from MeOH as white needles and was analysed for molecular formula CaHaoO10, mp 187-188%C and [M]* 468(EIMS). Elemental Analysis Found Calculated for C2sH2Oi0 C= 61.52% C= 61.53% H= 4.28% H= 4.27%137 % of acetyl group = 36.76 %of acetyl group = 36.75 Molecular weight 468 (By EIMS) ALKALINE DEGRADATION OF THE AGLYCONE (DG-A) The aglycone (60 mg) was treated with 50% KOH (50 ml) and EtOH (20 ml) in a 150 ml B-14 ground joint flask fitted with an air condensor for 24 hrs. and cooled. The reaction mixture was acidified with HCI and extracted with Et,O in a separating funnel. The ethereal layer was washed with water and separated into two parts. (i) The first part of the ethereal layer was treated with 50% NaHCOs and the aqueous part on acidification afforded compound (Ib} m.p. 214-215°c and was analysed for molecular formula C7HsOs, [M]* 138. It was identified as p-hydroxy benzoic acid. (i) The second part of the ethereal layer was treated with NaOH and the aqueous part on acidification afforded a compound (Ia) mp 215-2169 and was analysed for mf. CyHeOs, [MJ* 140. It was identified as 2,4,6-trinydroxy toluene. PARTIAL HYDROLYSIS OF THE GLYCOSIDE (DG) The glycoside (75mg) was treated with Kiliani mixture (HOAC:HCIH:O 35:15:50) in a 150 ml round bottomed flask for one week at room temperature. The contents were extracted with n-butanol.138 The n-butanol soluble extract on TLC examination showed two spots (Rr 0.42, 0.48). Which were separated by column chromatography over S#-gel 'G' using CHCls-MeOH in various proportions. The details of column chromatography are as follows : Length of the column 130 cm Diameter of the column 40cm Weight of the n-butanol soluble Part 200 mg Weight of the Sigel 20 gm TABLE - 7 Column Chromatography Elvants Fraction No. Spot on TLC | Remarks CHCls- MeOH (4:1) CHCIs - MeOH (4:2) CHCls - MeQH (4:3) STUDY OF FRACTIONS (1-8) one PDG-1 TWO PDG-1 & PDG-2 one PDG-2 The fractions (1-8) collected from CHCls - MeOH (4:1) were of same Rr values and so mixed together and on the removal of the solvent it yielded the compound PDG-1. Which was crystallised from MeOH and was analysed for molecular formula C22H20n, mp 189-190°c: and (M)* 462 (EIMS)139 ELEMENTAL ANALYSIS OF PDG-1 Found Calculated for C2zH22011 C= 57.12% C= 57.14% H= 475% H= 4.76% Molecular weight 462 (by EIMS) PERMETHYLATION AND HYDROLYSIS OF THE PROAGLYCONE PDG-1 The proaglycone PDG-1 {20 mg) was dissolved in DMF (6 ml) and treated with CHal (8 ml) and AgzO (75 mg} in a 150 ml conical flask at room temperature for 48 hrs. The reaction mixture was filtered and the residue was washed with DMF. The filtrate was concentrated under reduced pressure to yield a syrupy residue, which on hydrolysis with 10% HCI gave the methylated aglycone and methylated sugar. The aqueous hydrolysate after the removal of aglycone was neutralised with BaCOs and BaSOx filtered off. The concentrated filtrate was subjected to paper chromatography examination on Whatman filter paper no. 1 using BAW (4: 1 : 5) as solvent system and aniline hydrogen phthalate as spraying agent. The methylated sugar was identified as 2,3,4,6-tetra-O- methyl D-galactose.140 STUDY OF FRACTIONS (15-20) The fractions (15-20) were of the same Ri values, therefore they were combined together. On evaporation of the solvent it yielded compound PDG-2, which was crystallised from EfOH. It was analysed for molecular formula C2sHs2Ois, mp 280-281%c and [M]* 608 (EIMS). ELEMENTAL ANALYSIS OF PROAGLYCONE PDG-2 Found Calculated for CzsHs2015 C= 55.26% C= 55.24% H= 5.26% H = 5.27% Molecular weight 608 (by EIMS) PERMETHYLATION AND HYDROLYSIS OF THE PROAGLYCONE PDG-2 The permethylation and hydrolysis of the proaglycone PDG- 2 was cared out with the similar manner as described for PDG-] on page no. 139 of the thesis. It revealed the presence of methylated aglycone and methylated sugars. The methylated sugars were identified as 2,3,4-t-O-methyl-Lthamnose and 2,3,4+tr-O-methy-D- galactose. IDENTIFICATION OF THE SUGARS The aqueous hydrolysate obtained after the acid hydrolysis of the glycoside was neutralised with BaCOs and BaSO, filtered off. The141 filtrate was concentrated and examined by paper chromatography using the following two solvent systems; (i) ti) n-butanol - acetic acid- water (4:1:5) and \sopropanol - pyridine - acetic acid - water (8:8: 4: 1) and aniline hydrogen phthalate as spraying agent. The results are tabulated in table 8. Table -8 Identification of sugars S.No. | Solvent System Ri Value Sugar Reported” Found _| dentified 1. n-BuOH-acetic 0.37 0.38 L-rhamnose acid -water 0.16 0.17 D-galactose (4:1:5) 2. lsopropanol - 0.82 081 Lthamnose Pyridine - water | 9,62 0.41 | D-galactose - acetic acid - (8:8:4;1) PERIODATE OXIDATION OF THE GLYCOSIDE (DG) The glycoside (30 mg) was dissolved in MeOH (20 mi) and treated with sodium metaperiodate (10 ml) in a 150 ml conical flask and then the reaction mixture was left for 2 days at room temperature. Simultaneously a blank was also run in the same way. The quantity of consumed sodium metaperiodate and liberated formic acid were estimated by Jone’s method.142 ENZYMATIC HYDROLYSIS OF THE GLYCOSIDE (DG) The glycoside (70 mg) was dissolved in ethanol (30 ml) and treated with enzyme Takadiastase in a 150 ml conical flask fitted with a stopper. The contents were left at room temperature for 48 hrs. and filtered. The proaglycone PDG-1 and the hydrolysate were studied separately. The hydrolysate was concentrated and examined by paper chromatography using BAW (4:1:5) solvent system, Which suggested the presence of L-rhamnose (Rr 0.36, Co-Pe and Co-TLC). The proaglycone PDG-1 was dissolved in E(OH and treated with equal volume of almond emulsion. The contents were allowed to stay at room temperature for 48 hrs., and filtered. The aglycone was identified as 3,5,7,4'- tetra hydroxy - 8 - methyl flavone, mp 287°. 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