CHEMICAL INVESTIGATION OF SOME FERNS OF KUMAUN HILLS CHAPTER-III CHEMICAL INVESTIGATION AND STRUCTURAL ELUCIDATION. Estelar

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1 CHAPTER-III CHEMICAL INVESTIGATION AND STRUCTURAL ELUCIDATION [3.1] Chemical Investigation of the Flavonoids from Hymenophyllum polyanthos About 3kg air dried and powdered sample was extracted sequentially with 70% aq. ethanol and 50% aq. ethanol by cold percolation method for three days. The combined aq. ethanolic extracts was evaporated to dryness under reduced pressure at 60 0 C in Rota-evaporator until only a small H 2 O layer (approx. 50ml) remained. It was partitioned successively with CH 2 Cl 2 and BuOH. Each partition was examined on 2DPC for total flavonoidal constituents. In order to catalog all the flavonoids present in the fern fronds of H. polyanthos, 2DPC was applied by fresh aq. ethanolic extract on Whatman No. 1 PC using BAW(n-BuOH-AcOH-H 2 O,4:1:5, V/V, upper layer) and 30% HOAc solvent systems. After developing chromatogram, the spots were studied in visible and UV light both with and without the presence of NH 3 vapours and after spraying with NA reagent (Dissolving 1gm of 2-amino ethyl di phenyl borate in 100ml of absolute MeOH). Sums of twenty five fluorescent spots were discernible on 2DPC. Out of twenty five spots, nineteen were classified as flavonoids on the basis of their colour reactions. Other six spots were non-flavonoids in nature. The flavonoidal spots on PC were marked by soft pencil as compounds [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] and [19]. Out of the nineteen flavonoidal positive spots, nine {[6]-[13] and [15]} were dark purple UV fluorescent spots, five {[14], [16]-[18] and [19]} violet UV fluorescent spots, three {[1], [3] and [4]} purple UV fluorescent spots and remaining two [2] and [5] dull yellow UV fluorescent spots. After spraying 2DPC with methanolic solution of NA reagent, eight compounds {[4], [5], [8], [9], [10], [12], [13] and [19]} turned to orange and remaining eleven compounds turned to yellow. Using BAW as a suitable solvent for Rf values and sequence determination of flavonoidal components is being given in table [3.1(a)]. [3.1(i)] 2DPC Examination of CH 2 Cl 2 Fraction from Fresh Fronds of H. polyanthos CH 2 Cl 2 soluble derived from the partition of H 2 O layer of aq. ethanolic extract was evaporated to dryness and residue was dissolved in MeOH and chromatographed two dimensionally using BAW (4:1:5) and 30% HOAc solvent systems. The dried and developed 2DPC was examined under UV light. A total of seven fluorescent spots were discernible on PC, of which four spots could be identified as flavonoids on the basis of their colour reactions with UV, UV/NH 3, UV/NA and UV/ZrOCl 2 reagents, while remaining three spots could be identified as a non-flavonoids as they produce different shades of blue-green and yellow-green fluorescence with UV light. The Rf values and colour reaction of flavonoidal positive compounds are being given in table [3.1(b)]. 47

2 Table [3.1(a)]: Paper chromatography of H 2 O-EtOH extract from fresh fronds of H. polyanthos (on Whatman No. 1 PC) Components UV UV/NH 3 UV/NA Rf in BAW [A] [1] P YG Y 89 [B] [2] DY DY Y 80 [E] [3] P YG Y 76 [C] [4] P YG Or 70 [D] [5] DY DY Or 68 [F] [6] DP YG Y 65 [G] [7] DP YG Y 63 [H] [8] DP Y Or 62 [I] [9] DP Y Or 60 [J] [10] DP Y Or 58 [K] [11] DP YG Y 55 [L] [12] DP Y Or 54 [M] [13] DP Y Or 52 [N] [14] V LY Y 50 [O] [15] DP LY Y 47 [P] [16] V LY Y 44 [Q] [17] V LY Y 41 [R] [18] V LY Y 39 [S] [19] V LY Or 36 Table [3.1(b)]: Paper chromatography of CH 2 Cl 2 fraction from fresh fronds of H. polyanthos (on Whatman No. 1 PC) Components UV UV/NH 3 UV/NA Rf in BAW [A] P YG Y 89 [B] DY DY Y 80 [C] P YG Or 70 [D] DY DY Or 68 On comparing the Rf values and colour reactions of flavonoidal constituents of CH 2 Cl 2 fraction with the compounds of aq. ethanolic extract, it was concluded that the compounds [A], [B], [C] and [D] of CH 2 Cl 2 fraction were found similar to the compounds [1], [2], [4] and [5) respectively of H 2 O-EtOH extract. Thus, these four compounds were isolated from CH 2 Cl 2 fraction. [3.1(ii)] 2DPC Examination of BuOH Fraction from Fresh Fronds of H. polyanthos BuOH fraction of aq. ethanolic extract from fern fronds of H. polyanthos was evaporated to dryness and residue was dissolve in MeOH. The methanolic solution was chromatographed two dimensionally on Whatman No.1 PC using BAW (n- BuOH-AcOH-H 2 O, 4:1:5, V/V, upper layer) and 30% HOAc as a developing solvent systems. After development, the PC was air dried and inspected with UV light. A total of twenty fluorescent spots were inspected on 2DPC under UV light. Out of twenty fluorescent spots, fifteen could be identified as flavonoids on the basis of their colour reactions with UV, UV/NH 3, UV/NA, UV/ZrOCl 2 and UV/AlCl 3. The Rf values and colour reactions of these compounds is being summarized in table [3.1(c)]. 48

3 Table [3.1(c)]: Paper chromatography of BuOH fraction from fresh fronds of H. polyanthos (on Whatman No. 1 PC) Components UV UV/NH 3 UV/NA Rf in BAW [E] P YG Y 76 [F] DP YG Y 65 [G] DP YG Y 63 [H] DP Y Or 62 [I] DP Y Or 60 [J] DP Y Or 58 [K] DP YG Y 55 [L] DP Y Or 54 [M] DP Y Or 52 [N] V LY Y 50 [O] DP LY Y 47 [P] V LY Y 49 [Q] V LY Y 41 [R] V LY Y 39 [S] V LY Or 36 On comparing the Rf values and colour reactions of flavonoidal components from BuOH soluble from aq. ethanolic extract, it was concluded that the compounds [E], [F], [G], [H], [I], [J], [K], [L], [M], [N], [O], [P], [Q], [R] and [S] were found similar to the compound [3], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] and [19] respectively. Therefore, compounds [3], {[6]-[19]} were isolated from BuOH fraction of aq. ethanolic extract. The residue of BuOH solvent was further extracted with EtOAc in solvent extractor. The EtOAc soluble was also examined two dimensionally using BAW (n-buoh-acoh-h 2 O, 4:1:5, V/V, upper layer) and 30% HOAc solvents. The dried and developed 2DPC was examined under UV light (360nm). Fifteen UV fluorescent spots were observed on PC. Out of these, eleven spots gave positive flavonoidal tests. The Rf values and colour reactions of flavonoidal positive compounds of the EtOAc fraction were found similar to the compound {[E]-[O]} of BuOH fraction. Therefore these eleven flavonidal compounds were isolated from EtOAc fraction. [3.1(iii)] 2DPC Examination of Acid Hydrolysed Extract of Fresh Fronds of H. polyanthos About 6gm of powdered sample of fern fronds of H. polyanthos was extracted with 2N-HCl foran hour at C. The acid hydrolysed extract was filtered. The hydrolysate was neutralised with aq. BaCO 3 solution. The neutral solution was evaporated to dryness and residue was dissolved in isopropanol. The isopropanol soluble was chromatographed unidimensionally on Whatman No.1 PC using 30% HOAc as a developing solvent. The dried and developed paper chromatogram was inspected with UV light (360nm). A total of six fluorescent spots were noticed on PC. The Rf values and colour reactions of these compounds are being summarized in table [3.1(d)]. 49

4 Table [3.1(d)]: Paper chromatography of acid hydrolysed extract of fresh fronds of H. polyanthos (on Whatman No.1 PC) Components 30% HOAc UV UV/NH 3 UV/NA UV/ZrOCl 2 [1 ] 56 P YG Y f-yg [2 ] 53 YG YG YG YG [3 ] 46 DY DY Y f-yg [4 ] 43 BG BG Y I-BG [5 ] 40 P YG Or f-yg [6 ] 36 DY DY Or f-yg Out of six fluorescent spots, four could be identified as flavonoids on the basis of their colour reactions with UV, UV/NH 3, UV/NA and UV/ZrOCl 2. These four flavonoidal compounds are compaired with compounds [1 ], [3 ], [5 ] and [6 ].Out of these four flavonoidal compounds, two compounds [1 ] and [5 ] are purple fluorescent and remaining two compounds [3 ] and [6 ] are dull yellow fluorescent colour under UV light. On comparing the Rf values and colour reactions of these compounds with the flavonoidal compounds of CH 2 Cl 2 fraction, it was established that the compounds [1 ], [3 ], [5 ] and [6 ] of acid hydrolysed fraction are similar to the compounds [A], [B], [C] and [D] respectively of CH 2 Cl 2 fraction. No additional flavonoidal spots were observed on 2DPC acid hydrolysed extract of BuOH fraction. Thus, it has clearly been established that compounds [A], [B], [C] and [D] are free as well as aglycone derived from acid hydrolysis of glycoside of BuOH fraction. [3.1.1] Structural Elucidation of Flavonoids from H. polyanthos [3.1.1(I)] Structure Determination of Compound [1] The compound [1] was isolated from CH 2 Cl 2 fraction of CH 3 COCH 3 -H 2 O (1:1) extract of fern fronds of H. polyanthos. After concentration, methanolic elute of the compound gave a yellow needles, mp C. The MS of compound [1] exhibited a molecular ion at m/z 270 (100%) for C 15 H 10 O 5 in accordance with a flavone containing three hydroxyl groups. Flavone appeared as a purple fluorescent spot on PC under UV light and changed to yellow-green with NH 3 vapours, indicating the presence of a free 5- and 4 -hydroxyl groups. When PC was sprayed with NA reagent, the spot turned yellow, indicating a free 4 -hydroxyl group but no ortho-di-hydroxyl group in the B-ring. Its purple UV fluorescence on PC under UV light changed to bright yellow-green fluorescence after spraying the chromatogram with 1% AlCl 3 (ethanolic) and ZrOCl 2 indicating a free 5-OH group in the compound [1] (Fiegl,1960). Thus, these colour reactions of compound on PC, showed the presence of 4 - and 5-hydroxyl groups in the flavone skeleton. The ethonolic solution of the compound gave pink colour with vanilline+ HCl, indicating the presence of 5, 7-dihydroxyl group in the compound [1] (Hillis and Urbach, 1958). On the basis of above colour reaction the flavone skeleton has free hydroxyls at C-7, C-5 and C-4. The compound gave a tri-accetate derivative, mp C and a trimethyl ether derivative, mp C on acetylation and methylation respectively. Thus, confirmed the presence of three hydroxyl groups in the molecule. The position of hydroxyl groups in flavone skeleton was further confirmed by UV spectral study of compound [1]. The compound [1] exhibited UV maxima in MeOH at 336nm (band I) 267nm (band II) and shifts obtained with diagnostic reagents are given below in table [3.1.1(I) a]. 50

5 Table [3.1.1(I) a]: UV spectra of compound [1] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH NaOMe sh 392(Inc) AlCl AlCl 3 +HCl NaOAc sh 376 NaOAc+H 3 BO The flavone nature of compound was confirmed by its λ max in MeOH at 267nm (band II) and 336nm (band I). A bathochromic shift is 7nm ( ) nm of (band II) was obtained on addition of fussed sodium acetate and the presence of shoulder at 309nm with CH 3 ONa and 350nm with NaOAc, confirmed the presence of 7-OH group. A bathochromic shift of 48nm ( ) nm of (band I) was produced by the addition of AlCl 3 solution (free-oh). A bathochromic shift of 56nm ( nm) of (band I) was obtained on addition of sodium methoxide solution (free 4 -OH group). Thus UV spectra of the compound [1], revealed the presence of free hydroxyls at C-7, C-5 and C-4. The oxygenation pattern of compound [1] was further assigned on the basis of 1 HNMR spectra in (DMSO-d 6, 200MHz): In 1 HNMR spectra, [table 3.1(I) b], the presence of signals at δ 6.60 and δ 6.30 each with (1H, d, J=2.0 Hz) assigned to meta coupled A-ring Protons, H-8 and H-6 respectively, and signals at δ 7.94 and δ 6.98 each with (2H, d, J=8.8 Hz) assigned to B-ring, ortho coupled protons, H -2, H-6 and H-3 and H-5 respectively. One proton singlet at δ 6.77 (1H, s) represent to H-3 of flavone. Thus, the oxygenation pattern of the molecule was assigned at C-5, C-7 and C-4. Table [3.1.1(I) b]: 1 HNMR studies of compound [1] in DMSO-D 6, 200MHz Shift (δ) Multiplycity Protons identified H, d, J=8.8 Hz H-2 and H H, d, J=8.8 Hz H-3 and H H, s H H, d, J=2.0 Hz H H, d, J=2.0 Hz H-6 On the basis of above colour reaction, UV and 1 HNMR studies, compound [1] was identified as 4, 5, 7-tri-hydroxy flavone or apigenin representing structure (I) Fig. (I) Apigenin The structure (I) was further confirmed by its CoPC on Whatman No.1 PC with authentic apigenin by using these solvent systems: (i) BAW (n-buoh-acoh-h 2 O, 4:1:5, V/V, upper layer); (ii) t-baw (t-buoh-acoh-h 2 O, 3:1:1, V/V ); (iii) 30% HOAc; (iv) 3% NaCl; (v) Phenol saturated H 2 O and (vi) 2DPC: (a) BAW (4:1:5) and 51

6 (b) 15% HOAc. [3.1.1(II)] Structure Determination of Compound [2] The compound [2], a slower moving dull yellow UV fluorescent band on Whatman No. 1 PC at Rf 35 in 30% HOAc, was isolated from CH 2 Cl 2 fraction of CH 3 COCH 3 - H 2 O (1:1) extract of fern fronds of H. polyanthos, which was obtained from five sheets of Whatman No. 3 PC, afforded a yellow-light green crystalline compound m.p C, on standing the methanolic eluate for two days at room temperature 10 0 C. The MS of compound [2] exhibited a molecular ion at m/z 286 (100%) for C 15 H 10 O 6 in accordance with a flavone containing four hydroxyl groups. The compound appeared as dull yellow fluorescent spot on PC under UV light with and without NH 3 vapours, indicating the presence of a free 3- and 5- hydroxyl groups (Mabry et al., 1970). When PC was sprayed with NA reagent, the spot turned intense yellow colour, indicating the presence of a free 4 - hydroxyl group but no ortho-dihydroxyl groups in the B-ring. The ethanolic solution of the compound gave yellow colour on treatment with ZrOCl 2, which in presence of citric acid formed a white precipitate, indicating presence of free 3-OH group in flavone nucleus (Horhammer and Hansel, 1955). It gave red colour with Mg+HCl and Zn+HCl, which is specific for flavonol presence of 3-OH group (Shimizu, 1951; Geissman, 1955). The compound on treatment with solution of H 3 BO 3 in presence of citric acid in acetone gave yellow colour which produced yellow green fluorescence in UV light indicating the presence of free 5-OH group (Wilson, 1939). The paper strips spotted with compound [2] and sprayed with ethanolic solution of ZrOCl 2 and 1% AlCl 3, produced a yellow-green fluorescence in UV light indicating the presence of free 5-OH group (Feigl, 1960). It gave pink colour with vanilline+hcl, showed the presence of 5, 7-di-hydroxyl system in the molecule (Hillis and Urbach, 1958). On the basis of colour reactions the position of hydroxyl groups in flavone nucleus were assigned at C-3, C-5 and C-7. Compound [2] exhibited UV maxima in MeOH at 370nm (band I) and 266nm (band II) and shifts obtained with diagnostic reagents are given below in table [3.1.1(II) a] and fig. I-a. Table [3.1.1(II) a]: UV spectra of compound [2] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH NaOMe sh 440(inc) AlCl AlCl 3 +HCl NaOAc sh 430sh NaOAc+H 3 BO sh 372 UV spectrum of compound [2] in MeOH gave 266nm (band II) and 370nm (band I), suggested a flavonol compound.a bathochromic shift of 12nm (bandii) ( ) nm with NaOAc indicated presence of free 7-OH group in A-ring. A bathochromic shift of 70nm (band II) ( ) nm with increase intensity in the NaOMe spectrum relative to (band I) in the MeOH spectrum indicated the presence of a free 4 -OH grouping B-ring. A bathochromic shift of (band I) of 72nm ( ) nm with AlCl 3, which remain unchanged with AlCl 3 +HCl, indicated absence of ortho-di-hydroxyl 52

7 group in B-ring.These UV spectral data confirmed that the free 7-OH, 4 -OH, 5-OH and 3-OH group in the molecule. On the basis of UV spectral data and colour reactions of the compound [2], the position of OH groups will be at C-3, C-5, C-4 and C-7. Fig. (I-a) The assignments of the position of these hydroxyl groups were further confirmed on the basis of 1 HNMR spectra of the compound [2] in DMSO-d 6, 200 MHz: In 1 HNMR spectra, [3.1.1(II) b], gave two ortho coupled symmetrical doublets each with J=7.5 Hz at δ 6.95 and δ 8.06, which correspond to the H- 3, 5 and H- 2, 6 proton of the B-ring and the two meta coupled doublets each with J=1.8 Hz at δ 6.20 and δ 6.45 for H-6 and H-8 protons respectively in A-ring. A downfield signal showed 5-OH proton at (1H, s). Thus, 1 HNMR spectra revealed the presence of oxygenation at C-3, C-5, C-7 andc-4. Table [3.1.1(II) b]: 1 HNMR studies of compound [2] in DMSO-D 6, 200MHz Shift (δ) Multiplycity Protons identified H, d, J=7.5 Hz H 2 and H H, d, J=7.5 Hz H 3 and H H, d, J=1.8 Hz H H, d, J=1.8 Hz H H, s 5-OH In the EIMS spectrum of compound [2], the presence of molecular ion M + at m/z 286 (100%), and other prominent ions at 257 [M-CO-H], 153[A + +1] + and 121 [B 2 + ] suggested that the aglycone was kaempferol. The colour reactions, UV, MS and 53

8 1 HNMR spectral data of the compound [2] was identified as 3, 4, 5, 7-tetra-hydroxy flavone or kaempferol, representing structure (II). Fig. (II) Kaempferol Finally, the structure (II) was confirmed by its CoPC with its authentic kaempferol using these solvent systems: (i) BAW (n-buoh AcOH H 2 O, 4:1:5, V/V, upper layer); (ii) BEW (n-buoh EtOH H 2 O, 4:1:2.2, V/V ); (iii) 30% HOAc; (iv) 2-DPC : (a) BAW (4:1:5) and (b) 15% HOAc. [3.1.1(III)] Structure Determination of Compound [3] Compound [3] was isolated from EtOAc fraction of aq. ethanolic extract of fern fronds of H. polyanthos. It gave positive test with Mg+HCl (red), FeCl 3 (blue-green) and α-naphthol indicated a polyphenolic heterocyclic flavonoidal glycoside (Shinoda, 1928; Dean, 1961). EIMS of the compound gave a molecular ion, m/z at 270 [M] + represent the molecular formula of aglycone. FABMS (-ve) of the compound gave 431[M-H], 269 [M-163-H]. The compound appeared as a purple fluorescent on PC under UV light and changed to yellow-green after fuming with NH 3 vapours, indicating a flavone with a free hydroxyls at C-4 and C-5. When cellulose TLC of the compound was sprayed with NA reagent it turned to yellow, indicating presence of a free hydroxyl group. Further support the presence of a free hydroxyl group at C-4 and absence of ortho-di-hydroxyl group in the B-ring. It gave fluorescent yellow-green colour with methanolic solution of ZrOCl 2. Further, it supported the presence of free hydroxyl group in C-5 (Feigl, 1960). On the basis of chromatographic behaviour of the flavonoidal compound, it has been established as a flavone-mono-glycoside (Markham, 1982). The Rf values and colour reactions of th compound [3] are being given in table [3.1.1(III) a]. Table [3.1.1(III) a]: Rf values of the compound [3] (on Whatman No. 1 PC) using various solvent systems Solvent system Rf Values Locating reagent Visualization (V/UV) H 2 O 64 UV DP 15% HOAc 12 UV/NH 3 YG 30% HOAc 24 UV/NA YG 30%HOAc 44 UV/ AlCl 3 f-yg BEW 65 UV/ ZrOCl 2 f-yg t-baw (3:1:1) 62 UV/(CH 3 COO) 2 Pb (neutral) f-yg Compound [3] exhibited UV maxima in MeOH, table [3.1.1(III) b], at 336 (band II) and 267nm (band I) and shifts obtained with diagnostic reagents, suggested the presence of free hydroxyl groups at C-5 and C-4. 54

9 Table [3.1.1(III) b]: UV spectra of compound [3] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH NaOMe 245sh sh 440(inc) AlCl AlCl 3 +HCl NaOAc 286sh NaOAc+H 3 BO The compound [3] was hydrolysed with 2N-HCl at C for 45 minutes. The hydrolysate was neutralised with aq. solution of BaCO 3. The neutral solution was repeatedly evaporated to dryness on water bath. The residue was dissolved in isopropanol and it was chromatographed on Whatman No.1 using BAW (4:1:5) as a developing solvent. The dried and developed chromatogram was inspected under UV light, a dark purple fluorescent spot was observed on PC at Rf 89. It was isolated in milligram scale from 10 sheets of Whatman No. 1 PC using BAW (4:1:5) as an eluent. The non-fluorescent region on PC from Rf 0-80 was sprayed with sugar chromogenic reagent benzidine in acetic acid. The PC was dried in oven at C for 10 minutes. A single brown spot was observed on PC. It was identified as glucose by CoPC with its standard using following three solvent systems: (i) BAW (n-buoh- ACOH-H 2 O, 4:1:5, V/V, upper layer); (ii) t-baw (t-buoh-acoh-h 2 O, 3:1:1, V/V) and (iii) BEW (4:1:2.2, upper layer). The aglycone, a dark purple fluorescent compound on PC under UV light was observed at Rf 89 in BAW solvent derived from isoproponol soluble of acid hydrolysed mixture of compound [3] was identified as follows: The MS of compound [3] exhibited a molecular ion at m/z 270 (100%) for C 15 H 10 O 5 in accordance with a flavone containing three hydroxyl groups. Flavone appeared as a purple fluorescent spot on PC under UV light and changed to yellow green with NH 3 vapours, indicating the presence of a free 5 and 4 hydroxyl groups. When PC was sprayed with NA reagent, the spot turned yellow, indicating a free 4 - hydroxyl group but no ortho-di-hydroxyl group in the B-ring. Its purple UV fluorescent on PC under UV light changed to bright yellow-green fluorescence after spraying the chromatogram with ZrOCl 2 and 1% AlCl 3 (ethanolic), indicating a free 5-OH group in the compound [3] (Feigl, 1960). Thus, these colour reactions of compound on PC, showed the presence of 4 - and 5- hydroxyl groups in the flavone skeleton. The ethonolic solution of the compound gave pink colour with Vanilline+HCl, indicating the presence of 5, 7-di-hydroxyl group in the compound [3] (Hillis and Urbach, 1958). On the basis of above colour reaction the flavone skeleton has free hydroxyls at C-7, C-5 and C-4. The compound gave a tri-acetate dvt, mp C and a trimethyl ether derivative, mp C on acetylation and methylation respectively. The oxygenation pattern of compound [3] was further assigned on the basis of 1 HNMR spectra in DMSO-d 6, 400MHz: In 1 HNMR spectra the presence of signals at δ 6.60 (1H, d, J=2.0Hz) and δ 6.30 (1H, d, J=2.0Hz) were assignlde to meta coupled A-ring Protons, H-8 and H-6 respectively, and signals at δ 7.94 (2H, d, J=8.8Hz) and δ 6.98 (2H, d, J=8.8Hz) assignable to B-ring, ortho coupled protons, H-2, H-6 and H- 55

10 3, and H-5 respectively. One proton singlet at δ 6.77 (1H, s) represent to H-3 of flavone. Thus, the oxygenation pattern of the molecule assigned at C-5, C-7 and C-4. 1 HNMR spectra in DMSO-d 6, 400MHz is given in table [3.1.1(III) c] and fig. 2-a. Table [3.1.1(III) c]: 1 HNMR studies of compound [3] in (DMSO-d 6, 400MHz) Shift (δ) Multiplycity Protons identified H, d, J=8.8Hz H-2 and H H, d, J=8.8Hz H-3 and H H, s H H, d, J=2.0Hz H H, d, J=2.0Hz H-6 Fig. (II-a) On the basis of colour reactions, MS, 1 HNMR and derivatives formation, acetyl and methyl dvts, the aglycone could be identified as a 4, 5, 7-tri-hydroxy flavone or apigenin, representing structure (I). The structure (I) was finally confirmed by CoPC with the standard isolated form the petals of Dehlia variabilis (Nordstrom and Swain, 1953). [3.1.1(III-a)] Structure Determination of Glycoside Acid hydrolysis of the compound afforded an aglycone, apigenin and glucose (CoPC). The only point that now remains to be decided is the position of free hydroxyl group of aglycone involved in the formation of glycosidic linkage. Since there are three free hydroxyl groups in the aglycone molecule at position C-4, C-5 and C-7 glucose may be attached to any of them. It could be done as follows: On comparing the colour of the glycoside with its alglycone on PC under UV light in presence of NH 3 vapours, it was found that the aglycone and glycoside both appeared as a purple fluorescent on PC with UV light and turned to yellow-green with NH 3 vapours, indicating that both the molecules have free hydroxyl at C-4 and C-5. The methonolic solution of aglycone gave pinkish colour with vanilline+hcl while the glycoside did not give positive test with Vanilline+HCl. This clearly indicated that the C-7 hydroxyl group of apigenin is substituted. The UV spectra of compound aglycone in MeOH gave two peaks at 267nm, 336nm for (band II) and band (I) respectively. After addition of NaOAc, the 12nm bathochronic shift of band (II) clearly indicated that 7-OH group in the 56

11 A-ring of flavone molecule is free. While no bathochromic shift band (II) was observed after addition of NaOAc in the case of glycoside. Thus, it has been confirmed that the glucose is attached at C-7 of glycoside. Complete methylation of glycoside with dimethyl sulphate and aq.solution of K 2 CO 3 followed by hydrolysis with 2N-HCl gave a compound 7-hydroxyl-4, 5 -di-methoxy apigenin, which was finally confirmed by comparing with its standards on PC using three solvents systems: (i) 30% HOAc (ii) BAW (4:1:5) (iii) t-baw (3:1:1). Thus, the compound [3] was identified as apigenin-7-oglucoside, representing structure (III). Fig. (III) Apigenin-7-O-glucoside [3.1.1(IV)] Structure Determination of Compound [4] The compound [4] was isolated from CH 2 Cl 2 fraction of aq.alcoholic extractt of fern fronds of H. polyanthos. After concentration, methonolic elute of the compounds give light yellow needles, mp, C. The MS of the compound [4] exhibited a molecular ion at m/z 286 (100%) for C 15 H 10 O 5 in accordance with a flavone containing 4- hydroxyl group. It appeared as purple fluorescent spot on paper chromatogram under UV light and change to yellow-green with NH 3, indicating the presence of a free 5 - and 4 - hydroxyl group. When cellulose TLC of the compound was sprayed with methanolic solution of NA reagent the spot turned to orange, revealing the presence of ortho-di hydroxyl group in the B-ring. The paper chromatographically spotted compound was sprayed with ethonolic solution of ZrOCl 2, it produced fluorescent yellow-green colour, indicating the presence of free 5-OH group in the A-ring. Further, the methanolic solution of compound [4] gave pink colour with Vanilline+HCl reagent and showed the presence of 5, 7-di-hydroxyl system in the molecule (Hillis and Urbach, 1958). On the basis of colour reactions, the flavnoidal compound has free hydroxyl group at C-3, C-4, C-5, and C-7. The compound exhibited UV maxima in MeOH at 355nm (band I) and 258nm (band II) and shifts obtained with diagnostic reagents such as NaOMe, AlCl 3, AlCl+HCl, NaOAc and NaOAc+H 3 BO 3 further supported the presence of hydroxyl group at C-3, C-4, C-5 and C-7. The UV spectral data of compound [4] is being given in table [3.1.1(IV) a]. Table [3.1.1(IV) a]: UV spectra of compound [4] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH sh 349 NaOMe 266sh 329sh 401 AlCl sh AlCl 3 +HCl 266sh sh NaOAc sh 384 NaOAc+H 3 BO sh sh 57

12 1 HNMR spectra of the compound [4] in (DMSO-d 6, 400MHz) gave two meta coupled doublets each with J=2.0 Hz at δ 6.30 and δ 6.60 representing H-6 and H-8 respectively of A-ring. An ABX system was found for B-ring at δ 6.80 (1H, d, J=8.0 Hz), δ 7.26 (1H, dd, J=2.0 and 8.0 Hz) and δ 8.02 (1H, d, J=2.0 Hz) assignable to H- 5, H-6, and H-2, respectively of B-ring. A sharp signal appeared at δ 6.77 representing H-3 of flavone nucleus. Thus, on the basis of colour reactions, UV, MS and 1 HNMR spectral studies indicated the compound [4] was identified as 3, 4, 5, 7-tetra-hydroxy flavone or Luteolin, representing structure (IV). The structure (IV) was further confirmed by CoPC with its authentic using following three solvent systems: (i) BAW (BuOH- AcOH-H 2 O; 4:1:5, V/V upper layer); (ii) BEW (BuOH: EtOH: H 2 O; 4:1:2.2 V/V upper layer) and (iii) 30% HOAc. Fig. (IV) Luteolin [3.1.1(V)] Structure Determination of Compound [5] Compound [5], a pale yellow needles, mp C was isolated from CH 2 Cl 2 fraction of H. polyanthos. It gave molecular ion peak m/z 302 [M] + and was found to have molecular formula C 15 H 10 O 7. UV spectra of the compound indicated a flavonol nucleus, having UV maxima in MeOH λ max 370nm (band I) and 255nm (band II). Ethanolic solution of the compound gave olive green colour with FeCl 3 solution, red colour with Mg+HCl (Geissman, 1955). Ethanolic solution of the compound gave orange colour solution with characteristic fluorescence with concentrated sulphuric acid. It did not give positive test with 2, 4-Dinitrophenyl hydrazine indicating the absences of a hindered carbonyl group and no reduction with sodium borohydride was indicative of absence of flavanone skeleton. Therefore, the compound has a flavone nucleus (Horowitz and Gentile, 1960). The compound gave a penta acetate dvt, mp C and a penta methyl ether dvt, mp C an acetylation and methylation respectively. This confirmed the presence of five hydroxyl groups in the molecule. The relative positions of hydroxyl groups were assigned on the basis of various colour reactions, degradation and spectral studies. On PC it gave dull yellow UV fluorescence (Mabry et al., 1970). It gave red colour with Zn+HCl which is specific for flavone having a free 3-OH group (Shimizu, 1951). The ethanolic solution of the compound gave yellow colour on treatment with ZrOCl 2, which in presence of citric acid formed a white precipitate, indicating the presence of free 3-OH group in flavone nucleus (Harhammer and Hansel, 1955). The compound gave pink colour with vanilline+hcl showing the presence of 5, 7-di-hydroxyl system in the molecule (Hillis and Urbach, 1958). A deep red colour was produced when ethanolic solutions of boric acid and sodium acetate reagent were added to the ethanolic solution of the aglycone free catechol unit (Dean, 1963). The presence of a free 5-OH group in A-ring was further assigned on the basis of that it gave a yellowgreen fluorescence under UV light in the presence of boric acid solution, citric acid solution (Wilson, 1939) and ethanolic solution of ZrOCl 2 and 1% AlCl 3 (Feigl,1960). 58

13 The methanolic solution of the compound when mixed with solution of sodium molybdate (0.1m in 80% MeOH) indicated ortho-di-hydroxyl group in B-ring (Swain and Goldstein, 1964). The dull yellow colour of the compound on PC under UV light changed into orange colour when sprayed with NA reagent, indicated free 5-OH flavonol with ortho-di-hydroxyl system in B-ring (Sanggong et al., 1988). The position of ortho-di-hydroxyl system in B-ring was assigned on the basis of 1 HNMR studies: In the 1 HNMR spectrum two meta coupled doublets at δ 6.42 and δ 6.20 each with (1H,d,J =2.0 Hz ), assignable to the protons at H-8 and H-6 position in the A-ring and signals at δ 7.55 (1H, dd, J=8.5 Hz, 2.0 Hz), δ 7.69 (1H, d, J=2.0 Hz) and δ 6.90 (1H, d, J=8.5 Hz) assignable to the protons at H-6, 2 and 5 -positions respectively in the B-ring. The mass spectrum (see, fig. (IV-a)) of the compound established a flavone with five hydroxyl groups: [M] + at m/z 302 (100%), corresponding to C 15 H 10 O 7 and other prominent ions in mass spectra are: [M-1] (80%), [M-CO-H] (7%), and B- ring with two hydroxyls. [B 2 ] (20%), [B 2-28] +, 109 (10%) and A-ring with two hydroxyls. [A] (46%), [A1+H] (40%) and [A 1-28] (30%). Fig. (IV-a) Compound exhibited UVmaxima (table (V) and Fig. (IV-b)) in MeOH at λ max at 370 nm (band I), λ max at 255 nm (band II) and λ max at 300 nm (band III) and the shifts obtained with diagnostic reagent with AlCl 3, AlCl 3 +HCl, NaOAc, NaOAc+H 3 BO 3 and CH 3 ONa suggested that it has free hydroxyls at C-3, C-5, C-7, C-3 and C-4 (Mabry et al., 1970). Table [3.1.1(V) a]: UV spectra of compound [5] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH sh 370 NaOMe AlCl AlCl 3 +HCl sh NaOAc sh NaOAc+H 3 BO 3 257sh

14 Fig. (IV-b) These absorption maxima are indicative of a flavonol nucleus. A bathochromic shift of 58nm ( ) nm of (band I) was produced by the addition of 1% ethanolic AlCl 3 solution indicating the presence of free 5-OH group. A bathochromic shift of of 12nm ( ) nm of (band II) was obtained on addition of sodium acetate indicating the presence of free 7-OH group). The presence of (band III) and its bathchromic shifting of 25nm ( ) nm in presence of sodium acetate clearly indicated the prescence of 7-OH group. Bathochromic shifts of (band I) in presence of AlCl 3, AlCl 3 /HCl, and NaOAc/H 3 BO 3 indicated prescence of a free hydroxyl groups at position-5, 3 and 4. Bathochromic shifts of (band I) 370nm in presence of CH 3 ONa and NaOAc solution, confirms the presence of free 4 -OH group. A Bathochromic shifts of (band I) 40nm ( ) nm in presence of ZrOCl 2 +citric acid, indicated free 3-OH group (Sosa and Percheron, 1970). These UV Spectral data clearly indicated that the compound [5] may be 5, 7, tetra-hydroxyl flavonol. Further the position of hydroxyl groups in flavone skeleton wws assigned on the basis of of alkali degradation with aq. NaOH or KOH solution. The penta hydroxyl flavone was also assigned on the basis of alkali degradation of aglycone with 20% aq. NaOH solution. It gave two phenolic products phloro-glucinol and proto-catechuic acid and 60

15 were indentified by CoPC with their respective standards on Whatman No. 1 PC using BAW (4:1:5), BAW (4:1:1) and 30% HOAc, as a developing solvents. On the basis of colour reaction, alkali degradative products, UV, 1 HNMR and MS studies the aglycone was identified as 3, 5, 7, 3, 4 -penta hydroxyl flavone or quercetin, representing structure (V). Fig. (V) Quercetin The structure (V) was further confirmed by CoPC with its authentic, isolated from the flowers of Bassia butyraceae (Khetwal and Verma, 1986) using following solvent systems: (i) BAW( BuOH-AcOH-H 2 O, 4 :1:5, V/V, upper layer ); (ii) 30 % HOAc and (iii) 3 % NaCl. [3.1.1(VI)] Structure Determination of Compound [6] Compound [6] is a flavonoidal glycoside of EtOAc fraction of aq. ethanolic extract from fern fronds of H. polyathos. The flavonoidal glycosidic nature of the compound was confirmed by its colour reactions with Mg+HCl, FeCl 3, α-naphthol, Feigl s spot test for sugar, neutral lead acetate and Na 2 CO 3 (Geissman, 1956; Dean, 1961; Feigl, 1960). It appeared as dark purple fluorescent spot on PC and changed to yellow-green after fuming with NH 3 vapour indicating a flavone with free hydroxyls. On inspecting the paper chromatogram spoted with glycoside and its acid hydrolysed aglycone, it was found that the former compound appeared as dark purple fluorescent and latter as a dull yellow fluorescent under UV light, indicating a release of sugar moiety from C- 3 aglycone. Acetonic solution of compound was completely methylated with NaOH and dimethyl sulphate. The methylated brown waxy product was hydrolysed with 7% concentrated H 2 SO 4 for 2.5 hours at C. The hydrolysed product was extracted with diethyl ether and after evaporation a brown residue was obtained with mp C. It was identified as 5, 7, 4 - tri-methoxy kaempferol. It was finally confirmed by CoPC with its authentic (Glaxo) using following three solvent systems: (i) BAW (4:1:5, V/V, upper layer); (ii) t-baw (3:1:1) and (iii) 15% HOAc. Finally the compound was identified by 1 HNMR spectral studies in (DMSO-d 6, 400MHz),: In aromatic region, two meta coupled doublets appeared at δ 6.15 (1H, J=2.0 Hz) and δ 6.36 (1H, J=2.0 Hz) for H-6 and H-8 in A-ring protons respectively and two ortho coupled doublets at δ 6.82 (2H, d, J=8.5 Hz) and δ 8.03 (2H, d, J=8.5 Hz) for H-3 and 5 and H-2 and 6 protons of B-ring respectively. Anomeric proton appeared at δ 5.50 (1H, d, J=8.5 Hz) and other sugar protons were identified at δ 4.35, δ 3.85, δ 3.6 and δ 3.4 for H-2, H-3, H-4 and H-5 protons of sugar. The appearance of anomeric signals at δ 5.50 with very high coupling constant J=8.5 Hz indicated β- configuration in pyranose form of glucose Sugar at C-4 and C-5 (Mabry et al., 1950; Markham, 1982). When cellulose TLC of the compound was sprayed with methanolic solution of NA regent it turned to yellow, indicating the presence of free hydroxyl at 61

16 C-4 and absence of ortho-di-hydroxyl group in the B-ring. FABMS (-ve) of the compound gave a molecular ion at m/z 431 [M-1], representing molecular formula is C 21 H 20 O 10. EIMS of the compound gave molecular ion, m/z at 286 [M] +. UV spectra of compound in MeOH gave two maxima at 266nm (band II) and 349nm (band I) and shifts obtained with diagnostic reagents, NaOMe, AlCl 3, AlCl 3 +HCl, NaOAc and NaOAc+H 3 BO 3, indicated presence of free hydroxyls at C-5, C-7 and C- 4, (see, table [3.1.1(VI) a]. Table [3.1.1(VI) a]: UV spectra of compound [6] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH NaOMe AlCl AlCl 3 +HCl NaOAc NaOAc+H 3 BO ZrOCl 2 +Citric acid Rf values of compound obtained from various solvent systems, used on PC indicated the flavonol-mono-saccharide nature of the compound (Bate-Smith, 1949; Geissman, 1956; Harborne, 1958; 1959; Mabry et al., 1970; Harborne and Williams, 1971 and Markham, 1982). The Rf values and the colour reactions of the compound are being summarized in table [3.1.1(VI) b]. Table [3.1.1(VI) b]: Rf values of the compound [6] (on Whatman No.1 PC) using various solvent systems Solvent system Rf Values Locating reagent Visualization (V/UV) H 2 O 30 UV/(CH 3 COO) 2 Pb (neutral) % HOAc 49 UV DP 50% HOAc 78 UV/NH 3 LY BAW 65 UV/NA Y BEW 63 UV/AlCl 3 f-yg t-baw (3:1:1) 61 UV/ZrOCl 2 f-yg [3.1.1(VI-a)] Total Acid Hydrolysis of Compound [6] The compound [6] was hydrolysed with 2N-HCl at C for an hour. The hydrolysate was neutralised with aq. solution of BaCO 3. The neutral aq. solution was repeatedly evaporated to dryness and the residue obtained was dissolved in isopropanol. The isopropanoal soluble was chromatographed on Whatman No. 1 PC using BAW (n-buoh-acoh-h 2 O, 4:1:5, V/V, upper layer) as a developing solvent. On inspecting dried and developed PC with UV light, a dull yellow fluorescent spot was observed on PC, it was identified as kaempferol on the basis of colour reaction with UV, UV/NH 3, UV/NA, UV/ZrOCl 2 and UV/AlCl 3. The paper chromatographically isolated compound gave UV, 1 HNMR and MS data similar to kaemperol, isolated from CH 2 Cl 2 of aq. methanolic extract of H. polylanthos. Thus, the aglycone was identified as kaempferol, representing structure (II). 62

17 The isopropanol soluble was further chromatographed on Whatman No.1 PC using BAW (4:1:5, V/V) as a developing solvent. After spraying PC with Benzidine reagent a brown spot was visualised on PC, it was identified as rhamanose by CoPC with its authentic using following systems: (i) BAW (n-buoh-acoh-h 2 O, 4:1:5 V/V, upper layer); (ii) BEW (n-buoh-etoh-h 2 O, 4:1:2.2, upper layer) and (iii) t-baw (t- BuOH-AcOH-H 2 O 3:1:1). Complete methylation of glycoside with dimethyl sulphate and aq. NaOH, an oil viscous product was obtained. It was hydrolysed with 5% Conc. H 2 SO 4 for 3 hours at C gave a diethyl ether souble methylated product was identified as kaempferol-5, 7, 4 -tri-methoxy ether C and it was finally identified by CoPC with its authentic. Thus, the compound [6] was identified as kaempferol-3- O-α-L-rhamnopyranoside. Fig. (VI) Kaempferol-3-O-α-L-rhamnopyranoside Finally, it was confirmed by 1 HNMR studies (fig. VI-a) in (DMSO-d 6, 400MHz): In aromatic region two meta coupled doublets appeared at δ 6.15 (1H, J=2.0 Hz) and δ 6.36 (1H, J=2.0 Hz) for H-6 and H-8, A-ring protons respectively and two ortho coupled doublets at δ 6.82 (2H, d, J=8.5 Hz) and δ 8.03 (2H, d, J=8.5Hz) for 3, 5 and 2, 6 proton of B-ring respectively. Anomeric proton appeared at δ 5.50 (1H, d, J=8.5 Hz) and other sugar protons were identified at δ 4.35, δ 3.85, δ 3.6 and δ 3.4 for H-2, H-3, H-4 and H-5 protons of sugar. The appearance of anomeric signals at δ 5.50 with very high coupling constant J=8.5 Hz, indicated β-configuration in pyranose form of glucose sugar (Overend, 1972; Altona and Haasnoot, 1980; Markham et al., 1987). Fig. (VI-a) [3.1.1(VII)] Structure Determination of Compound [7] Compound [7], a polyphenolic heterocyclic flavonoidal glycosidic compound was isolated from EtOAc fraction of aq. ethonolic extract of fern fronds of H. polyanthos. The polyphenolic heterocyclic flavonoidal glycosidic nature of the compound [7] was confirmed on the basis of its positive colour reactions with Mg+HCl, FeCl 3 and α- naphthol (Geissman, 1956; Dean, 1961). It appeared as a purple fluorescent on paper chromatogram under UV light and changed to yellow-green with NH 3 vapours, indicating the presence of 4 and 5 hydroxyl groups (Mabry et al., 1970; Markham, 1982). When cellulose TLC of the compound [7] was sprayed with methanolic 63

18 solution of NA it turned to yellow, indicating the presence of free OH group in C-4 and absence of ortho-di-hydroxyl group in the B-ring. Two UV maxima of the compound were observed at 266nm (band II) and 349nm (band I) in methanolic solution and shifts obtained with diagnostic reagents, NaOMe, AlCl 3 +HCl, NaOAc, NaOAc+H 3 BO 3 and ZrOCl 2 +Citric acid, indicated presence of free hydroxyls at C-4, C-5 and C-7. The UV spectral data of compound are being given in table [3.1.1(VII) a]. Table [3.1.1(VII) a]: UV spectra of compound [7] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band MeOH NaOMe AlCl AlCl 3 +HCl NaOAc NaOAc+H 3 BO ZrOCl 2 +Citric acid The Bathochromic shift of (band II) with NaOAc from (9nm), indicated presence of 7-OH group in the A-ring. Bathochromic shift of (band I) with NaOMe from nm (50nm) and appearance of intense peak suggested the presence of free OH in the C-4 of B- ring. Presence of a peak for (band II) at 325nm with NaOAc and 47nm and 46nm shift of (band II) with AlCl 3 and AlCl 3 /HCl respectively, indicating the presence of free OH group at C-5. On the basis of colour reactions and UV spectra studies, compound [7] has free hydroxyls at C-5, C-7 and C-4. Rf value of compound [7] on paper chromatogram using various solvent systems, indicated flavonol-mono-saccharide nature of the compound (Bate-Smith, 1949; Geissman, 1956; Harborne, 1958; 1959; Mabry et al., 1970; Harborne and Williams, 1971 and Markham 1982). The Rf values and colour reactions of the compound are being given in table [3.1.1(VI) b]. Table [3.1.1(VI) b]: Rf values of the compound [7] (on Whatman No.1 PC) using various solvent systems Solvent system Rf Values Locating reagent Visualization(V/UV) H 2 O 25 UV DP 15% HOAc 36 UV/NH 3 Y 30% HOAc 60 UV/NA Y 50% HOAc 79 UV/AlCl 3 f-yg BAW (4:1:5) 63 UV/ZrOCl 3 f-yg BEW 61 UV/ZrOCl 2 +Citric Acid DP t-baw 58 UV/Lead acetate (Neutral) IY 64

19 [3.1.1(VII-a)] Total acid hydrolysis of compound [7] Compound [7] was hydrolysed with 2N-HCl at C for an hour. The hydrolysed mixture was neutralised with aq. solution of BaCO 3 and neutral solution was repeatedly evaporated to dryness and residue was dissolve in isopropanol. It was chromatographed on Whatman No.1 PC using BAW (n-buoh-acoh-h 2 O, 4:1:5, V/V, upper layer) as a developing solvent. After inspecting dried and developed PC with UV light, a dull yellow fluorescent spot was observed. It was representing the acid hydrolysed aglycone of compound [7]. The structure of the aglycone was elucidated as follows: The compound was isolated from EtOAc soluble of acid hydrolysed mixture of compound [7] by RPPC using BAW (4:1:5, upper layer) as eluent. EIMS of the compound gave a molecular ion m/z at 286 calculated for C 15 H 10 O 6. The compound appeared as a dull yellow fluorescent spot on PC under UV light and remains unchanged after fuming with NH 3 vapour indicating a flavonol with free 5-OH in the A-ring (Mabry et al., 1970). When cellulose TLC of the compound sprayed with methanolic solution of NA, the dull yellow fluorescence of compound turned to yellow, indicating the presence of a free hydroxyl at C-4. 1 HNMR of the compound in (DMSO-d 6, 400MHz) gave four aromatic signals of which two signals appeared at δ 6.20 (1H, d, J=2 Hz) and δ 6.45 (1H, d, J=2 Hz) representing H-6 and H-8 of A-ring and remaining two signals, at δ 6.95 (1H, d, J=9.0 Hz) and δ 8.05 (1H, d, J=9.05 Hz) correspond to H-3, H-5 and H-2, H-6 of symmetrically substituted B-ring. A low field broad singled appeared at δ 12.50, represent proton of chelated 5-OH of A-ring. UV maxima of the compound in MeOH appeared at 260nm (band II) and 370nm (band I). The MS, UV, 1 HNMR and colour reactionis of compound on PC are closely resemble with compound kaempferol, representing structure (II), isolated as compound [2] from CH 2 Cl 2 fraction of aq. ethanolic extract of fern fronds of H. polyanthos.the compound was finally identified by CoPC with its standard using following five solvent systems: (i) 30% HOAc; (ii) 50% HOAc; (iii) 30% NaCl; (iv) BAW (n- BuOH-AcOH-H 2 O, 4:1:5, V/V upper layer) and (v) t-baw (t-buoh-acoh-h 2 O; 3:1;1). [3.1.1(VII-b)] Identification of Sugar Constituents The isopropanol soluble of acid hydrolysed mixture of compound [7] was chramatographed on Whatman No. 1 PC and developed with BAW (4:1:5). The dried and developed paper chromatogram was sprayed with benzidine reagent. A dark brown spot was visualised at Rf 21. It was identified as glucose by CoPC with its authenticity. [3.1.1(VII-b) (i)] Structural Determination of Glycoside Acid hydrolysis of the glycoside gave an aglycone, kaempferol and a sugar, glucose. The only point that now remains to be decided is the position of free hydroxyl group of the kaempferol involved in glycosidic linkage. Since there are four free hydroxyl groups in the aglycone at position C-3, C-5, C-7 and C-4 and rhamnose may be attached any of them. It could be done as follows: 65

20 Table [3.1.1(VII-b) (i)]: 1 HNMR of compound [7] in DMSO-d 6, 400MHz Shift (δ) Multiplicity Identification H, d, J=2.0 Hz H H, d, J=2.0 Hz H H, d, J=8.5 Hz H-3, H H, d, J=8.5 Hz H-2, H H, d, J=8.5 Hz H -1, anomeric proton of glucose H, s, br 5-0H On the basis of chromatographic behaviour, UV, MS, 1 HNMR and hydrolytic studies the compound [7] was identified as kaempferol-3-o-β-d-glucoside, representing structure (VII). Fig. (VII) Kaempferol-3-O-β-D-glucoside [3.1.1(VIII)] Structure Determination of Compound [8] The compouund [8], a polyphenolic heterocyclic flavonoidal glycoside was isolated from EtOAc fraction of aq. ethanolic extract of fern fronds of H. polyanthos. The polyphenolic heterocyclic flavonoidal glycosidic nature of the compound was confirmed by observing its colour reactions with Mg+HCl, alcoholic solution of FeCl 3, alcoholic solution of α-naphthol and Feigl spot test for sugar (Geissman, 1956; Dean, 1961; Feigl, 1960; Mabry, 1970; Markham, 1982). FABMS (-ve) gave a molecular ion m/z at 447 [M] -, for C 21 H 20 O 11. Flavone appeared as a purple fluorescent spot on a paper chromatogram under UV light and changed to yellowgreen with NH 3 vapours, indicating the presence of free 5- and 4 - hydroxyl groups (Mabry et al., 1970; Markham, 1982). When a cellulose TLC of the compound was sprayed with methonolic solution of NA reagent, the spot turned yellow, indicating the presence of ortho-di-hydroxyl group in the B-ring. Compound [8] exhibited UV maxima in MeOH, table [3.1.1(VIII) a] and fig. (VII-a), at 255nm (band II) and 351nm (band I) indicated a 3-O-substituted flavonal (Mabry et al., 1970). The UV maxima in MeOH of compound did not alter with ZrOCl 2 +Citric acid reagent, indicated that the 3-OH group of flavonal was substituted (Sosa and Percheron, 1970). The shifts of band (II) and band(i) in MeOH of compound obtained with diagnostic reagents, indicated presence of free hydroxyl at C-5, C-7, C-3 and C-4. Table [3.1.1(VIII) a]: UV spectra of compound [8] in MeOH (λ max, nm) Shift Reagent Shift (λ max, nm ) band II band I MeOH sh NaOMe dec 270 AlCl sh 274 AlCl 3 +HCl sh 273sh NaOAc sh 268 NaOAc+H 3 BO sh

21 Fig. (VII-a) [3.1.1(VIII-a)] Total Acid Hydrolysis of the Compound [8] Total acid hydrolysis of the compound was made with 2N-HCl at C for an hour. The acid hydrolysed extract was cooled and neutralised with aq. solution of BaCO 3. The neutral solution was repeatedly evaporated to dryness and residue was dissolved in isopropanol. The isopropanol soluble was chromatographed on Whatman No. 1 and developed with BAW (4:1:5). The dried and developed paper chromatogram was inspected with UV light. A dull yellow fluorescent spot was observed and representing compound [8(a)]. The compound [8(a)], an aglycone generated after the acid hydrolysis of the compound was isolated and identified as follows: The isopropanol soluble from acid hydrolysed extract of the compound was adsorbed on small column of Sephadex LH-20 and eluted with aq. MeOH (40: 60). The column was inspected with UV light (360nm). A dull yellow fluorescent band was observed of column with UV light and it was eluted and collected separately. The aq. methanolic elute of the compound was evaporated to dryness and residue was recrystallised with petrol ether and MeOH. The structure of the aglycone [8(a)] was determined as follows: EIMS of the compound gave a molecular ion, m/z at 302 for C 15 H 10 O 7. It appeared as a dull yellow fluorescent spot on PC under UV light and its fluorescence remain unchanged after fuming with NH 3 vapours, indicating a flavonol with free 5-OH group (Mabry et al.., 1970; Markham, 1982). When cellulose TLC of the compound was sprayed with NA reagent, it turned to orange indicated a flavonol with ortho-dihydroxyl group in the B-ring. UV, 1 HNMR and MS data of the compound were found similar to the compound [5], isolated from diethyl ether fraction of the fern, H. polyanthos. Thus, the aglycone could be identified as quercetin, representing structure, (V). 67