Indian Journal of Chemistry Vol. 438 . October 2004. pp. 22 17-2222

Phytochemical investigation of Shilajit

M AIi *. I Sahrawat & 0 Singh

Phytochemi stry Research Laboratory. Faculty of Ph armacy Jamia Hamdard (Hamdard University). P.O. Hamdard Nagar. New De lhi 110 062. Indi a

*E- mail: mali che m @yahoo.co. in

Received 29 September 2003; accepted (revised) 1 lllne 2004

T he phytochemical in vesti gation of Shiic!iit has afforded six new compounds named as shil aj ityl acetate. shil ajitol, shil aca techol, shilaxanthone, shil anth ranil and naphsil aj itone along with pyrocatechol and their stereostructures have been

eluc idated correspondingly as 4a, Sa, 6a-trihydroxygerany l acetate, 6-(9, 9-d imethylbutyl) phenol. l -cyc lohexy l-3. 4-dihydroxybenzene, 2. 3. 12, I 3-tetrahydroxy- 1 0, IS-[a.Jl-phenylxanth- 17-one, 2, 3, 13, 14-tetrahydroxy- ll , 16- [a.Jl -phenyl-7 H­<llllhraccn- 18-one and 3-hydroxynaphthaleny l-6, 7-y-lactone on the bas is of spectral data analyses and chemical reactions.

IPC: Int.CI7 A 61 K 35/00

Shilajit is a blackish-brown organic exudation of vari able consistencies obtained from the steep rocks of differe nt fo rmations fo und in the Himalayas at alti tudes between 1000-5000m, in Afganisthan, Austra lia and the fo rmer USSRI.2. Shilajit is believed to arrest ageing and also produces rej uvenation, the two important attribu tes of an Ayurvedic rasayan medicine. It is used in the treatment of bronchial asthma, d iabetes, genito­uri nary in fect ions and stomach ulcers". Considerable controversy has ex isted in the literature concerning the nature and chemical character of Shilaj it. It has been descri bed as bitumen (Asphalt), a mineral res in , a pl ant foss il , a substance of mixed plant and animal orig in, or an inorganic substance3

.4 . Shilaj it has urinous odour and slightl y bitter, sa line, somewhat pungent and astringent taste. The purified substance is nearly completely soluble in water and has an acid reaction. It was reported to contain res ins, fatty ac ids, benzoic and hippuric ac ids, albuminoids, amino ac ids and a number of minerals l

. T hree oxygenated biphenyls and three oxygenated 3, 4-benzcoumarins, ful vic acids (FAs) and 4-methoxy-6-carbomethoxybiphenyl (MCB), several phenolic and amino ac ids and triterpenes in Shilaj it have been reported 4-9 . The present paper describes the isolation and characteri zation of five new phenolic constituents and a geranyl acetate type monoterpenes along with pyrocatechol from the drug.

Results and Discussion Compound 1, designated as shilajity l acetate, was

obtained as a colourless semi-solid. Its IR spectrum

showed absorption bands fo r chelated hydroxy l groups (3 100 cm-I) and ester group (1725 cm-I). The +ve ion FAS mass spectrum di splayed a molecular ion peak at mlz 248 consistent with the molecular formul a of an acycl ic trihydroxymonoterpenic ester, C1 2H240 S. it indicated one double bond equi valent adj ustable to the ester group. The important ion fragments appeared at m/z 205 [M-Acr, 162 [205-C3H7r, 187 [205-H20r, 144 [1 87-CJH7r, 169 [1 87-H20 r, 126 f 169-CJH7f, 15 1 [1 69-H20 r and 133 [15 1-H20 r suggesting the presence of three hydroxyl groups and an isopropy l chain in the molecule. The ion peaks at mlz 2 19 fM ­C2Hsr, 176 [2 19-CJH7r, 20 1 [2 19- H20 f, 158 1201-C3H7r, 183 f20 1-H20r, 140 [1 83- C3H7r, 165 f 183-H20r and 122 [165-C3H7r supported the presence of an ethyl and the isopropyl moieties in the molecule. The generation of prominent ion peaks at mlz 1'15 and 133 due to cleavage of CJ-C4 linkage indicated the location of the hydroxyl groups at CA, C-5 and C-6 and acetoxyl group at C-3/C- IO. The ion peaks at mlz 145 [C4-Cs fi ssionr, 116 [145-C2Hsr and 175 [C S-C6

fi ssionr also supported the existence of the acetoxyl group at C-3/C- IO.

The IH NMR spectrum of 1 exhibited two one­

proton doublets at 8 4 .89 (J=IO.8 Hz) and 4.85 (1=10.8 Hz) ass igned to oxygenated C-1O methylene

protons. Three one-proton double doublets at 8 4.30 (1=6,6, 6 .9 Hz), 3 .87 (J=6.9, 6.2 Hz) and 3.78 (J=5. 1, 6.2 Hz) were ascribed correspondingly to H-4~, H-5 ~

and H-6~ carbinol protons. Two three-proton doublets at 8 1.33 (J=6.0 Hz) and 1.28 (J=6.0 Hz) were

2218 INDIAN 1. CHEM., SEC B, OCTOBER 2004

attributed to C-8 and C-9 secondary methyl protons, respectively. A broad signal at 8 1.25 and a triplet at 8 0.85 (1=6.5 Hz), integrating three protons each, were associated with correspondingly to acetyl and C-I primary methyl protons. The remall1l11g C-2 methylene and C-3 and C-7 methine protons resonated as multiplets at 8 0.93 (2H), 1.72 (lH) and 2.20 (1H), respectively. The 13C NMR spectrum of 1 exhibited signals for oxygenated methylene carbon at 865.31 (C-I0) and carbinol carbons at 8 63.76 (C-4), 62.83 (C-5), 61.43 (C-6). The carbon signals at 8 171.13 and 21.83 supported the location of the acetyl group in the molecule. The methyl carbon . ignal s appeared at 8 11.17 (C-I), 14.39 (C-8) and 22.79 (C-9). The degree of protonation was determined by OEPT experiments. On the basis of these evidences the structure of 1 has been elucidated as 4a, Sa, 6a­trihydroxygeranyl acetate. This is an unknown monoterpenic ester isolated from a natura l or synthetic source for the first time.

Compound 2, named shilajitol, was obtained as pale yellow crystalline product from chloroform-methanol (49: I) mixture. It responded positively to phenolic tests. Its IR spectrum exhibited absorption bands for hydroxyl group (3320 cm"l) and unsaturation (1602 cm" I). It had a molecular ion peak at mlz 178 in its +ve ion FAS mass spectrum corresponding to a C6-substituted phenol, C I2HISO. The 'H NMR spectrum of 2 displayed a one-proton arrha-coupled doublet at 8 7.92 (1=7.47 Hz) assigned to H-2. Two one-proton multiplets at 8 7.58 and 7.49 were accounted to H-4 and H-3 , respectively . A one-proton double doublet at 87.44 (1=8.67, 3.0 Hz) was associated with arrha- and mela- coupled H-5. A two-proton double doublet at 8 2.29 (1=10.5, 10.5 Hz) was ascribed to C-7 methylene protons attached to aromatic ring. Three broad signals at 8 1.22, 1.14 and 0.66, all integrated for three protons, were associated with C-lO, C-ll , C-12 tet1iary methyl protons, respectively . The C-8 methylene protons appeared as a multiplet at 8 0.87. A 0 20 washable broad signal at 8 7.90 was due to hydroxyl group. In 13C NMR spectrum of 2 the carbon signal at 8 167.97 was assigned to C-l attached to a phenolic group. The signals at 8 21.06, 25.97 and 19.11 were attributed to C-lO, C-ll and C-12 methyl carbons, respectively. The remaining aromatic carbon signals resonated between 8 136.43-128.98. On the basis of these evidences, the structure of 2 has been characterized as 6-(9, 9-dimethylbutyl) phenol. This is an unknown phenolic constituent.

Compound 3, obtained from chloroform-methanol (49: I) eluents, was identified as a pyrecatechol on the basis of spectral data analysis, chemical test, melting point and ca-TLC.

Compound 4, designated as shilacatechol , showed positive tests for phenol s and characteristic [R

absorption bands for hydroxyl groups (3407, 3203 cm"I), unsaturation (1662, 16 11 cn,- I) and aromatic ring (1515 , 775 cm"I). It had a molecular ion peak at mlz 192 in its +ve ion FAB mass spectrum corresponding to cyclohexyl substituted pyrocatechol , C I2H 1602. The other important ion fragments appeared at mlz 178 [M-CH 2t, 164 [178-CH zt, 150 [164-CH2t, 136 f150-CH2t and 122 [136-CH2t. The IE NMR spectrum of 4 exhibited two one-proton doublets at 8 7 .92 (1=7.9 Hz) and 7.58 (J=3.0 Hz) assigned to arrha-coupled H-5 and meta-coupled H-2, respectively . A one-proton doubl e d(;Ub let at 8 7.49 (1=7.9,3.0 Hz) were associated with orlha- and mew­coupled H-6. Two 0 20 exchangeable one-proton signals at 8 5.73 and 4.0 were due to hydroxyl groups. A one-proton multiplet at 8 2.96 was due to C-- I' methine proton. A lO-proton broad signal at 8 2.73 was accounted to five methylene proton of a cyclochexyl ring. The I3C NMR spectrum of 4 displayed signals for aromatic carbons between 8 159.97-128.97, C-l' methine carbon signal at 8 40.14 and five cyclic methylene carbons at 8 36.29. Treatment of 4 with diazomethane yielded a dimethoxy derivative. On the basis of these evidences the structure of 4 has been formulated as 1-cyclohexyl-3,4- dihydroxybenzene. This is unknown natural product reported from a natural or synthetic source for the first ti me.

Compound 5, named shilaxanthone, was obtained as a brown coloured crystals from chloroform-methanol (19: 1) mixture. It gave positive tests of a phenolic compound. Its UV spectrum showed absorption maxima at 220, 281 and 360 nm for conjugated compounds. The characteristic IR absorption bands appeared at 3326, 3157 (OH), 1702 (C=O) and 1615 (C=C) cm"l. The +ve ion FAB mass spectrum showed a molecular ion peak at mlz 310 consistent with the molecular formula, C I7 H 1006. The important ion fragments at mlz 108 [C6-0, C:-C I7 fiss iont, 136 rC6-

0, C w C 17 fissiont, 124 [C7 -0, C I-C I7 fissiont and 152 [CrO,CI6-CI7 fissiont suggested the presence of pyrocatechol and a, ~-dihydroxynaphthalenc moiety in the molecule. The 'H NMR spectrum of 5 exhibited four one-proton artha-coupled doublets at 8 8.04

ALI el at.: PHYTOCHEMICAL INVESTIGATION OF SHILAJIT 2219

(J=8 .52 Hz), 7.95 (J=8.52 Hz), 7.30 (J=8.39 Hz) and 6.79 (1=8.39 Hz) assigned to H-4, H-5, H-8 and H-9, respectively. Two one-proton p-coupled broad signals at 8 7.46 and 6.69 were attributed correspondingly to H-14 and H- ll. The hyrdoxyl protons appeared at 8 3.95 (2H), 3.89 (1H) and 2.50 (lH). The 13C NMR spectrum of 5 displayed signals for carbonyl group (8 184.31) and 16 carbon signals for aromatic ring. On the basis of the foregoing account the structure of 5 has been established as 2, 3, 12, 13-tetrahydroxy-1O, 15-[a,f]-phenylxanth-17-one. This is an unknown xanthone derivative reported as a constituent of Shilajit for the first time.

Compound 6, named shilanthralin , was obtained as a brown coloured crystalline mass from CHClrMeOH (19: I). It responded positively to phenolic tests . Its IR spectrum di splayed absorption bands for hydroxyl groups (3407, 3361, 3200 cm-I), carbonyl group (1706 cm-I), unsaturation (1611 cm-I) and aromatic rings (1514, 774 cm-I). The +ve ion mass spectrum of 6 showed a molecular ion peak at m/z 308 corresponding to a tetracycl ic phenolic ketone, C1 8H 120 S. The

10 OAc

HO',4 3 2

, , HO

9 8

2

4

OH

HO

6

important ion fragments at m/z 108, 208 [C I. IS-C6. 7 fissiont, 136 [C6. r C 17.18 fissiont , 122 [C7,8-C I.I R fissiont and 150 [C7.8-CI7.18 fissiont supported the existence of the carbony l group at C-18 and two each phenolic groups in different rings_ The IH NMR spectrum of 6 exhibited four one-proton ortho-coupled doublets at 8 8.09 (J=8 .8 1 Hz), 7 .97 (1=8 .8 1 Hz), 7.33 (J=7 .5 Hz) and 6.78 (J=7.5 Hz) assigned to H-4, H-5 , H-9 and H-lO, respectively. Two one-proton broad signals at 8 7.48 and 6.71 were attri buted to con'espondingly para-coupled protons H-12 and H-15 _ A two-proton unshielded broad signal at 8 2.73 was accounted to methylene protons Hr 7 . Four 0 20 exchangeable protons at 8 5.71, 3.85, 3.82 and 3.79 were due to hydroxyl protons_ The 13C NMR spectrum of 6 showed signals for C-18 carbonyl carbon (8 183.7), C-7 methylene carbon (8 35.95) and aromatic carbons between 8 159.11-113.22. These data led to formu late the structure of 6 as 2, 3, 13, 14-tetrahydroxy-ll , 16-[a,j]-phenyl-7 H-anthracen-1 8-one. This is a new phytoconstituent isolated from a natural or synthetic source for the first time.

12

11

t5 HO 1 10 ~6

3~ Is

3

OH

HO

5

OH

o

7

2220 INDIAN 1. CHEM., SEC B, OCTOBER 2004

Compound 7, named naphsilajitone, was obtained as a co lourless crystalline mass from chloroform extract. It gave blue colour with fe rric chloride for phenoli c group. Its IR spectrum showed characteri stic absorpti on bands for hydroxyl group (3343 cm-'), y­lactone ( 1755 cm-') and aromatic ring (1555, 999, 847 cm-') . Its +ve ion FAB mass spectrum showed a molecul ar ion peak at mlz 200 correspondi ng to C'2HSOJ' The important ion fragments appeared at mlz 183 [M-OHr, 153 [183-CH20r, 155 [L83-COr and 125 [155-CH20r supporting the ex istence of a y­lac tone in the molecule. The 'H NMR spectrum of 7 exhib ited a one-proton ortho- , meta-coupled double doublet at 8 8.83 (J=7.75, 2.14 Hz), ass igned to H-2. Three one-proton ortho-coupled doublets at 8 7.83 (1=7.75 Hz), 7.53 (1=7.22 Hz) and 7.46 (1=7.22 Hz) were associated with H-I , H-8 and H-9, respectively. A one-proton meta-coupled doublet at 8 7.43 (1=2.14 Hz) was accounted to H-4. The oxygenated C-12 methylene protons appeared as broad signals at 84.02 (Hr 12a) and 3.93 (Hr I2b). In the l3C NMR spectrum of 7 the signals at 8 171.72 and 41.58 were attributed to lactone C-Il and oxygenated C- 12 methylene carbons. The signals for aromatic carbons resonated between 8 167.44-127.50. Acetylation of 7 yielded a monoacety l product. On the basis of these informati ons, the structure of 7 has been formulated as 3-hydroxynaphthalenyl-6, 7-y-lactone. This is a new phytoconstitutent isolated from a natural or synthetic source.

Experimental Section

Melting points were determined on Perfit melting point apparatus and are uncorrected. Fr IR: Jasco Fr/IR-5000; UV: Lambda Bio 20 Spectrophotometer, MeOH ; 'H NMR (400 MHz): Advance DRY 400, Bruker Spectrospin, CDCb; '3C NMR (75 MHz) : Advance DRY 100, Bruker Spectrospin, CDCI] with TMS as an internal standard; MS: FAB ionization on JEOL-JMS-DX 303 ; CC: Silica gel (Qualigens), 60-120 mesh; TLC: Silica gel G (Qualigens). Spots were visualized by exposure to iodine vapours, UV radiation and by spraying with ceric ammonium sulphate and perchloric acid.

Collection of material

Shilajit was procured from the Khari Baoli market of Delhi and was identified by Dr M P Sharma, Taxonomist, Department of Botany, Jamia Hamdard. New Delhi. A voucher specimen is deposited in the

herbarium of the Phytochemical Research Laboratory, Faculty of Pharmacy.

Extraction and Isolation

The dried drug (850 g) was coarsely powdered and exhaustively extracted wi th ethanol (95%). The combined extracts were then concentrated on a water­bath and dried under reduced pressure to get 3 10 g (36.47% yie ld) of dark brown mass. The viscous dark brown mass was di ssolved in a little quantity of methanol and adsorbed on silica gel (60-1 20 mesh) fo r the preparation of sl urry. It was dried, packed and chromatographed over sil ica gel column packed in petroleum ether. T he column was el uted with petroleum ether, chloroform and methanol success­sively in order of increasing poiarity to isolate fo llowing compounds.

Shilajityl acetate 1. Elution of the column with CHCI3 furni shed colourless semi solid mass of 1, recrystallized from CHCIJ-MeOH ( I: I); yield 383.6 mg (0. 1237%); Rr 0.59 (petroleum ether-chloroform, 1:1); m.p . 75-77°C; UV: 226 nm (log E 4.6) ; IR (KBr): 3100, 2926, 2856, 1725, 1447, 1379, 1279, 1124, 1074, 967 , 928, 738 cn'-'; 'H NMR (DMSO­d6): 84.89 (1 H, d, J= I 0.8 Hz, Hr 10 a), 4.85 (1 H, d, J=1O.8 Hz, H2-10 b) , 4.30 (1 H, dd, J=6.6, 6.9 Hz, H-4~), 3.87 (1 H, dd, J=6.9 , 6.2 Hz, H-5~) , 3.78 (I H, dd, J=5.1, 6.2 Hz, H -6~), 2.20 (1 H, 1, H -7), 1.72 (1 H, m, H-3), 1.33(1 H, d, J=6.0 Hz, Me-8) , 1.28 (3H, d, J=6.0 Hz, Me-9), 1.25 (3H, brs, Ac), 0.93 (2H, m, Hr2), 0.85 (3H, t, J=6.5 Hz, Me- I) ; '3C NMR (DMSO-d6) : 0 1l.l7 (C-l), 29.81 (C-2), 35.15 (C-3), 63.76 (C-4), 62.83 (C-5), 61.43 (C-6), 32.73 (C-7), 14.39 (C-8), 22.79 (C-9), 65.3 1 (C-I0), 21.82,171.13 (Ac); +ve ion FAB-MS: mlz (rel.int.) 248 [Mt (C'2H240S) (9.3), 219 (12.3), 205 0 8.7), 201 (11.6), 187 (16.5), 183 (12.3), 176 (28.6), 175 (25.1), 169 (18.3), 165 (24.8), 162 (43. L) , 158 (33.9), 151 (34.6), 145 (47.6) , 144 (36.8), 140 (26.3) , 133 (56.3), 126 (32.3), 122 (89.7), 116 (50.3), 115 (38. 1), 108 (100).

Shilajitol 2. ELution of the coLumn with CHCb­MeOH (49:1) (fraction 292-294) furnished pale yellow crystals of 2, recrystallized from MeOH, yield 482 mg (0.1555 %); Rr 0 .38 (CHCI3-MeOH, 95:5); m.p. 84-85°C; UV: 246 nm (log £: 5.3) ; IR (KBr): 3320, 2937, 2870, 1602, 1423, 1326, 1289, 1180, 1125, 1070, 1025, 832, 707 cm"; 'H NMR (DMSO­d6) : 87.92 (l H, d, J=7.47 Hz, H-2), 7.90 (1 H, brs, D20 exchangeable, OH), 7.58 (1 H, m, H-4), 7.49 (l H, m, H-3), 7.44 (l H, dd, J=8.67, 3.0 Hz, H-5), 2.29

ALI et al.: PHYTOCHEMICAL INVESTIGATION OF SHILAJIT 222 1

(2H, dd, J=1O.5, 10.5 Hz, Hr 7), 1.22 (3H, brs, Me-10), 1.14 (3H, brs, Me-ll), 0.87 (2H, m, Hr 8), 0.66 (3H, brs, Me-12); \3C NMR (OMSO-d6): 8 167.97 (C-1), 129.61 (C-2), 128.98 (C-3), 130.88 (C-4), 133.36 (C-5 ), 136.43 (C-6) 31.90 (C-7), 31.09 (C-8), 40.95 (C-9), 21.06 (C-I0), 25.97 (C-ll ), 19.11 (C-12); +ve ion FAB-MS: mlz (rel.int.) 178 [Mt (C I2H ISO) (5.2), 148 (100), 107 (13.1 ).

Pyrocatechol 3. Elution of the column with CHCI3-MeOH (49: 1) gave pyrocatechol 3, recrystal­lized from MeOH, yield 2.78 g (0.8967 %) ; Rr 0.75 (CHCI3-MeOH, 95 :5); m.p. 101-04°C; UV (MeOH): 274 nm (log E 6.1 ); IR (KBr): 3350,2965, 2855, 1648, 1602, 1423, 1326, 1290, 1180, 1125, 1070, 1024, 932, 805, 707 cm·l; IH NMR (OMSO-d6 ): 8 7.92 (2H, d, J=7.52 Hz, H-3, H-6), 7.61 (l H, m, H-4), 7.49 (l H, m, H-5), 7.45 (1 H, brs, 0 20 exchangeable, OH), 4.01 (l H, brs, 0 20 exchangeable, OH); 13C NMR (OMSO-d6 ): 8 167.99 (C-l), 167.99 (C-2), 133.28 (C-3 ), 129.62 (C-4), 128.91 (C-5), 130.29 (C-6) ; +ve FAB-MS: m/z. (ret.int. ) 110 [Mt (C6H60 2) (18.3 ).

Shilacatechol 4. Elution of the column with CHCI3-MeOH (95:5) gave colourless semi-solid mass of 4, yield 265 .3 mg (0.0855 %); Rr 0.57 (toluene-ethyl acetate-formic acid, 4:5: 1); m.p. 269-70°C; UV (MeOH): 242 nm (log E 5.1); IR: 3407, 3203, 2936, 2877, 1662, 1611 , 1515, 1412, 1273, 1101 , 1071,1025, 775 cm-I; IHNMR (OMSO-d6): 8 7.92 (l H, d, J=7.9 Hz, H-5 ), 7.58 (1 H, d, J=3.0 Hz, H-2), 7.49 (l H, dd, J=7 .9, 3.0 Hz, H-6), 5.73 (1 H, brs, 0 20 exchangeable, OH), 4.0 (1H, brs, 0 20 exchangeable, OH), 2.96 (l H, m, H-l') , 2.73 (l0 H, brs, 5 x CH2); 13C NMR (OMSO-d6) : 8 133.26 (C-l), 129.62 (C-2), 159.97 (C-3), 159.97 (C-4), 128.97 (C-5), 129.62 (C-6), 40 .14 (C-l'), 36.29 (C-2'), 36.29 (C-3'), 36.29 (C-4'), 36.29 (C-5'), 36.29 (C-6'); +ve ion FAB-MS: mlz (rel.int.) 192 [Mt (C 12HI60 2) (5.2), 178 (18.9), 164 (7.3), 150 (23 .6), 136 (72 .5) , 122 (5.6), 109 (8.9).

Methylation of 4. A solution of 4 (25 mg) in diethyl ether was treated with ethereal solution of diazomethane. Evaporation of the solvent yielded a dimethoxy derivative 4a, mp 201-03°; IR: 2940, 2865, 1660, 1610, 1515 cm-I; different spot on a TLC plate from that of 4.

Shilaxanthone 5. Elution of the column with CHCI3-MeOH (19: 1) yielded brown coloured crystalline mass of 5, recrystallized from MeOH; yield 124.8 mg (0.0402 %); Rr 0.72 (toluene-ethyl

acetate-formic acid, 4 :5: 1); m.p. 320°C (dec.); UV: 220, 281, 360 nm (log E 6.3, 3.1, 1.2); JR (KB r): 3326, 3157, 1702, 1615, 1459, 1315, 1270, 11 64, 1120,809 cm-I; IH NMR (OMSO-d6): 88.04 (l H, d, J=8 .52 Hz, H-4), 7.95 (1 H, d, J=8 .52 Hz, H-5), 7.46 (1 H, brs, H-14), 7.30 (l H, d, J=8.39 Hz, H-8), 6.79 (l H, d, J=8 .39 Hz, H-9), 6.69 (l H, brs, H- ll ), 3.95 (2H, brs, 0 20 exchangeable, 2 OH), 3.89 (1 H, brs, 0 20 exchangeable, OH), 2.50 (1 H, brs, 0 20 exchangeable, OH); \3C NMR (OMSO-d6 ): 8 119.13 (C-l), 161.22 (C-2), 158.76 (C-3), 124.15 (C-4), 120.33 (C-5), 127.33 (C-6), 124.72 (C-7), 123.94 (C-8), 113.84 (C-9), 103. 16 (C-lO), 11 3.56 (C- II ), 157.17 (C-12), 151.12 (C-13), 110.89 (C-14), 110. 15 (C-15), 109.51 (C-16), 184.31 (C-17); +ve ion FAB­MS : mlz (rel.int) 310 [Mt (C I7H I00 6) (3. 1), 152 (9.9), 136 (73 .7), 124 (7.5), 108 (26.3).

Shilanthralin 6. Elution of the column with CHCl r MeOH (19 : 1) gave light brown coloured crystalline mass of 6, recrystalized from CHCI3-MeOH (l: 1), yield 158 mg (0.0509%); Rr 0.48 (toluene-ethyl acetate-formic acid, 4 :5:1); m.p. 189-90°C; UV (MeOH): 274 (log E 6.1) ; IR (KBr): 3407, 3361, 3200, 2937, 2874, 1706, 1660, 1611 , 1514,1461,1412,1275,1239,1102, 1071 , 1026, 774 cm- I; IH NMR (OMSO-(h): 8 8.09 (l H, d, J= 8.81 Hz, H-4), 7.97 (IH, d, J=8.81 Hz, H-5 ), 7.48 (lH, brs, H-13) , 7.33 (IH, d, 1=7.5 Hz, H-9), 6.78 (lH, d, J=7.5 Hz, H- I0), 6.71 (lH, brs, H-15 ), 5.71 (I H, brs, 0 20 exchangeable, OH), 3.85 (I H, brs, 0 20 exchangeable, OH), 3.82 (l H, brs, 0 20 exchangeable, OH) , 3.79 (I H, brs, 0 20 exchangeable OH) , 2.73 (2H, brs, H2-7); 13C NMR (OMSO-d6): 8 135.16 (C-l), 159.11 (C-2) , 158.65 (C-3), 126.36 (C-4), 126.15 (C-5), 125.19 (C-6), 35.95 (C-7), 102.85 (C-8) , 124.39 (C-9), 123 .88 (C-10), 121.63 (C-l1) , 113.53 (C-12), 157.19 (C-13), 151.33 (C-14), 113.22 (C-15) , 123.61 (C-1 6), 134.92 (C-17), 183.7 (C-18); +ve ion FAB-MS: (reI. int.) 308 [Mt (Cl sHI 20 S) (17.6), 208 ( 13.5 ), 150 (12.8), 136 (100), 122 (14.8), 108 (47. 8).

Naphsilajitone 7. Chloroform extract obtained by di ssolving Shilajit in sodium bicarbonate solution and then extracting the acidified extract with chloroform furnished 7, recrystalized from MeOH, yield 465 mg (0.15 %); Rr 0.61 (toluene­ethyl acetate-formic acid, 4:5: 1); m.p. 157-58°C; UV: 226 nm (log E 4.6); IR (KBr): 3343, 3091, 2936,1755,1605 , 1555, 1487, 1415,1310, 1256, 1181,1077, 999,847 cm- I; IH NMR (OMSO-(h): 8

2222 INDIAN 1. CHEM., SEC B, OCTOBER 2004

8.83 (lH, dd, J=7.75, 2.14 Hz, H-2), 7.83 (lH, d, J =7 .75 Hz, H-1), 7.53 (l H, d, J=7 .22 Hz, H-8), 7.46 (l H, d, J=7.22 Hz, H-9), 7.43 (lH, d, J=2.14 Hz, H-4), 4.02 (lH, brs, Hr 12 a), 3.93 (l H, brs, H2-12 b), 3.99 (l H, brs , D20 exchangeable, OR); 13C NMR (DMSO-d6): 8 127.57 (C-l), 128.8 (C-2), 167.44 (C-3), 127.50 (C-4), 133.90 (C-5), 131.99 (C-6), 133.90 (C-7), 128.8 1 (C-8), 127.50 (C-9), 128.81 (C-IO) , 171.72 (C-ll), 41.58 (C-12); +ve ion FABMS: mJz 200 [Mt (C 12 HS0 3) (2. I) , 183 (28 .6),155 (28.6),153 (100),125 (11.0) .

Acetylation of 7. Compound 7 (5 mg) was heated with AC20 (3mL) and pyridine (1 mL) for 3 hr. Water (10 mL) was added , extracted with CHCI3 (3x5 mL) , the organic phase was washed with water (2x5 mL), dried (Na2S04) and evaporated to get monoacety I product, different spots on TLC, IR: 1755, 1725 cm- I

.

Acknowledgement

The authors are thankful to the Head, Instrumenta­tion Center, All India Institute of Medical Sciences, New Delhi for recording the NMR spectra.

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