Indian Journal of Chemistry Vol. 42B, November 2003, pp. 2858-2862

Isolation of some novel phytoconstituents from Anaphalis araneosa roots

S K Sharma, M Ali* & S R Mir

Faculty o f Pharmacy, l ami a Hamdard, Hamdard University , P.O. Hamdard Nagar, New Delhi - l 10062, INDIA *E-mail : mali_chem @yahoo.com

Received 22 November 2002; accepted (revised) 3 May 2003

Three new phytoconstituents, named anaphal isoleanenoic acid, anaphali soic acid and araneosoic ac id have been iso­lated from Anaplialis aralleosa roots and their structures have been charact.erized as 3~-hydroxyolean - 12-eJl -30-o i c ac id, 5, 9. 13-trimelhy l-eicos-5-en- 17, 18-d iol -24-oic acid and bisabol-I (7),2, 4 (13), II ( 12)- lelraen- 15-o1- 14-oic acid by spec tral data analysis and chemical reactions.

Anaphalis is a genus of perennial, rarely annual , erect, cottony or wooly, pubescent or glabrate herbs distri b­uted in Asia, Europe and America. About 32 species are reported fro m India. Anaphalis araneosa, DC. sy n. A. busua (D. Don) DC. (Asteraceae) is di stri b­uted from altitude 1200 to 2400 mI . The herb is used locally as an antiseptic for cuts and wounds . The f1 a­vones, araneol and araneosol2

, f1avonoids3.4, helipy­

rone4, sterols and phthalides5 from the aerial parts of

the plant and 5-methyl-n-hexatriacontane, anaphali­sone, acycl ic triterpenes, dulcioic acid and areneosol 6

from the flowers of the plant have been reported. This paper describes the isolation and characteri zation of the phytoconstituents from the roots of the herb.

Results and Discussion Compound 1, named anaphalisoleanenoic acid, was

obtained as colourless crystals from chloroform­methanol (98:2) eluants. It responded positively to the Liebermann-Burchard, TAC and TNM tests, suggest­ing that it was a pentacyclic triterpenoid possess ing olefinic linkage. The compound gave effervescence with sodium bicarbonate solution . Its mass spectrum showed a molecul ar ion peak at mJz 456 consisting with a molecular formula C30H4S0 3 that was sup­ported by its \3C-NMR spectrum. Its IR spectrum ex­hibited absorptions at 3400 (OH), 3140, 1665, (COOH), 1415, 1340 (gem-dimethyl) and 1630, 795 cm-I (tri-substituted double bond). The IH NMR spec­

trum of 1 accounted for one olefinic proton at 8 5.00 (brs) assigned to H-12, one carbinol proton at 8 3.73 as a doubl e doublet (J = 5.50, 9.50 Hz) ascribed to H-3a and seven methyl groups as broad singlets at 8 1.20 (Me-24), 1.03 (Me-25), 0.90 (Me-26, Me-27), 0.86 (Me-28), 0.75 (Me-23) and 0.70 (Me-29) . The

30 HOOC 29

2 ",

1

HOQH

_1B ~ 16 14

19 ~ 17 15

H £OOH

2

14 COOH

OH

3

mass spectrum of 1 indicated that the double bond triggered the typical retro-Diels-Alder (RDA) frag­mentation of ring C resulting in a fragment at mlz 207. The base peak at mlz 248 was also derived by RDA fragmentation . T hese fragments are characteri stic of to l2 amyrin derivatives containing a hydroxyl group in

SHARMA elof.: NOVEL PHYTOCONSTITUENTS FROM ANA PHA LIS ARANEOSA ROOTS 2859

ring A or B and a carboxylic group in ring D or E7. Elimination of 44 m.u. (C02) and HCOOH from mlz 248 gave ion peaks at mlz 203 and 202. The ion frag­ments at mlz 176 and 175 were generated by cleavage of C I6. 17, C 17. IS, C22.17 linkages from mass units at mlz 203 and 202, respecti vely . Expulsion of methyl group from mass unit 202 yielded an ion peak at mlz 187. Removal of water molecule from mlz 207 produced a peak at //'lIz 189. The ion fragment at mlz 133 arose due to elimination of methyl group and 70 m.u. from the ion fragment at mlz 248. The 13C_NMR spectrum of 1 showed characteristic signals for C-12 and C-13 olefinic carbons (8 122.6, 142.4), C-3 carbinol carbon (8 70.3) and C-30 carboxy li c carbon (8 183.3). The carbon 8 values were compared with the re lated pen­tacyclic triterpenes8

. The DEPT spectra of 1 exhibited the existence of seven methyl, ten methylene, five methine and eight quaternary carbons. Compound 1 on treating with acetic anhydride and pyridine formed a monoacetyl derivative la, [M t at mlz 498. Its IR absorption showed absorpti ons due to acetate group (1725, 1240 cm-I

) . The compound 1 on treatment wi th diazomethane fo rmed monomethyl este r lb . Jones oxidati on of 1 yielded 3-oxo derivative Ie that gave a posi ti ve Zimmermann test9 conforming the presence of a secondary hydroxy l group at C-3. The compound 1 failed to form a 12-bromo-y-Iactone with bromine ruling out the presence of carboxy lic group at C-29 10. Moreover, the H-1 8 proton appeared as a broad singlet in place of a doublet as in case ursane type­triterpenes . Therefore, compound 1 could be 3-epikatonic acids isolated from Cyampsis letra­gonoloba II. A comparison of the physical properties (m.p. and I H NMR ) of 1 with those reported for 3-epikatoni c acid indicated that they were not identica l. Therefore, the carboxyl function in 1 must be at C-30. This assignment was further substanti ated by the chemical shi fts of the olefinic protons at C-12. Usu­all y, in members of urs-12-ene series, thi s signal ap­pears in the vicinity of 85.25. In maprounic acid con­rain ing C-29 carboxylic acid 12, it appeared downfield at 8 5.50. In 1 it resonated 8 5.00. Molecular models indicated that a carboxylic fu nction at C-29 could de­shield the proton at C-12 whil e C-30 carboxyli c group had little effect on C-12 proton. Based on these find­ings the structure anapha li soleanenoic ac id 1 has been established as 3~-hydroxyolean-12-en-30-oic ac id . This is a new oleanane type triterpenic ac id .

Compound 2, named anapha lisoic acid, was ob­tained from chloroform-methanol (95:5) e luants. The

compound gave effervescences with NaHC03 solu­tion . Its IR spectrum showed absorption bands at 3435 (OH), 3050, 1695 (COOH) and 1610 cm·1 (C = C). Its mass spectrum showed molecular ion peak at mlz 398 corresponding to C24H460 4. It indicated two double bond equivalents; one each was adjusted in the vinylic linkage and in carboxy lic group. The mass spectrum of 2 exhibited important ion fragments at mlz 369 [M­C2Hst, 339 [C 17-C IS fis siont and 59 [C3H60H, M-339t suggesting the hydroxyl group at C-18; and the carboxy lic and hydroxyl groups at C-17. The ion peaks at mlz 153 [C9-C lOf issiont, 125 [153-C2H4t, 111 [125-CH2t, 97 [C6-C7 fissiont and 57 [C4-CS

fiss iont reflected the presence of o lefinic bond at Cs carbon6

. The I H NMR spectrum of 2 displayed signals

for the olefinic proton at 8 5.10 ass igned to H-6. One proton double-doublet at 8 3.30 (J = 5.5, 9.5 Hz) was ascribed to a-oriented C- 18 carbinol proton. A three­proton broad signal at 8 1.65 was associated with C-21 methyl group attached to an unsaturated carbon (C-5) . Two three- proton doublets 8 1.00 (J = 6.5 Hz) and 8 0 .98 (J = 6.0 Hz) were a cribed to C-22 and C-23 secondary methyl protons. Two three-proton tri p­lets at 8 0.91 (J = 6.5 Hz) and 8 0.85 (J = 6. 1 Hz) were accounted to C-l and C-20 termi nal primary methyl protons. The remai ning methylene and me­thine protons resonated between 8 2.25- 1.25 . The I3C_ NMR spectrum of 2 displ ayed 24 carbon signals in­cluding two olefinic carbons (8 139.3, 121.3), two carbinol carbons (8 81.6, C-17 ; 71.1 , C- 18) and one carboxy lic carbon (8 183.2). The I3C NMR carbon shifts were compared with the re lated sesterterpenes7

.

The compound produced a monoacetyl derivati ve 2a indicating the presence of a terti ary hydroxyl group in the mol ecule. Treatment of 2 with diazomethane formed a monomethyl ester 2b. These data led to formu late the structure of anaphali soic ac id 2 as 5, 9, 13-trimethyl eicos-5-en-17 , 18-d io l-24-oic acid. Th is is a new acyclic sesterterpene carboxylic acid and the first report of such natural product fro m A. araneosa.

Compound 3, named araneosoic acid, was obtained from ch loroform-methanol (9: 1) e luants of the col­umn . The compound produced effervescences in NaHCO} so lution disclosing the presence of carboxyl group. Its mass spectrum ex hibited a molecular ion peak at mlz 248 corresponding to a sesquiterpenic formula Cl sI-h o0 3. Its IR spectrum howed absorption bands at 34 10, 3300, 1655 (COOH) and 1625 cm·1 (C = C). Absorption maxima at 242, 302, 353 and 392 nm in the UV spectrum indicated conjugation in the

2860 INDIAN J. CHEM., SEC B, NOV EMBER 2003

molecule. The mass spectrum of 3 di splayed impor­tant ion fragments at mlz 203 [M-COOHt, 217 [203-CH20Ht, 57,191 [CI O-C II fissiont, 177, 171 [CrC lo fissiont, 163,85 [Cg-C9 fi ssiont, 149, 99 [Cr Cg fi s­siont, 83 [113-CH20Ht and 56 [113-CH2=C­CH20H, CIO-C II fission , 57 m.u.t. These data indi­cated that the compound was a bi sabolene type ses­quiterprene containing an exocyclic methylene group at C-4, another methylene group at C-Il, carboxy l functio nal group at C-7 and a C-I 5 hydroxyl methyl­ene group. The IH NMR spectrum of 3 demonstrated the presence of one-proton each two down field dou­blets at 8 7.10 and 6.73, with coupl ing interaction of 6.0 Hz each, assigned to H-3 and H-2, respectively; two broad signa ls at 8 5.82 and 5.25, integrat ing fo r two protons each associated correspondingly with C-13 and C-12 methylene protons. Two one-proton doublets at 4.65 (J = 4.5Hz) and 4.62 (J = 4 .5Hz) were accounted to C-15 hydroxymethylene protons. The remaining methylene protons appeared at 8 2.40-1.25 . The 13C NMR of 3 spectrum showed eight downfield signals for olefinic carbons and one each signal for carboxy lic (8 183.6) and hydroxy methylene (8 62.3) carbons. Methylation of 3 with diazomethane yielded a methoxy derivative 3a reflecting the pres­ence of carboxylic group. Monoacety l derivative 3b was obtained when 3 was allowed to react with acetic anhydride-pyridine at room temperature. The data led to establish the structure of araneosoic acid (3) as bis­abol-I (7), 2, 4 (13), 11 ( 12)-tetraen-15-01- 14-oic acid. Thi s is an unknown natural product and the first report of sesquiterpene from Anaphalis araneosa.

Experimental Section M.ps uncorrected ; IR: Perkin-Elmer 1710, KBr;

UV: Beckman OU-64, MeOH ; IH NMR (400 MHz): Varian T 300 A, COCl 3; 13C-NMR ( 100 MHz) : Perki n-Elmer 32 with TMS as an in terna l standard; MS: l eol JMS-DS 303. GC mass spectrometer; CC: Silica gel (Quali gens), 60-120 mesh; TLC: Sil ica gel G (Qualigens). Spots were visualized by exposure to iodine vapours , UV radiation and by spray ing with ceric ammoni um sulphate and perchloric acid .

Plant material The fresh roots of A. araneosa were collected from

the Kullu valley (H imachal Pradesh) and identified by Dr. M. P. Sharma, Taxonomist, Department of Bot­any, lamia Hamdard, New Delhi. A voucher speci­men is retained in the DepaI1ment of Pharmacognosy and Phytochemistry , Jamia Hamdard, New Delhi.

Extraction and Isolation

Air-dried and coarsely powdered roots (0.95 kg) of A. araneosa were extracted exhaustively with EtOH (95%) in a Soxhlet apparatus. The ex tract was dried under reduced pressure to obtain a brown-coloured viscous res idue (30 g). The dried ethanolic ex tract was di ssolved in minimum amount of MeOH and ad­sorbed on silica gel to form slurry. The air-dried s lurry was chromatographed over a si lica gel column packed in petroleum ether. The column was eluted with petroleum ether, petroleum ether-CHCI J (9: I , 3: I , I: 1, 1:3 v/v), CHCb, CHCl r MeOH (99: I , 95:5, 9: 1,3: I, I: I, 1:3 v/v) and MeOH to isolate the fo llow­ing compounds:

Anaphalisoleanenoic acid 1: Elution of the col­umn with CHCI3: MeOH (98:2) furn ished colourless compou nd 1, crys tall ized from MeOH 50 mg (0.0055 % yie ld), Rr 0.854 (CHCI3-MeOH, 8.5: 1.5), m.p. 105- 107°, fa]I RD + 3.33°, (C=0.030, MeOH); UV: 215 nm (log E 6.1 ); lR (KBr): 3400,3 140,2900, 2840, 1665, 1630, 14 15, 1340, 1240, 1010, 975,795 cm' l; IH NMR (DMSO-d6): 8 5.00 (tH, brs, H-12), 3.73 ( IH , dd, J = 5.50,9.50 Hz, H-3a) , 2.45 (l H, brs, H-18), 1.20 (3 H, br , CH3-24), 1.03 (3H, brs, CHr 25), 0.90 (6H, brs, CH3-26, CHr 27), 0 .86 (3H, brs, CH3-28), 0.75 (3H, brs, CHr 23), 0.70 (3H, S, CH)-29); ElMS //lIz (rel.int.): 456 [Mt (C30H.1S0 3) (4.3), 248 (100), 207 (25.8), 203 (33 .1 ). 202 (5.0), 191 (6. 1), 190 (13.1) , 189 (10.8), 187 (5 .0), 176 (7.3),175 (5.0), 146 (8.7),133 (23.5), 119 (13 .6), 107 (10.0),95 (12.0),85 (18.6), 71 (25 .0),57 (38.4); 13C MR (DMSO-d6) : 838.7 (C-l), 27. 1 (C-2), 70.3 (C-3), 38.8 (C-4), 55.3 (C-5), 18.2 (C-6), 32.9 (C-7), 39.6 (C-8), 47.3 (C-9), 36.9 (C- lO), 23.4 (C- l l), 122.6 (C-12), 142.6 (C-13), 42 .7 (C- 14), 26.2 (C-15), 27.3 (C- 16), 3 1.9 (C- 17), 47.5 (C- 18), 42.3 (C- 19), 43.9 (C-20), 3 1.9 (C-21), 37.6 (C-22), 28.3 (C-23), 15.3 (C-24), 15.9 (C-25), 16.1 (C-26), 25.8 (C-27), 27 .7 (C-28), 27.5 (C-29), 183.3 (C-30).

Acetylation of 1. The compound 1 (5 mg) was dis­so lved in pyridine (2 mL) and acetic anhydride (5 mL) added to it. The reaction mixture was heated for 30 min on a steam bath. The resulting mixture after being left overnight was quenched by addition of co ld water (20 mL) and extracted with CHCb (3x 10 mL). The CHCI)-I ayer was washed with water (2 x 5mL), dried over anhydrous sodium sulphate and evaporated to get viscous res idue of monoacety l product la, 1240 It was crystalli zed from MeOH, m.p. 81-82°; TR (KBr): 3300,1725,1665 cm' l; [Mt mlz498.

SHARMA et al.: NOVEL PHYTOCONSTITUENTS FROM ANAPHALIS ARANEOSA ROOTS 2861

Esterification of 1. The compound 1 (5 mg) was dissolved in solvent ether (30 mL). To this ethereal solution of diazomethane (10 mL) was added. The reaction mixture was kept overnight. The solvent was evaporated to get monomethyl ester of lb. TLC was compared with that of 1 (different spots); m.p. 93-94° .

Jones oxidation of 1. A solution of 1 (10 mg) in Me2CO (15 mL) was cooled to 0° and excess of Jones reagent was added. The reaction mixture was stirred at 0° for 30 min . Excess of water (LOO mL) was added and extracted with CHCI3 (3 x 20mL). The CHCl 3-layer was washed with water (3 x 20 mL), dried (Na ZS04) and evaporated to get 3-oxo derivative 1c; m.p. 81-83°; IR (KBr): 3300, 1710, 1690 cm-'.

Anaphalisoic acid 2: Elution of the column with CHCI3-MeOH (95:5) yielded colourless crystals of 2, crystallized from MeOH-EtOAc (95:5), 50 mg (0.0052% yield), Rr 0.82 (CHClrMeOH, 9: I); m.p. 91-92°, [a]' 8o 0°; (C = 0.01, MeOH); UV : 212,225 nm (log E 5.3 , 5.4); IR (KBr): 3435, 3050, 2940, 2870, 1695 , 1610, 1460, 1420, 1255 , 1190, lOSS , 1010,795 cn'-'; 'H NMR (CDCI 3): 85.10 (1H, m, H-6),3.30 (1H, dd , J = 5.5, 9.5 Hz, H-18a), 2.55 (2H, m, H2-7), 2.10 (2H, m, Hr 4), 1.90 (1H, m, H-13) , 1.87 (1 H, m, H-9),1.65 (3H, brs, Me-21), 1.36 (4H, brs, H2-19, H2- 16), 1.25 (l8H, brs, 9 x CH2 ),1.00 (3H, d, J = 6.5Hz, Me-22), 0.98 (3 H, d, J = 6.0 Hz" Me-23), 0.9 1 (3H, t, J = 6.5 Hz" Me-I), 0.88 (3H, d, J = 6.1 Hz" Me-20); ElMS mlz (rel.int.): 398 [Mt (C24H4604) (7 .2), 383 (36.0),369 (19.3), 355 (18.1), 339 (5.2), 248 (7.0) , 157 (8.4), 153 (4.9), 139 (8.1), 127 (11.20), 125 (13.2), 113 (23.2), III (27.5), 99 (19.30),97 (50.9), 85 (33.7), 83 (60.2),71 (52.3),69 (54. 1) , 59 (7.2), 57 (100), 55 (62.8); '3C-NMR (CDCI3): 8 ]4.3 (C-I), 28.9 (C-2), 28.9 (C-3), 31.3 (C-4), 139.3 (C-5), 121.3 (C-6), 33.6 (C-7), 28.6 (C-8),39.3 (C-9), 27 .8 (C-I0), 28.9 (C-ll), 28.9 (C-12), 38.7 (C-l3), 28.7 (C-I4), 28.8 (C-15), 33.6 (C-16), 81.6 (C-17), 71.1 (C- 18), 33.7(C-19) , 22.3 (C-20), 21.5 (C-21), 22 .1 (C-22), 21.3 (C-23), 183.2 (C-24).

Acetylaton of 2. The compound 2 (10 mg) was re­acted with AczO (3 mL) and pyridine (1 mL) at room temperature for 24 hrs. Water (10 mL) was added, extracted with CHCI3 (3 x 10 mL), the organic phase washed with water (2 x 10 mL) , dried (Na2S04) and evaporated to get monoacetyl product 2a, m.p. 73-74°; IR (KBr): 3400,3100,1725,1690 cm-' .

Methylation of 2. The compound 2 (5 mg) was dissolved in solvent ether (10 mL). The ethereal solution of CH2N2 (5 mL) was added and left overnight. The solvent was dried to obtain mono­methyl ester 2b.

Araneosoic acid 3: Elution of the column with CHCI3-MeOH (9: I) furnished 3 as colourless product, crystallized from MeOH-EtOAc (95:56), 60 mg (0.0062% yield), Rr 0.44 (CHCI3-MeOH, 7:3); m.p. 78-79°, [a]' 8o + 1.56°, (C = 0.64, MeOH); UV: 242, 302, 353, 392 nm (log E 6.5 , 6.4, 6.6, 6.1); IR (KBr): 3410, 3300, 2905, 1655, 1625, 1590, 1540, 1445 , 1390, 1225, 1010,935,790 cm·'. 'H NMR (DMSO­d6): 7.10 (I H, d, J = 6.0 Hz, H-3), 6.73 (l H, d, J = 6.0 Hz, H-2), 5.82 (2H, brs, H2-l3), 5.25 (2H, brs, H2-12), 4.65 (l H, d, J = 4.5 Hz, CH20H-I5a), 4.62 (1 H, d, J = 4.5 Hz, CH20H-I4b) , 2.40 (2H, m, Hz-5), 2.38 (2H, m, Hr 6), 2.25 (2H, m, H2-8), 1.73 (2H, m, Hz- IO), 1.25 (2 H, m, Hz-9); ElMS mJz (reI. int.): 248 [Mt (C' 5H200 3) (77.6), 217 (11.6),203 (28.8),191 (19.7), 177 (17.5),163 (9.3), 149 (22.6), 133 (29.1), 119 (28 .1 ), 113 (25.0), 107 (34.3), 105 (22.0),99 (2 1.8), 97 (43 .6), 95 (27.3), 85 (65.0), 83(31.2) 71 (54.3),68 (64.2), 57 (100), 56 (98.9); '3C-NMR (CDCI3): 8 141.3 (C-l), 131.7 (C-2), 129.6 (C-3), 139.9 (C-4), 39.7 (C-5), 31.3 (C-6), 143.5 (C-7), 29.5 (C-8), 28.3 (C-9), 28.2 (C-lO), 137.6 (C-ll), 109.5 (C-I2), 110.1 (C-l3), 183.6 (C-14), 62.3 (C-15) .

Methylation of 3. Compound 3 (10 mg) was treated with ethereal CH2Nz for 12 hrs. The solvent was evaporated to dryness to get methoxy derivative 3a. TLC was compared with 3, m.p. 73-74°; IR (KBr): 3410,1710 cm-' .

Acetylation of 3. Compound 3 (10 mg) was treated with Ac20 (3 ml) and pyridine (1 mL) at room tem­perature overnight. Water (10 mL) was added and the reaction mixture was extracted with CHCh (3 x 10 mL). The CHCh-layer was washed with water (2 x 5 ml), dried (Na2S04) and evaporated to get monoacety­lated product 3b, m.p. 71 -72°; IR (KBr): 3410,1725, 1725, 1690 cm-'.

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2862 INDIAN J. CHEM., SEC B, NOVEMBER 2003

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