Indian Journal of Chemistry Vol. 43B, March 2004, pp. 655-659

Phytoconstituents from Saussurea Zappa roots

Vijender Singht & Mohd Ali*

*Dcpartment of Pharmacognosy & Phytochemi stry, Faculty of Pharmacy, lamia Hamdard (Hamdard Un ivers ity), P.O. Hamdard Nagar, New Delhi 110062, Indi a.

e-mail : maliche m@redi lTmail.com

Received 26 December 2002; accepted (revised) 6 January 2003

Two new phytosterols, named lappasterol and 3-epi-lappasterol and an unreported lanosterol type triterpene, lap­palanasterol , along with j3-sitosterol-o-glucopyranoside have been isolated from the roots of Saussurea lappa C.B. Clarke and their structures have been establi shed as (245) -stigmasta-5 , 9( 11 )-dien-20, 28-endo-3j3-01 1, (24S)-stigmasta-5, 9(11)­dien-20, 28-endo-3a-ol 2 and lanosta-20(22), 24-dien-2-one-3j3. 9a, 12a-triol-21-al 3, respectively on the basis of spectral analyses and chemical reactions.

IPC: Int.CL7 A 61 K 31/00

Saussurea lappa C.B. Clarke, syn. S. costus (Falc) Lipsch (Asteraceae) is a Himalayan species and oc­curs in the region from 2700-4000 m in Kashmir, La­hul valley in Himachal Pradesh and Garhwal in Ut­tranchalt.2. The roots possess carminative, analgesic, anthelmintic and emmenagogic properties stimulate the brain 1.3 and cure blood diseases and li ver and kid­ney disorders . They are prescribed in advance stages of typhous fever, rheumatism, nervous di sorders, ir­regular menstruation, heart diseases, to improve com­plexion, as hair wash to kill lice and to turn grey hair to black3-6. The roots of S. lappa contain 22-dihydro­stigmasterol7

, dehydrocostuslactone, costic, palmitic and linoleic acids, f)-sitosterol, u- and f)-cyclocostun­olide, isoalantolactone8

, 4-f)-methoxy dehydrocostus lactone9

, sesquiterpenoid-saussureal lO, amino acids,

saussureamines A-E, lignan glycoside (-) massoni­resinol-4"-O-f)-D-glucoside ll , two guaianolides-iso­dehydrocostus lactone, isozaluzanin-C I2

, two ses­qui terpene lactones with an u, f)-unsaturated aldehy­dic groupl3 and is-hydroxy dehydrocostus lactone '1 .

This paper describes the isolation and characterization of two new phytosterols and a lanostane-type triter­pene from the roots of the plant.

Results and Discussion Compound 1, designated as lappasterol , was

obtained as cream coloured needles from petroleum ether-chloroform (I: I ) eluents. It responded positively

tResearch & Development Division, AIMIL Pharmaceutical s (India) Ltd., 2994/4, Street No.17, Ranjeet Nagar, Ncw Delhi 1 \0 008

H _ H

29 28

22

I n

...c-:-:-- 15

1. 313 - OH 2. 3 a - OH

H

:lO

3.

2()

27

to Liebermann- Burchard test for sterol and exhibited IR absorption bands for hydroxyl group (3450 cm-I

)

and unsaturation (1640 cm-I) . Its molecular weight

was estab lished as 410 (C29H460) on the basis of mass and 13C NMR spectra. It indicated seven double bond equivalents; four of them were adjusted in the tetracyclic carbon skeleton of sterol, two in olefinic linkages and the remaining one in a cyclized side chain. The diagnostically important mass ion peaks in the mass spectrum appearing at rnIz 395 [M-Met,

656 lNDIAN J. CHEM., SEC B, MARCH 2004

380 [M -2 x Mer, 365 [M-3 x Mer, 392 [M-H20r, 377 [392-Mer, 271 [M-CIOHI9, side chainr, 253 [271-H20r, 230 [271 -ring-Dr, 212 [230- H20 rand 197 [212-Mer suggested a steroidal carbon framework of the molecule with two vinylic linkages, the hydroxyl group in rings A-D and a cyclized side chain at C-17 . The prominent ion peaks at rnJz 83 [C2,3-CS,IO -C7,8 fissionr, 72 [CI.l0-C4,s-fissionr, 54 [72- H20r, 124, 286 [C6.rC9.10 fissionr, 106 [124-H20r, 147 [286-CIOHI9, side chainr and 132[147-Mer indicated the location of the hydroxyl group in ring-A, placed at C-3 on the basis of biogenetic considerations, and one of the olefinic linkage in ring­B at C- 5. The ion peaks at mlz 176 [C8, WCI1.I2 fissionr, 158 [176- H20r, 95 [M-176-sidechainr, 80 [95-Mer, 190 [CI 2.1 3.C8.14 fissionr and 172 [190-H20r supported the existence of another vinylic linkage at C-9 (l1)I S-17. The IH NMR spectrum of 1 displayed two one-proton signals as a doublet at 85 .28 (J = 4.6 Hz) and as a multiplet at 85.04 assigned to C-6 and C-ll vinylic protons, respectively. A one-proton carbinol broad multiplet at 83.47 (w v,=15 .24 Hz) was ascribed to 3a methine proton . Three singlets at 80.61, 1.18 and 0.93, integrating for three protons each, were attributed correspondingly to C-18, C-19 and C-21 tertiary methyls, respectively. Three doublets at 80.77 (J = 6.12 Hz), 0.73 (J = 4.23 Hz) and 0.86 (J = 6.32 Hz), integrating for three protons each, were associated with C-26, C-27 and C-29 secondary methyl s, respectively. The appearance of all the methyls in the region 8 1.18-0.61 suggested that these functiona li ties were attached to the saturated carbons Ig.20. The remaining methylene and methine protons appeared in the region 8 2.20-1.01. The H3 -29 resonance of 24R­

configuration (80.83) was more unshielded as compared to the 24S-resonance (8 0.86)21. The assignment of the chemical shifts was made by comparison with 8 values of the corresponding protons in the structurally similar sterols 22-2S.

Further evidence for the structure 1 was provided by its I3C NMR spectral data, which showed the existence of 29 carbon atoms in the molecule. Four downfield signals at 8 1:)9.8, 120.7, 137.3 and 128.3 were assigned to C-5, C-6, C-9 and C-l1 unsaturated carbons, respectively . The C-3 carbinol proton appeared at 8 70.8. The assignments of the carbon chemical shifts were made by comparison wi th the be values of the con'esponding carbon atoms of ~­sitosterol22, and lawsaritols23.24 and pleuchinol 18. In the

'H_ 'H cosy spectrum of 1, the co-relations of 3 a H with 2 a Hand 4H, 6H with H-4, H-7 and H-8 , H-ll with H-12, H3-21 with Hr29 and H-17 and H-24 with H-25, Hz-23 and Hz-22 were observed. The zero specific rotation of 1 indicated racemic mixture of the compound. On the basis of the spectral data, the structure of 1 was characterized as (24S)-stigmasta -5 , 9(ll)-dien-20, 28-endo-3 ~-ol. This is a new member of the phytosterols.

Compound 2, named 3-epi-lappasterol, was obtained as colourless crystalline mass from petroleum ether-chloroform (l :3) eluents . Its spectral data were almost identical to that o f lappasterol. However, difference in physical constants, eluent ' s polarity and crystal shape suggested it to be an epimer of the previous compound. In the IH NMR spectrum of 2, the C-3 carbinol proton appeared as a one -proton multiplet and its half-width of 10.92 Hz indicated its ~ -orientation. Jones oxidation of 2 yielded lappasterone 2a. The latter gave a positive Zimmermann test indicating the presence of the 3-oxo group26. The NaBH4 reduction of 2a regenerated lappasterol 1, confirming axial orientation of the hydroxyl group in 3-epi- lappasterol 2. On the basis of these findings, compound 2 was identified as (24S)­

stigmasta-5 , 9(11) -dien-20, 28-endo- 3a-ol. This is a new phytosterol.

Compound 3, named lappalanasterol , was obtained as pale yellow crystals from chloroform-methanol (99: 1) eluents. It gave positive Liebennann-Burchard test for triterpenes and sterols and had JR absorption bands at 3460 (hydroxyl), 1710, 1680 (carbonyls) and 1630 cm- I (C=C). Its mass and 13C NMR spectra establi shed the molecular weight 502 consistent with a tetracyclic triterpene C30H4606. It had eight degrees of unsaturation, which were adjusted in the lanostane­type carbon framework with two carbonyl groups and two .olefinic linkages. The mass spectrum exhibited significant ion peaks at mlz 346 IM-CgHIIO, side chain, -H20, ··Mer, 328 [346- H20r, 313 [328-Mer, 271 [313-ring-D fissionr and 123 [CgHIlOr reflecting the presence of Cg-side chain with two olefinic linkages and an aldehydic group. The ion peak ~~ at rnJz 83 [C3. 4-CS. IO-C7.8 fiss iont, 114 [CI.IO­C 4.S fi ssionr, 96 [114-H20r, 182 [C7.8-C9. 10 fis~ionr , 167 1182-Mer, 149 [167-H20r, 168 [C6.TC9. 10 fi ssio~/l82-CH2r, 154 [CS.6- C9.10 fi ss ionll68- CH2r and 139 [15 t :-Mer indicated saturated nature of rings A and B, one hydroxyl group in ring-A, placed at C-3 on the basis of biogenetic grounds and a keto group at C-2. The' ion peaks at mlz 224 [C9.WCS.14 fissionr ,

SINGH et al. : PHYTOCONSTITUENTS FROM SAUSSUREA LAPPA ROOTS 657

206 [224-H20t, 191 [206-Met, 238 [CII ,lrCs.14 fissiont, 205[238-Me-H20t and 268 [CI2.13-CS.14 fissiont supported the saturated nature of the ring-C and existence of hydroxyl groups at C-9 and C- 12. The IH NMR spectrum of 3 was consistent with the proposed structure and clearly showed a one-proton downfield multiplet at 8 6.58 and a one-proton downfield doublet at 8 5.85 (J = 7. 69 Hz) assigned to vinylic H-22 and H-24, respectively. A one-proton broad signal at 8 3.31 was due to 3 a-carbinol proton. A one-proton double doublet at 8 4.23 with coupling interactions of 3.51 and 3.48 Hz was associated with C-12 B-carbinol proton. A one-proton broad signal at 8 3.11 and a one-proton doublet at 8 3.03 (J = 3.74 Hz) were ascribed to C- 18 hydroxy methylene protons. The presence of one-proton downfield signal at 8 9.63 suggested the existence of aldehydic group at C-21 in the molecule. Two three -proton signals at 8 l.96 and l.90 were attributed to C-26 and C- 27 methyl protons attached at C- 25 unsaturated carbon. Four tertiary methyls resonated as broad signals at 8 l.33 (Me-19, Me -28), l.19 (Me-29) and 0.98 (Me-30). The remaining methylene and methine protons resonated between 8 2.65-l.04. The 13C NMR spectrum of 3 displayed signals for C-2 oxocarbon (8 210.1), C-21 aldehydic carbon (8 205.2), carbinol carbons (8 75 .1, 81.1, 78.2, 73.2) and unsaturated carbons (8 148.5, 122.2, 128.6, 139.4). In the IH_IH cosy spectrum of 3, there were co-relations of 3 a-H with H3-28 and H-5a, 12 B with 11 Band Hr18, H-21 with 17 a-H and H-22, 22-H with 23-H2, H-21 and 24-H and 24-H with H2-22, H3-26 and H3-27. Based on these evidences, the structure of 3 was characterized as lanosta-20 (22), 24-dien-2-one- 3B, 9a, 12a-triol-21-al. This is a new member of lanostane-type triterpenes.

Experimental Section Melting points were determined in open-end

capillary tubes and are uncorrected. UV spectra were recorded on a Beckman DU-64 spectrophotometer in methanol; IR spectra on a Shimadzu-480 instrument using KBr pellets; IH and I3C NMR spectra on Jeol FT 300 MHz and 75 MHz instruments, respectively, in CDCI3 (chemical shifts in 8 ppm and J values in Hz). Mass spectra were scanned by effecting electron impact ionization at 70 eV on a Jeol JMS-DX 303 GC-MS. Silica gel and silica gel G (60-120 mesh) were used for TLC and column chromatography, respectively. The collected fractions of the column

eluents were subjected to TLC to check homogeneity. Chromatographically identical fractions were combined and concentrated. The TLC spots were visualized under UV light or exposure of plate to h vapours or spraying with vanillin -H2S04 or ceric sulphate followed by heating at 120°C for 15 minutes.

Plant material The roots of S. lappa were purchased from the

Khari Baoli Market, Delhi and were authenticated by Dr M P Sharma, Taxonomist, Department of Botany, Jamia Hamdard, New Delhi. A specimen voucher of the sample is deposited in the herbarium of the Phytochemistry Lab, Faculty of Pharmacy, Jamia Hamdard.

Extraction and isolation of metabolites Air-dried pulverized roots of S. lappa (1.95kg)

were extracted exhaustively in a Soxhlet apparatus with EtOH(95%). The combined extracts were dried under reduced pressure to secure a viscous brownish coloured residue (400 g) (20.51 %). The dried alcoholic extract was dissolved in minimum amount of MeOH and adsorbed on silica gel. The slurry was air-dried and chromatographed over silica gel column. The column was eluted with petroleum ether, petro­leum ether-CHCh (9:1, 3:1,1:1,1:3), CHCh, CHCb­MeOH (99:1, 98:2, 95:5, 3:1,1:1,1 :3) and MeOH in order of increasing polarity to isolate the following compounds.

Lappasterol 1. Elution of the column with petroleum ether-chloroform (1: 1) afforded cream­coloured needles of 1, recrystallized from chloroform -methanol (1: 1); yield 170 mg (0.0087%); mp 130-32°; Rr 0.70 (petroleum ether: chloroform: methanol ; 5:3:0.5); [a] g 0.00 (CHCI3, c 0.14); IR (KBr) 3450,

2965, 2870, 1640, 1480, 1455, 1395, 1275, 1115, 1075, 1035, 810 cm-I. IH and I3C NMR (Table I). ElMS rnIz (rel.int.): 410 [Mt (C29H460) (74.3), 396 (100), 395 (34.5), 392 (40.8), 380 (34.0), 379 (68 .8), 377 (32 .5), 365 (38 .5), 347 (24.9), 286 (36.2), 271 (33.5),253 (46.2),230 (19.3), 212 (33.8), 197 (14.2), 190 (8.2), 176 (1l.0), 172 (10.8), 158 (28.8), 147 (28.9), 132 (33.1), 124 (1l.8), 118 (27 .6), 106 (43 .1), 95 (44.7), 83 (43.5), 80 (56.2), 72 (2 .3), 70 (6l.8), 56 (93 .5),54 (83.2), 43(91.0).

3-epi-Lappasterol 2. Elution of the column with petroleum ether-chloroform (1 :3) furnished colourless crystals of 2,recrystallized from chloroform: methanol (1:1); yield 180 mg (0.0092%); mp 126-28°C; Rr O.61 (petroleum ether: chloroform: methanol; 5:3:0.5);

658 INDIAN J. CHEM .. SEC B, MARCH 2004

Table I_ 'H and DC NMR speclral dala of 1, 2 and 3

Posilion IH NMR DC NMR

2 3 1 2 3 Alpha Bela Alpha Bela Alpha Bela

1.37 dddd 2.20 dddd 1.36 dddd 2.2 1 dddd 2.6 brs 2.36 38.8 37.3 35.3 (16. 12,9.78, (1 2. 10, 4.58, ( 11.4, 3.05, (5.39, 7.00, 5.37, 4.58) 9.72,11.31) 9.58,5.38) 6.44,4.43)

2 1.93 m 1.88 m 1.93 m 1.87 m 32.2 31.7 2 10.1

3 3.47 brm 3.43 brm 3.3 1 brs 70.8 7.8 75. 1 (wo;, 15.24) (WO!, 10.92)

4 2. 16 d 1.79d 2. 12 d 1.75 d 41.3 42 .3 40.6 ( I 1.32) (5.34) ( 11.35) (5.92)

5 1.59 dd 139 .8 140.8 54.7 (\ 1.32, 5.6)

6 5.28 d 5.27 d 2.1 0 m 1.62 m 120.7 121.7 20.4 (4.6) (4.71 )

7 1.75 m 1.75 m 1. 79 111 1.79111 2.04 m l .4m 30.7 31.9 29 .7

8 2.75111 2.75111 2.40 brs 30.9 31.7 44.0

9 137.3 138.3 8 1.1

10 36.3 36.2 36.9

II 5.04 m 5.00111 152 d 1.44d 128.3 2 1.1 23.2 (9.4 1 ) (5.4)

12 1.89 d 1.75 111 1.87 d 1.80 m 4.23 dd 3().5 129.3 78.2 (5.21) (5.36) (3.5 1,

3.48)

13 44.9 45.9 52.1

14 1.50m 1.55 III 55.S 56.8 57.2

15 1.09 III 1.41 In 1.11 In 1.48 In 1.04 In 1.40 In 21.3 23 .9 31.7

16 1.61 III 1.4 1 111 1.58 III I .48 III 1.06 111 1.75 111 28.7 28.2 35 .8

17 1.47 dd 1.44 dd 2.53 dd 55. 1 56.0 56.4 (8.52, ( 11.4, (5.03 , 5.56) 3.05) 5. 15)

18 0.61 s 0.1 6 ., 3.11 brs 11 .0 12.0 73.2 3.03 d (3.74)

19 1.1 8 s I.I S s 1.33 s 192 19.4 13.8

20 35 .2 36.2 14S .5

2 1 0.93 s 0.93 s 9.63 s I .8 18.S 205.2

22 1.4 1 s 1.0 1 m 1.26 m 1.01 m 6.58 m 32.9 33 .9 122.2

23 1.14 III 2. 13 m 1.14 m 2. 12 m 2.74 In 2.47 m 27.2 26. 1 36.2

24 1.05 m 1.07 m 5.85 d 45.7 45.9 128.6 (7.69)

25 1.50 m 1.58 111 29 .3 29.2 139.4

26 0.77 d 0.77 d 1.96 s 20. 1 20.2 2 1.9 (6. 12) (3.37)

27 0.73 d 0.73 d 1.90 s 17.3 18.3 22.0 (4.23) (6. 12)

28 1.14 m 1.I4m 1.33 s 25 .1 25.4 14.7

29 0.86 d 0.85 d 1.19 s 10.9 11.9 21. 1 (6.32) (3.27)

30 0.98 s 12.4

Coueling constants in Hertz are given in earcnlheses

SINGH et ai.: PHYTOCONSTITUENTS FROM SAUSSUREA LAPPA ROOTS (

659

[a] ~8 (+) 345.2° (CHCl3, c 0.04); IR (KBr): 3450,

2915, 2850, 1640, 1465, 1375, 1265, 1105, 1045, 1015, 800 em-I . IH and I3C NMR (Table I). ElMS mlz (reI. int.): 410 [Mt (C29H460) (81.3), 395 (53.7), 392 (47.0), 380 (34 .8), 377 (28.5), 365 (18.0), 347 (20.0),286 (15.2), 272 (9.1), 270 (27.5), 253 (43.5), 230 (17 .7), 212 (30.3), 197(12.1), 176 (9.4), 172 (14.5), 158 (35.8), 147 (16.4), 132 (35.6), 124 (6.3), 118 (3 1.6), 106 (63.8), 95 (41.3), 83 (68.0), 80 (58.1), 70 (21.5), 68 (62.8), 56 (53.4), 54 (100), 43 (71.8) .

Jone's oxidation of 3-epi-lappasterol 2. Compound 2 (50 mg) was dissolved in acetone (60 mL) and treated with freshly prepared Jone's reagent (5 mL). The reaction mixture was stirred at room temperature till the reaction was complete (TLC monitoring) . It was diluted with water and extracted with Et20 . Removal of the solvent from the extract yielded lappasterone 2a (32 mg), mp Ill-12°, IR (KBr): 1705 em-I , IH NMR (CDCI3): 05 .3 1 (lH, d, J = 4.7 Hz, H-6), 5.03 (1H, m, H-ll ), 1.33 (3H, brs, Me-19), 0.93 (3H, brs, Me-21), 0.85 (3 H, d, J = 6.3 Hz, Me-29), 0.77 (3H, d, J = 3.4 Hz, Me-26), 0.73 (3H, d, J = 6.1 Hz, Me-27), 0.17 (3H, brs, Me-IS) ; ElMS mlz (re\. int.) : 408[Mt (C29H440) (3.6), 393 (47.8),390 (43 .1 ), 378 (37.2), 375 (21.6), 363 (2 1.5), 286 (11.7), 269 (24.5), 251 (48.3), 228 (15.2), 210 (33.5), 195 (11.6), 174 (11.3), 122 (7.1), 104 (63 .5), 81 (67.8), 70 (27.2).

Reduction of lappasterone 2a. Lappasterone 2a (15 mg) was dissolved in ethanol (10 mL) and NaBH4 (1 g) was then added with stirring for I hr. After dilution with water, the reaction mixture was extracted with chloroform, the chloroform layer was washed with water, dried and evaporated to obtain lappasteroll (12 mg), mp and mmp 131-32°, co-TLC comparable.

Lappalanasterol 3. Elution of the column with chloroform-methanol (99: 1) furni shed pale yellow crystals of 3, recrystallized from chloroform-methanol (\ :3), yield 230 mg (0.011 %); mp 280-82°; Rr 0.64 (petroleum ether: chloroform:methanol; 5:3: 1); [a] ~8

336.8° (CHCI3, c 0.16); IR (KBr): 3460, 2990, 2930, 2875, 1710,1680, 1630, 1465, 1420,1370, 1310, 1290, 1275, 1210,1130,1095, 1075,1025,910, 790,735 cm­I. IH and I3C NMR (Table I). ElMS mlz (rei. int.) : 502 [Mt (C30~606) (1.0), 346 (6.3), 328 (33.0), 313 (100), 271(10.1),268 (5.3), 238 (5.2),224 (9.5), 222 (6.1), 206 (9.3), 205 (9.2), 191 (13.6), 186 (12.8), 182 (4.5), 168 (7 .8), 167 (8 .5), 154 (6.3), 149 (10.2), 145 (11.5), 141 (7.9), 139 (9.8),135 (13.1),129 (12.0), 126 (23.7),

123 (10.9), 114 (8.3), 111 (18 .5), 96 (18.2), 91 (22.1), 83 (22.0), 77 (31.2), 69 (41.3), 55 (43.0),43 (89.4).

Acknowledgement The authors are thankful to the Regional

Sophisticated Instrumentation Centre, CDRI, Lucknow for scanning spectra and Aimil Pharmaceuticals (India) Ltd. , New Delhi 110 008 for providing necessary facilities.

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