Andrographolide and Other Constituents of Holmskioldia Sanguinea ~ (C H 2)Tiyh 2 'CH 20 R , O ~ · 1 R = H

Indian lournal of Chemistry Vol. 38B, May 1999, pp. 632-634

Andrographolide and other constituents of Holmskioldia sanguinea ~ (C H 2)tIYH 2 'CH 20 R , o ~ · 1 R = H . 7 ~ 1 P K Chaudhuri*, M. Singh, M. Pal, R.P. Sharma & 1a R = A c S.P.lain+ OH 0 2 R = H Phytochemical Technology Di vision and +Botany Division 3 R = G IJC A ... Central Institute of Medicinal and Aromati c Plants, P.O. CIMAP, Lucknow 226 015, India triplet at (54.0 (2H, J=6 Hz) showing the presence of Received 20 Augusl 1998; accepled (revised) 12 March 1999 a terminal acetoxy methylenej protons in la. Compound 1 in its DC NMR spectrum ( 100 MHz) A new compound 27-methylnonaeicosanol 1 together with known lipids, lIiterpenoids, tl avonoids, bi oacti ve andrographolide showed two methyl signals at (5 13.95 (C- 29) and and neoandrographol ide have been isolated from Holmskioldia 16.20 (CHr 27), and the absence of double bond . The sanguinea and identi fi ed by thei r spectral and chemical study. This other DC NMR signals at (5 32.35 (CH), 33.40, 29.90, is the second natural occurrence of andrographolide and 26.30 and 23.20 due to CH? 's are characteri stics of a neoandrographolide, and their first occurrence from H. sanguinea is 1 of chemotaxollomic importance. long chain moiety . Twin pe;ks at 720 ~ nd 730" em· in its IR spectrum were also consistent wi th the presence of a long chain moiety. A tri plet at (50.80 (1= 5 Hz) HoLmskioLdia sanguinea Retz (F. Verbenaceae) and a broad doublet at (50. 90 (}= 4 Hz) were assigned distributed from Kumaon to Bhutan is reported to be l to terminal methyl (C-29) and methyl groups at C-27 used for the cure of different ailments . The presence respecti vely. The presence of a methyl substitution at of antiinflammatory activity against carrageenin 2 C-27 was shown by the base peak at rnlz 57 ( 100%) induced albino rats prompted us to carry out and the peak at mlz 363 (2. 1%) in its ElMS chemical screening of H. sanguinea which led to the 3 spectrum . The ion peaks at mlz 83, 97, III , 125, characterization of known active diterpenoids 139, 153,167, 181 and 195 with decreasin g intensity andmgrapholide and neoandrographolide, twelve due to the loss of successive meth ylene groups are known lipids, wogonin, oroxindin, fri edelin, friede characteristics of a long chain compound" The above linol, B-sitosterol, B-sitosterolglucoside, B-amyrin and spectral data of compound 1 led to the characteri a new lipid, 27-methylnonaeicosanol 1. The twelve zation of its stucture as 27-methylnonae icosanol. known lipids which are present as an in separabl e mi xture were identi fied by GC-MS. The known A homogenous TLC fracti on MHS-9, Rr 0.7 diterpenoids and triterpenoid s were identified by thei r (CHCI3 : MeOH, 47:3), mp. 68-70° was isolated as a spectral data and compari son to their respecti ve white solid from column chromatography and authentic samples isolated earlier in our laboratory. repeated PTLC (CHCI 1: MeOH, 94:6) on E-Merck Compound 1, isolated as a white solid from the precoated silica gel plates. Its IR spectrum showed I CHCl3-hexane fraction of column chromatography on absorption band at 720 cm- due to stra ight chain repeated PTLC on silica gel, m.p. 72°, showed the moiety and at 1704 and 3550 cm·1 for a carboxylic • presence of molecular ion peak at mlz 438 group. Its 400 MHz IH NMR spectrum showed corresponding to molecul ar formula C 30H620 and ion chemical shift values at (5 0.85 (br.t, CH ,), 1. 23- 1.50 peak at mlz 420 (3. 1%) due to the loss of water (m, CH2'S ) and a triplet at (5 2.28 (-Cl-l 2C02H). The (M+-18 ). The presence of a terminal hydroxyl above spectral data of MHS-9 were in dicati ve of a methylene protons was evident from its two proton straight chain fatty acid . Its I'C NMR study showed triplet at (5 3.60 (1=6 Hz ) in its IH NMR spectrum methyl carbon at (5 14.68 and meth ylene carbons at and a DEPT edited I3C NMR signal at (5 62.10. 29.72, 3 1.98 and 34.64 wi th carbonyl carbon at Compound 1 on acetylation gave la which showed a 179.78 whose DEPT edited L1C NMR spectra showed singlet for acetyl meth yl protons at (5 2.0 I (3 H) and a no secondary carbon (CH). However ElMS showed NOTES 633

MHS-9 as a mixture of compounds. Its methylated CHCb and n-BuOH soluble portions. The CHCI, part derivative on GC-MS analysis showed the presence (55.5 g) on column chromatography over sili ca gel of twelve known lipids reported first time from this (60-120 mesh) using solvents of increasin g polarities plant. The compounds identified by comparison of afforded friedelin (50 mg), friede linol (20 mg), B-s ito their ion peaks present in GC-MS with those of the sterol (30 mg), B-amyrin (50 mg) and B-sito compounds reported in literature were propionic sterol-3B-D-glucoside (60 mg). Wogonin (60 mg) was (0.3%), maloni c (0.3 %), succinic (0.3%), myristic purified by preparative TLC (CHC I.,: MeOH, 49: I) ( 1.0%), azelaic ( 1.0%), tricosanoic ( 1.3 %), arachic whereas oroxindin (40 mg) was separated by paper

(2.8%), heneicosanoic (1.0), beheni c (2.5%), li gno chromography ( n-BuOH: AcOH:H}O, 4: I :5 ). H2S04 ceric (10.8%), pentacosanoic (6.0%) and hexaco ( 10%) and FeCI., ( I % in MeOH) were used as spray sanoic (70%) acids . reagents. Oroxindin was hydrolysed by 10% HC I to • The known compounds andrographolide, neo give wogonin and glucuroni c acid . andrographolide, friedelin, fri edelinol, B-s itosterol, 27-Methylnonaeicosanol 1. CHCI,-hexane ( I: I) B-sitosterol-3B-D-glucoside and B-amyrin were fractions of the column chromatography were purified identified either by compari son of their reported by preparati ve TLC on silica gel (CHCI,) to give spectral and physical data or by their direct matching compound 1 (30 mg), R, 0.25 (CHCI,-hexane, 2: I, with the respecti ve compound isolated ealier in our E-Merck precoated TLC silica gel G). IR (KBr): laboratory. High resolution NMR study of wogonin 2 34 14,2849, 1470, 1410, 1062 cm' l; El MS (re I. int): and oroxindin 3 having antiinflammatory, anti viral mlz 438 (M +, 0 .9%), 420 (3. 1% ), 279 (2%), 265 4 5 and oestrogenic activit ies . were reported for the first (2 .6%), 251 (3.9%), 237 (3.3%), 223 (4.2 (/0), 209 time (experimental). Isolat ion of andrographolide and (7.9%), 195 (8.8%), 18 1 ( 11 .9 %), 167 (23 .0%), 153

' -.j neoandrographolide from f-J. sanguinea reports their (22.3%), 139 (33.3%), 125 (40.6%), III (46.5%). 97 second natural occurrence and first from thi s plant. f-J. (70%), 83 (75 %). Anal. Calc. for C 'oH(,10 : C, 0.82; sanguinea which elaborates compounds similar to H, 0.14. Found: C, 0.80; H, 0. 15%. Acetylati oll of those of Andrographis pallicul(lta6 may be used as a compound 1 (5 mg) with Py/Ac10 after usual work substitu te of A. paniculata. Occurrence of similar up and purification by prep-TL.C gave la .HMS compounds both in H. sanguinea and A. paniculata is (rel.int.): mlz 480 (0.4%, M+) . of chemotaxonomic importance as th ey belong to MHS-9. Column fractions from I o/e MeOH - closely related fami li es. C HCI , of the CC of the CHCI, extract were purified by repeated PTLC (CHC I,: MeOH, 47:3) on E-Merck Experimental Section precoated si lica gel plates to yie ld MHS-9 as

General. Mps are un corrected. 'H and "c NMR amorphous sol id (60 mg); R,0.35 (CHC I1 : MeOH. 23 spectra wel\' rxcrded on 400 and 500 MHz (B ru cker) : 2, E-Merk precoated silica gel G ); lR (KBr): 355() , NMR' in strurnents. ElMS were obtained on l eol DX 29 18, 1704, 1463,720 em"; ElMS (re l.int) mlz : 4 10 300 MS at 70eV. GLC was run on FFAP column with (4% ),396 ( 1. 2% ),382 ( 1.5 %), 340 (0.8%). N2 as carrier gas ( 14 psi ). GC-MS data were obtained Andrographolide. Column chromatograph y of th e on Shimadzu OP-2000 instrument at 70eV on CHCI, extract over sili ca gel (60-120 mesh) lI sing ULBON-HR- I column usin g fused Si capilary (0.25 CHCI, : MeOH ( 19 : I) as e lu ent furnished bioaeti ve mm x 50 m) with film thi ckn<.: ss 0.25 mm. Helium andrographolide, m.p. 2300 (lit7 230-3 1°. I g) and R, was used as carri er gas in GC-MS at 20 psi. The 0.4 (EtOAc :Hexane, 85: 15); [all) - 124° (AcOH{ o • temperature programme was as follows: 150 for 2 Neoandrographolide. Column chromatograph y of mi n., then increased at SO/min to 250° and maintained the CHCb extract usi ng CHCI, :MeOH (9: I) as elu ent at this temperature for 80 mi n. fumished 'leoandrographolide, m.p. 201 0 (lit X 20 1-

Collection, exh'action and isolation. Aerial parts 03°, 0.20 g) and R, 0.5 ( CHCl1 : MeOH, 9: I): [a]1) 0 of Holmskioldia sanguinea were coll ected from _64 .0 (pyridine)8 . district Shillong, India in Augu t' 1993. A vouchar Wogonin. CHCIJ :MeOH (9: 1) elu ents of th e specimen has been deposited in the ClMAP column chromatography of the C HCI .. extract 0 herbarium. Powdered air dried plant material (2 kg) fumished wogonin (60mg), m.p 20 1-03 ( lit.') 202-04\

vas percolated with colel MeOH. The concentrate IH NMR (DMSO-d6, 400 MHz): (5 3.8 (3H,s,OMe), tfter distill ati on was fracti onated into n-hexane, 6.20 (I H,s,H-6), 6.8 ( I H, s, H-3 ), 7.4 (3H. d. J= 8 Hz, 634 INDIAN J. CHEM., SEC. B, MAY 1999

H-3', 4',5'), 8.1 (2H, d, 1=8 Hz, H-2', 6'), 8.4 (OH), Acknowledgement . 12.3 (5-0H); I3C NMR (DMSO-d , 100 MHz): 6 The authors are thankful to the Director, CIMAP 861.40 (OMe), 99.3 (C-6), 104.5 (C· I 0), 105.1 (C-3), for constant support and to RSIC, CDRI, Lu ckn ow 126 (C-2', 6'), 127.6 (C:8), 129.0 (C-3', 5'), 131.0 for GC-MS and other spectral analysis. (C-4'), 131.8 (C-I '), 149.6 (C-9), 156.0 (C-5), 156.7 (C-7), 163.6 (C-2), 182.4 (C-4). References Oroxindin. Paper chromatography of an yellow precipitate of CHCI, extract furnished oroxindin (40 I Wealth of India, (PID, New Delhi), 1959, p. l OX . 9 2 Pal M, Singh M, Chaudhuri P K, Sharma R P & Si ngh N, mg), m.p. 210-16° ( lit. 210°); Rr 0.7 (B :A:W, 4: 1:5); Phytotherapy Research, 10, 1996,357. IH NMR (DMSO-d6, 500 MHz): 8 3.2-3.6 (br.m, 3 Pande A, Shukla Y N & Tripathi A K, Phvtochemistr\" 39, sugar protons), 3.8 (3H, s, OMe), 5.10 (IH, br.s, 1995, 709. H- I ") , 6.1 (I H, s, H-3), 7.0 (I H, s, H-6), 7.6 (3H, d, 4 Singh P, Jain S, Bhala M, Goyal R B, Yaya Prakas h D & Lohiya N K, Phytotherapy Research, 4, 1990, 86. 1=7.8 Hz, H-3', 4' & 5'), 8.1 (2H, d, 1=7.8 Hz, H-2', 5 Pinye Z & Guoquirao L, Zhong Cao),o{), 15 , 1984, 375; Chelll 6'), 12.5 (br, 5-0H & C02H). 13C_NMR (DMSO-d6, Abstr, 101 : 226842s 125 MHz): 8 61.41 (OMe), 67.20 (C-4"), 71.87 6 Kuroyanagi M, Sato M , Ueno A & Nishi A, Chl'1Il Phorlll BIIII, (C·2"), 73.11 (C3"), 76.62 (C-5"), 99.12 (C-6), 35, 1987, 429 100.31 (C-1 "), 105.21 (C-IO), 105.23 (C-3), 126.42 7 Chan M, Chan W R, Hynes L J, John son L F & Weinstei n B, Tetrahedron, 18, 1962, 397. (C-2',6'), 129.32 (C-3', 5',8), 130.76 (C-4'), 132.88 8 Chan W R, Taylor D R, Willis C R, Bodder R L & Fehlh aber (C-l'), 149.12 (C-9), 156.02 (C-5), 156.67 (C-7), H W, Tetrahedron, 27, 1971,508 1. 163.55 (C-2), 180.37 (C-4, C-6"). 9 Nair G K & Joshua C P, Curr Sci, 55, 1986, 402.