Indian Journal of Chemistry Vol. 438 , October 2004, pp. 2223-2227

Phytochemical investigation of Gomphrena globosa aerial parts

Biswanath Dinda* BipJab Ghosh, Shiho Arima" Nariko Sato" & Yoshihiro Harigaya" , .

Department of Chemi stry.Tr ipura Uni versity, Suryamaninagar, Agartala 799 130, India.

E. mail: dindabtu @rediffmail. colll

"Schoo l of Pharmaceutical Sciences, Kitasato University, Minato-ku , Tokyo 108 8641 , Japan.

Received 9 September 2003; accepted (revised) 18 March 2004

One new triterpenoid saponin (gomphrenoside, 1) and one new hopane derivative (hopan-7p-ol. 2) along with known compounds, p -sitosterol-p-D-glucoside and I-triacontano l have been isolated from the aerial parts of Gompizrena globosa . On the basis of chemical and spectroscopic(including 20 NMR) methods, the structure of 1 has been elucidated as 3p-0-a­L-arabinopyranosy l-6P-hydroxyolean-1 2-ene-23,29-dioi c acid-29-0-P-D-glucopyranoside and structure of 2 is determined.

IPC: Int.C!.7 A 61 K 35/00

Gomphrena globosa L.(Amaranthaceae), a tropical American species is grown in Indian gardens for its beautiful red tinged leaves and stem as well as for the use of its leaf extract in traditional medicine for preparation of cough syrup and to stop local haemorrhagel.2 Earlier investigations on flower and whole plant reported3

,4 the presence of betalain , betacyanin and flavone glycoside. We reinvestigated the aerial parts of the plant and isolated two new triterpenoids, gomphrenoside 1, hopan-7 fJ-ol 2 along with two known compounds, fJ-sitosterol - fJ-D­glucoside 3 and I-triacontanol 4.

Results and Discussion

A methanolic extract of the aerial parts of G. globosa was dissolved in a minimum volume of water and partitioned between benzene, chloroform, and n­butanoL The benzene soluble fraction on silica gel chromatography afforded I-triacontanol 4 and hopan-7 fJ-ol 2. The n-butanol soluble fraction on silica gel chromatography gave fJ-s itosterol- fJ-D-glllcoside 3 and gomphrenoside 1. Compounds 3 and 4 were identified by comparing their physical and spectral data (see Experimental) with literature values5 .. Both the compounds 3 and 4 are reported from this plant for the first time.

Gomphrenoside 1, amorphous powder, responded positively to the Liebermann Burchard and Molish tests, which indicated that it could be a triterpenoid saponin.The FAB-mass spectrum showed a quasi-

molecular ion peak at rnIz 819 [M + Nat as well as elemental analysis corresponds to the molecu lar formula C4IH6401 5. The IR spectrum in KBr revealed absorption bands at 3400(OH), 1720 (COOR), 1690(COOH) and 1645 (C=C) cm-I. The IH NMR spectrum of 1 showed six three-proton singlets at (5

0.97 , 1.06, 1.09, 1.14, 1.29 and l.32 ass igned to the C-24, C-25 , C-26, C-28, C-30 and C-27 methyl groups, respectively . The downfield shift of C-30 methyl protons was possibly due to presence of ester group at C-20 carbon. The upfield shifts of C-24, C-25 and C-26 methyl protons were due to 1,3-diaxial interaction with fJ-hydroxyl group at C-6 carbon7

. A one-proton double-doublet at 0 3.47 (J = 9.5 and 6.5 Hz) and a one-proton multiplet at 0 4.39 (W 1/2 = 9.5 Hz) were assigned to the 3a- and 6a-protons, respectively. The IH NMR spectrum also displayed signals for typical 18fJ-proton at 0 2.87 (dd, J = 14.0 and 4.0 Hz), one olefinic proton at 0 5.28 (t, J = 3.5 Hz), one a-arabinopyranosyl unit [0 5.3 1 (d, J = 6.0 Hz), H-l '] , one fJ-glucopyranosyl unit [b 5.3 1 (d, 1=7.0 Hz), H-ll and one carboxyl proton at 0 11.20 (brs). The I3C NMR spectrum of 1 supported the presence of fJ-amyrin skeletal structure with arabinose and glucose units (Table I) . The normal value of chemical shift of C-13 carbon (bc 144.5 ppm) simil ar to that of p-amyrin (bC 145.1 ppm)8 discarded the possibility of ester/carboxyl group at C-14 or C-17 carbons as these positions could result shielding effect on C-13 carbon due to y and 0 effects, respectively . The HMBC correlation of C-3a-proton with C-4,

2224 INDIAN J. CH EM., SEC B, OCTOBER 2004

Table I - IH and J3C NMR data of 1 (400 MHz for IH and 100 MHz for DC)

Carbon No

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29 30

Ara-I' -2' -3' -4' -5'

Glc-I" -2" -3" -4" -5" -6"

2.04 Ill , 1.26 III 1.59 Ill , 1.38 III 3.47 dd (9.5, 6.5)

1.75 d (4.5) 4.39 III (9.5)*

3.26 Ill , 3.06 III 5.28 t (3.4)

1.68 Ill, 1.30 III 1.18 Ill , 1.04 III

2.87 dd (14.0, 4.0) 1.86 dd (14.0, 3.5) 2.52 dd (7.5, 4)

1.80 Ill, 1.34 III

I 1.20 brs 0.97 s 1.06 s 1.09 s 1.32 s 1.14 s

1.29 s 4 .87 d (6.0) 3.20 t (9.0) 3.54 t (9.0) 3.071ll (6.0) 3.35 dd (10.0, 8.0) 3.91 dd(lO.O. 3.5) 5.31 d (7 .0) 3.21 l (9.0) 3.49 t (9.0) 3.34 t (9.0) 3.45 ddd(9.0, 5.0, 1.5) 3.73 dd (12.0,5.0) 3.60 dd ( 12.0, 1.5)

39.0 t 27.6 t 82.5 d 54.4 s 49.6 d 68.6 d 40.4 t 38.2 s 47 .7 d 37.6 s 24.1 t

124.0d 144.5 s 41.4 s 28.6 t 24.1 t 36.0 s 42.7 d 41.7t

43.4 s 30.7 t 33.9 t 181.9 s 14.1 q 16.6 q 17.6 q 26.4 q 18.8 q 182.3 s 23.9 q 100.6d 72.9 d 73.8 d 69.6 d 66.7 t

100.2 d 75.4 d 78.2 d 72.1 d 76.2 d 66.8 t

HMBC (H->C)

C-2, C-4, C-23, C-24, C-I'

C-4, C-6, C-IO, C-23 C-5 , C-7 , C-8

C-9, C-12 C-II, C-13. C-14. C-18

C-17, C-19, C-20, C-29, C-30 C-20, C-29, C-30

C-3. C-4. C-5. C-23 C-I, C-9. C-IO C-8, C-9, C-14 C-8, C-13, C-14, C-15, C- 16, C-17, C-18

C-19, C-20, C-21 C-3, C-2'

C-3', C-4'

C-20, C-2"

C-4", C-5"

Coupling constants in Hertz are given in parentheses * Coupling constant of W 112.

C-23, C-24 and C-l' carbons; of C-19 protons with C-20, C-29, and C-30 carbons; of C-18 ,8-proton with C-19, C-20, C-29 and C-30 carbons as well as of C-1 ' anomeric proton of arabinosyl unit with C-3 carbon and of C-l " anomeric proton of glucosyl unit with C-29 and C-20 carbons and of C-6a-proton with C-7, and C-8 carbons suggest the attachment of arabinosyl unit at C-3 hydroxyl group, carboxyl group at C-4,

glucosyl ester group at C-20 and hydroxyl group at C-6 position, respectively (Table J) . The FAB (positive) mass spectrum exhibited some significant mass peaks at mlz 664 [M-arabinosyl +Hr, 634 [M-gluco­pyranosyl + Hr, 503 [aglycone + Hr, 488 [503-CH3r, 467 [aglycone-2H20 + Hr, 453 [467-CH3+ Hr, 411 [a + Hr, 368 [br, 248, 236, 218, 203 and 173 [c+Hr, which could be rationalized by structure

DINDA el at.: PHYTOCHEMICAL INVESTIGATION OF GOMPHRENA GLOBOSA AERIAL PARTS 2225

1. The mass peaks at mJz 410 and 386 generated due to retro-Diels-Alder fragmentation indicated ~­

amyrin skeletal structure of the compound with a f'..1 2 double bond 9. 10. The facile loss of H 20 from mlz 386 to mJz 368 supported the existence o f the hydroxyl group at C-6 as it involved the formation of stable 1,3-diene. Acid hydrolysis of the compound I in 2M HCI in aqueous methanol yielded an aglycone, 3~ ,6~­

dihydroxyolean-12-ene-23,29-dioic acid , C30H460 6 (M+502) (named gomphrenic acid) la, D( + )-glucose and L(+)-arabinose. The aglycone la was identified by IH , 13C NMR and HR-EIMS studies as well as elemental anal ys is . Sugars were identified by TLC and PC comparison with authentic sugar samples. On the basis of these evidence, the structure of gom­phrenoside has been elucidated as 3,B-0-a-L-arabino­pyranosyl-6,B-hydroxyolean-12-ene-23 , 29-dioic acid-29-0-,B-D-glucopyranoside 1. It is a new saponin and its aglycone la is a new triterpenoid.

Hopan-7~-01 2, C30H520 (M+ 428) was obtained as colourless crystals. Its lR spectrum showed an absorption band at 3430 cm·1 for hydroxyl group. The IH NMR spectrum of 2 showed six tertiary methyl singlets at 6 0.78, 0.84, 0.88, 0 .94, 0.95 and 0.96 assigned to C-26, C-24, C-25 , C-28, C-23 and C-27 methyl groups, respectively ; two secondary methyl doublets at 0 0.87 and 0.91 (each J = 6.0 Hz), ascribed to C-29 and C-30 methyl groups, respectively and a one-proton double-doublet appeared in quartet shape at

HO

H

H

6 3.72 (J = 8.5 and 4 .0 Hz) accounted for 7a-proton. The presence of ,B-hydroxyl group at C-7 caused the upfield shift of C-26 and C-25 methyl protons compared to those of hopan-3,B-ol, where the values are 0.83 and 0.97 ppm, respectivel/ I The 13C NMR spectral data with DEPT experiment (see Experimental) also supported this assignment of hydroxyl group by revealing upfield chemical shift value of C-26 and C-25 carbons as well as downfield shift of C-6 carbon. The HMBC correlation of C-7a­proton with C-8, C-9, C-26 carbons and of C-9 proton with C-8 and C-26 carbons also accounted this assignment. The chemical shift values of the carbons in A to D rings are very similar to those reported 12 for hopan-7~, 22-diol, isolated from Pseudocyphellaria mongeotiana (Stictaceae) 13. The EI-MS of the compound exhibited a molecular ion peak at mlz 428 cOITesponding the molecular formula, C30H520 and other mass peaks at mJz 413 [M-CH3t , 410 [M­H20t,395 [M-H20-CH3t , 367 [395-CH2=CH2t , 207, 205, 191, 189, 175 and 43 (base peak), which could be rationalized by hopane-like skeletal structure 2 for the compound. The compound on acetylation with AC20 in pyridine yielded one monoacetate 2a, C32H540 2 (M+ 470). Therefore, in the light of all the above evidence, the structure of the compound was assigned as hopan-7,B-ol or 7,B-hydroxyhopane 2. To the best of our knowledge it is a new hopane derivative. Study on the biological activities of I and 2 is in process.

30

21 ( "~I .,,. H 22

29

23 2

H

2226 INDIAN J. CHEM., SEC B, OCTOBER 2004

m/z 410

Experimental Section

General. Silica gel 60- I 20 mesh (Merck) and silica gel G (Merck) were used for column chromatography (CC) and thin layer chromatography (TLC), respectively . In case of saponin the spot on TLC pl ate was visuali zed by spraying with 10% ethanolic H2S04

and heating the plate at 110°C. UV, JR , MS and NMR spectra were recorded on Spectronic 21 0, Perkin­Elmer 577, Jeol JMS-AX505 HA and Varian Unity 400 spectrometers, respectively. GC was carried out on Pye Unicam 104 gas chromatograph. Petroleum ether lI sed had the bp 60 ·80°C.

Plant material. The aerial parts of Gomphrena globosa were collected from the garden of College Tilla area of Agartala in September, 2002 and identified by Dr N K Chakraborty, retired Professor of Botany , M.B.B . College, Agartala. A voucher specimen has been preserved in the herbarium of Shibpur Botanical Garden , Howrah, West Bengal , India.

Extraction and Isolation. Air-dried powdered aerial parts of the flowering plant (3 kg) were extracted with MeOH in a percolator. The MeOH extract was concentrated and partitioned between H20 and C6H(j, H20 and CHCb, H20 and n-BuOH, successively. The C6H6, CHCI3, n-BuOH layers were washed with H20 and concentrated under reduced pressure. The residue obtained from C6H6 so! uble fraction was subjected to Cc. The petrol-C6H6 (1: 1) eluate gave 4 in colourless granu les (0.06 g; yield 2.0 x 10.3 %). The C6H6-CHCI3 (85: 15) eluate furni shed 2 in colourless needles (0.05 g; yield 1.67 x 10-3 %). The residue from n-BuOH soluble fraction was subjected to CC with solvents of increasing polarity. The CHCI3-MeOH (95:5) eluate furnished 3 in colourless needles (0.03 g; yield 1.0 xlO-3 %). The CHCI3-MeOH (70:30) eluate gave 1 (0.09 g; yield 3xlO-3%).

Compound 1: An amorphous white powder, mp 185°C; Anal. Found: C, 61.45; H,7_76. Calcd for C4 ,H640,s: C, 61.79; H, 8.09%; FAB-MS (positive) rnlz (rel.in t.) : 819 (3), 664 (16), 634 (12), 503 (12),

b m/z 368

c m/z 173

488 (11) , 467 (14),453 (19),4 11 (28),386 ( 13), 467 (14),453 (19), 411 (28), 386 (13),368 (12),248 (47), 236 (30) , 2 18 (44), 203 (87), 191 (86), 173 (100).

Compound 2: Colourless need les, mp 220"C, ra] o + 20.8° (c = 0.22, CHCI3); Anal. Found: C, 83.85; H, 11 .78. Cal cd for C]oHS20: C, 84.04; H, 12.22%. 'H NMR (CDCl3): 80.78 (3 H, s, Hr 26), 0.84 (3 H, s, Hr 24), 0.87 (3 H, d, J = 6.0 Hz, H3-29), 0.88 (3 H, s, Hr 25), 0.9 1 (3H, d, J = 6.0 Hz, Hr 30), 0.94 (3 H, s, H]-28), 0.95 (3 H, s, H3-23), 0.96 (3 H. s, Hr 27), 1.43 (lH, dd, J = 11.5 and 6.5 Hz, H-5 ), 1.72 (1H, dd, J = 11.0 and 6.5 Hz, H-9), 1.90 (1 H, dd, j = 10.5 and 6.0 Hz, H-I 7),3.72 (1H, dd , J = 8.5 and 6.0 Hz, H-7 ); I3C NMR (CDCb): 84 1.9 (t, C-I) , 17 .9 (t, C-2), 4 1.9 (t , C-3), 33.7 (s, C-4), 51.1 (d, C-5), 29.5 (t, C-6) , 73.0 (d, C-7), 41.4 (s, C-8), 49.4 (d,C-9), 37.6 (s, C-IO), 20.2 (t, C-Il), 24.0 (t, C-12), 48.2 (d., C-13) , 39.2 (s, C-14), 35.4 (t, C-15), 23.2 (t, C-16), 54.4 (d, C-17), 41.3 (s, C-18), 43 .2 (t, C-19), 27.9 (t, C-20), 48.2 (d, C-21), 38.9 (d, C-22), 33.7 (q, C-23), 2 1.4 (q, C-24), 15.9 (q, C-25), 11.2 (q, C-26), 16.7 (q, C-27), 16.6 (q, C-28), 28.6 (q, C-29), 3 1.0 (q, C-30); EI - MS mlz (rel.int): 428 (48), 413 (39), 410 (I I), 395 (10), 367 (5), 207 (30), 205 (60), 191 (45), 189 (30), 175 (42), 43 (100).

Compound 3: Colourless needles, mp 279° (lit5 mp 280-82°C); FAB-MS (positive) rnl2. (rel.int.): 599 [M(C3SH600 6) + Nat (l00), 415 [aglycone + Ht (19), 397 [M-glucose + Ht, (46), 329 (46). It was identical (m mp and co-TLC) with an authentic sample of fJ­si tosterol-fJ-D-gl ucoside.

Compound 4: Colourless needles, mp 89° (lit6 mp 88°C); 'H NMR (CDCI]): 80.91 (3H, t, J = 7.5 Hz), 1.28 (54 H, brs, 27xCH2), 1.60 (2H, quintet, CH2),

3.66 (2H, t, J = 7.0 Hz); FAB-MS (positive) mlz (reI. int.) : 461 [M(C30H620 ) + Nat (100). It was identical (co-GC) with an authentic sample of I-triacontanol.

Acid hydrolysis of saponin 1. Saponin 1 (50 mg) was dissolved in 2M HCI in aqueous MeOH (15 mL) and refluxed for 5 hr on a water-bath. The solution was concentrated and diluted with H20 and extracted with CHCh. The CHCb layer was dried over

DINDA et al.: PHYTOCHEMICAL INVESTIGATION OF GOMPHRENA GLOBOSA AERIAL PARTS 2227

anhydrous Na2S04 and evaporated to dryness. The residue on column puri fication gave colourless needles of la, yield 16 mg, mp 285°C; Anal.. Found: C, 71.48 ; H, 8.93. Calcd for C30H460 6: C, 71.68; H, 9.22%; IR (KBr): 3430 (OH) , 1690 (COOH), 1650 cm·1 (C=C); IH NMR(400 MHz, CD30D): J 0.96, 1.03, 1.08,1.21 , 1.30, 1.34 (each 3H, s), 2.68 (1H, dd , J = 13 .0 and 5.0 Hz, H-18), 3.32 (1H, dd, J = 9.5 and 6.5 Hz, H-3), 4.42 (1H, m, WI/2 = 10.0 Hz, H-6), 5.30 (IH, t, J = 3.5 Hz, H- 12); 11.20-11.38 (brs, HOOC-23,29); 13C NMR (100 MHz, CD30D): b 39.0 (t,C-I), 27.1 (t,C-2), 74.0 (d, C-3), 53 .2 (s, C-4), 48.2 (d, C-5), 68.9 (d, C-6), 40.5 (t, C-7) , 38.2 (s, C-8) , 47.2 (d, C-9) , 37.6 (s , C-IO), 23 .7 (t, C-II), 125.1 (d, C-12), 143.9 (s, C-13), 41.3 (s, C-14), 28.8 (t, C-15), 24.4 (t, C-16), 34.0 (s , C-17), 42.8 (d, C-18), 38.1 (t, C-19), 43.1 (s, C-20), 27.5 (t, C-21), 35 .0 (t, C-22), 181.6 (s, C-23), 14.0 (g,C-24), 17 .5 (g,C-25), 18.4 (g, C-26) , 25.2 (g, C-27), 21.5 (g, C-28), 181.8 (s, C-29), 23.7 (g, C-30); HR-EIMS: mlz 502 [Mt(1), 487 (3), 484 (2),469 [M-H20-CH3t (4), 451 [469-H20t (4),248 (l00), 236 (15), 218 (16), 203 (98), 191 (19), 189 (26), 173 (23).

The agueous layer was neutralized with Ag2C03 and filtered. The filtrate was concentrated under reduced pressure and examined for carbohydrates by TLC and PC with authentic sugars. Two spots were detected corresponding to L (+ )-arabinose and D( +)­glucose. TLC was performed on silica gel G using solvent system EtOAc-HOAc-H20-MeOH (6: 1:2: I), spray reagent ethanolic a-naphthol-H2S04 and heating the plate at 110° e. PC was done on Whatmann 1 MM paper using solvent system n-BuOH-Me2CO-H20 (4:5: 1) and methanolic ammoniacal AgN03 as spray reagent and heating the paper for 2 min at 11 O°e.

Acetylation of compound 2. Compound 2 (15 mg) was dissolved in 4 drops of pyridine and 2 rnL of acetic anhydride was added to it. The resultant

mixture was kept for 48 hr at room temperature. Usual work-up of the reaction mixture afforded 2a in colourless needles ( 10 mg), mp 238° C; Anal. . Found: C, 81.31; H, 11.22. Calcd for C32 H540 2: C, 8 1.64; H, 11.56%; IR (KBr) : 1735 cm·1 (COOR ); IH NMR (400 MHz, CDCl3): J 0.78, 0.84, 0.92, 0.94, 0.96, 0.99 (each 3H, s), 0.88 and 0.93 (each 3H, d, J = 6.0 Hz), 4.22 (lH, m, W I/2 = 9.5 Hz, H-3).

Acknowledgement

The authors thank Dr N K Chakraborty , retired Professor of Botany , M B B College, Agartala for identification of the plant species and to RSIC, Lucknow for microanalysis. The authors are grateful to DST, New Delhi for financial assistance and for providing JRF to one of the authors (BG).

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