Journal of Nataral Products Vol. 55, No. 2 , p p . 163-167, February 1992
163
CHEMICAL INVESTIGATION AND ANTI-INFLAMMATORY ACTIVITY OF VZTEX NEGUNDO SEEDS A.S.CHAWLA,+A.K.SHARMA,S.S. HANDA, Department of Pharmarartical Sciences, Panjab University, Chandigarh I60014 , India and K.L.DHAR
Regional Resecrrch Liabwatwy,Jammu Tawi 180001, India bSTRACT.--The CHCI, extract of the defatted seeds of V i t a negundo exhibited anti-inflammatory activity and yielded four triterpenoids: 3&acetoxyolean- 12-en-27-oic acid 111, 2 a , 3a-dihydroxyoleana-5,12dien-28-oic acid {2], 2~,3a-diacetoxyoleana-S, 12-dien-28-oic 12-dien-28-oicacid f51.This is the first reacid 131, and 2c1,3f3-diacetoxy-18-hydroxyoleana-5, port of the isolation of compounds 2, 3, and 5 from a natural source.
In the Indian system of medicine, Vitex negundo L. (Verbenaceae) has been used for the treatment of rheumatoid arthritis (1). Continuing our investigation of the seeds of V. negundo, the CHCI, extract has yielded four triterpenoids. Their characterization and biological activity are reported in this paper.
RESULTS AND DISCUSSION The CHCI, extract of defatted seeds of V. negundo exhibited 34.8% anti-edema activity at 500 mglkg p.0. in the carrageenan-induced paw edema test in albino rats. The extract was fractionated by chromatographic methods followed by crystallization, yielding compounds 1-3 and 5. Spectral characteristics of compound 1 and of its hydrolysis product identified the compound as 3P-acetoxyolean-12-en-27-oic acid ( 2 4 ) . Compound 2 did not show absorption above 224 nm in the uv spectrum. The ir spectrum showed bands at 3200 cm-' (-OH), 1700 cm-' (C=O, -COOH), and 1390 and 1375 cm-' (gem-dimethyl). The 'H-nmr spectrum gave a multiplet at 6 5.27 for two vinylic protons. The multiplet at 6 3.87 and a doublet at 6 3.34 (d,J= 3.0 Hz), one proton each, were assigned to H-2P and H-3P, respectively. The spectrum also gave singlets at 6 1.14, 1.09, 0.98, 0.96, 0.92, 0.84, and 0.81, corresponding to seven methyl groups, which suggested the oleanane skeleton. The eims gave a molecular ion peak at m/z 470 (1%) and fragments at m/z 452 [M - H,0}+ (l%), 434 [M2 X H201+ (2%), 425 [M - COOHI+ (2%), 248 (loo%), 203 (65%),and 189 (20%). Retro-Diels-Alder fragmentation was observed in the mass spectrum of 2 , with ions at mlz 248 and 203, indicative of a triterpenoid having A'*-unsaturation. The higher intensity of peak mlz 203 suggested an oleanane skeleton with a 28-carboxyl group (5,6).
Me Me
1
Journal of Natural Products
164
Ma
2
Wol. 5 5 , No. 2
Ma
R'=R2=a-OH, R 3 = H
3 R'=P-OAc, R2=a-OAc, R3=H 4 R'=p-OH, R2=a-OH, R3=H 5 R'=a-OAc, R2=P-OAc, R3=OH
Compound 2 formed an acetonide (7), and its eims gave peaks at m/z 5 10 [MI+ (l%),464 [M - H - COOHI+ (1%), 452 [M - MeCOMe)+ (1%), 406 (5%), 248 (loo%),and 203 (48%).Formation of an acetonide supported the cis configuration or the diequatorial conformation of the hydroxyl groups (8). Acetylation of compound 2 yielded the product as a gum. The Occurrence of two sharp three-proton singlets at 6 2.11 and 1.96 in the 'H-nmr spectrum revealed the presence of two acetyl groups. Other signals were observed at 6 5.28 (2H, vinylic protons), 4.98 (H-2), and 4.70 (H-3). The 'H-nmr spectrum also showed seven C-Me singlets at 6 1.25, 1.18, 1.12, 1.04,0.97, 0.87, and0.76. Thesimilarityinchemical shifts of the seven methyl groups in the 'H-nmr spectrum of 2 and its diacetate suggested that the two oxygen hnctionalities in the molecule were both a.If the hydroxyl groups were p, the chemical shifts of the C-24 and C-25 methyls of 2 would appear downfield in comparison to those of the diacetate (5,8). Methylation of 2 with CH,N, yielded a crystalline methyl ester, mp 285-286'. Thus compound 2 was shown to be 2a,3a-dihydroxyoleana-5,12-dien-28-oicacid. This is the first report of the isolation of compound 2 from a natural source. The uv spectrum of compound 3 did not show absorption above 225 nm. The 'Hnmr spectrum of 3 gave signals at 6 5.28 (m, 2H, vinylic protons), 4.72 ( l H , H-2a), and 4.64 ( l H , H-3p). The sharp three-proton singlets at 6 2.05 and 1.99 revealed the presence of two acetyl groups. The spectrum displayed C-Me singlets at 6 1.26 (3H), 1.07 (6H), 0.90 (9H), and 0.76 (3H), suggesting an oleanane skeleton. The eims o f 3 gave the molecular ion peak at m/z 554 (1%) and other major peaks at m/z 434 [M - 2 X HOAC)+ (4%), 419 (20%), 248 (loo%), 203 (66%), 189 (23%), and 133 (44%). These data suggested that a C-12 double bond and a C-28 carboxyl group were present in the molecule. Signals at 6 143.8 (C-13) and 122.2 (C-12) in the 13C-nmrspectrum supported the presence of A1*double bond. The spectrum also indicated the presence of two additional olefinic carbons at 6 138.2 (C-5) and 125.5 (C-6). Singlets at 6 184.0, 17 1.O, and 170.8 confirmed the presence of a carboxyl group and two acetoxyl groups. Hydrolysis of compound 3 with ethanolic KOH afforded a crystalline material, mp 245-246' (dec) [4].Compound 4 did not form an acetonide, indicating that the hydroxyl groups at C-2 and C-3 were diaxial. Methylation of 4 with CH,N, yielded a crystalline compound, mp 205-206', and the eims showed the molecular ion peak at m/z 484 (1%). The spectral data of compound 3 and its derivatives indicate that compound 3 is 2p, 3a-diacetoxyoleana-5,12-dien-28-oic acid. To our knowledge compound 3has not been reported previously. Compound 5 showed absorption at 225 nm in the uv spectrum. The 'H-nmr spec-
February 19921
165
Chawla et al. : Anti-inflammatory Activity
trumexhibitedsignalsat65.41(m, 2H,vinylicprotons), 5.13(dd, 1H,]= 10.0,4.0 H t , H-2P), 4.76 (d, l H , J = 10.0 Hz, H-3a), 2.06 (3H, s, OAc), and 1.98 (3H, s, OAc). These values are in close agreement with known 2a,3P-diacetoxy triterpenoids (9). A one-proton singlet at 6 2.55 may be due to a hydroxyl group, the presence of which was indicated in the ir spectrum. The hydroxyl group appears to be tertiary as it could not be acetylated. The spectrum showed seven C-Me singlets at 6 1.24, 1.2 1, 1.06, 0.99, 0.96, 0.93, and 0.73 consistent with an oleanane skeleton. The eims showed the molecular ion at m/z 570 (1%). Other peaks observed at d z 305 (1%) and 264 (2%) were attributed to fragments a and b. These data revealed the presence of a C-
Me 'me
a
b
mlz 305
mlz 264
12 double bond, a C-28 carboxyl group, and a C- 18 hydroxyl group in the molecule. Thus, compound 5 has been identified as 2a,3P-diacetoxy-18-hydroxyoleana-5,12dien-28-oic acid. This is the first reported isolation of 5 from natural sources. The CHC1, extract and triterpenes 2 and 4 (hydrolyzed products of 3) obtained from the seeds of V. negundo exhibited anti-inflammatory activity as shown in Table 1. TABLE 1.
Percent Inhibition of Edema (after 3.5 h) Exhibited bv V i t a negundo CHCI, Extract and Tritemenes 2 and 4.
Dose m g k p.0.
Mean increase in paw volume ml 2 SD
% inhibition
Group
of edema
Significance
Control . . . . . . . . . . . . . . Standard (ibuprofen) . . . . . . . . CHCI, extract . . . . . . . . . . Compound 2 . . . . . . . . . . . Compound4 . . . . . . . . . . .
50 500 50 50
0.6620.091 0.24 2 0.045 0.3120.043 0.54 2 0.087 0.43 2 0.071
63.2 34.8 18.7 34.3
P
163
CHEMICAL INVESTIGATION AND ANTI-INFLAMMATORY ACTIVITY OF VZTEX NEGUNDO SEEDS A.S.CHAWLA,+A.K.SHARMA,S.S. HANDA, Department of Pharmarartical Sciences, Panjab University, Chandigarh I60014 , India and K.L.DHAR
Regional Resecrrch Liabwatwy,Jammu Tawi 180001, India bSTRACT.--The CHCI, extract of the defatted seeds of V i t a negundo exhibited anti-inflammatory activity and yielded four triterpenoids: 3&acetoxyolean- 12-en-27-oic acid 111, 2 a , 3a-dihydroxyoleana-5,12dien-28-oic acid {2], 2~,3a-diacetoxyoleana-S, 12-dien-28-oic 12-dien-28-oicacid f51.This is the first reacid 131, and 2c1,3f3-diacetoxy-18-hydroxyoleana-5, port of the isolation of compounds 2, 3, and 5 from a natural source.
In the Indian system of medicine, Vitex negundo L. (Verbenaceae) has been used for the treatment of rheumatoid arthritis (1). Continuing our investigation of the seeds of V. negundo, the CHCI, extract has yielded four triterpenoids. Their characterization and biological activity are reported in this paper.
RESULTS AND DISCUSSION The CHCI, extract of defatted seeds of V. negundo exhibited 34.8% anti-edema activity at 500 mglkg p.0. in the carrageenan-induced paw edema test in albino rats. The extract was fractionated by chromatographic methods followed by crystallization, yielding compounds 1-3 and 5. Spectral characteristics of compound 1 and of its hydrolysis product identified the compound as 3P-acetoxyolean-12-en-27-oic acid ( 2 4 ) . Compound 2 did not show absorption above 224 nm in the uv spectrum. The ir spectrum showed bands at 3200 cm-' (-OH), 1700 cm-' (C=O, -COOH), and 1390 and 1375 cm-' (gem-dimethyl). The 'H-nmr spectrum gave a multiplet at 6 5.27 for two vinylic protons. The multiplet at 6 3.87 and a doublet at 6 3.34 (d,J= 3.0 Hz), one proton each, were assigned to H-2P and H-3P, respectively. The spectrum also gave singlets at 6 1.14, 1.09, 0.98, 0.96, 0.92, 0.84, and 0.81, corresponding to seven methyl groups, which suggested the oleanane skeleton. The eims gave a molecular ion peak at m/z 470 (1%) and fragments at m/z 452 [M - H,0}+ (l%), 434 [M2 X H201+ (2%), 425 [M - COOHI+ (2%), 248 (loo%), 203 (65%),and 189 (20%). Retro-Diels-Alder fragmentation was observed in the mass spectrum of 2 , with ions at mlz 248 and 203, indicative of a triterpenoid having A'*-unsaturation. The higher intensity of peak mlz 203 suggested an oleanane skeleton with a 28-carboxyl group (5,6).
Me Me
1
Journal of Natural Products
164
Ma
2
Wol. 5 5 , No. 2
Ma
R'=R2=a-OH, R 3 = H
3 R'=P-OAc, R2=a-OAc, R3=H 4 R'=p-OH, R2=a-OH, R3=H 5 R'=a-OAc, R2=P-OAc, R3=OH
Compound 2 formed an acetonide (7), and its eims gave peaks at m/z 5 10 [MI+ (l%),464 [M - H - COOHI+ (1%), 452 [M - MeCOMe)+ (1%), 406 (5%), 248 (loo%),and 203 (48%).Formation of an acetonide supported the cis configuration or the diequatorial conformation of the hydroxyl groups (8). Acetylation of compound 2 yielded the product as a gum. The Occurrence of two sharp three-proton singlets at 6 2.11 and 1.96 in the 'H-nmr spectrum revealed the presence of two acetyl groups. Other signals were observed at 6 5.28 (2H, vinylic protons), 4.98 (H-2), and 4.70 (H-3). The 'H-nmr spectrum also showed seven C-Me singlets at 6 1.25, 1.18, 1.12, 1.04,0.97, 0.87, and0.76. Thesimilarityinchemical shifts of the seven methyl groups in the 'H-nmr spectrum of 2 and its diacetate suggested that the two oxygen hnctionalities in the molecule were both a.If the hydroxyl groups were p, the chemical shifts of the C-24 and C-25 methyls of 2 would appear downfield in comparison to those of the diacetate (5,8). Methylation of 2 with CH,N, yielded a crystalline methyl ester, mp 285-286'. Thus compound 2 was shown to be 2a,3a-dihydroxyoleana-5,12-dien-28-oicacid. This is the first report of the isolation of compound 2 from a natural source. The uv spectrum of compound 3 did not show absorption above 225 nm. The 'Hnmr spectrum of 3 gave signals at 6 5.28 (m, 2H, vinylic protons), 4.72 ( l H , H-2a), and 4.64 ( l H , H-3p). The sharp three-proton singlets at 6 2.05 and 1.99 revealed the presence of two acetyl groups. The spectrum displayed C-Me singlets at 6 1.26 (3H), 1.07 (6H), 0.90 (9H), and 0.76 (3H), suggesting an oleanane skeleton. The eims o f 3 gave the molecular ion peak at m/z 554 (1%) and other major peaks at m/z 434 [M - 2 X HOAC)+ (4%), 419 (20%), 248 (loo%), 203 (66%), 189 (23%), and 133 (44%). These data suggested that a C-12 double bond and a C-28 carboxyl group were present in the molecule. Signals at 6 143.8 (C-13) and 122.2 (C-12) in the 13C-nmrspectrum supported the presence of A1*double bond. The spectrum also indicated the presence of two additional olefinic carbons at 6 138.2 (C-5) and 125.5 (C-6). Singlets at 6 184.0, 17 1.O, and 170.8 confirmed the presence of a carboxyl group and two acetoxyl groups. Hydrolysis of compound 3 with ethanolic KOH afforded a crystalline material, mp 245-246' (dec) [4].Compound 4 did not form an acetonide, indicating that the hydroxyl groups at C-2 and C-3 were diaxial. Methylation of 4 with CH,N, yielded a crystalline compound, mp 205-206', and the eims showed the molecular ion peak at m/z 484 (1%). The spectral data of compound 3 and its derivatives indicate that compound 3 is 2p, 3a-diacetoxyoleana-5,12-dien-28-oic acid. To our knowledge compound 3has not been reported previously. Compound 5 showed absorption at 225 nm in the uv spectrum. The 'H-nmr spec-
February 19921
165
Chawla et al. : Anti-inflammatory Activity
trumexhibitedsignalsat65.41(m, 2H,vinylicprotons), 5.13(dd, 1H,]= 10.0,4.0 H t , H-2P), 4.76 (d, l H , J = 10.0 Hz, H-3a), 2.06 (3H, s, OAc), and 1.98 (3H, s, OAc). These values are in close agreement with known 2a,3P-diacetoxy triterpenoids (9). A one-proton singlet at 6 2.55 may be due to a hydroxyl group, the presence of which was indicated in the ir spectrum. The hydroxyl group appears to be tertiary as it could not be acetylated. The spectrum showed seven C-Me singlets at 6 1.24, 1.2 1, 1.06, 0.99, 0.96, 0.93, and 0.73 consistent with an oleanane skeleton. The eims showed the molecular ion at m/z 570 (1%). Other peaks observed at d z 305 (1%) and 264 (2%) were attributed to fragments a and b. These data revealed the presence of a C-
Me 'me
a
b
mlz 305
mlz 264
12 double bond, a C-28 carboxyl group, and a C- 18 hydroxyl group in the molecule. Thus, compound 5 has been identified as 2a,3P-diacetoxy-18-hydroxyoleana-5,12dien-28-oic acid. This is the first reported isolation of 5 from natural sources. The CHC1, extract and triterpenes 2 and 4 (hydrolyzed products of 3) obtained from the seeds of V. negundo exhibited anti-inflammatory activity as shown in Table 1. TABLE 1.
Percent Inhibition of Edema (after 3.5 h) Exhibited bv V i t a negundo CHCI, Extract and Tritemenes 2 and 4.
Dose m g k p.0.
Mean increase in paw volume ml 2 SD
% inhibition
Group
of edema
Significance
Control . . . . . . . . . . . . . . Standard (ibuprofen) . . . . . . . . CHCI, extract . . . . . . . . . . Compound 2 . . . . . . . . . . . Compound4 . . . . . . . . . . .
50 500 50 50
0.6620.091 0.24 2 0.045 0.3120.043 0.54 2 0.087 0.43 2 0.071
63.2 34.8 18.7 34.3
P
