INTRODUCTION:

Carissa carandas Linn. (Syn. Carissa congesta Wight) is a large dichotomously branched evergreen shrub with short stem and strong thorns in pairs, belonging to family Apocynaceae. The plant is native and common throughout much of India, Sri Lanka, Java, Malaysia, Myanmar and Pakistan. In traditional system of medicine the plant is used as an anthelmintic, astringent, appetizer, antipyretic, in stomach disorder, rheumatism, disease of the brain, in biliousness and biliary dysfunction¹. Earlier studies have shown that the extract of the plant possesses cardiotonic², antipyretic³, anticonvulsant⁴, hepatoprotective⁵ and antiviral activity⁶. Various cardiac glycosides like odoroside H, odoroside G, evomoside, rhamnoside, rhamno glycoside², ursolic acid, lupa-12, 20(29)-dien-3β, 28-diol, urs-12-ene-3β,22β-diol⁷, carissone and β-sitosterol⁸ were reported from the root extract of the plant. In the present study, the roots of the plant have been chemically characterized.

MATERIALS AND METHODS:

All the melting points were determined in a Toshniwal melting point apparatus and were uncorrected. The IR spectra of the compounds were recorded using KBr pellet method on Rx-1 Perkin-Elmer FTIR. ¹HNMR and ¹³CNMR spectra were run on Bruker Avance II 400 spectrophotometer using CDCl₃ as a solvent. Mass spectra (FAB-MS) were obtained on a JEOL SX 102/DA-6000 mass spectrometer.

The roots of C. carandas were collected from Udupi, Karnataka, during April 2007. It was authenticated by Dr. Gopalakrishna Bhat, Department of Botany, Poorna Prajna College, Udupi, Karnataka, India. The dried root bark (1 Kg) was powdered and soaked in 95 % ethyl alcohol and extracted in cold for 4 days with occasional shaking. After 4 days the ethanol layer was decanted off. The process was repeated for 4 times. The solvent from the total extract was filtered by using Whatman No. 1 filter paper, the concentrate was evaporated to dryness under reduced pressure and low temperature (40^o C) on a rotary evaporator to give the ethanolic extract (16% w/w yield), The ethanol extract (125 g) was suspended in distilled water and then extracted with petroleum ether (60-80^oC, 8x500 ml) and chloroform (8x500 ml) in succession. All fractions were then washed with distilled water (30 ml), dried over anhydrous sodium sulphate and freed of solvent by distillation to give petroleum ether (60-80^oC) soluble fraction (40 g) and chloroform soluble fraction (55 g). Petroleum ether fraction (25 g) was saponified by refluxing for 6 h in 500 ml 5% methanolic KOH and then allowed to stand at room temperature for 20 h. The unsaponifiable portion was then extracted with diethyl ether. All the ethereal fractions were combined and washed with distilled water. The solvent was evaporated and dried over anhydrous sodium sulphate to afford a yellowish residue (10 g).

The residue (8 g) was dissolved in chloroform (10 ml) and adsorbed on to silica gel (60-120 mesh, 20 g). After evaporation of the solvent, it was subjected to column chromatography over silica gel (150 g) prepared in petroleum ether (60-80^oC). The elution carried out with petroleum ether (60-80^oC)-chloroform graded mixture (80:20) and chloroform-methanol graded mixture (85:15) afforded three compounds, compound I (610 mg), II (390 mg) and III (52 mg). The individual compounds were purified by preparative TLC on silica gel G and further purified by recrystallization with chloroform.

The chloroform soluble fraction (8 g) was dissolved in chloroform (10 ml) and adsorbed on to silica gel (60-120 mesh, 20 g). After evaporation of the solvent, it was subjected to column chromatography over silica gel (150 g) prepared in chloroform. The elution carried out with chloroform-methanol graded mixture (95:5) afforded two compounds, compound IV (100 mg) and V (95 mg). The individual compounds were purified by preparative TLC on silica gel G and further purified by recrystallization with methanol.

RESULTS:

Compound I (lupeol):

Pearl white crystals; R_f 0.55 in petroleum ether-chloroform (80:20); m.p. 213-215^oC; IR v_max (KBr): 3314, 2943, 2870, 1642, 1458, 1378, 1035, 880 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.75, 0.78, 0.82, 0.91, 0.94, 1.02 (m, 18H, Me-28, Me-23, Me-24, Me-25, Me-26, Me-27), 1.67 (3H, d, J= 0.6 Hz, Me-30), 3.23 (1H, dd, J= 9.4, 6.1 Hz, H-α3), 4.56 ( s, 1H, H-3), 4.68 (s, 1H), 4.69 (s, 1H); ¹³CNMR (CDCl_3, 400 MHz): δ 38.80 (C-1), 27.02 (C-2), 78.59 (C-3), 38.97 (C-4), 54.89 (C-5), 18.34 (C-6), 34.30 (C-7), 39.99 (C-8), 51.09 (C-9), 37.59 (C-10), 20.32 (C-11), 25.74 (C-12), 37.39 (C-13), 42.53 (C-14), 27.18 (C-15), 36.40 (C-16), 42.93 (C-17), 48.21 (C-18), 47.99 (C-19), 150.05 (C-20), 28.56 (C-21), 40.09 (C-22), 28.89 (C-23), 15.58 (C-24), 16.37 (C-25), 15.93 (C-26), 14.54 (C-27), 17.96 (C-28), 109.65 (C-29), 19.13 (C-30); FAB-MS m/z (rel.int): 426 [M⁺] (C₃₀H₅₀O) (53), 409 (100), 395 (10), 318 (7), 218 (80), 203 (74), 187 (40), 175 (32), 161 (41), 147 (60), 125 (77), 121 (77), 93 (80).

Lupeol

Compound II (β-sitosterol):

White crystals; R_f 0.36 in petroleum ether-chloroform (80:20); m.p. 138-140^oC; IR v_max (KBr): 3454, 2940, 1643, 1461, 1374, 1045 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.68 (s, 3H, Me-18), 0.82 (3H, d, J= 7.1 Hz, Me-27), 0.80 (3H, d, J= 7.1 Hz, Me-26), 0.85 (3H, m, Me-29), 0.91 (3H, d, J= 6.1 Hz, Me-21), 1.00 (s, 3H, Me-19), 3.51 (m, 1H, H-3a), 5.35 (1H, d, br, J=5.4 Hz, H-6); ¹³CNMR (CDCl_3, 400 MHz): δ 32.98 (C-1), 31.73 (C-2), 72.90 (C-3), 40.05 (C-4), 150.96 (C-5), 124.46 (C-6), 33.40 (C-7), 34.33 (C-8), 50.21 (C-9), 35.63 (C-10), 22.50 (C-11), 40.66 (C-12), 43.04 (C-13), 29.89 (C-14), 24.22 (C-15), 32.98 (C-16), 55.23 (C-17), 12.89 (C-18), 19.90 (C-19), 36.23 (C-20), 19.37 (C-21), 36.94 (C-22), 33.79 (C-23), 31.31 (C-24), 28.80 (C-25), 31.12 (C-26), 28.15 (C-27), 79.01 (C-28), 28.05 (C-29); FAB-MS m/z (rel.int): 414 [M⁺] (C₂₉H₅₀O) (50), 397 (100), 329 (9), 301 (5), 288 (15), 271 (15), 255 (22), 231(4), 199 (17), 161 (30), 147 (35), 133 (42), 105 (58), 91 (57), 81 (40).

β-sitosterol

Compound III (16β-hydroxybetulinic acid):

White amorphous powder; R_f 0.69 in chloroform-methanol (85:15); m.p. 310-312^oC; IR v_max (KBr): 3423, 2915, 1682, 1448, 1373, 885 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.82 (m, H-1), 1.87 (m, H-2), 3.46 (t, br, J= 8.3 Hz, H-3), 0.83 (m, H-5), 1.41 (m, H-6), 1.49 (m, H-7), 1.27 (t, J= 9.1 Hz, H-9), 1.25 (m, H-11), 1.08 (m, H-12), 2.48 (m, H-13), 2.18 (t, J= 12.0 Hz, H-15), 4.20 (1H, dd, J= 11.2, 4.5 Hz, H-16), 1.83 (m, H-18), 3.49 (m, H-19), 1.69 (m, H-22), 1.26 (s, 3H, Me-23), 1.08 (s, 3H, Me-24), 0.88 (s, 3H, Me-25), 1.10 (s, 3H, Me-26), 1.17 (s, 3H, Me-27), 4.89 (s, H-29), 1.79 (s, 3H, Me-30); ¹³CNMR (CDCl_3, 400 MHz): δ 38.09 (C-1), 28.52 (C-2), 78.11 (C-3), 39.79 (C-4), 55.84 (C-5), 18.91 (C-6), 34.03 (C-7), 40.82 (C-8), 49.96 (C-9), 37.53 (C-10), 21.02 (C-11), 24.42 (C-12), 38.01 (C-13), 43.92 (C-14), 39.71 (C-15), 75.22 (C-16), 62.43 (C-17), 49.37 (C-18), 47.95 (C-19), 150.46 (C-20), 32.11 (C-21), 36.83 (C-22), 28.67 (C-23), 15.95 (C-24), 16.11 (C-25), 16.79 (C-26), 15.99 (C-27), 176.52 (C-28), 109.59 (C-29), 20.09 (C-30); FAB-MS m/z (rel.int): 472 [M⁺] (C₃₀H₄₈O₄) (55), 454 (60), 246 (70), 207 (63), 189 (100).

16β-hydroxybetulinic acid

Compound IV (α-amyrin):

White crystals; R_f 0.62 in chloroform-methanol (95:5); m.p. 179-181^oC; IR v_max (KBr): 3444, 2948, 2891, 1639, 1451, 1384 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.80 (s, 3H, Me-24), 0.88 (s, 6H, Me-29, Me-30), 0.89 (s, 3H, Me-28), 0.99 (s, 3H, Me-23), 0.95 (s, 3H, Me-25), 1.08 (s, 3H, Me-26), 1.16 (s, 3H, Me-27), 3.85 (m, 1H, H-3a), 5.35 (1H, t, J= 3.4 Hz, H-12); ¹³CNMR (CDCl_3, 400 MHz): δ 38.95 (C-1), 27.51 (C-2), 78.41 (C-3), 38.95 (C-4), 55.34 (C-5), 18.40 (C-6), 33.24 (C-7), 40.07 (C-8), 47.42 (C-9), 36.96 (C-10), 23.63 (C-11), 124.28 (C-12), 138.48 (C-13), 42.28 (C-14), 26.16 (C-15), 28.16 (C-16), 33.88 (C-17), 59.22 (C-18), 39.68 (C-19), 39.38 (C-20), 31.32 (C-21), 41.58 (C-22), 28.25 (C-23), 15.20 (C-24), 15.56 (C-25), 16.91 (C-26), 23.27 (C-27), 28.74 (C-28), 18.40 (C-29), 21.46 (C-30); FAB-MS m/z (rel.int): 426 [M⁺] (C₃₀H₅₀O) (70), 393 (50), 218 (47), 207 (70), 203 (45), 189 (66).

α-amyrin

Compound V (β-sitosterol glycoside):

White crystalline powder; R_f 0.68 in chloroform-methanol (95:5); m.p. 279-281^oC; IR v_max (KBr): 3420, 2925, 2823, 1672, 1478, 1370, 1031 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.68 (s, 3H, Me-18), 0.85 (3H, d, J= 8.6 Hz, Me-26), 0.88 (6H, d, J= 7.6 Hz, Me-27, Me-29), 0.95 (3H, d, J= 5.4 Hz, Me-19), 1.02 (s, 3H, Me-21), 3.88 (m, 1H, H-3), 4.58 (m, 1H, H-6), 5.25 (1H, d, J=7.3 Hz, C-1'H); ¹³CNMR (CDCl_3, 400 MHz): δ 38.21 (C-1), 30.35 (C-2), 80.58 (C-3), 41.57 (C-4), 142.83 (C-5), 121.80 (C-6), 35.25 (C-7), 32.50 (C-8), 50.21 (C-9), 38.21 (C-10), 21.29 (C-11), 40.57 (C-12), 42.38 (C-13), 56.95 (C-14), 24.38 (C-15), 28.32 (C-16), 56.85 (C-17), 12.12 (C-18), 19.90 (C-19), 37.33 (C-20), 19.47 (C-21), 34.02 (C-22), 26.15 (C-23), 46.91 (C-24), 30.35 (C-25), 19.47 (C-26), 20.28 (C-27), 23.14 (C-28), 12.33 (C-29), 102.82 (C-1'), 76.77 (C-2'), 78.41 (C-3'), 71.88 (C-4'), 78.09 (C-5'), 62.91 (C-6'); FAB-MS m/z (rel.int): 577 [M+H]⁺ (C₃₅H₆₁O₆) (65), 415 (40), 397 (63), 396 (50), 382 (35), 354 (26), 339 (45), 275 (37), 255 (54), 149 (64), 147 (42), 109 (40), 97 (100).

β-sitosterol glycoside

DISCUSSION:

Compound I was obtained as pearl white crystals and it gave characteristic color reaction for triterpenoids. The FAB-MS spectrum showed a molecular ion peak at m/z 426 corresponding to molecular formula C₃₀H₅₀O. The IR spectrum exhibited strong absorptions at 3314 cm^-1 (hydroxyl group) and 2943, 1642, 880 cm^-1 (exomethylene group). The ¹H-NMR spectrum exhibited six tertiary methyl groups at [δ 0.75, 0.78, 0.82, 0.91, 0.94, 1.02], a methane group at [δ 1.67 (3H, d, J= 0.6 Hz)], a secondary carbinol group at [δ 3.23 (1H, dd, J= 9.4 and 6.1 Hz)] and an exomethylene group at δ 4.56 and 4.68 implies a typical pentacyclic triterpenoid of the lupeol. Based on the spectral data the compound was identified as lupeol^{9, 10}.

Compound II obtained as white crystals and it gave characteristic color reaction for sterol. The FAB-MS spectrum showed a molecular ion peak at m/z 414 corresponding to molecular formula C₂₉H₅₀O. The most downfield signals at d 150.96 (C-5) was accommodated for sp₂ (olefinic) and the next downfield signal at d 124.46 (C-6). The oxygenated carbon at C-3 gave a down field signal at d 72.90. The next downfield d 55.23 was accommodated for C-17. ¹³CNMR spectral data matched exactly with that of b-sitosterol¹¹. Its identity as b-sitosterol was further confirmed by IR, ¹HNMR spectral properties identical to those described for b-sitosterol¹².

Compound III obtained as white amorphous powder and it gave characteristic color reaction for triterpenoids. The molecular formula was established as C₃₀H₄₈O₄ from FAB-MASS spectrum which shows molecular ion peak at m/z 472. The IR spectrum exhibited absorption bands assigned to the hydroxyl groups (3423cm^-1) and carbonyl at (1682 cm^-1). The ¹HNMR spectrum showed five tertiary methyl groups at δ 1.26 (Me- 23), 1.08 (Me- 24), 0.88 (Me- 25), 1.10 (Me- 26), 1.17 (Me- 27), and one vinylic methyl at δ 1.79 (Me-30), two protons of an isopropenyl moiety at δ 4.89. Based on the IR, ¹HNMR and ¹³NMR spectral data, the compound was identified as 16β-hydroxybetulinic acid¹³.

Compound IV obtained as white crystals; it gave characteristic color reaction for triterpenoids. FAB-MS spectrum displayed the molecular ion peak at m/z 426 [M⁺] corresponding to the molecular formula (C₃₀H₅₀O). The IR spectrum exhibited strong absorption at 3444 cm^-1 (OH), 2948 cm^-1 (C-H), 2891 cm^-1 (C-H), 1639 cm^-1 (C=C). The ¹HNMR spectrum of this compound exhibited the presence of methyl group at δ 0.80 (H-24), δ 0.88 (H-29, 30), δ 0.89 (H-28), δ 0.95 (H-25), δ 0.99 (H-23), δ 1.08 (H-26), δ 1.16 (H-27) and HC-OH at δ 3.85 (H-3) and 5.35 (H-12). The ¹³CNMR spectrum revealed 30 signals which were duly assigned as CH₃, CH₂, CH, -C- group represent in the compound. Based on the IR, ¹HNMR and ¹³NMR and MS spectral data, the compound was identified as α-amyrin¹⁴.

Compound V obtained as white crystalline powder; it gave a charateristic colour reaction for steroidal glycoside. The IR spectrum exhibited strong absorption at 3420 cm^-1 (OH), 2925 cm^-1 (C-H), 2823 cm^-1 (C-H), 1672 cm^-1 (C=C). FAB-MS spectrum displayed the molecular ion peak at m/z 577 [M+H]⁺ corresponding to the molecular formula (C₃₅H₆₁O₆). The mass of the aglycone (β-sitosterol) was determined at m/z 415 [M+H]⁺indicating the presence of β-sitosterol and glucose unit. The ¹HNMR spectrum signals exhibited the presence of methyl signals at δ 0.68 (H-18), δ 0.85 (H-26), δ 0.88 (H-27, 29), δ 0.95 (H-19), δ 1.02 (H-21). A signal present at δ 4.58 was assigned to H-6. the signal of the proton of anomeric carbon of glucose unit was found as doublet at δ 5.25. The ¹³CNMR spectrum also showed the signals exactly like that of β-sitosterol moiety and a glucose unit. The point of attachment of glucose unit to β-sitosterol was at C-3, and the ¹³CNMR value of this carbon was found at δ 80.58 , and the value of glucose C-1 was observed at δ 102.82¹⁵. Therefore the compound V was confirmed as β-sitosterol glycoside.

From the spectral data, the compounds I-V was analyzed as lupeol, β-sitosterol, 16β-hydroxybetulinic acid, α-amyrin and β-sitosterol glycoside respectively. However, the compounds lupeol, 16β-hydroxybetulinic acid and α-amyrin were reported for the first time from the roots of this plant.

ACKNOWLEDGEMENT:

The authors are thankful to the authorities of A. Shama Rao Foundation Mangalore, Karnataka, India and Nitte Education Trust Mangalore, Karnataka, India for the facilities.

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Received on 08.09.2009 Modified on 03.11.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 217-220