INTRODUCTION:

Aquilaria malaccensis (Agar wood) belongs to the family Thymelaeaceae, locally known as Eagle wood distributed in India, Burma, Malaysia, Philippines and Indonesia. Agar wood leaves are reported to possess antidiabetic, anti-inflammatory, antioxidant, antibacterial, antidepressant and antiviral activities^1-3. They have a wide use in medicines like general pain reducer, kidney and rheumatic diseases, as a venom repellent and free radical scavenging properties. Further the agar wood leaves are reported to possess anticancer and radioprotective activities^4,5. The extract of the leaves of agar wood is reported to contain more than a dozen chemical constituents belongs to steroids, flavonoids, alkaloids, terpenoids and glycosides class of secondary metabolites that can be extracted². Since the chemical investigation of the leaves of Aquilaria malaccensis has not been dealt in detail, in the present study an effort was made to establish the chemical investigation of the extract of the leaves of this plant.

MATERIALS AND METHODS:

Chemicals and Instruments:

All the chemicals and solvents were of analytical grade and procured from Ranbaxy Fine Chemicals Ltd., Mumbai, India. 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.

Plant Material:

The leaves of A. malaccensis were collected from the local areas and authenticated by Taxonomist. The collected leaf materials were cleaned, shade dried and stored in an air tight container.

Extraction and Isolation:

The coarse leaf powder (1000 g) were soaked in 1 L of 95% ethyl alcohol and extracted in the 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, the concentrate was evaporated to dryness under reduced pressure and low temperature (40^oC) on a rotary evaporator to give the ethanolic extract (16% w/w yield), which was stored at 4^oC until use. The ethanol extract (100 g) was suspended in distilled water and then extraction with petroleum ether (60-80^oC, 8x500 ml). All the 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 (28 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 (8 g)⁶.

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 n-hexane. The elutions carried out with n-hexane: ethyl acetate graded mixture (95:5) afforded compound I (60 mg) and n-hexane: ethyl acetate graded mixture (90:10) resulted into compound II (85 mg) and III (100 mg). Further, the elutions carried out with n-hexane: ethyl acetate graded mixtures (85:15) resulted into compound IV (85 mg) and V (80 mg) respectively. The individual compounds were purified by preparative TLC on silica gel G and further purified by recrystallization with n-hexane.

RESULTS:

Compound I (stigmasterol):

Pearl white crystals; R_f 0.63 in n-hexane: ethyl acetate (95:05); m.p. 167-170^oC; IR v_max (KBr): 3423, 2952, 2850, 1668, 1619, 1465, 1070 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.79, 0.78, 0.81, 0.91, 0.94, 1.08 (m, 18H), δ 1.13 to δ 2.53 (m, 18H, 9x CH₂ and 8H), 3.54 (1H, dd, J= 9.1, 6.0 Hz, H-α3), 5.33 ( s, 1H, H-3), 5.11 and 5.15 (2H, br); ¹³CNMR (CDCl_3, 400 MHz): δ 33.79 (C-1), 26.48 (C-2), 778.98 (C-3), δ 40.57 (C-4), δ 145.83 (C-5), δ 121.81 (C-6), δ 32.50 (C-7), δ 35.35 (C-8), δ 45.99 (C-9), δ 35.95 (C-10), δ 21.19 (C-11), δ 38.93 (C-12), δ 51.32 (C-13), δ 30.02 (C-14), δ 37.33 (C-15), δ 32.08 (C-16), δ 56.93 (C-17), δ 18.36 (C-18), δ 19.91 (C-19), δ 50.23 (C-20), δ 18.61 (C -21), δ 138.39 (C-22), δ129.35 (C-23), δ 30.35 (C-24), δ 29.03 (C-25), δ 31.78 (C-26), δ 28.31 (C-27), δ 18.89 (C-28), δ 27.15 (C-29); FAB-MS m/z (rel.int): 412 [M⁺] (C₂₉H₄₈O) (100), 397 (20), 369 (10), 351 (70), 329 (65), 300 (40), 299 (23), 273 (31), 254 (28).

Compound II (β-sitosterol):

White crystals; R_f 0.36 in n-hexane: ethyl acetate (90:10); 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).

Compound III (lupeol):

Pearl white crystals; R_f 0.56 in n-hexane: ethyl acetate (90:10); m.p. 213-215^oC; IR v_max (KBr): 3436, 2928, 2852, 1649, 1455, 1374, 1038, 880 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 0.76, 0.78, 0.82, 0.91, 0.94, 1.02 (m, 18H, Me-28, Me-23, Me-24, Me-25, Me-26, Me-27), 1.66 (3H, d, J= 0.7 Hz, Me-30), 3.21 (1H, dd, J= 9.5, 6.2 Hz, H-α3), 4.57 ( s, 1H, H-3), 4.69 (s, 1H), 4.83 (s, 1H); ¹³CNMR (CDCl_3, 400 MHz): δ 38.55 (C-1), 27.01 (C-2), 78.49 (C-3), 38.98 (C-4), 54.34 (C-5), 18.44 (C-6), 34.38 (C-7), 39.68 (C-8), 49.87 (C-9), 37.96 (C-10), 20.12 (C-11), 25.24 (C-12), 37.36 (C-13), 42.28 (C-14), 27.16 (C-15), 36.30 (C-16), 42.98 (C-17), 48.24 (C-18), 47.42 (C-19), 150.48 (C-20), 28.25 (C-21), 40.07 (C-22), 28.74 (C-23), 15.50 (C-24), 16.51 (C-25), 15.96 (C-26), 14.40 (C-27), 17.40 (C-28), 109.29 (C-29), 19.74 (C-30).; FAB-MS m/z (rel.int): 426 [M⁺] (C₃₀H₅₀O) (50), 409 (10), 395 (13), 318 (9), 218 (74), 203 (65), 187 (46), 175 (30), 161 (39), 147 (64), 125 (71), 121 (70), 93 (100).

Compound IV (ursolic acid):

Pale yellow powder; R_f 0.51 in n-hexane: ethyl acetate (85:15); m.p. 271-274^oC; IR v_max (KBr): 3453, 2926, 2854, 1636, 1458, 1381, 1034 cm^-1; ¹HNMR (CDCl₃, 400 MHz): δ 1.00 to 1.91 (m, 22H), 3.20 (1H, dd, J= 10, 5.7 Hz, H-3), 2.17 (1H, d, J= 4.52 Hz, H-18), 1.03 (s, 3H, Me-23), 0.92 (s, 3H, Me-24), 0.77 (s, 3H, Me-25), 0.94 (s, 3H, Me-26), 0.95 (s, 3H, Me-27), 0.87 (s, 3H, Me-29), 0.84 (s, 3H, Me-30), 2.28 (m, 1H, OH), 5.34 (s, 1H, H-12); ¹³CNMR (CDCl_3, 400 MHz): δ 37.30 (C-1), 28.19 (C-2), 78.49 (C-3), 39.98 (C-4), 41.18 (C-5), 22.87 (C-6), 29.25 (C-7), 39.05 (C-8), 51.87 (C-9), 30.32 (C-10), 22.59 (C-11), 125.25 (C-12), 137.48 (C-13), 39.38 (C-14), 29.25 (C-15), 26.16 (C-16), 55.39 (C-17), 38.95 (C-18), 32.54 (C-19), 37.30 (C-20), 27.91 (C-21), 38.06 (C-22), 14.92 (C-23), 14.20 (C-24), 18.45 (C-25), 16.95 (C-26), 23.69 (C-27), 177.48 (C-28), 20.12 (C-29), 16.16 (C-30); FAB-MS m/z (rel.int): 456 [M⁺] (C₃₀H₄₈O₃) (71), 428 (50), 289 (49), 277 (35), 248 (100), 149 (27), 85 (11), 83 (44).

Compound V (α-amyrin):

White crystals; R_f 0.62 in n-hexane: ethyl acetate (85:15); 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).

Stigmasteral

β-sitosterol

Lupeol

Ursolic acid

α-amyrin

DISCUSSION:

Compound I was obtained as pearl white crystals and gave characteristic color reaction for sterol. The FAB-MS spectrum showed a molecular ion peak at m/z 412 corresponding to molecular formula C₂₉H₄₈O. The IR spectrum exhibited strong absorptions at 3423 cm^-1 (hydroxyl group), 2952 and 1668 cm^-1. The ¹HNMR spectrum exhibited six tertiary methyl groups at δ 0.79, 0.78, 0.82, 0.91, 0.94, and 1.08, one vinylic proton at δ 5.33 and two olefinic protons at δ 5.11 and 5.15 respectively. In ¹³CNMR spectrum the most down field signals at δ 145.83 was accommodated for sp² (olefinic) carbon at C-5 and the next downfield signal at δ 138.39 ppm and δ 129.35 ppm to C-22 and C-23. The downfield signal at δ 121.81 is to C-6. The oxygenated carbon at C-3 gave a downfield signal at δ 78.98 ppm. The next downfield signal at δ 56.93 ppm was accommodated for C-17. Other carbon atoms of the steroidal skeleton except that in the side chain appeared in the range δ 45.99 to δ 30.02 ppm. The angular methyl groups and the side chain methyl carbons gave signals in the region δ 19.91 to δ 18.89 ppm. ¹³CNMR spectral data matched exactly with that of Stigmasterol⁷. Its identity as stigmasterol was confirmed by m.p., IR, ¹HNMR, ¹³CNMR and mass spectral data and by co-chromatography with an authentic sample.

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-sitosterol⁸.

Compound III 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 3436 cm^-1 (hydroxyl group) and 2928, 1649, 880 cm^-1 (exomethylene group). The ¹HNMR spectrum exhibited six tertiary methyl groups at δ 0.76, 0.78, 0.82, 0.91, 0.94 and 1.02, a methane group at δ 1.66, a secondary carbinol group at δ 3.21 and an exomethylene group at δ 4.56 and 4.68 implies a typical pentacyclic triterpenoid of the lupeol. Based on the m.p., IR, ¹HNMR, ¹³CNMR and mass spectral data the compound was identified as lupeol^9,10.

Compound IV was obtained as pale yellow powder and it gave characteristic color reaction for triterpenoids. The FAB-MS showed a molecular ion peak at m/z 456 corresponding to a molecular formula C₃₀H₄₈O₃. It was unambiguously identified as ursolic acid on the basis of its m.p., IR, ¹HNMR, ¹³CNMR and mass spectral data¹¹.

Compound V 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-amyrin¹².

CONCLUSION:

From the spectral data, the compounds I-V was analyzed as stigmasterol, b-sitosterol lupeol, ursolic acid and α-amyrin respectively. However, the compounds stigmasterol, ursolic acid and α-amyrin were reported for the first time from this plant.

ACKNOWLEDGEMENT:

The authors are thankful to the Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore for financial assistance to this work.

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Received on 28.12.2018 Modified on 13.01.2019

Res. J. Pharmacognosy and Phytochem. 2019; 11(1): 34-37.

DOI: 10.5958/0975-4385.2019.00007.4