Phytochemical investigation of Calotropis procera

2.1 Plant collection

Fresh or dried plant material can be used as a source for the extraction of secondary plant components. Freshly harvested and dried material is more commonly used since old dried material stored for a long period may undergo some qualitative changes. Latex of C. Procera was collected from the rural areas of Rewa and Bhopal districts in different month of 2009. These plants were identified by the Botany Department of Bareily College, Bareily. The pooled frozen latex was thawed a fortnight after collection when the serum separated and the solid part of the latex could easily be removed and dried between filter paper sheets.

2.2 Extraction

The dried latex of plant (∼5 kg) was soaked in with ethanol at room temperature for two weeks. The filtrate was evaporated under reduced pressure to obtain a dark brown gunmy material (750 g), partitioned between n-hexane, chloroform, acetone, ethyl acetate and n-butanol, yielding petrol–ether soluble (80 g), chloroform soluble (285 g), ethyl acetate soluble (112 g) and n-butanol soluble (159 g) fractions. The petrol–ether soluble fraction was evaporated under vacuum on rotatory evaporator below 50 °C temperature to yield 60 gm of petrol–ether extract. A well-stirred suspension of silica gel (100–150 g in petrol–ether 60–80 °C) was poured into column (150 cm long and 50 mm in diameter). When the absorbent was well settled, the excess of petrol–ether was allowed to pass through column. Slurry was made to petrol–ether extract with 5 g of silica gel in petrol–ether and was digested to well settle column. The column was successively eluted with the solvents and solvent mixtures of increasing polarity. Elution with chloroform/ethyl acetate (3:5) yielded compound CP-1. Whereas on elution with ethyl acetone/acetate (1:1) afforded compound CP-2. Elution with chloroform/EtOAc (3:2) provided compound CP-3. Similarly the acetone soluble fraction was also subjected to column chromatography. The column was successively eluted with the solvents and their mixtures of increasing polarity. Elution with petrol/acetone (8:2) yielded compound CP-4. The ethyl acetate soluble thus obtained was successively eluted with the solvents and their mixtures of increasing polarity. Elution with benzene/ethyl acetate (8:2) afforded CP-5 (Table 1).

2.3 Compound CP-1

White powder; m.p.: 198–201 °C; IR (CHCl₃) λ_max: 1720, 1320, 1635 and 885 cm⁻¹ Mass spectra m/z: 468 [M]⁺, 453 [M−Me]⁺, 408 [M−AcOH]⁺, 393 [M−Me−AcOH]⁺, 272 [M−C₁₂H₂₀O₂]⁺, 257 [M−C₁₃H₂₃O₂]⁺, 249 [M−C₁₆H₂₇]⁺, 218 [M−C₁₆H₂₅O₂]⁺, 189 [M−C₁₆H₂₇−AcOH]⁺; ¹H NMR (300 MHz, CDCl₃): δ 4.52 (1H, dd, J_ax,ax = 9.8, J_ax,eq = 4.5 Hz, H-3), 2.12 (3H, s, O–C–Me), 1.04 (3H, s, 27-Me) 1.02 (3H, s, 26-Me), 1.01 (3H, s, 23-Me), 0.91 (3H, d, J = 6.6 Hz 30-Me), 0.83(3H, s, 25-Me), 0.82 (6H, s, 24,28-Me), 4.67–4.62 (2H, br s H-29).

2.4 Compound CP-2

Colorless needles; R_f (silica gel): 0.38 (MeOH–CH₂Cl₂) 1:9; MS m/z (rel. int.%): 414 (76), 412 (92), 396 (22), 369 (17), 351 (14), 329 (10), 300 (43), 283 (10), 271 (43), 255 (47), 231 (18), 213 (22), 69 (87), 51 (100); ¹H NMR (CDCl₃): δ 5.01 (1H, dd, J = 8.4, 15.2 Hz, H-23), 5.15 (1H, dd, J = 8.4, 15.2 Hz, H-22), 5.35 (2H, d, J = 5.2 Hz, H-6), 3.51 (2H, m, H-3), 1.00 (3H, s, 19-Me), 0.69 (3H, s, 18-Me), 1.03 (3H, d, J = 6.60 Hz, 21-Me), 0.85 (3H, d, J = 6.60 26-Me), 0.81 (3H, d, J = 7.53 Hz, 29-Me), 0.80 (3H, d, J = 6.41 Hz, 27-Me).

2.5 Compound CP-3

White crystals; m.p.: 189–191 °C; IR (CHCl₃) λ_max: 3510 (hydroxyl group), 3045, 1640, 811 (tri substituted double bond) cm⁻¹; MS m/z (rel. int.%): 426 [M+H]⁺, (10), 411 [C₃₀H₅₀O−CH₃]⁺ (15), 408 [C₃₀H₅₀O−H₂O]⁺ (25), 393 [C₃₀H₅₀O−CH₃−H₂O]⁺ (30), 273 [a]⁺ (42), 259 [b]⁺ (35), 247 [c (R.D. fragment)]⁺ (42), 220 [d]⁺ (50), 205 [e]⁺ (100).

2.6 Compound CP-4

Colorless shining needles; m.p.: 167–168 °C; IR ν_max (CHC1₃): 3420, 3075, 1645, 890 cm⁻¹; MS m/z (rel. int.%): 426 (30) [M]⁺, 411 (10) [M−Me]⁺, 408 (16) [M−H₂0]⁺, 393 (8) [M−Me−H₂0]⁺, 272 (10) [ion d]⁺, 257 (20) [ion c]⁺, l218 (6) [ion b]⁺, 207 (100) [ion a]⁺, 189 (62) [ion a−H₂0]⁺.

2.7 Compound CP-5

Colorless crystals; m.p.:165–166 °C; IR ν_max: 3400 (OH), 2980 (CH), 1760 (five-membered lactone ring) and 1570 (C⚌C) cm⁻¹; MS m/z (rel. int.%): 470 [M+H]⁺ (29), 452 [M−H₂0]⁺ (I2.0), 263 [C₁₆H₂₃O₃; fragment a] (23.6), 249 [C₁₅H₂₁O₃; fragment b] (8.8), 235 [C₁₆H₂₃O; (fragment a)−CO] (18.5), 220 [C₁₅11₂₄O; (fragment a)−CO₂+H] (13.3), 207 [C₁₄H₂₃O; fragment c] (19.5), 205 [C₁₄H₂₁O; (fragment b)−CO₂] (22.0), 189 [C₁₄H₂₁; (fragment c)−H₂O] (33.4) and 187 [C₁₄H₁₉; (fragment b)−CO₂−H₂O] (12.6); ¹H NMR (CDC1₃, 300 MHz) 3.18 (H-3, 1H,dd, J = 11.4, 4.8 Hz), 3.55–3.73 (each 1H, d, J = 11.0 Hz, H-27a, 27b), 1.05 (3H, d, J = 7.1 Hz, H-30), 0.99 (3H, s, 23-Me), 0.97 (3H, s, 24-Me), 0.94 (3H, s, 25-Me), 0.88 (3H, s, 29-Me).

3.1 Compound CP-1

Compound CP-1 was isolated as white needle, m.p.: 198–201 °C, on elution with chloroform/ethyl acetate (3:5). Mass spectrum showed [M] ⁺ ion at m/z 468 corresponding to molecular formula C₃₂H₅₂O₂. In the ¹H NMR of CP-1 a pair of broad singlets observed at δ 4.67 and 4.62 was indicative of the exomethylene group. The acetoxy group was placed at C-3 on the basis of biogenetic consideration. Furthermore a two proton double doublet observed at δ 4.52 (1H, s, J = 9.8, 4.5 Hz, H-3) was attributed to the proton attached to the carbon bonded to the acetoxy group, showing α- and axial configuration, thus confirming that acetoxy group must be β- and equatorial at the usual C-3 position (Alam and Ali, 2009).

The ¹H NMR of CP-1 revealed signals for six singlet methyl at δ 1.04 (s, H-27), 1.01 (s, H-23), 1.02 (s, H-26), 0.83 (s, H-25) and 0.82 (s, H-24 and H-28), a doublet methyl at δ 0.91 (d, J = 6.6 Hz, H-30), A signal observed at δ 1.12 assignable to the methyl attached to the acetoxy group. The ¹³C NMR spectrum of this compound showed 32 signals clearly indicating that the compound contains eight methyl, eleven methylene, six methine groups and seven quaternary carbons. On the basis of these spectral data, the compound CP-1 was identified as urs-19(29)-en-3-yl acetate. All these spectral data were in good concurrence with those reported in the literature for calotropenyl acetate (Ali, 2001).

3.2 Compound CP-2

Compound CP-2 was isolated as colorless needles, on elution with ethyl acetone/acetate (1:1). This compound gave purple coloration upon spraying with anisaldehyde reagent. The sample showed a dark green colour with Liebermann–Burchard reagent, which is typical of a steroid. In the ¹H NMR spectrum, the signal at δ 5.01 (1H, dd, J = 8.4, 15.2 Hz), 5.15 (1H, dd, J = 8.4, 15.2 Hz) and 5.35 (2H, d, J = 5.2 Hz) could be assigned to H-23 and H-22 of stigmasterol and H-6 of β-sitosterol and stigmasterol, respectively. The integration value for H-6 was twice those of H-22 or H-23. Therefore, it could be deduced that the compound was a 1:1 mixture of β-sitosterol and stigmasterol. The ¹³C NMR spectrum of this compound displayed 43 signals.

The compound CP-2 in its EI-MS showed two major molecular ion peaks at m/z 412 and 414. The fragmentation pattern with significant peaks at m/z 396, 369, 351, 329, 300, 273, 271, 231 and 213 was reminiscent of a sterol mixture of stigmasterol and β-sitosterol. The NMR data of this compound were in full agreement with the published valued mixture of β-sitosterol and stigmasterol (Wright et al., 1978).

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3.3 Compound CP-3

The compound was isolated as white crystals, m.p.: 189–191 °C, by eluting the column with chloroform/EtOAc (3:2). The molecular ion peak of this compound in mass spectra m/z at 426 corresponds to the molecular formula C₃₀H₅₀O. The IR spectrum of the compound showed absorption bands at 3450 (hydroxyl group) and 3045, 1640, 811 (trisubstituted double bond). The mass spectrum of this compound showed characteristic peaks of bauerene and multiflorene type triterpenes having double bond at 7(8) (Budzikiewicz et al., 1963). An eminent fragment ion at m/z 247 arises by retro Diels Alder decomposition of ring; ‘c’ proceeding the initial rearrangement of C-9 proton (Budzikiewicz et al., 1963). The ¹H NMR spectrum provided signals at δ 0.85 (3H), 0.93 (3H), 0.97 (6H), 0.95 (3H), 1.05 (3H), 1.07 (3H) and 1.02 (3H) were assignable for eight tertiary methyl groups. In the ¹H NMR a pair of doublets at δ 3.21(J_ax,ax = 9.7 Hz, J_ax,eq = 4.2 Hz) were assigned to α-axial proton germinal to hydroxyl group, one proton doublet observed at δ 5.47 (d, J = 8.9, 4.1 Hz) assignable for olefinic proton. The ¹³C NMR spectrum exhibited 30 carbon signals for different type carbon atoms. Survey of the literature showed that this compound is similar to multiflorenol (Bhandari and Rastogi, 1984).

3.4 Compound CP-4

The compound was isolated, from acetone extract by eluting column with petrol/acetone (8:2). In its electro spray mass spectrum the molecular ion peak of the compound at m/z 426 [M+H]⁺ corresponded to the molecular formula C₃₀H₅₀O. This compound responded to Liebermann–Burchard test and showed violet coloration with ceric sulphate, suggesting its triterpenic nature. The IR spectrum showed absorption bands at 3420, 3075, 1645 and 890 cm⁻¹ assignable for the hydroxyl and a methylene groups, respectively.

The ¹H NMR spectrum showed signals for vinylic proton at δ 4.65–4.70 (2H, br s) and a proton geminal to the hydroxyl group at δ 3.23. In addition there were signals at δ 0.93 (3H, d, J = 6.2 Hz) allocated to one secondary and at δ 1.05 (3H), 1.02 (3H), 0.99 (3H), 0.84 (3H) and 0.82 (6H) six tertiary methyl groups (Rahman, 1986). The mass spectrum of this compound was characteristic of pentacyclic triterpene of the ursane series with saturation in rings A, B, C and D. The major ions ‘a’ and ‘b’ were generated by the transfer of hydrogen from C-26 to C-11 accompanied by cleavage of 9–11 and 8–14 bonds with charge retention on either of the resulting fragments (Benn and Gunther, 1983; Karline and Djerassi, 1966; Barton and de Mayo, 1954). Oxidation of CP-4 with Jones reagent gave calotropenone (CP-4a). The latter gave positive Zimmermann test indicating the presence of 3-oxo group (Panosyan et al., 1977). The NaBH₄ reduction of CP-4a regenerated the parent alcohol CP-4, confirming the equatorial orientation of the hydroxyl group in CP-4. The configuration of the hydroxyl group in CP-4 was also indicated by the characteristic downfield shift of the 3-α-H which appeared as double doublets at J_ax,ax = 9.8 Hz and J_ax,eq = 4.7 Hz owing to coupling with proton at C-2. The catalytic reduction of CP-4 afforded the corresponding dihydro derivative calotropanol (CP-4b) which was identified as 3-hydroxy ursane through comparison of spectral data with these reported in the literature (Domingelez et al., 1974; Hassall and Reyle, 1959). Its ¹H NMR spectrum showed the absence of olefinic proton but gave a signal at δ 0.86 (3H, d, J = 6.9 Hz) assignable for an additional secondary methyl group. The reduction of the methylene group was expected to form a methyl group with α- or β-orientation. However, the former was formed in trace amount due to steric crowding of the β-oriented methyl group at C-8. These observations left only one position for the vinylic double bond, i.e., C-19 (Khan et al., 1988). In the ¹³C NMR spectrum the signals of C-3, C-12, C-13, C-21, C-29 and C-30 could easily be correlated with the corresponding protons in the ¹H NMR spectrum. The ¹³C NMR spectrum of the compound showed signals for 23 carbons atoms, which revealed the presence of seven methyl, 11 methylene and six methine carbon atoms. On the basis of these spectral data the compound was identified as urs-19(29)-en-3-β-ol. Its acetate was been isolated from the same plant source reported in literature (Kojima and Ogura, 1989).

3.5 Compound CP-5

The compound (m.p.: 165–166 °C) was isolated from ethyl acetate soluble fraction by eluting the column with benzene/ethyl acetate (8:2). The mass spectrum exhibited a molecular ion peak at m/z 470 which consistent with the molecular formula C₃₀H₄₆O₄. The IR absorption at 1760 cm⁻¹ and a carbon signal at δ_C 178.9 suggested the presence of a lactone moiety. The IR spectrum showed absorptions at 1760 cm⁻¹ suggesting the presence of a five membered lactone ring and a band at 1620 cm⁻¹ for C⚌C. The molecular formula showed eight double bond equivalents, five of which were accounted for by the pentacyclic ring nucleus, two by the lactone ring and one by a C⚌C. These features suggested that the remaining two oxygen atoms are present as hydroxyl groups (3400 cm⁻¹). The NMR spectrum showed a double doublet at δ 3.18 (J = 11.4 and 4.8 Hz) and δ_C 78.9 attributable to the proton germinal to the hydroxyl group placed at C-3 on biogenetic consideration (Jacobs and Hoffman, 1928). The coupling constants and the chemical shifts of H-3 favored the β-orientation (equatorial) of the hydroxyl group at C-3 (Siddiqui et al., 1990). The ¹H NMR spectrum further showed two AB doublets at δ 3.73 and 3.55 each with a geminal coupling of 11.0 Hz (δ_C 62.5), indicating a methylene carbon linked to an oxygen function. Further, in the ¹H NMR spectrum six methyl signals were observed, five as singlets at δ 0.78, 0.94, 0.97, 0.99 and 1.88 and one as a doublet at δ 1.05 (J = 7.1 Hz). These data pointed out that compound CP-5 belongs to the ursane series of triterpcnes, with two methyls functionalized, one involved in the lactone ring and the other forming a –CH₂OH function. The chemical shift of one of the methyls (δ 1.88) indicated that it is vinylic and presence of only one methyl as a doublet (δ 1.05) along with absence of any vinylic proton led to place a double bond at C-18 (δ_C 136.7). In mass spectra the fragments a–c exemplified that the lactone ring and the remaining hydroxyl group are present in rings C and D. The ¹³C NMR spectrum also favors the presence of lactone ring between C-13 and C-28 and the hydroxyl group at C-27. Thus on the basis of the above spectral evidences, the structure of the isolated compound CP-5 was finally concluded to be 3β,27-dihydroxy-urs-18-en-13,28-olide, good concurrence with those reported in the literature (Kang, 1987; Numata et al., 1990; Siddiqui et al., 1992; Begum et al., 1993).

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