New Compounds from Zizyphus vulgaris

Abstract

A lanostane-type triterpene, zizyphulanostane-21-oic acid, and a terpenic δ-lactone, zizyphulanostan-18-oic acid, have been isolated from the roots of Zizyphus vulgaris Lam. (syn. Z. sativa Gaertn., Z. mauritina. Lam.) and characterized as lanosta-25 (26)-en-9α-ol-21-oic acid and lanosta-25 (26)-en-22β-ol-18-oic acid 3(19)-olide, respectively, on the basis of spectroscopic techniques and chemical means.

Keywords:

• Isolation
• lanosta-25 (26)-en-9α-ol-21-oic acid
• lanosta-25 (26)-en-22β-ol-18-oic acid 3(19)-olide
• structure elucidation
• Zizyphus vulgaris Lam.

Introduction

The genus Zizyphus belongs to the family Rhamnaceae that consists of 45 genera and 550 species that are widely distributed in tropical and subtropical climates (Nasir & Ali, 1972). Zizyphus vulgaris Lam. (syn., Z. sativa Gaertn., Z. mauritiana Lam.), a small deciduous tree, is found in Punjab, Himalayas, up to 2100 m, eastwards to Bengal, Baluchistan, Iran, and the Mediterranean region. Its bark is used to heal ulcers, wounds, scabies, throat trouble, and burning sensation of the body (Kirtikar & Basu, 2000). Its fruit purifies and enriches blood, treats chronic bronchitis, fever, and enlargement of the liver. Seeds are used to treat dry cough and skin eruptions (Anonymous, 1988). The juice of the root is used as a purgative and externally in gout and rheumatism (Nadkarni, 1985). The plant contains anthocyanidins (Rastogi & Mehrotra, 1990), triterpenoids (Rastogi & Mehrotra, 1993, 1995), saponins and flavonoids (Rastogi & Mehrotra, 1993, 1998), alphitolic acid (Rastogi & Mehrotra, 1990), and β-sitosterol (Rastogi & Mehrotra, 1991, 1993, 1998). The current paper describes the isolation and structure elucidation of a new lano stane-type triterpene and terpenic δ-lactone from the roots of Zizyphus vulgaris.

Materials and methods

Melting points: uncorrected. UV spectra (γ max [nm]): Beckman DU 650 spectrophotometer (S.D. Fine-Chem Limited, Mumbai, India) MeOH. IR spectra (cm⁻¹): Perkin-Elmer FTIR 1720 X instrument; KBr pellets. ¹H NMR and ¹³C NMR spectra (δ [ppm], J [Hz] at 300 MHz [¹H] and at 100 MHz [¹³C]) in FT mode using a Bruker DRX-300 instrument (S.D. Fine-Chem Limited, Mumbai, India) using DMSO-d₆ with TMS as an internal standard. MS (m/z): Hewlett-Packard Model 8450 spectrophotometer (S.D. Fine-Chem Limited, Mumbai, India) in MeOH. TLC: Silica gel G, with solvent systems; petroleum ether-toluene-ethyl acetate (10:5:3), benzene-chloroform (1:1), and chloroform-methanol (1:1); detection by iodine vapor, perchloric acid, ceric ammonium sulfate, and UV fluorescence.

Plant material

The roots of Z. vulgaris Lam. were purchased from the local market of Khari Baoli, Delhi, and identified by Dr. M.P. Sharma, Taxonomist, Faculty of Science, Hamdard University (New Delhi). A voucher specimen of the drug is preserved in the Pharmacognosy Laboratory, Faculty of Pharmacy, Hamdard University.

Extraction and isolation

The pulverized sample (3500 g) was extracted exhaustively in a Soxhlet apparatus with chloroform. The extract was concentrated in vacuo to yield a thick, viscous, dark reddish brown mass (105 g). This material was adsorbed on silica gel (100 g) with constant stirring until completely dried and subjected to a silica gel column prepared in benzene. Compound 1 (96 mg) was eluted in C₆H₆–CHCl₃ (3:1), and compound 2 (80 mg) was eluted from C₆H₆–CHCl₃ (3:1) and crystallized from CHCl₃–MeOH (1:1) (Fig. 1).

Figure 1 Structures of compounds 1 and 2.

Zizyphulanostan-21-oic acid (1)

Compound 1 was obtained as colorless amorphous powder, m.p. 330–331°C, , (0.1, MeOH), UV λmax (MeOH), 240 nm (log iε 5.6), IR λ max (KBr), 3490, 3085, 2945, 2855, 1690, 1640, 1455, 1380, 1240, 1190, 1030, 880 cm⁻¹. ¹H NMR: iδ 4.67 (1H, brs, H-26a), 4.54 (1H, brs, H-26b), 2.49 (1H, m, H-1b), 2.49 (1H, brs, H-8β), 2.24 (2H, m, H-2a, H-16a), 2.20 (2H, m, H-12a, H-12b), 2.12 (2H, m, H-20, H-24), 2.09 (2H, m, H-6a, H-11a), 1.79 (1H, m, H-16b), 1.77 (2H, m, H-6b, H-17), 1.62 (3H, brs, Me-27), 1.56 (1H, m, H-5), 1.52 (1H, m, H-2b), 1.49 (2H, m, H 2a, H-22), 1.42 (2H, m, H-3b, H-12b), 1.36 (2H, m, H₂-23), 1.30 (1H, m, H-15a), 1.24 (1H, m, H-15b), 1.21 (1H, m, H-3a), 1.08 (2H, m, H₂-7), 0.91 (3H, brs, Me-19), 0.85 (6H, brs, Me-28, Me-30), 0.75 (3H, brs, Me-29), 0.63 (3H, brs, Me-18).¹³C NMR: iδ 38.43 (C-1), 30.09 (C-2), 38.26 (C-3), 40.04 (C-4), 54.88 (C-5), 18.90 (C-6), 29.16 (C-7), 39.48 (C-8), 76.75 (C-9), 39.20 (C-10), 27.11 (C-11), 36.68 (C-12), 39.76 (C-13), 55.36 (C-14), 31.70 (C-15), 37.55 (C-16), 41.94 (C-17), 15.88 (C-18), 18.62 (C-19), 33.91 (C-20), 177.12 (C-21), 36.32 (C-22), 29.01 (C-23), 38.92 (C-24), 150.20 (C-25), 109.52 (C-26), 25.05 (C-27), 17.93 (C-28), 15.70 (C-29), 14.34 (C-30). EIMS m/z (rel. int.) 458 [M]⁺ (C₃₀H₅₀O₃) (15.8), 440 (8.6), 414 (4.2), 396 (5.3), 317 (5.6), 250 (15.3), 236 (16.8), 222 (19.4), 220 (13.4), 208 (45.9), 206 (12.0), 204 (22.2), 194 (6.3), 192 (20.9), 190 (100), 176 (24.3), 152 (12.1), 148 (31.7), 138 (33.3), 125 (6.3), 124 (29.5), 123 (19.4), 109 (33.2), 95 (13.6), 83 (17.5), 81 (48.6), 69 (16.2), 55 (47.2).

Zizyphulanostan-18-oic acid (2)

Compound 2 was obtained as colorless amorphous powder, m.p. 322–323°C, [α], (0.1, MeOH), UV λmax (MeOH), 220 nm (log iε 5.2), IR λ max (KBr) 3500, 3080, 2930, 2855, 1740, 1695, 1640, 1470, 1365, 1310, 1230, 1200, 1030, 880 cm⁻¹. ¹H NMR: δ 4.69 (1H, brs, H-26a), 4.58 (1H, brs, H-26b), 3.92 (1H, brs, W1/2 = 15.5 brs, Hz), 3.00 (1H, ddd, J = 4.5, 5.2, 11.2 Hz, H-22iα), 2.50 (1H, m, H-2a), 2.38 (2H, m, H-1a, H-24a), 2.30 (2H, m, H-16a, H-12a), 2.19 (1H, m, H-24b), 2.23 (2H, m, H-12b, H-6a), 2.13 (1H, m, H-17), 2.10 (1H, m, H-11a), 1.81 (1H, m, H-2b), 1.78 (1H, m, H-1a), 1.64 (1H, m, H-15a), 1.56 (3H, brs, Me-27), 1.54 (1H, m, H-15b), 1.51 (1H, m, H-5), 1.47 (1H, m, H-11a), 1.43 (1H, m, H-11b), 1.39 (1H, m, H-8), 1.34 (1H, m, H-9), 1.28 (3H, brs, H-20), H₂-23), 1.14 (1H, m, H-7a), 1.09 (1H, m, H-7b), 1.06 (1H, m, H-6b), 0.99 (3H, d, J = 5.8 Hz, Me-21), 0.90 (3H, brs, Me-28), 0.87 (3H, brs, Me-29), 0.79 (3H, brs, Me-30).¹³C NMR: iδ 38.92 (C-1), 30.74 (C-2), 83.25 (C-3), 48.34 (C-4), 55.89 (C-5), 19.02 (C-6), 25.05 (C-7), 43.78 (C-8), 42.67 (C-9), 39.75 (C-10), 23.06 (C-11), 37.96 (C-12), 46.50 (C-13), 55.57 (C-14), 31.74 (C-15), 30.14 (C-16), 48.56 (C-17), 176.11 (C-18), 177.15 (C-19), 33.63 (C-20), 18.12 (C-21), 65.52 (C-22), 42.52 (C-23), 36.52 (C-24), 150.26 (C-25), 109.53 (C-26), 19.39 (C-27), 29.42 (C-28), 16.14 (C-29), 14.51 (C-30). EIMS m/z (rel. int.) 486 [M]⁺ (C₃₀H₄₆O₅) (15.2), 468 (23.0), 450 (15.0), 440 (36.3), 422 (20.4), 407 (10.1), 315 (6.5), 280 (5.6), 266 (6.2), 259 (19.8), 252 (10.5), 236 (35.5), 234 (27.4), 220 (23.8), 219 (62.9), 206 (38.3), 203 (48.8), 191 (29.5), 189 (80.0), 180 (10.5), 166 (12.9), 165 (23.5), 161 (33.5), 152 (17.3), 151 (27.0), 147 (41.6), 137 (22.2), 133 (60.0), 121 (85.6), 107 (78.2), 95 (76.2), 81 (77.6), 69 (75.8), 55 (91.3), 43 (100).

Results and Discussion

Compound 1 responded positively to the Liebermann-Burchard's test and yielded effervescence with sodium bicarbonate solution. Its IR spectrum exhibited absorption bands for hydroxyl group (3490 cm⁻¹), carboxylic group (3085, 1690 cm⁻¹), and unsaturation (1640 cm⁻¹). Its molecular formula as C₃₀H₅₀O₃ was established on the basis of a molecular ion peak at m/z 458 and ¹³C NMR signals. It had six double-bond equivalents, four of them were adjusted in a tetracyclic carbon framework and one each in an olefinic linkage and a carboxylic function. The mass spectrum displayed characteristic ion fragments of a lanostane-type triterpene at m/z 440 [M-H₂O]⁺, 414 [M-CO₂]⁺, 317 [M-side chain, C₈H₁₃O₂]⁺, and 396 [414-H₂O]⁺. The ion peaks at m/z 83 [C_3,4-C_5,10,-C_7,8 fission],⁺, 69 [83-CH₂]⁺, 55 [69-CH₂]⁺, 125 [C_1,10-C_5,10-C_7,8 fission]⁺, and 152 [C_9,10-C_7,8 fission]⁺ indicated the location of the hydroxyl group at C-9 and the saturated nature of rings A and B. The ion peaks at m/z 194 [C_8,14-C_9,11 fission]⁺, 176 [194-H₂O]⁺, 250, 208 [C_8,14-C_11,12 fission]⁺, 206 [250-CO₂]⁺, 109 [250-side chain]⁺, 190 [208-H₂O]⁺, 222, 236 [C_8,14-C_12,13 fission]⁺, 192 [236-CO₂]⁺, and 95 [236-side chain]⁺ supported the saturated nature of ring C. From this information, it was inferred that compound 1 possessed an unsaturated side-chain with a carboxylic group, and the hydroxyl group was located at C-9. The ¹H NMR spectrum of 1 showed two one-proton downfield singlets at δ 4.67 and 4.54 assignable to C-26 methylene protons. Three broad signals at iδ 0.91, 0.75, and 0.63, integrating for three protons each, were ascribed to tertiary Me-19, Me-29, and Me-18, respectively. A three-proton de-shielded signal at iδ 1.62 was associated with C-27 methyl function attached to the C-25 olefinic carbon. A six-proton broad signal at δ 0.85 was attributed to C-28 and C-30 methyl protons. The absence of any signal between δ 3.00 and 4.54 supported the tertiary nature of the hydroxyl group (Ali, 2001). The carboxylic group was located at C-21 due to disappearance of a doublet near iδ 0.95 for a C-21 secondary methyl functionality, which is commonly found in triterpenes of the lanostane type. The remaining methylene and methine protons resonated between δ 2.49 and 1.08. The ¹³C NMR spectrum of 1 displayed 30 carbon signals of the molecule. The important carbon signals appeared at iδ 177.12 (C-21), 150.20 (C-25), 109.52 (C-26), and 76.75 (C-9). Distortionless Enhancement by Polarization Transfer (DEPT) spectra indicated the signals for C-21, C-25, and C-9 as quaternary carbon and C-26 as a methylene carbon. These data led to establishing the structure of 1 as lanosta-25 (26)-en-9α-ol-21-oic acid. This is a new lanostene-type triterpene isolated from Z. vulgaris.

Compound 2 gave a positive test for Liebermann-Burchard reagent and effervescence with sodium bicarbonate solution. Its IR spectrum exhibited absorptions for hydroxyl group (3500 cm¹), carboxylic group (3080, 1695 cm⁻¹), δ-lactone (1740 cm⁻¹), and unsaturation (1640 cm⁻¹). Its mass and ¹³C NMR spectra indicated the molecular weight as 486, consistent with a tetracyclic triterpenic formula, C₃₀H₄₆O₅. The characteristic ion fragments appeared at m/z 468 [M-H₂O]⁺, 315 [M-CO₂]⁺, 440 [M-HCOOH]⁺, 422 [440-H₂O]⁺, and 407 [422-Me]⁺. The ion fragments at m/z 152 [C_5,6-C_9,10 fission]⁺, 137 [152-Me]⁺, 166 [C_6,7-C_9,10 fission]⁺, 151 [166-Me]⁺, 180 [C_7,8-C_9,10 fission]⁺, and 165 [180-Me]⁺, suggested a δ lactone structure in ring A bridging 19-CO-3-Oβ. The ion peaks at m/z 280, 206 [C_8,14-C_9,11 fission]⁺, 191 [206-Me]⁺, 236 [280-CO₂]⁺, 266 [C_8,14-C_11,12 fission]⁺, 205 [220-Me]⁺, 220 [266-CO₂]⁺, 234, 252 [C_8,14-C_12,13 fission]⁺, and 219 [234-Me]⁺ indicated the saturated nature of ring C. The ion peaks at m/z 109 [C₈H₁₅O, side chain]⁺ and 259 [C_14,15-C_13,17-fission]⁺ suggested (Ali, 2001) the presence of unsaturated side chain with hydroxyl group and carboxylic function at C-18. The ¹H NMR spectrum of compound 2 displayed two one-proton broad signals at δ 4.69, 4.58 assigned to C-26 methylene protons. A one-proton broad multiplet at iδ 3.92 (w1/2 15.6 Hz) was ascribed to C-3 oxygenated methine proton. A one-proton doublet of double doublets at iδ 3.00 (J = 4.5, 5.2, 11.2 Hz) was ascribed to C-22 iα carbinol proton. A three-proton broad signal at iδ 1.56 was ascribed to C-27 methyl protons attached to C-25 olefinic carbon. A three-proton doublet at δ 0.99 (J = 5.8 Hz) was attributed to C-21 secondary methyl protons. Three broad signals at iδ 0.90, 0.87, and 0.79, integrating for 3 protons each, were attributed to C-28, C-29, and C-30 tertiary methyl functionalities. The remaining methylene and methine protons appeared between iδ 2.50 and 1.06.

Among the 30 carbon signals of compound 2 in its ¹³C NMR spectrum, the important signals appeared at iδ 177.15 (19-lactone CO), 176.11 (18-COOH), 150.26 (C-25), 109.53 (C-26), 83.25 (carbinol C-3), and 65.52 (C-22 carbinol). The DEPT spectra of 2 exhibited the presence of 5 methyl, 12 methylene, 6 methine, and 7 quaternary carbons in the molecule. All of this evidence led to assign the structure of zizyphulanostan-18-oic acid (2) as lanosta-25(26)-en-22β-ol-18-oic acid, 3(19)-olide. This is the first report concerning the existence of δ-lactone containing triterpene in plants of the Zizyphus species.

Acknowledgments

The authors are grateful to the Head, Instrumentation Center, CDRI, Lucknow, and IIT, New Delhi, for recording spectroscopic data of the compounds.