Three new physalins from Physalis alkekengi var. franchetii

Introduction

The genus of Physalis (Solanaceae), including about 120 species, is widely distributed throughout South and North America, which also includes five species, and two variations which also can be found in China¹. The plant Physalis alkekengi var. franchetii, is used in traditional Chinese medicine², and is chiefly used for the treatment and prevention of tumors, leishmaniasis, sore throat, cough, eczema, hepatitis and urinary problems³. The chemical constituents of P. alkekengi var. franchetii are mainly composed of alkaloids, flavonoids, sterols, fatty acids, amino acids, and steroids⁴.

Physalins, as one of the characteristic constituents from P. alkekengi var. franchetii, is a type of steroids with 16, 24-cyclo-13, 14-seco-ergostane skeleton which has been established by X-ray crystallographic analysis^5-7. Neophysalins, are another characteristic structure, and are transformed from physalins through an acid-induced benzilic acid-type rearrangement reaction⁸. Until recently, more than 50 physalins and neophysalins have been identified from P. alkekegi var. franchetii, P. agulata, P. lancifolia and P. minima. Physalins and neophysalins have tremendous biological activities, including anti-tumor⁹, anti-microbial¹⁰, anti-malarial¹¹, immunosuppressive¹², anti-inflammatory^{13, 14}, immunomodulatory¹⁵, cytotoxic¹⁶, and trypanocidal^{17, 18} effects.

In this study, five physalins (1, 2, 4-6) and three neophysalins (3, 7, 8) were isolated from P. alkekengi var. franchetii, three of which (1-3) were new. Their chemical structures and their elucidation are described herein.

Results and Discussion

Compound 1 was obtained as a white powder. The molecular formula of 1 was determined to be C₂₈H₃₂O₁₂ by HREIMS (m/z 560.1885, [M]⁺; calcd 560.1894) and NMR data (Table 1). The ¹H NMR spectrum of 1 (in pyridine-d₅) showed four methyl signals at δ_H 1.29, 1.47, 1.65, and 2.02, four oxygenated signals at δ_H 4.01 (H-4), 3.54 (H-6), 5.70 (H-7) and 4.73 (H-22), and two olefinic proton signals at δ_H 6.24 (H-2) and 7.19 (H-3). The ¹³C DEPT spectrum of 1 (in pyridine-d₅) displayed the presence of twenty-eight carbons, including four methyls, three methylenes, ten methines (two olefinic carbons and four oxymethanes), eleven quaternary carbons (two ketone groups, two lactone groups, four oxygenated carbons, and one ketal carbon). These signals indicated the structural similarity of 1 and physalin J¹⁹. The chemical shift of the ketal carbon at C-14 (δ_C 103.4) of 1 differently from that (δ_C 107.8) of physalin J, together with one more methyl signal and less an oxygenated CH₂ in 1 indicated that the C(27)-O-C(14) ether bridge was ruptured. This was further determined by the HMBC correlations (Figure 1) from Me-27 (δ_H 1.29) to C-25 (δ_C 42.4) and C-26 (δ_C 173.0). Moreover, the HMBC correlations from δ_H 1.65 (H-19), 6.24 (H-2), and 7.19 (H-3) to δ_C 202.9 (C-1) demonstrated the presence of an conjugated 2-en-1-one group, and the ¹H-¹H COSY correlations of δ_H 7.19 (H-3)/δ_H 4.01 (H-4) revealed that there existed an hydroxyl group at C-4. An oxymethine in 1 instead of a methene in physalin J suggests that one hydroxyl group could be located at C-7, which was confirmed by the ¹H-¹H COSY correlations of δ_H 3.54 (H-6)/δ_H 5.70 (H-7) and δ_H 5.70 (H-7)/δ_H 2.93 (H-8). The relative configurations of H-8 and Me-28 in the physalin skeleton were established to be β-oriented by X-ray.^{20, 21} Thus, the NOE correlations (Figure 1) of H-4/H-6, H-6/H-7, H-7/H-8β, and H-27/Me-28 established the relative configuration of OH-4 and OH-7 to be α-oriented and Me-27 to be β-oriented. Hence, the structure of compound 1 was identified to be physalin Ⅲ (1).

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Compound 2 was obtained as a colorless needle crystal. The molecular formula of 2 was determined to be C₂₈H₃₂O₁₂ on the basis of HREIMS (m/z 560.1854, [M]⁺; calcd 560.1894) and NMR data (Table 1). The NMR data of 2 were similar to those of physalin Z²², and the differences between them were two of the double-bond carbon signals in physalin Z were replaced by two oxygenated methine signals in 2 located at C-6 and C-7, which were deduced by ¹H-¹H COSY correlations of H-8 (δ_H 3.39)/H-7 (δ_H 5.23) and H-7 (δ_H 5.23)/H-6 (δ_H 4.79) and by HMBC correlations from H-6 to C-10, C-4, C-8, C-7, and C-5, from H-7 to C-6, C-5, C-8, and C-14. The ROESY correlations (Figure 2) of H-6/H-7 and H-7/H-8β revealed that OH-C(6) and OH-C(7) were α-oriented. So the structure of compound 2 was deduced and named as physalin Ⅳ (2).

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Compound 3 was obtained as an amorphous yellow powder. The molecular formula of 3 was determined as C₂₉H₃₂O₁₀ using HREIMS and NMR data (Table 1). These NMR data of 3 were similar to those of physalin X²³, except the presence of an additional methoxyl group. Comparing with physalin X, the chemical shift of C-3 (δ_C 76.7) at down field and the HMBC correlation (Figure 3) of OMe (δ_H 3.27) with C-3 in 3 indicated a methoxyl group at C-3. The relative configuration of Me-19 and H-16 in physalin skeleton were established to be β-oriented by X-ray.^{20, 21} So the relative configuration of 3-OMe could be determined as α-oriented by the NOE correlations of Me-19/H-3 and H-16/H-3. Hence, the structure of compound 3 was identified to be 3-O-methylphysalin X (3).

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The structures of five known steroidal compounds, physalin Z (4)²², physalin C (5)²⁴, physalin I (6)²⁵, physalin P (7)²⁶, 25, 27-dihydro-4, 7-dedehydro-7-deoxyneophysalin A (8)²⁷, were elucidated in comparison to their spectroscopic data with those reported in the literature.

Experimental Section

General Experimental Procedures

Column chromatography (CC): silica gel (100-200 mesh, 200-300 mesh; Qingdao Marine Chemical Co., Ltd.), Sephadex LH-20 (Pharmacia). Thin-layer chromatography (TLC): silica gel 60F₂₅₄ (Qingdao Marine Chemical Co., Ltd.). Optical rotations: Jasco P-1020 polarimeter. UV Spectra: Shimadzu UV-2401 PC spectraphotometer. IR Spectra: Bruker Tensor-27 infrared spectrophotometer, with KBr pellets in cm^-1. NMR Spectra: Bruker Bruker Avance Ⅲ-600MHz spectrometer; chemical shifts δ were recorded as ppm relative to TMS, coupling constants J in Hz. ESIMS: API QSTAR Pulsar spectrometer; HREIMS: Waters Autospec Premier P776.

Plant Material

The calyces of Phsalis alkekengi var. franchetii were purchased in Kunming CTM market, Kunming city, Yunnan province at December 2011 and were identified by Prof. Xi-Wen Li who was botanic taxonomist in Kunming Institute of Botany.

Extraction and Isolation

Dried calyces (15 kg) of P. alkekengi var. franchetii were extracted with 80% EtOH under reflux. The extract was suspended in water (5 L) and then partitioned successively with PE (petroleum ether) (8 L × 4) and EtOAc (8 L × 5). The EtOAc-soluble layer (evaporated under vacuum to get 250 g) eluted successively with CHCl₃, CHCl₃-MeOH (100:1), CHCl₃-MeOH (20:1), and CHCl₃-MeOH (5:1). Then, the CHCl₃-MeOH (100:1) portion (80 g) was chromatographed on a silica gel column using a gradient of CHCl₃-CH₃OH (100:0 to 0:100), which yielded five fractions (1-5). Fraction 2 was successively subjected to RP-18, Sephadex LH-20 and silica gel to derive subfractions Ⅰ-Ⅳ. Subfraction Ⅱ was further separated over silica gel with CHCl₃-CH₃OH (120:1) to yield compound 3 (5 mg), 6 (4 mg), 7 (10 mg) and 8 (3 mg). Subfraction Ⅲ was subjected to semipreparative HPLC (CH₃CN-H₂O, 50:50) to produce 4 (1 mg) and 5 (1 mg). Fraction 3 was successively subjected to RP-18, Sephadex LH-20 and separated over silica gel with CHCl₃-CH₃OH (80:1) to yield 1 (2 mg) and 2 (6 mg).

Physalin Ⅲ (1)

white powder; [α]_D²¹-29.33 (c 0.2, CHCl₃); UV (CHCl₃): λ_max (log ε): 240 (3.36) nm; IR (KBr): v_max 3430, 2924, 1787, 1758, 1728, 1462, 1376, 1218, 1073, 1041 cm^-1; ¹H and ¹³C NMR: see Table 1. Negative ESIMS: 559 ([M-H]^-). HREIMS: 560.1885 [M]⁺ (calcd. for C₂₈H₃₂O₁₂, 560.1894).

Physalin Ⅳ (2)

colorless needle crystal (CHCl₃); [α]_D¹⁶ -114.40 (c 0.1, MeOH); UV (MeOH): λ_max (log ε): 201 (4.23) nm; IR (KBr): v_max 3431, 1781, 1729, 1632, 1383, 1374, 1167, 1134, 1065 cm^-1; ¹H and ¹³C NMR: see Table 1. Positive ESIMS: 583 ([M + Na]⁺). HREIMS: 560.1854 [M]⁺ (calcd. for C₂₈H₃₂O₁₂, 560.1894).

3-O-Methylphysalin X (3)

yellow powder; [α]_D¹⁸ -16.00 (c 0.1, pyridine); UV (MeOH): λ_max (log ε): 225 (3.77), 201 (3.80) nm; IR (KBr): v_max 3432, 2923, 1786, 1629 cm^-1; ¹H and ¹³C NMR: see Table 1. Positive ESIMS: 563 ([M + Na]⁺). HREIMS: 540.1933 [M]⁺ (calcd. for C₂₉H₃₂O₁₀, 540.1995).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0021-z and is accessible for authorized users.

Notes

Acknowledgements

This project was financially supported by the National Major Basic Research Program (No. SB2007FY400) of the National Ministry of Science and Technology of China, the National Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX2-YW-G-038), and the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).

Compliance with Ethical Standards