Four New Cucurbitane Glycosides from Hemsleya jinfushanensis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A phytochemical study of the tubers of Hemsleya jinfushanensis L. T. Shen resulted in the isolation of four new cucurbitane glycosides, jinfushanosides A - D (1 - 4), as well as four known compounds 5 - 8. Compounds 1 - 7 were tested for bioactivity against rabbit platelet aggregation induced by PAF, ADP, or AA. Among them, compounds 1, 5, 6 and 7 weakly inhibited PAF-induced platelet aggregation.

A number of plants of the Hemsleya spp. (Cucurbitaceae) grow abundantly in the Yunnan and Sichuan Provinces of China [1], [2], [3]. The tubers of Hemsleya species have been used as an antibacterial remedy for the treatment of sore throat, toothache, diarrhea, dysentery, bronchitis, and chronic cervicitis [4], [5]. It has been discovered that cucurbitane triterpene glycosides are efficient in the treatment of acute dysentery, tuberculosis, and bronchitis [4]. Until now, thirty-nine cucurbitane triterpene compounds have been isolated from the genus Hemsleya [5], [6], [7], [8], [9], [10], [11], but there has been no report on phytochemical studies of Hemsleya jinfushanensis.

From the ethanolic extract of the tubers of H. jinfushanesis L. T. Shen, eight cucurbitane triterpene compounds, including four new saponins, jinfushanosides A (1), B (2), C (3) and D (4), were isolated. Their structures were determined as 11α,26,27-trihydroxy-3-O-(β-D-glucopyranosyl)-cucurbit-5,24-diene, 26,27-dihydroxy-3-O-(β-D-glucopyranosyl)-cucurbit-5,24-dien-11-one, 27-hydroxy-3-O-(β-D-glucopyranosyl)-26-O-(β-D-glucopyranosyl)-cucurbit-5,24-dien-11-one, and 3β,27-dihydroxy-26-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-cucurbit-5,24-dien-11-one, respectively, by means of spectral analysis and chemical methods (Fig. [1]). The four known compounds were identified as carnosifloside I (5) [12], scandenoside R8 (6) [10], dihydrocucurbitacin F (7) [13] and 25-O-acetyl-23,24-dihydrocucurbitacin F (8) [13] by comparison of their spectral data to those reported in literature (Fig. [1]).

A close comparison of the NMR data (see Tables [1] and [2]) of jinfushanoside A (1) and those of a known compound, carnosiflogenin C (9) [12], indicated that the two compounds were very similar except for one additional hydroxy group and one additional sugar unit in 1. The chemical shift value of C-3 in 9 was downfield shifted to δ_C = 87.9 in 1, which indicated that the sugar unit was linked to C-3 in 1. The correlation of H-3 (δ_H = 3.66, br s) with C-1 (δ_C = 107.4, d) of the sugar unit and H-1 of the sugar with C-3 in the HMBC spectrum of 1 supported this conclusion. A methyl carbon signal at δ_C = 14.0 (q) due to C-27 in 9 [12] was replaced by a hydroxymethyl signal at δ_C = 58.5 (t) in 1, along with the signal of C-25 being shifted downfield by 4.6 ppm, which manifested one hydroxy group attached to C-27 in 1. The HMBC correlations of the proton signal at δ_H = 4.72 (2H, br s) with C-24 (δ_C = 127.6, d), C-25 (δ_C = 140.7, s) and C-26 (δ_C = 65.4, t) in 1 further confirmed our elucidation. The only difference between 1 and 2 was that the hydroxymethine [δ_H = 4.18 (1H, m) and δ_C = 77.8 (d)] due to C-11 in 1 was replaced by a ketone carbonyl (δ_C = 214.1, s) in 2. The carbon correlations of the ketone with H-8, 10, methyl-19 in the HMBC spectrum of 2 confirmed that the ketone carbonyl was positioned at C-11. As shown in Tables [1] and [2], the NMR signal patterns of jinfushanosides C (3) and D (4) were very similar to those of 2 except for one additional sugar unit in 3 and 4 respective. The carbon signal  δ_C = 64.6 (t, C-26) in 2 was downfield shifted 8.2 ppm to δ_C = 72.8 (t) in 3 suggesting that the additional sugar was attached to C-26. This suggestion was further supported by the correlations of H-1 of the additional sugar with C-26 in the HMBC spectra of 3. The carbon signal δ_C = 62.5 (t) attributable to the C-6 of the sugar in 2 was up-field shifted to δ_C = 70.0 (t) in 4, which indicated that the additional sugar was attached to the C-6 of this sugar in 4. The correlation between H-6 of the inner sugar and C-1 of the terminal sugar in the HMBC spectrum of 4 suggested that the two sugars form a gentiobiosyl unit. Partial acid hydrolysis of 4 and 3 also yielded 2, together with D-glucose, respectively. The sugars were determined to be a D-glucose on the basis of the NMR spectra and by comparison with trade samples run on GC and the acid hydrolysate of the compounds [14]. Hence, the structures of jinfushanosides A, B, C and D were deduced as above.

Compounds 1 - 7 were tested for their in vitro inhibition activity on rabbit platelet aggregation induced by PAF (platelet activating factor), ADP (adenosine diphosphate), or AA (arachidonic acid) using the bioassay methods described [15]. Ginkgolide B (BN52021) and acetylsalicylic acid (ASA) were used as positive controls, and 2 % PEG (polyethylene glycol) was used as contrast. Compounds 1, 5, 6 and 7 weakly inhibited rabbit platelet aggregation induced by PAF (see Supporting Information).

Materials and Methods

General: Melting points were measured on an XRC-1 micromelting point apparatus and are uncorrected. IR spectra were obtained on a Bio-Rad FTS-135 infrared spectrometer with KBr pellets. UV spectra were recorded on a Shimadzu double-beam 210A spectrophotometer in MeOH. Optical rotations were taken on a SEPA-300 polarimeter. Mass spectral data were obtained on a VG Autospec-3000 spectrometer. NMR spectra and 2D-NMR, including H¹-H¹ COSY, HMQC, HMBC and ROESY experiments, performed and used for signal assignment, were measured in pyridine-d ₅ on Bruker AM-400 and (or) DRX-500 instruments at 25 °C with TMS as internal standard. The authentic sample of β-D-(+)-glucose (G5250) was obtained from Sigma. Spots were visualized by heating silica gel plates sprayed with 20 % H₂SO₄ in H₂O.

Plant material: The tubers of H. jinfushanensis were collected in Jinfushan, Congqing City, China, in 2000. The plant material was identified by Prof. Wen-Jin Zhan, and a specimen has been deposited in the Herbarium of the Kunming Institute of Botany.

Extraction and isolation: Air-dried and powdered tubers (2.0 kg) of Hemsleya jinfushanensis were extracted with 95 % ethanol under reflux (3 × 8 L) and filtered. The combined filtrate was concentrated under vacuum resulting in 411 g residue. The residue (322 g) was absorbed on 800 g of silica gel and chromatographed over a silica gel (10 × 150 cm, 2.5 kg) column eluted with a gradient system of CHCl₃ (10 L), CHCl₃-Me₂CO (15 : 1/12 L, 9 : 1/12 L) and CHCl₃-EtOH (9 : 1/10 L, 7 : 3/10 L) to give five fractions (Frs. 1 - 5). Compound 8 (39.1 g) was recrystallized from Fr. 2 in acetone. Fraction 3 (2.83 g) was fractioned on silica gel (10 g) and developed with a gradient system of CHCl₃-MeOH (20 : 1, 15 : 1, 12 : 1, each 800 mL) resulting in three fractions (Fr. 3.1 - 3.3). Fr. 3.2 (1.6 g) was rechromatographed over silica gel (8 g) and eluted with CHCl₃-CH₃OH (15 : 1, 1200 mL) to afford two subfractions. The first one (900 mg) was purified on Sephadex LH-20 using acetone (200 mL) as eluent to give 7 (49 mg). Fr. 4 (12.8 g) was subjected to CC (20 g) resulting in four fractions (Frs. 4.1 - 4.4) eluted with CHCl₃-CH₃OH (12 : 1, 9 : 1, 85 : 15, each 1 L). Fr.4.2 (600 mg) was rechromatographed on silica gel (5 g) and developed with CHCl₃-MeOH (12 : 1, 500 mL) to give subfraction 4.2.1 and subfraction 4.2.2. Subfraction 4.2.2 (200 mg) was repeatedly subjected to CC on RP₁₈ silica gel (4 × 30 cm, 85 g) developed with aqueous MeOH 60 % → 70 % → 100 % (each in 1000 mL) to afford compounds 1 (35 mg) and 2 (19 mg). Fr. 4.3 (8.43 g) was rechromatographed on silica gel (50 g) developed with CHCl₃-CH₃OH (12 : 1/1.5 L, 9 : 1/2.0 L, 85 : 15/1.5 L) resulting in parts A and B. Compound 5 (500 mg)was obtained from part A (1 g), which was purified over Sephadex LH-20 (100 g) using MeOH (300 mL) as eluent. Part B (5.51 g) was repeatedly subjected to CC on RP₁₈ silica gel (4 × 30 cm, 85 g) and developed with aqueous MeOH 50 % → 60 % → 70 % → 100 % (each 3000 mL) to give parts B1 and B2. Compound 6 (100 mg) was isolated from part B1 (1.5 g) by silica gel CC (20 g) eluted with CHCl₃-CH₃OH (9 : 1, 600 mL). Part B2 (2.5 g) was further rechromatographed over RP₁₈ silica gel (4 × 30 cm, 85 g) and developed with aqueous MeOH 50 % → 60 % → 70 % → 100 % (each 1800 mL) to afford compounds 3 (45 mg) and 4 (25 mg).

Acid hydrolysis of 1 - 4: Compounds 1 - 4 (10, 9, 15, 10 mg, respectively) were boiled under reflux with 1 mol L^-1 HCl-CH₃OH (1 : 1, v/v, 4 mL) in a water bath at 90 °C for 6 h. After 3 h, compound 2 was detected in the reaction mixture of compounds 3 and 4 by TLC [3: R_f = 0.50 (CHCl₃-CH₃OH, 3 : 1), 2: R_f = 0.39 (CHCl₃-CH₃OH, 5 : 1), 4: R_f = 0.46 (CHCl₃-CH₃OH, 3 : 1)]. After the hydrolysis was finished, the water-soluble part was evaporated to dryness. The dry sugar residue (10 mg) and an authentic sample of d-(+)-glucose (Sigma, G5250) were diluted in 1 mL pyridine without water and treated with 0.4 mL hexamethyldisilazane and 0.2 ml trimethylchlorosilane (TMCS, Fluka) at 0 °C. The reaction mixture was then incubated for 6 h at 20 °C. After that, the upper layer of the reaction mixture was analyzed by GC. (GC conditions: AC-5 capillary column (30 m × Ø0.25 mm); column temperature: 180 - 260 °C; column head pressure: 12 Pa; carrier gas: N₂). GC of all samples showed an identical retention time, R_t (m):11.521.

Compound 1: C₃₆H₆₀O₉, white powder, m. p. 146 - 149 °C; [α]_D ²²: 38.46 (c 0.13, MeOH); UV (MeOH): λ_max (log ε) = 202.8 nm (5.08); IR (KBr): ν_max = 3428, 2934, 2875, 1634, 1464, 1380, 1073, 1027 cm^-1; ¹³C-NMR and ¹H-NMR data, see the Tables [1] and [2]. HR-FAB-MS: m/z = 635.4157 [M - H]^- (calcd. for C₃₆H₅₉O₉ : 635.4159).

Compound 2: C₃₆H₅₈O₉, white powder, m. p. 117 - 121 °C; [α]_D ²⁴: 115.79 (c 0.095, MeOH); UV (CH₃OH): λ_max (logε) = 209.0 nm (5.66) IR: ν_max = 3440, 2925, 2877, 1690, 1630, 1462, 1384, 1074, 1017 cm^-1; HR-FAB-MS: m/z = 633.4016 [M - H]^- (calcd. for C₃₆H₅₇O₉ : 633.4002).

Compound 3: C₄₂H₆₈O₁₄, white powder, m. p. 149 - 152 °C; [α]_D ²¹: 72.73 (c 0.11, MeOH); UV (MeOH): λ_max (log ε) = 202.2 nm (5.11); IR (KBr): ν_max = 3428, 2928, 2877, 1693, 1634, 1588, 1540, 1463, 1383, 1266, 1075 cm^-1; HR-FAB-MS: m/z = 795.4542 [M - H]^- (calcd. for C₄₂H₆₇O₁₄ : 795.4530), 633 (20).

Compound 4: C₄₂H₆₈O₁₄, white powder, m. p. 156 - 159 °C; [α]_D ²²: 63.16 (c 0.095, MeOH); UV (MeOH): λ_max (logε) = 202.0 nm (4.98); IR (KBr): ν_max = 3429, 2927, 2877, 1683, 1634, 1558, 1539, 1384, 1166, 1073, 1031 cm^-1; HR-FAB-MS: m/z = 785.4502 [M - H]^- (calcd. for C₄₂H₆₇O₁₄ : 795.4530), 633 (16), 341 (35).

Acknowledgements

This work was supported by the NKIP Foundation of CAS (KSCZX-SW-301 - 08) and NSFC (No.39970086) and SFC of Yunnan Province (No.98C089M). The authors are grateful to Prof. Ling Li (Yunnan Pharmacological Laboratory of Natural Products, Kunming Medical College, Kunming, China) and Mr. Xian-Wen Yang for bioassays.

References

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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 14 Hu K, Dong A J, Yao X Sh, Kobayashi H, Iwasaki S. Antineoplastic agents; II. Four furostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca .  Planta Med. 1997;  63 161-5
  PubMedSearch in Google Scholar
• 15 Wang B G, Hong X, Li L, Zhou J, Hao X J. Chemical constituents of two Chinese Magnoliaceae plants, Tsoongiodendron odorum and Mangietiastrum sinicum, and their inhibition of platelet aggregation.  Planta Med. 2000;  66 511-5
  Thieme ConnectPubMedSearch in Google Scholar

Prof. Dr. Ming-Hua Qiu

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

The Chinese Academy of Sciences

610 Longquan Road

Kunming 650204

Yunnan Province

People's Republic of China

Fax: +86-871-5150227

Email: mhchiu@mail.kib.ac.cn

Email: mhchiu@public.km.yn.cn

References

• 1 Chang W J, Shen L D. The genus Hemsleya cogn. in Sichuan.  Acta Phytotax Sin. 1983;  21 182-93
  PubMedSearch in Google Scholar
• 2 Zheng C Z. A new species of Hemsleya cogn. from Zhejiang.  Acta Phytotax Sin. 1985;  23 67-8
  PubMedSearch in Google Scholar
• 3 Wu C Y. Materia ad floram cucurbitacearum sinensium-Hemsleya cogn.  Acta Phytotax Sin. 1985;  23 121-43
  PubMedSearch in Google Scholar
• 4 Jiangsu Medical C ollege. Encyclopedia of Chinese Materia Medica. Shanghai; Shanghai Scientific Technology Press; 1977. Vol. 1:: p. 1358-9.
  Search in Google Scholar
• 5 Pharmacopoeia of  China. Part 1. Beijing; People’s Health Press; 1977.: p. 531-4.
  Search in Google Scholar
• 6 Chen W X, Nie R L, Chen Y C, Hsia K M. The structure of 23,24-dihydrocucurbitacin F-25-acetate and 23,24-dihydrocucurbitacin F.  Acta Chem Sin. 1975;  33 49-52
  PubMedSearch in Google Scholar
• 7 Riu H K, Yuan M Y, Yu Q M, Ye X Q, Qian G H, Wang K L. The structure of cucurbitacin II glycoside.  Acta Pharm Sin. 1981;  16 445-7
  PubMedSearch in Google Scholar
• 8 Meng X J, Chen Y Z, Nie R L, Zhou J. A new cucurbitacin from Hemsleya graciliflora .  Acta Pharm Sin. 1985;  20 446-9
  PubMedSearch in Google Scholar
• 9 Kasai R, Matsumoto K, Nie R L, Zhou J, Tanaka O. Glycosides from Chinese medical plant, Hemslera panacis-scandens, and structure-taste relationship of cucurbitane glycosides.  Chem Pharm Bull. 1988;  36 234-43
  PubMedSearch in Google Scholar
• 10 Kubo H, Ohtani K, Kasai R, Yamasaki K, Nie R L. Cucurbitane glycosides from Hemsleya panacis-scandens rhizomes.  Phytochemistry. 1996;  41 1169-74
  CrossrefPubMedSearch in Google Scholar
• 11 Chen Y, Qiu M H, Gu K, Chen J C. A new cucurbitacin and a new oleanolic acid glycosides from Hemsleya gigantha .  Acta Bot Yunnan. 2003;  25 613-9
  PubMedSearch in Google Scholar
• 12 Kasai R, Matsumoto K, Nie R L, Morita T, Awazu A, Zhou J, Tanaka O. Sweet and bitter cucurbitane glycosides from Hemsleya carnosiflora .  Phytochemistry. 1987;  26 371-6
  CrossrefPubMedSearch in Google Scholar
• 13 Nie R L, Morita T, Kasai R, Zhou J, Wu C Y. Saponins from Chinese medicinal plants. Isolation and structures of hemslosides.  Planta Med. 1984;  50 322-7
  PubMedSearch in Google Scholar
• 14 Hu K, Dong A J, Yao X Sh, Kobayashi H, Iwasaki S. Antineoplastic agents; II. Four furostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca .  Planta Med. 1997;  63 161-5
  PubMedSearch in Google Scholar
• 15 Wang B G, Hong X, Li L, Zhou J, Hao X J. Chemical constituents of two Chinese Magnoliaceae plants, Tsoongiodendron odorum and Mangietiastrum sinicum, and their inhibition of platelet aggregation.  Planta Med. 2000;  66 511-5
  Thieme ConnectPubMedSearch in Google Scholar

Prof. Dr. Ming-Hua Qiu

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

The Chinese Academy of Sciences

610 Longquan Road

Kunming 650204

Yunnan Province

People's Republic of China

Fax: +86-871-5150227

Email: mhchiu@mail.kib.ac.cn

Email: mhchiu@public.km.yn.cn

• Supplementary Material