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Marine Drugs 2004, 2(4), 185-191;

Article
Philinopgenin A, B, and C, Three New Triterpenoid Aglycones from the Sea Cucumber Pentacta quadrangulasis
Shi-Long Zhang , Ling-Li , Yang-Hua Yi *, Zheng-Rong Zou and Peng-Sun
Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, P. R. China. Fax: (+86)-21-65483662
*
Author to whom correspondence should be addressed; E-mail: yiyanghua@hotmail.com
Received: 17 May 2004 / Accepted: 16 October 2004 / Published: 25 November 2004

Abstract

: Three new triterpenoid aglycones named Philinopgenin A (1), B (2), and C (3) were isolated from the acid hydrolysate of the crude glycoside mixture prepared from the whole sea cucumber Pentacta quadrangulasis Lesson. The corresponding structures were determined as 16β-acetoxyholosta-8(9), 24(25)-diene-3β-ol (1), 20, 25-epoxy-lanosta-9(11)-ene-3β-ol 18(16)–lactone (2) and 16β-acetoxyholosta-9(11), 24(25)-diene-3β-ol (3), respectively, on the basis of spectral evidence.
Keywords:
Pentacta quadrangulasis Lesson; Philinopgenin A; Philinopgenin B; Philinopgenin C; triterpenoid aglycone

Introduction

Investigations of triterpene glycosides in sea cucumbers (Holothuroidea, Echinodermata) have a long history. In the search for new biologically active substances from marine organisms, we have been investigating antitumour constituents in the sea cucumber Pentacta quadrangulasis Lesson, which is widely distributed throughout the South China Sea, especially in the area near Guangdong. Before examining the components of this sea cucumber to elucidate the structure of the biologically active glycosides, we investigated the aglycones. We report herein the isolation, purification, and structural elucidation of three new aglycones named Philinopgenin A (1), B (2), and C (3) from the acid hydrolysate of the crude glycoside fraction.

Marinedrugs 02 00185f2 1024

Results and Discussion

The molecular formula of Philinopgenin A (1) was determined as C32H48O5 (m/z 512) by the pseudomolecular ion peaks at m/z 512 ([M]+) and 535 ([M + Na]+) in the HRESI-MS (positive-ion mode). The IR spectrum showed absorptions due to hydroxyl (3443 cm−1), γ-lactone (1770 cm−1) and carbonyl (1748 cm−1) functions. The structure of compound 1 was deduced from extensive NMR spectroscopy studies (1H- and 13C-NMR, DQCOSY, HMQC, and HMBC, Table 1). These spectra indicated that compound 1 is a triterpenoid compound with two olefinic bonds, one ester, and one lactone carbonyl group. These features showed a close similarity to those reported for the triterpenoid lactone aglycone stichopogenin A2, isolated from the acid hydrolysate of holotoxin A [1]. The 13C-NMR and DEPT spectra exhibited 32 carbon signals (8×CH3, 8× CH2, 7×CH, 9×C). The 1H-, 13C- and DEPT spectra of compound 1 showed resonances for a 9(11)-double bond [δC 110.8 (s, C-11) and 151.2 (S, C-9)] and those due to an acetoxy group [δC 170.0(s) and 21.4(q); δH 1.93(3H, s)]. The location of the acetoxy group at C-16 was deduced from the chemical shift of the H-16 signal (δ 5.76), which showed coupling to signals at δ 2.57 (H-17α), 2.19 (H-15α), and 1.43 (H-15β) in the DQCOSY spectrum. This conclusion was also confirmed by the cross peak at δ 5.76/170.0 (H-16/CH3CO) in HMBC spectrum. The 16β configuration of the acetoxy group was confirmed by NOESY experiments and by coupling constant analysis for the C-16 proton with the C-17α proton (see Figure 1a). The calculated coupling constant value of 9.6Hz (J16α, 17α) for the most stable conformation of 16β-acetoxy-holosta-9,24-diene-3β-ol obtained by Molecular Mechanics (Hyperchem) coincided with the experimental and reported values [2] and differed considerably from those calculated for 16α-isomer [1.2Hz (J16β, 17α)]. The 1H-NMR spectrum showed two vinyl methyl signals at δ 1.50 and 1.57 and one olefinic proton signal at δ 5.04, assigned to the terminal isopropenyl group of the side chain. The 13C-NMR spectrum confirmed the presence of this group: two methyl groups at δ 25.5 (C-26) and 17.7 (C-27) attached to olefinic carbons at δ 131.8 (C-25) and 124.4 (C-24). This conclusion was also confirmed in the HMBC spectrum. Therefore the structure of compound 1 was determined as 16β-acetoxyholosta-9(11), 24(25)-diene-3β-ol.

The molecular formula of Philinopgenin B (2) was determined as C30H46O4 (m/z 470) by pseudomolecular ion peaks at m/z 471 ([M+H]+) and 493 ([M+Na]+) in the HRESI-MS (positiveion mode). The IR spectrum showed absorptions due to hydroxyl (3446cm−1) and γ-lactone (1770cm−1) groups. The 13C-NMR and DEPT spectrum exhibited 30 carbon signals (7×CH3, 9× CH2, 6×CH, 8×C). The 1H- and 13C-NMR spectra of compound 2 (Table 1) showed some similarities to those of compound 1 and to those of the aglycone moieties of cucumarioside G2[3]. The main difference from compound 1 being the replacement of the 16 OAc-substitutuent (δC 75.0, δH 5.63) by one oxygen-bearing methine group (δC 79.2, δH 4.77) forming the 18(16)-lactone with the C-18 carbonyl group, in agreement with the chemical shifts for C15 and C17, and the replacement of the side chain by a 2,2-dimethyl-6-pyranyl moiety. The quaternary carbons at δC 73.0 and 71.8 suggested that the side chain is in a pyran form. The correct assignments for the side chain are based the fact that the carbon peaks at δC 73.0 (C-20) and 28.2 (C-22) ppm correlate with the methyl protons H-21 and the carbon peaks at δC 71.8 (C-25) and 36.5 (C-24) ppm correlate with the methyl protons H-26 and H-27 in the HMBC spectrum. Furthermore, the HMQC spectrum of compound 2 shows that H-22, H-23 and H-24 correlate with the carbon peaks at δC 33.8 ppm, 36.5 and 71.8 ppm respectively. The chemical shifts of the H-22 signals (δ 1.48) and H-24 signals (δ 1.36, 1.51) showed coupling to signals at δ 1.58 and 1.76 (H-23) in the DQCOSY spectrum. This conclusion was also confirmed in the NOESY spectrum (see Figure 1b). Thus, compound 2 was determined as 20,25-epoxylanosta-8(9)-ene-3β-ol 18(16)-lactone.

The molecular formula of Philinopgenin C (3) was determined as C32H48O5 (m/z 512) by pseudomolecular ion peaks at m/z 535 ([M+Na]+) in the HRESI-MS (positive-ion mode). Compound 3 shows spectral features (Table 1) similar to those of compound 1, except for the signals due to the carbon atoms at the B/C-ring junction. These findings suggest that compound 3 is an olefinic function regioisomer of compound 1. The two olefinic carbon signals at δ=130.2 (s) and 135.6 (s) in the 13C-NMR spectrum of compound 3 indicate that compound 3 is the Δ8(9) isomer of 1. Thus, compound 3 can be identified as 16β-acetoxyholosta-8(9), 24(25)-diene-3β-ol.

About 40 genuine and artifact triterpenoid aglycones have been obtained by acidic or enzymatic hydrolysis of crude sea cucumber glycosides [4]. The newly obtained aglycones, described in this paper, differ from previously reported ones in their substitution patterns. It should be noted, however, that compounds 1, 2, and 3 may be artifacts arising from the extraction treatment, because the coexistence of minute amounts of other not identified regioisomers was noted in the acid hydrolysate of the glycoside mixture, and signals due to a trisubstituted olefin group (Δ7(8)) were observed in the NMR spectra of the parent oligoglycoside sulfates of this sea cucumber.

Experimental

General

Melting points were determined with a XT5-XMT micro melting point apparatus and are uncorrected. IR spectrum was measured on a BRUKER VECTOR-22 spectrophotometer. The ESIMS (positive and negative ion modes) was obtained on Micromass Quattro mass spectrometer. 1HNMR and 13C-NMR spectra were recorded in CDCl3 (at 100 MHz and 400 MHz for 13C- and 1HNMR, respectively) with an Inova-400 spectrometer. Chemical shifts are given in δ values with tetramethylsilane (TMS) as internal standard in 1H-NMR. Silica gel (Yantai, 200–300 mesh) was used for column chromatography. Pre-coated silica gel plates (Yantai, HSG, 0.15–0.20mm) were used for analytical TLC. HPLC was carried out with Zorbax 300 SB-C18 reverse phase.columns (9.4×250mm, Zorbax).

Animal material

Specimens of P. quadrangnlaris were collected at different locations around the South China Sea (near Guangdong Province, China) and identified by Prof. J. R. Fang of Fujian Institute of Oceanic Research, China in May 2002. A voucher specimen (No. SA200042) is deposited at the Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, China.

Extraction and Purification

Air-dried body walls of the sea cucumber Pentacta quadrangulasis (10kg) were extracted with 50% EtOH (50 L). The EtOH was evaporated in vacuo to give a crude extract (23.1g), which was partitioned between water (15 L) and chloroform (15 L). The water layer was extracted with nbutanol (15 L) and the organic layer was evaporated in vacuo to give the n-butanol extract (16.3g). A portion of this extract (6.0g) was heated at reflux in aqueous 15% H2SO4 (2000 mL) and the cooled reaction mixture was then extracted with three portions of chloroform (800 mL, 600 mL and 600 mL), the combined chloroform extracts were washed with water (300 mL), dried over Na2SO4 and concentrated to give an aglycone mixture (1.890g). This mixture was separated by flash chromatography on silica gel (3×40cm column, eluent: 4:1 n-hexane/EtOAc, flow rate: 1.5 mL/min) with detection of eluates by TLC (SiO2, 2:1 n-hexane/EtOAc; SCRC reagent) to yield a main fraction (600.9 mg, vR: 80mL–160mL). This fraction was further separated by HPLC (85% MeOH/H2O, flow rate 1.5 mL/min) to afford Philinopgenin A (1) (54.4 mg, tR: 15.91min), Philinopgenin B (2) (63 mg, tR: 16.86min) and Philinopgenin C (3) (77.7 tR: 17.50min).

Spectral Data

  • Philinopgenin A (1): a white powder, mp 208.5–208.5 °C; IR (KBr): ν = 3443, 2970, 2937, 2869, 1770, 1748, 1378, 1240 cm−1; EIMS: m/z = 512 [M]+, 493[M-H2O, A], 479 [A-CH3, B], 435, 69 [C5H9, base peak]; 13C-NMR and 1H-NMR: see Table 1.

  • Philinopgenin B (2): a white powder, mp 212.5–213.5°C; IR (KBr): ν = 3446, 2968, 2934, 2869, 1770, 1649 cm−1; EIMS: m/z = 470 [M+], 452 [m+-H2O], 391, 109, 69 [C5H9, base peak]; 13C-NMR and 1H- NMR: see Table 1.

  • Philinopgenin C (3): a white powder, mp 216.5–217.5 °C; IR (KBr): ν = 3466, 2968, 2935, 1869, 1770, 1748, 1649, 1030 cm−1; EIMS: m/z = 513 [M+1], 494 [M+1-H2O, A], 479 [A-CH3, B], 435, 69 [C5H9, base peak]. 13C-NMR and 1H-NMR: see Table 1.

Marinedrugs 02 00185f1 1024
Figure 1. Key NOESY connections in compounds 1 (a), 2 (b) and 3 (c)

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Figure 1. Key NOESY connections in compounds 1 (a), 2 (b) and 3 (c)
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Table Table 1. 13C- and 1H NMR data for compounds 1, 2, and 3

Click here to display table

Table 1. 13C- and 1H NMR data for compounds 1, 2, and 3
Compound123

PositionδC multaδH multb (J in Hz)δC multaδH multb (J in Hz)δC multaδH multb (J in Hz)
136.6tHα 1.44 m35.9tHα 1.43 m36.0tHα 1.45 m
Hβ 1.80Hβ 1.87 mHβ 1.84 m
228.0tHα27.8tHα1.68 m27.8tHα
HβHβ 1.72 mHβ
379.1d3.34, dd (5.2, 11.6)78.3d3.20 dd, (4.6, 11.6)79.0d3.21 dd, (5.2, 11.6)
440.0s39.2s38.5s
552.6d0.90, d, 11.052.6d0.86 tr52.5d0.90 m
621.4tHα 1.58 m21.1tHα 1.46 m21.0tHα 2.07 m
Hβ 1.75 mHβ 1.70 mHβ 2.07 m
728.0tHα28.3tHα 1.78 m27.8tHα 1.36 m
HβHβ 1.78 mHβ 1.62 m
839.6d3.22, dd (2.4, 13.2)43.4d130.2s
9151.2s151.3s135.6s
1040.5s39.4s39.3s
11110.8d5.16s113.4d5.40 d, (6.0)27.8t
1234.0tHα 2.36 m24.9tHα 2.33 m32.3tHα 2.40 m
Hβ 2.36 mHβ 2.40 mHβ 2.45 m
1358.6s54.8s59.6s
1443.2s40.4s44.7s
1544.1tHα 2.19 m42.0tHα1.56 m41.1tHα 2.22 m
Hβ 1.43mHβ 1.74 mHβ 1.42m
1675.0d5.76 m79.2d4.77 tr75.0d5.63 m
1752.9d2.57 d (9.6)63.5d2.25 m52.3d2.51 d, (9.6)
18176.7s178.1s176.8s
1922.3q1.30 s21.5q1.25 s21.8q1.19 s
2084.8s73.0s84.4s
2128.8q1.36 s26.0s1.25 s28.2q1.50 s
2238.5tHα 1.96 m33.8tHα1.48 m38.4tHα 1.76 m
Hβ 2.30mHβ 1.48 mHβ 2.42 m
2323.8tHα 1.98 m16.3tHα 1.58 m23.5tHα 1.92 m
Hβ 2.02 mHβ 1.76 mHβ 2.11 m
24124.4d5.04 d (11.2)36.5tHα 1.36 m)123.7d5.05 d, (11.2)
25131.8s71.8s132.1s
2625.5q1.57 s32.7q1.13 s25.5q1.69 s
2717.7q1.50 s28.4q1.20 s18.6q1.60 s
3016.4q0.98 s15.2q0.80 s15.4q0.85 s
3128.6q1.10 s28.3q0.98 s28.1q1.02 s
3221.4q0.80 s21.7q1.07 s27.0q1.10 s
CH3COO170.0s170.3s
CH3COO21.4q1.93 s21.0q2.02 s

aRecorded at 100MHz in CDCl3; multiplicity by DEPT.bRecorded at 400MHz in CDCl3

Acknowledgments

This work was supported by the State Foundation for High-Projects Grant “863” from the Ministry of Science and Technology, P. R. China awarded to Y-H Yi.

  • Sample Availability: Samples are available from the authors.

References

  1. Katagawa, T.; Sugawara, I.; Yosioka, K. Saponin and Sapogenol. XIV. Antifungal Glycosides from the Sea Cucumber Stichopus japonicus Selenka. Chem. Pharm. Bull 1976, 24, 266–274. [Google Scholar]
  2. Girard, M.; Bélanger, J.; ApSimon, J.W.; Garneau, F-X.; Harvey, C.; Brisson, J-R. Frondoside A. A novel triterpene glycoside from the holothurian Cucumaria frondosa. Can. J. Chem 1990, 68, 11–18. [Google Scholar]
  3. Avilov, S.A.; Antonov, A.S.; Drozdova, O.A.; Kalinin, V.I.; Kalinovsky, A.I.; Stonik, V.A.; Riguera, R.; Lenis, L.A.; Jiménez, C. Triterpene Glycosides from the Far-East Sea Cucumber Pentamera calcigera. 1. Monosulfated Glycosides and Cytotoxicity of Their Unsulfated. J. Nat. Prod 2000, 65–71. [Google Scholar]
  4. Burnell, DJ; Apsimon, JW. Marine Natural Products — Chemical and Biochemical Perspectives; Scheuer, P.J., Ed.; Academic Press: New York, 1983; Volume 5, pp. 287–389. [Google Scholar]
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