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Four new labdane-type diterpenoid glycosides from Diplopterygium laevissimum

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Authors and affiliations

Ming-Ming LI ,
Kou WANG ,
Juan HE ,
Li-Yan PENG ,
Xuan-Qin CHEN ,
Xiao CHENG ,
Qin-Shi ZHAO Email author

Received date: 13 March 2012; Accepted: 24 February 2013

Abstract

Four new labdane-type diterpenoid glycosides, laevissiosides A-D (1-4) were isolated from the 95% ethanol extract of Diplopterygium laevissimum (Christ) Nakai, along with two known analogues, 18-β-D-glucopyranosyl ester-sclareol (5) and 18-hydroxy-sclareol (6). The structures of compounds 1-4 were elucidated by extensive 1D and 2D NMR spectroscopy as well as high-resolution MS analyses. All isolated compounds were evaluated for their cytotoxic effects.

Keywords

Diplopterygium laevissimum labdane-type diterpenoid glycosides laevissiosides

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Introduction

Diplopterygium laevissimum (Christ) Nakai, belonging to the Gleicheniaceae family, is widely distributed in south China. Its rhizome has been used for treating hemostasis, stomach, and epistaxis as Chinese herbal medicine.¹ Many clerodaneand labdane-type diterpenoid glycosides, which are commonly glycosidated at C-13 in ferns, have been isolated from this family.^2–8 Previous research showed that some clerodane-type diterpenoid glycosides isolated from Dicranopteris species could accelerate the growth of the stems of lettuce and inhibit the root growth.⁵

Our previous chemical studies have led to the isolation of two highly oxygenated phenolic derivatives and some clerodane-type diterpenoid glycosides from Dicranopteris and an ent-kaurene diterpenoid glycoside from Hicriopteris.^{6, 9-11} As a systematic research work on the bioactive constituents from the ferns, the whole plant of D. laevissimum had been studied, which led to the isolation of four new labdane-type diterpenoid glycosides (1–4), along with two known analogues (5 and 6). All of these showed no in vitro cytotoxicity against five human cancer cell lines (HL-60, SMMC-7712, A-549, SK-BR-3 and PANC-1). Herein, the isolation and structure elucidation of compounds 1-4 were described.

Results and Discussion

Compound 1 was obtained as a white amorphous powder. The molecular formula C₃₅H₅₂O₁₀ was established by the HRESIMS (631.3471 [M – H]^–; calcd. 631.3482), corresponding to ten degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3428 cm^–1) and carbonyl (1704 cm^–1) groups. The ¹H and ¹³C NMR (Tables 1 and 2) indicated the existence of a set of signals for a hexose [anomeric signals at δ_H 4.30 (d, J = 8.0 Hz); δ_C 104.6] and other 20 carbon resonances, including two olefinic carbons (δ_C 147.5 and 110.8) and three oxygen-bearing carbons (δ_C 73.4, 73.9 and 79.2). These data were very similar to those of 18-β-D-glucopyranosyl ester-sclareol (5), a known compound also isolated from this plant. However, detailed comparison the MS and NMR data of 1 with those of 5 revealed that 1 had one more p-coumaroyl group, which was attached to C-4′ of the sugar moiety as concluded from the HMBC (Figure 1) correlations of H-4′ (δ_H 4.84) with C-1′′ (δ_C 167.4). The double bond of the p-coumaroyl group was suggested as trans-due to the coupling constant (J = 15.6 Hz). Acidic hydrolysis of 1 gave D-glucose as sugar residue. The coupling constants of the anomeric proton (J = 8.0 Hz) indicated the β configuration of glucosyl moiety. Assignment of glycosidic protons system was achieved by analysis of ¹H-¹H COSY and HSQC. The location of the sugar unit was established by the HMBC correlations of H-1′ (δ_H 4.30) with C-18 (δ_C 79.2).

Table 1

¹H NMR data of compounds 1–4 (δ in ppm, J in Hz)

no	1^a	no	2^a	3^b	4^{a, c}
1	1.66 (m); 0.95 (m)	1	1.59 (m); 1.09 (m)	1.59 (m); 1.04 (m)	1.79 (m); 1.13 (m)
2	1.63 (m); 1.42 (m)	2	1.59 (m); 1.41 (m)	1.60 (m); 1.45 (m)	1.69 (m); 1.51 (m)
3	1.56 (m); 1.27 (m)	3	1.59 (m); 1.18 (m)	1.50 (m); 1.30 (m)	1.48 (m); 1.40 (m)
5	1.48 (m)	5	1.36 (m)	1.38 (m)	1.32 (m)
6	1.55 (m); 1.25 (m)	6	1.52 (m); 1.27 (m)	1.55 (m); 1.36 (m)	1.62 (m); 1.32 (m)
7	1.70 (m); 1.26 (m)	7	1.74 (m); 1.52 (m)	1.75 (m); 1.47 (m)	1.80 (m); 1.50 (m)
9	1.16 (m)	9	1.35 (m)	1.24 (m)	1.00 (m)
11	1.33 (m)	11	1.47 (m); 1.29 (m)	1.50 (m); 1.36 (m)	1.43 (m)
12	1.73 (m); 1.55 (m)	12	1.78 (m); 1.56 (m)	1.62 (m); 1.51 (m)	1.76 (m); 1.54 (m)
14	5.91 (dd, 10.8, 17.6)	14	5.89 (dd, 10.5, 17.0)	5.91 (dd, 10.8, 17.6)	5.88 (dd, 10.8, 18.0)
15	4.92 (d, 10.8); 5.19 (d, 17.6)	15	5.15 (d, 17.0); 4.91(d, 10.5)	5.17 (d, 17.6); 5.15 (d, 10.8)	5.18 (m); 5.16 (m)
16	1.20 (s)	16	1.05 (s)	1.09 (s)	1.09 (s)
17	1.10 (s)	17	1.19 (s)	1.32 (s)	1.32 (s)
18	3.45 (m); 3.25 (m)	18	3.38 (m); 3.20 (m)	3.48 (m); 3.21 (m)	3.44 (m); 3.35 (m)
19	0.75 (s)	19	0.70 (s)	0.76 (s)	0.81 (s)
20	0.82 (s)	20	0.79 (s)	0.82 (s)	0.85 (s)
1′	4.30 (d, 8.0)	1′	4.27 (d, 7.5)	4.25 (d, 7.2)	4.42 (d, 7.2)
2′	3.33 (m)	2′	3.24 (m)	3.45 (m)	3.53 (m)
3′	3.68 (m)	3′	3.52 (m)	3.46 (m)	3.68 (m)
4′	4.84 (t, 9.6)	4′	3.26 (m)	3.24 (m)	3.36 (m)
5′	3.48 (m)	5′	3.48 (m)	3.19 (m)	3.35 (m)
6′	3.54 (m); 3.58 (m)	6′	3.79 (m); 3.62 (m)	3.64 (m); 3.83 (m)	3.68 (m); 3.84 (m)
2″	6.36 (d, 15.6)	1″	5.51 (d, 1.5)	5.42 (d, 1.5)	5.62 (d, 0.8)
3″	7.64 (d, 15.6)	2″	3.87 (m)	3.91 (m)	5.32 (m)
5″/9″	7.55 (d, 8.8)	3″	3.77 (m)	3.74 (m)	5.15 (m)
6″/8″	6.90 (d, 8.8)	4″	3.43 (m)	3.40 (m)	5.12 (m)
		5″	4.08 (m)	4.06 (m)	4.46 (m)
		6″	1.25 (d, 6.5)	1.27 (d, 6.0)	1.18 (d, 6.4)
		1′′′		4.28 (d, 8.0)	4.52 (d, 8.0)
		2′′′		3.41 (m)	5.37 (m)
		3′′′		3.40 (m)	3.87 (m)
		4′′′		3.55 (m)	4.92 (m)
		5′′′		3.52 (m)	3.82 (m)
		6′′′		1.20 (d, 6.4)	1.08 (d, 6.4)
^aMeasured in acetone-d₆. ^bDetermined in CD₃OD. ^cAcetyl groups δ_H: (1.98, 2.06, 2.13, 2.13, 2.16).

Table 2

¹³C NMR data of compounds 1-4 (δ in ppm)

no.	1^a	no.	2^a	3^b	4^{a, c}
1	39.8 CH₂	1	39.7 CH₂	40.4 CH₂	39.5 CH₂
2	18.5 CH₂	2	18.4 CH₂	18.8 CH₂	18.4 CH₂
3	36.6 CH₂	3	36.5 CH₂	37.0 CH₂	36.4 CH₂
4	37.9 C	4	37.8 C	38.3 C	38.2 C
5	49.6 CH	5	48.9 CH	50.7 CH	51.2 CH
6	21.0 CH₂	6	20.9 CH₂	21.4 CH₂	21.5 CH₂
7	44.6 CH₂	7	44.7 CH₂	44.8 CH₂	45.4 CH₂
8	73.9 C	8	74.6 C	75.4 C	73.8 C
9	62.1 CH	9	60.9 CH	62.0 CH	62.9 CH
10	40.0 C	10	40.0 C	40.4 C	40.0 C
11	20.1 CH₂	11	20.4 CH₂	20.5 CH₂	19.9 CH₂
12	46.5 CH₂	12	45.7 CH₂	44.8 CH₂	46.7 CH₂
13	73.4 C	13	73.7 C	81.8 C	80.7 C
14	147.5 CH	14	147.0 CH	145.0 CH	143.5 CH
15	110.8 CH₂	15	111.1 CH₂	115.2 CH₂	115.5 CH₂
16	28.1 CH₃	16	23.6 CH₃	23.0 CH₃	23.8 CH₃
17	24.6 CH₃	17	27.3 CH₃	23.7 CH₃	24.6 CH₃
18	79.2 CH₂	18	78.2 CH₂	79.4 CH₂	79.0 CH₂
19	17.9 CH₃	19	18.2 CH₃	18.2 CH₃	17.9 CH₃
20	16.2 CH₃	20	16.2 CH₃	16.5 CH₃	16.3 CH₃
1′	104.6 CH	1	102.9 CH	103.5 CH	102.5 CH
2′	75.2 CH	2	77.5 CH	78.0 CH	75.9 CH
3′	75.5 CH	3	79.6 CH	79.8 CH	79.3 CH
4′	72.3 CH	4	71.9 CH	72.0 CH	72.1 CH
5′	75.8 CH	5	76.8 CH	77.9 CH	77.5 CH
6′	62.4 CH₂	6	62.6 CH₂	62.7 CH₂	62.5 CH₂
1″	167.4 C	1	100.8 CH	101.5 CH	97.8 CH
2″	115.2 CH	2	72.0 CH	72.3 CH	70.5 CH
3″	146.1 CH	3	71.7 CH	72.1 CH	71.0 CH
4″	121.8 C	4	73.7 CH	74.0 CH	74.0 CH
5″/9″	116.7 CH	5	69.2 CH	70.0 CH	67.1 CH
6″/8″	131.0 CH	6	18.3 CH₃	18.8 CH₃	17.6 CH₃
7″	160.8 C	1′′′		99.6 CH	97.1 CH
		2′′′		72.4 CH	70.1 CH
		3′′′		75.2 CH	71.1 CH
		4′′′		73.0 CH	73.4 CH
		5′′′		71.5 CH	69.5 CH
		6′′′		17.0 CH₃	16.9 CH₃
^aMeasured in acetone-d₆. ^bDetermined in CD₃OD. ^cAcetyl groups (δ_C: C=O 170.0, 170.4, 170.6, 171.0, 171.5; Me: 20.7, 20.8, 21.2, 21.2, 21.2).

Fig. 1
Selected HMBC correlations of compound 1 and 2

The relative configuration of the aglycone was established on a ROESY experiment. The ROESY correlations (Figure 2) between H-5 and H-9 confirmed that these hydrogen atoms were α-oriented, while correlations of H-11/Me-17, H-11/Me-20, Me-17/Me-19, Me-17/Me-20, and Me-19/Me-20 indicated they were β-orientation. The absolute configuration of C-13 was inferred as S according to the chmical shift of C-13 (δ_C 73.4).^12-14 Therefore, the structure of 1 was determined as shown, named laevissioside A.

Fig. 2
Significant ROESY correlations of compounds **1-4**

Compound 2, a white amorphous powder, and its molecular formula, C₃₂H₅₆O₁₂, was determined on the basis of the HRESIMS (667.3454 [M + Cl]^–; calcd. 667.3460). The ¹H and ¹³C NMR spectroscopic data of 2 were very similar to those of 5, except for one more sugar moiety signals (δ_C 100.8, 72.0, 71.7, 73.7, 69.2, 18.3) presented in 2 which was further confirmed by mass spectra. Acidic hydrolysis of 2 gave Dglucose and L-rhamnose as sugar residues. The coupling constants (δ_H 4.27, J = 7.5 Hz and δ_H 5.51, J = 1.5 Hz) of anomeric protons of the two sugar moieties indicated the β configuration glucose and α configuration of rhamnose. The HMBC correlations between H-1″ (δ_H 5.51) and C-2′ (δ_C 77.5) identified a rhamnosyl (1→2) glucopyranosyl linkage. Furthermore, the sugar chain was linked to C-18 of the aglycone as inferred from the HMBC (Figure 1) correlation of H-1′ (δ_H 4.27) with C-18 (δ_C 78.2). Thus, the structure of 2 was elucidated and named as laevissioside B.

Compound 3, obtained as a white amorphous powder, had the molecular formula C₃₈H₆₆O₁₆ as determined by the analysis of ¹H, ¹³C, and DEPT NMR data and verified by the HRESIMS (813.4042 [M + Cl]^–; calcd. 813.4039). Compared with 2, compound 3 had one more sugar moiety (δ_C 99.6, 72.4, 75.2, 73.0, 71.5, and 17.0) which was attached to C-13 as inferred from the HMBC correlations from H-1′′′ to C-13. Acidic hydrolysis of 3 gave D-glucose, L-rhamnose, and Dfucose as sugar residues. They were in β, α and β configurations, respectively, by the coupling constants (δ_H 4.25, J = 7.2 Hz, δ_H 5.42, J = 1.5 Hz, and δ_H 4.28, J = 8.0 Hz) of their anomeric protons. The sugar moiety linked to C-13 was supposed to be fucose by the HMBC correlations of the anomeric proton H-1′′′ (δ_H 4.28) with C-13 (δ_C 81.8). The linkage and location of the other two sugar moieties were suggested the same as compound 2 deduced from the HMBC correlations. Accordingly, the structure of 3 was established as shown, named laevissioside C.

The molecular formula of compound 4 was deduced as C₄₈H₇₆O₂₁ by the HRESIMS (1023.4557 [M + Cl]^–; calcd 1023.4567). The ¹H and ¹³C NMR features of 4 were closely related to those of 3. The only difference was that there were five more acetyl groups in 4. The locations of acetyl groups were confirmed by the HMBC experiments. The correlations of H-2′′′ (δ_H 5.37) with δ_C 170.4, H-4′′′ (δ_H 4.92) with δ_C 170.0, H-2″ (δ_H 5.32) with δ_C 171.5, H-3″ (δ_H 5.15) with δ_C171.0, and H-4″ (δ_H 5.12) with δ_C 170.6 indicated the five acetyl groups attached to C-2′′′, C-4′′′, C-2″, C-3″, and C-4″, respectively. Therefore, the structure of 4 was identified and named as laevissioside D.

Since the known compounds 18-β-D-glucopyranosyl estersclareol (5, [α]_D^24.3 -27.7) and 18-hydroxy-sclareol (6, [α]_D^24.3 -4.7), whose physical properites were quite difference with that reported, ^{15, 16} were also isolated from this plant, compounds 1–4 should be labdane-type diterpenoid glycosides from the biogenic view.

All compounds isolated were evaluated for their cytotoxic activity against five human cancer cell lines, HL-60 myeloid leukemia, SMMC-7721 hepatocellular carcinoma, A-549 lung cancer, SK-BR-3 breast cancer, PANC-1 pancreatic cancer, applying the MTT method. However, all of the compounds were inactive, and they showed IC₅₀ values > 40 μM.

Experimental Section

General Experimental Procedures. Optical rotations were measured on a Horiba SEPA-300 polarimeter. IR spectra were obtained by Tensor 27 FT-IR spectrometer with KBr pellets. The ¹H and ¹³C NMR spectra were recorded on Bruker AV-400 spectrometers in acetone-d₆ at room temperature (δ in ppm, J in Hz). FABMS was carried out on a VG Autospec-3000 spectrometer. HRESIMS was recorded with an API QSTAR Pulsar i spectrometer. Silica gel (200–300 mesh), Silica gel H (Qingdao Marine Chemical Ltd., China), and LiChroprep RP-18 silica gel (40–63 μm, Merck, Dramstadt, Germany) were used for column chromatography. Fractions were monitored by TLC and spots visualized by heating silica gel plates immersed with 15% H₂SO₄ in ethanol. Solvents were distilled prior to use. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with Shimadzu PRCODS (K) column. Sephadex LH-20 (Amersham Pharmacia biotech, Sweden).

Plant Material. The aerial parts of D. laevissimum were collected from Pingbian, Yunnan Province, China in July 2007 and identified by Professor Xiao Cheng at Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 200707A03) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation. The dried and powdered plant materials (2.6 kg) were extracted with 95% ethanol (15.0 L, each 2 d) for three times. After evaporation of the solvent in vacuo, the concentrate was suspended into H₂O and partitioned successively with ethyl acetate. The ethyl acetate extract (120 g) was chromatographed on a silica gel column eluted with CHCl₃-MeOH (1:0 to 5:5) to give five fractions 1-5. Fraction 2 was subjected to column chromatograph (CC) over silica gel (petroleum ether-acetone 8.5:1.5) and further purified by recrystallization to obtain 6 (20 mg). Fraction 3 was eluted with CHCl₃-MeOH (9:1) over silica gel CC then further purified by RP-18 and Sephadex LH-20 to yield 1 (3 g), 4 (500 mg), and 5 (2 g). Fraction 4 was subjected to (CHCl₃:MeOH = 8.5:1.5) and further purified by RP-18 and Sephadex LH-20 to afford 2 (8 mg) and 3 (20 mg).

Laevissioside A: amorphous powder; [α]_D^19.2 -50.4 (c = 0.14, MeOH). UV (MeOH) λ_max (log ε): 314 (4.18), 227 (3.94), 211 (3.95), 200 (3.99), 192 (3.91) nm. IR (KBr): 3428, 2931, 1704, 1630, 1604, 1515, 1450, 1387, 1162, 1066, 1031 cm^–1. ¹H and ¹³C NMR: see Table 1. FABMS (neg.) m/z: 631 [M-H]^-. HRESIMS (neg.) m/z: 631.3471 (C₃₅H₅₁O₁₀; calcd. 631.3482).

Laevissioside B: amorphous powder; [α]_D^26.4 -62.2 (c = 0.18, MeOH). UV (MeOH) λ_max (log ε): 203 (3.38), 194 (3.12) nm. IR (KBr): 3423, 2927, 1069, 1052 cm^–1. ¹H and ¹³C NMR: see Table 2. FABMS (neg.) m/z: 631 [M-H]^-. HRESIMS (neg.) m/z: 667.3454 (C₃₂H₅₆O₁₂Cl; calcd. 667.3460).

Laevissioside C: amorphous powder; [α]_D^18.9 -11.2 (c = 0.10, MeOH). UV (MeOH) λ_max (log ε): 201 (3.31), 191 (3.10) nm. IR (KBr): 3431, 2926, 1704, 1638, 1384, 1169, 1128, 1054 cm^–1. ¹H and ¹³C NMR: see Table 2. FABMS (neg.) m/z: 777 [M-H]^-, 631 [M-146-H]^-. HRESIMS (neg.) m/z: 813.4042 (C₃₈H₆₆O₁₆Cl; calcd. 813.4039).

Laevissioside D: amorphous powder; [α]_D^26.6 -58.3 (c = 0.25, MeOH). UV (MeOH) λ_max (log ε): 199 (3.07), 192 (3.12) nm. IR (KBr): 3442, 2935, 1749, 1373, 1228, 1062, 1062 cm^–1. ¹H and ¹³C NMR: see Table 2. Acetyl groups (δ_C: C=O 170.0, 170.4, 170.6, 171.0, 171.5; Me: 20.7, 20.8, 21.2, 21.2, 21.2) δ_H: (1.98, 2.06, 2.13, 2.13, 2.16). FABMS (neg.) m/z: 987 [M-H]^-. HRESIMS (neg.) m/z: 1023.4557 (C₄₈H₇₆O₂₁Cl; calcd. 1023.4567).

Acidic Hydrolysis of Compounds 1-3. Compounds 1-3 (6-8 mg) were hydrolyzed with 2 M HCl-dioxane (1:1, 4 mL) under reflux for 6 h. The reaction mixture was extracted with CHCl₃ five times (4 mL × 5). The aqueous layer was neutralized with 2 M NaHCO₃, and was evaporated to dryness. The dry powders were dissolved in pyridine (2 mL). Then L-cysteine methyl ester hydrochloride (about 1.5 mg) was added and kept at 60 ℃ for 1 h. Next, trimethylsilylimidazole (about 1.5 mL) was added to the reaction mixture in ice water and kept at 60 ℃ for 30 min. The mixture was subjected to GC analysis, run on a Shimadzu GC-14C gas chromatograph equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5 quartz capillary column and an H₂ flame ionization detector with the following conditions: column temperature, 180-280 ℃; programmed increase, 3 ℃/min; carrier gas, N₂ (1 mL/min); injector and detector temperature, 250 ℃; injection volume, 4 μL; and split ratio, 1/50. The configuration of D-glucose, L-rhamnose, and D-fucose were determined by comparison of the retention time of the corresponding derivatives with those of standard D-glucose, L-rhamnose, and D-fucose, giving a peak at 18.576, 16.173, and 14.865 min, respectively.

Cytotoxic Bioassay. The cytotoxicity assay was performed according to the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method, ¹⁷ by use of the following five human cancer cell lines: Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer SK-BR-3, and pancreatic cancer PANC-1. The IC₅₀ values were calculated by the Reed and Muench method.¹⁸

Notes

Acknowledgments

This work was financially supported by the National Basic Research Program of China (973 Program Nos. 2009CB522303 and 2011CB915503), the National Natural Science Foundation of China (Nos. 90813004 and U0932602), the projects from Chinese Academy of sciences (Nos. 2009311211011 and 2009312311024), and the State Key Laboratory of Phytochemistry and Plant Resources in West China (No. P2010-ZZ05).

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Authors and Affiliations

Ming-Ming LI
Kou WANG
Juan HE
Li-Yan PENG
Xuan-Qin CHEN
Xiao CHENG
Qin-Shi ZHAO
Email author

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China