Clerodane-type Diterpene Glycosides from Dicranopteris pedata

1 Introduction

The genus Dicranopteris is consists of about 10 species, distributed mainly in Asia and widely in Japan, India, Vietnam and China. There are six species in China and they mainly distribute in the south of the Yangtze River such as Yunnan, Sichuan and Guizhou provinces ^[1]. The whole plant of D. pedata is commonly used as a folk medicine in ancient China for the treatment of hemorrhage, dysentery and empyrosis ^{[2, 3]}. Previous researches revealed a diversity of pharmacological properties of the extract of D. pedata, such as antioxidant, antibacterial, antinociceptive, anti-inflammatory and antipyretic activities ^[4-8]. Up to date, various secondary metabolites including flavonoids ^{[9, 10]}, phenols ^[11], proanthocyanidins ^[12] and clerodane-type diterpene glycosides ^{[10, 13]} have been reported from D. pedata. Our previous investigation on this species revealed the presence of two oxygenated phenolic derivatives ^[14], dichotomains A and B and some clerodane-type diterpene glycosides, in which dichotomain A has exhibited weak anti-HIV activity ^{[15, 16]}. As part of our ongoing search for new bioactive metabolites from fern plants, a chemical investigation on the whole plant of D. pedata led to the discovery of three previously undescribed compounds (1–3) (Fig. 1) and two known analogues (compounds 4 and 5) from the acetone extract ^{[15, 16]}. This paper describes the details of isolation, structure identification, and cytotoxicity of compounds 1–3.

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2 Results and Discussion

Compound 1 was isolated as pale yellow powder. Its molecular formula was deduced as C₃₂H₅₂O₁₀ by HRESIMS analysis (found m/z 619.3454 [M + Na]⁺, calcd for 619.3458), suggesting seven degrees of unsaturation. The ¹³C and ¹H NMR spectra displayed two set of signals ascribed to a diterpene and two hexoses. Five methines (δ_C 103.2, 72.6, 72.7, 83.5, 68.8) and a methyl (δ_C 17.9) revealed the presence of rhamnose. In addition, the spectra displayed signals for a glucopyranosyl unit (five methines at δ_C 105.7, 76.1, 78.1, 71.5, 78.2 and a methylene at δ_C 62.7). Assignment of each glycosidic proton system was achieved by analysis of ¹H-¹H COSY, HMBC and HSQC spectra (Fig. 2). The ¹H NMR spectrum of the diterpene displayed the presence of four methyls at δ_H 0.73 (s), 1.08 (s), 0.84 (d, J = 6.7 Hz) and 1.73 (d, J = 1.1 Hz, an olefinic methyl), and six olefinic protons at δ_H 5.24 (br s), 6.37 (dd, J = 17.6, 10.9 Hz), 5.20 (d, J = 17.6 Hz), 5.04 (d, J = 10.9 Hz), and 4.97 (2H, d, J = 4.4 Hz). The signal at δ_H 3.43 (dd, J = 11.2, 4.7 Hz) could be attributed to a proton on the oxygenated carbon. Twenty carbons of the diterpene could be classified to four methyls, seven methylenes (two olefinic at δ_C 113.3, 116.2), five methines (one oxygenated at δ_C 87.7; two olefinic at δ_C 123.6, 140.1) and four quaternary carbons (two olefinic at δ_C 144.6, 148.9). The structure of 1 was identified as a clerodane-type diterpenoid glycoside with two sugar moieties. Aforementioned 1D NMR resonances of 1 showed good agreement with the known compound 5 ^[16], except for the presence of one olefinic methylene and one olefinic quaternary carbon, together with the absence of a methyl and hydroxyl. The difference of compounds 1 and 5 lie in C-13, C-14, C-15 and C-16. The HMBC correlations (Fig. 2) from H₂-16 (δ_H 4.97) to C-12 (δ_C 25.5), C-13 (δ_C 148.9), C-14 (δ_C 140.1), and H₂-15 (δ_H 5.20, 5.04) to C-13 and C-14, accompanied with the ¹H-¹H COSY correlations (Fig. 2) between H-14 (δ_H 6.37) and H₂-15 (δ_H 5.20, 5.04) confirmed the existence of conjugated diene at C-13/C-16 and C-14/C-15. Thus the planar structure of compound 1 was determined.

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The relative configurations of α-rhamnopyranosyl and β-glucopyranosyl moiety were identified by the coupling constants of their anomeric protons of the rhamnopyranosyl (H-1ʹ, δ_H 4.79, J = 1.5 Hz) and glucopyranosyl (H-1ʺ δ_H 4.59, J = 7.8 Hz), respectively. The location of the sugar chain was determined at C-6 of the aglycone by HMBC correlations (Fig. 2) from the anomeric proton (H-1ʹ, δ_H 4.79) of the rhamnopyranosyl unit to C-6 (δ_C 87.7). HMBC correlation from H-1ʺ (δ_H 4.59, J = 7.8 Hz) to C-4ʹ (δ_C 83.5) defined a glucopyranosyl (1 → 4) rhamnosyl linkage. The absolute configurations of sugar moieties were determined by the experiments of acid hydrolysis, together with sugar derivatization and analysis with HPLC ^[16]. The retention times of the product in the test sample are consistent with that of standard sugar derivatives in HPLC (Rha derivative: t_R = 6.68 min; Glc derivative: t_R = 12.78 min), which gave d-glucose and l-rhamnose.

The relative configuration of 1 was assigned by analysis of its ROESY data (Fig. 4). The correlations of H-6/H-8, and H-6/H-10 indicated that they are spatially close and were thus assigned arbitrarily as β-oriented. Consequently, the correlations of CH₃-19/CH₃-20 revealed that they are cofacial and adopt a α-orientation. The correlation of H-16/H-14 disclosed the configuration of s-(E)-13(16), 14-diene. The similarity of the NMR results between 1 and 5 suggests the same (5R, 6S, 8R, 9S, 10R) absolute configurations for the aglycone as previously reported. Finally, the structure of 1 was determined as (5R, 6S, 8R, 9S, 10R)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]cleroda-3, 13(16), 14-diene.

Compound 2 was purified as pale yellow powder with a molecular formula of C₃₂H₅₂O₁₂, which was revealed by the [M + Na]⁺ ion at m/z 651.3355 (calcd for 651.3357), implying seven indices of hydrogen deficiency. The analysis of 1D NMR spectrum revealed the presence of a diterpene and two hexoses. Each glycosidic proton system was confirmed by analysis of ¹H-¹H COSY, HMBC and HSQC (Fig. 2). The ¹H NMR spectrum of the diterpene displayed the presence of five methyls, one olefinic methylene at δ_H 5.16 (dd, J = 17.4, 1.5 Hz) and 5.03 (dd, J = 10.8, 1.5 Hz), two olefinic at δ_H 5.69 (s), 5.84 (dd, J = 17.4, 10.8 Hz). Detailed analysis of its ¹³C NMR and DEPT spectra revealed 20 carbons resonance which were assigned as five methyls, five methylenes (one olefinic at δ_C 112.5), five methines (two olefinic at δ_C 127.1, 146.1) and five quaternary carbons (one olefinic at δ_C 175.3 and one carbonyl at δ_C 202.6). The ¹H and ¹³C NMR of 2 were highly analogous to 5 ^[16], except for the following difference: in the ¹³C NMR spectrum, the downfield of 2 in chemical shifts of C-3 (δ_C 127.1) and C-4 (δ_C 175.3), together with one methylene in 5 was replaced with one carbonyl (δ_C 202.8). This might be caused by an additional carbonyl at C-2. In the HMBC spectrum, correlations could be observed from δ_H 5.69 (H-3) to δ_C 202.8 (C-2), δ_C 175.3 (C-4) and from δ_H 1.85 (H-10) to δ_C 202.8 (C-2), δ_C 175.3 (C-4). Thus, the carbonyl group was ascertained to be at C-2 position.

The presence of α-l-rhamnose and β-d-glucose were identified by the same method as compound 1. By the HMBC correlations of C-6 (δ_C 85)/H-1ʹ (δ_H 4.86) and C-4ʹ (δ_C 83.3)/H-1ʺ (δ_H 4.86, d, J = 7.8)(Fig. 2), the linkage positions of the sugar moieties were confirmed. The relative configurations of the diterpene were determined the same for their similar ROESY correlations as 1: H-6/H-10, H-6/H-8, and CH₃-19/CH₃-20 (Fig. 3). Furthermore, the absolute configuration of 2 was determined by electronic circular dichroism (ECD) experiment which fitted well with the calculated ECD (Fig. 4). Finally, the structure of 2 was determined as (5R, 6S, 8R, 9S, 10R, 13S)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]-2-ox-oneocleroda-3, 13-dien-15-ol.

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Compound 3 was obtained as pale yellow powder. It showed a molecular ion peak at m/z 651.3356 [M + Na]⁺ (calcd for 651.3351), corresponding to the molecular formula of C₃₂H₅₂O₁₂, which indicating seven degrees of unsaturation. The 1D NMR spectra demonstrated two hexoses and a diterpene. The spectroscopic data of the sugar moieties were consistent with 1 and 2, suggesting the presence of α-l-rhamnopyranosyl moiety and β-D-glucopyranosyl moieties. The diterpene part involved five methyls (one at δ_C 23.8), five methylenes (one oxygenated at δ_C 59.1), five methane (one olefinic at δ_C 125.5) and five quaternary carbons (one olefinic at δ_C 140.2). Those spectra suggested that the structure of 3 was highly similar to that of 2. In comparison with 2, the chemical shifts of C-14, C-15 and C-16 decreased from δ_C 146.1, 112.5 and 27.7 to δ_C 125.5, 59.1 and 23.8 respectively, while C-13 increased from δ_C 73.8 to 140.2, which testified the position of the double bond of 3 at C-13 and C-14. This was demonstrated again by the HMBC correlations of H₂-15/C-13, C-14 and CH₃-16/C-13, C-14. In the meantime, C-15 was determined to be a terminal oxymethylene by the chemical shift (δ_C 59.1) and ¹H-¹H COSY correlation of H₂-15/H-14. Besides, the linkage of the sugar moieties was determined by the identical way with 1 and 2. Thus the plannar structure of compound 3 was determined.

The relative configuration of 3 was assigned by ROESY correlations of H-6/H-10, H-6/H-8, and CH₃-19/CH₃-20. The ROESY correlations of CH₃-16/H₂-15 established the E configuration of the C-13/C-14 double bond. The experimental 1D and 2D NMR results of 2 and 3 were similar, which suggesting that they had the same (R, S, R, S, R) absolute configuration at C-5/6/8/9/10, respectively. Finally, the structure of 3 was determined as (5R, 6S, 8R, 9S, 10R)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]-(13E)-2-oxoneocleroda-3, 14-dien-13-ol.

Additionally, the cytotoxicities of compounds 1–3 were evaluated against five human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7 and SW-480, with cisplatin as the positive control. Compound 1 (40 μM) showed weak inhibitory activities in vitro against SMMC-7721, MCF-7 and SW480 with the inhibition ratio of 48.1%, 48.8% and 39.0%, respectively. However compounds 2 and 3 did not display any activities against the tested cell lines.

3 Conclusion

In conclusion, this research led to the isolation of five compounds including three new and two known clerodane-type diterpene glycosides from D. pedata. Compounds 1–3 were assayed for their cytotoxicitives against five human tumor cell HL-60, SMMC-7721, A-549, MCF-7 and SW-480 with cisplatin as the positive control, in which compound 1 showed weak inhibitory activities in vitro against SMMC-7721, MCF-7 and SW480. This research enriched the chemical diversities of ferns.

4 Experimental Section

4.1 General Experimental Procedures

Optical rotations were carried out on Autopol VI automatoc polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. A Thermo Nicolet iS10 spectrometer was used for measuring IR spectroscopy, which used KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-600 and spectrometers with SiMe₄ (TMS) as an internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESI and HRESIMS were performed on an UPLC-IT-TOF spectrometer. Semi-preparative HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 150 mm) column. Column chromatography was carried out using silica gel (100–200 mesh, Qingdao Haiyang Chemical Co. Ltd, Qingdao, People's Republic of China) and macroporous absorption resin (DM 130, Tianjin Haoju Resin Technology Co. Ltd, people's Republic of China). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH. The reagents used for acid hydrolysis and derivatization are listed: Trifluoroacetic acid (TFA, Beijing Yinuokai Technology Co, Ltd, Beijing, people's Republic of China); 1-Phenyl-3-methyl-5-pyrazolon (PMP, analytical reagent, Tianjin Damao Chemical Reagent Factory); sodium dihydrogen phosphate (NaH₂PO_4, analytical reagent, Tianjin Damao Chemical Reagent Factory, Tianjin, people's Republic of China); d-glucose and l-rhamnose (Qingdao Jieshikang Biological Technology Co., Ltd, Qingdao, people's Republic of China).

4.2 Plant Material

The fronds of Dicranopteris pedata were collected from Huanglian Mountain, Lvchun County, Yunnan Province, People's Republic of China, in September 2019, identified by Prof. Xiao Cheng (Kunming Institute of Botany, Chinese Academy of Sciences). The voucher specimen (No. 20190921) has been deposited in State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, the Chinese Academy of Sciences.

4.3 Extraction and Isolation of Compounds 1–5

The dry fronds of Dicranopteris pedata (9 kg) were powdered and extracted with acetone (3 × 25 L) for 24 h at room temperature. The acetone extract was concentrated under reduced pressure to yield the residue (700 g), which was then suspended in H₂O and subjected to column chromatography over DM 130 eluting with EtOH-H₂O (0:10, 5:5, 7:3, 5:95, v/v), EtOH/H₂O (5:5, v/v) fraction (230 g) was subjected to silica gel chromatography (CC) using silica gel (200–300 mesh) eluting with CHCl₃-MeOH (from 12:1 to 0:1) to yield fractions A-D. Fraction B was subsequently separated on a Sephadex LH-20 CC (MeOH) to give two subfraction Fr.B₁ (27 g) and Fr.B₂ (25 g). Fr.B₁ was divided into ten fractions (from Fr.B₁-1 to Fr.B₁-10) by using RP-C18 eluting with MeOH-H₂O (from 1:9 to 1:1). Fr.B₁-10 was separated by CC on silica gel (CHCl₃-MeOH, 10:1) and further purified by semi-preparative HPLC with MeCN-H₂O (23:77, v/v) to obtain compound 2 (6 mg, t_R = 44.11 min). Compound 4 (4 mg, t_R = 26.70 min) and 5 (3 mg, t_R = 31.35 min) were obtained by MeOH-H₂O (40:60, v/v). Compound 3 was separated and purified from Fr.B₁-8 by semi-preparative HPLC with MeOH-H₂O (48:52, v/v). Fr.B₁-9 was separated by CC on silica gel (CHCl₃-MeOH, 20:1 to 15:1) and further purified by semi-preparative HPLC with MeOH-H₂O (75:25, v/v) to give compound 1 (4 mg, t_R = 36.11 min).

4.4 Spectroscopy Data of Compounds 1–3

(5R, 6S, 8R, 9S, 10R)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]cleroda-3, 13(16), 14-diene (1): pale yellow powder; [α]_D²⁶ − 25.8 (c, 0.26, MeOH); UV (MeOH) λ_max (log ε): 196.5 (3.26), 208.0 (3.17), 223.5 (3.27); IR (ν_max): 3402, 3090, 2956, 2926, 2875 cm⁻¹; HRESIMS at m/z 619.3454 [M + Na]⁺ (calcd for 619.3458). ¹H and ¹³C NMR data, see Tables 1 and 2.

(5R, 6S, 8R, 9S, 10R, 13S)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]-2-oxoneocleroda-3, 13-dien-15-ol (2): pale yellow powder; [α]₂₆^D − 37.98 (c, 0.10, MeOH); UV (MeOH) λ_max (log ε): 195.5 (3.13), 209.0 (3.02), 238.5 (3.35); ECD (MeOH) λ_max (Δε) 245 (21.24); IR (ν_max): 3387, 2964, 2926, 2878, 1652 cm⁻¹; HRESIMS at m/z 651.3355 [M + Na]⁺ (calcd for 651.3357); ¹H and ¹³C NMR data see Tables 1 and 2.

(5R, 6S, 8R, 9S, 10R)-6-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl]-(13E)-2-oxoneocleroda-3, 14-dien-13-ol (3): pale yellow powder; [α]₂₆^D − 18.9 (c, 0.26, MeOH); UV (MeOH) λ_max (log ε): 196.0 (3.32), 215.0 (2.99), 238.0 (3.19); IR (ν_max): 3404, 2964, 2928, 1716, 1651 cm⁻¹; HRESIMS at m/z 651.3356 [M + Na]⁺ (calcd for 651.3351). ¹H and ¹³C NMR data see Tables 1 and 2.

4.5 Acid Hydrolysis and Derivatization

Compounds 1–3 were hydrolyzed with trifluoroacetic acid (TFA) by the procedure of the previous reported ^[17], with minor modifications. Compound 1 (2 mg, 3.35 μmol) was dissolved in 2 M TFA (2 mL), after the reaction mixture was heated to 120 ℃ and stirred for 2 h before cooling to room temperature. Then, extracted with CHCl₃ (3 × 1 mL) and the aqueous layer was concentrated for next derivatization. Next, adding 0.3 M NaOH (1 mL) and 0.5 M PMP (1 mL) to the aqueous layer. The reaction was heated to 75 ℃ and stirred for 1.5 h before cooling to room temperature. Then the reaction mixture was quenched with 0.3 M HCl (1 mL) and extracted with CHCl₃ (3 × 2 mL). Further analysis the aqueous layer over HPLC [t_R = 6.67, 12.78 min; 40% MeOH: 60% sodium phosphate (pH 6.8); 1.5 mL/min]. The acid hydrolysis and HPLC analysis of 2 and 3 were manipulated by the same way as that of 1. Likewise, the standard monosaccharides, d-Glc (2 mg) and l-Rha (2 mg) were derivatized with PMP as compounds 1–3, then HPLC analysis in the same condition as 1–3. The sugar moieties of compounds 1–3 were identified as d-Glc (t_R = 12.78 min) and l-Rha (t_R = 6.67 min), showing retention times consistent with the standard monosaccharide derivatives.

4.6 Cytotoxicity Assay

MTT assays ^[18] were used to evaluating the cytotoxicities of compounds 1–3. Five human tumor cell lines including leukemia HL-60, liver cancer SMMC-7721, lung cancer A-549, breast cancer MCF-7 and colon cancer SW-480, were incubated in RMPI-1640 or DMEM medium supplemented with 10% fetal bovine serum and seeded at 3–15 × 10³ cells each well of a 96-well cell culture plate. After 12–24 h of incubation at 37 ℃, the tested compound (40 μM, dissolved in DMSO) was added. After incubated for 48 h, each well were added 20 μL MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. Then, they were cultured for further 4 h. The cisplatin was applied as the positive control. The MULTISKAN FC was used to measure the OD value at 492 nm.

Notes

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s13659-021-00315-y.

Acknowledgements

This work is supported by the Natural Science Foundation of Yunnan province. (No. 202001AT070052)

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Declarations

Conflict of interest

All authors declare no conflict of interest.