Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima

Meng, Qian-Qian; Tong, Shun-Yao; Peng, Xing-Rong; Zhao, Yu-Qing; Li, Zheng-Hui; Chen, He-Ping; Liu, Ji-Kai

doi:10.3390/molecules28062705

Open AccessArticle

Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima

¹

School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China

²

School of Pharmaceutical Sciences, South-Central Minzu University, Wuhan 430074, China

³

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

^*

Authors to whom correspondence should be addressed.

Molecules 2023, 28(6), 2705; https://doi.org/10.3390/molecules28062705

Submission received: 20 February 2023 / Revised: 10 March 2023 / Accepted: 15 March 2023 / Published: 16 March 2023

(This article belongs to the Special Issue Terpenes, Steroids and Their Derivatives)

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Abstract

:

The ethnobotanical plant Marsdenia tenacissima has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed that pregnane glycosides were the main biological constituents. Nine new pregnane glycosides, marsdeosides A–I (1–9), were isolated from cultivated dried stems of the medicinal plant Marsdenia tenacissima in this study. The structures were analyzed by extensive spectroscopic analysis, including 1D, 2D NMR, HRESIMS, and IR spectroscopic analysis. The absolute configurations of the sugar moieties were identified by comparing the R_f values and specific optical rotations with those of the commercially available standard samples and the data reported in the literature. Marsdeosides A (1) featured an unusual 8,14-seco-pregnane skeleton. Compounds 1, 8, and 9 showed activity against nitric oxide production in lipopolysaccharide-activated macrophage RAW264.7, with IC₅₀ values of 37.5, 38.8, and 42.8 μM (L-NMMA was used as a positive control, IC₅₀ 39.3 μM), respectively. This study puts the knowledge of the chemical profile of the botanical plant M. tenacissima one step forward and, thereby, promotes the sustainable utilization of the resources of traditional folk medicinal plants.

Keywords:

Marsdenia tenacissima (Roxb.) moon; asclepiadaceae; pregnane glycosides; structural elucidation; nitric oxide production inhibition

Graphical Abstract

1. Introduction

The plant Marsdenia tenacissima (Roxb.) Moon. (M. tenacissima) mainly grows in the Yunnan, Guizhou, Sichuan, and Guangxi Provinces in China and other tropical regions, such as India, Myanmar, Sri Lanka, Indonesia, Vietnam, Laos, and Cambodia in Southeastern Asia [1]. In China, it was named as “Tong Guan Teng” and was first recorded in Dian Nan Ben Cao (Herbal Medicine of Southern Yunnan), a medicinal classic published during the Ming dynasty in 1436 A.D. This plant, belonging to the Asclepiadaceae family, is a medicinal herb used in traditional Chinese Medicine, as recorded in the 1977, 2010, 2015, and 2020 editions of Chinese Pharmacopoeia, and is also widely used in ethnic Dai Medicine by Dai people living in Laos, Myanmar, and Yunnan Province in China as detoxification, swelling-decreasing, and pain-alleviating agents [2]. Nowadays, it is noteworthy that M. tenacissima is extensively applied to treat tracheitis, asthma, rheumatalgia, and cancer [3,4,5]. The water extract of the stems and roots of M. tenacissima is a commercially available drug in China under the trademark of “Xiao-Ai-Ping”, which is applied in prescribed combinational chemotherapy for treating different cancers, such as liver cancer, stomach cancer, colon cancer, and non-small cell lung cancer [6]. Furthermore, the water extract of M. tenacissima is also a main ingredient of many other Chinese prescriptions, such as Shi-Iiao-Cao-Ke-Chuan granule used for treating bronchitis, Lv-Ji-Ke-Chuan granule used for treating cough, Ya-Jie tablets used for relieving stomach illness, and Bai-Jie capsules used for curing swollen throat [7]. The heavy demand for the plant resources of M. tenacissima has spawned large-scale cultivation of this plant in the southwest areas of Yunnan Province.

Marsdenia tenacissima has been chemically investigated previously and is rich in polyoxygenated pregnane glycosides. So far, 166 polyoxygenated pregnane glycosides have been reported [7,8,9,10,11,12]. Many of them displayed a multidrug resistance reversal effect, antitumor effect, immunomodulatory activity, antiviral effect, and anti-angiogenic effect, and the components 11α-O-2-methylbutyryl-12β-O-2-tigloyltenacigenin B [9], 11α-O-2-methylbutyryl-12β-O-2-benzoyltenacigenin B [9], and 11α,12β-O,O-ditigloyl-17β-tenacigenin B [9] exhibited cytotoxicity against KB-VI cells, with ED₅₀ values of 4.1, 2.5, and 3.4 μg/mL, respectively. In a continuation of efforts searching for biologically active natural products from traditional medicines, the ethyl acetate fraction of the ethanol extract of the stems of M. tenacissima was chemically investigated. As a result, nine new pregnane glycosides, marsdeosides A–I (1–9), have been isolated (Figure 1). Herein, the structure elucidation and inhibitory activities on nitric oxide production by lipopolysaccharide-activated macrophages (RAW264.7) of marsdeosides A–I (1–9) are described.

2. Results and Discussion

2.1. Structural Elucidation of Compounds 1–9

Compound 1, obtained as white amorphous powders, had the molecular formula of C₄₁H₅₈O₁₄, as determined by the sodium-adduct ion peak at m/z 797.37292 ([M + Na]⁺, calculated for C₄₁H₅₈O₁₄Na, 797.37188) in HRESIMS analysis. The absorption bands for the hydroxy group (3411 cm⁻¹), carbonyl group (1691 cm⁻¹), and double-bond (1549 cm⁻¹) groups were shown in the IR spectrum of 1. The ¹H NMR spectrum of 1 (Table 1 and Table 2; Figure S1, Supplementary data) showed 3 singlet methyls (δ_H 1.19, s, 3H; δ_H 0.89 s, 3H; δ_H 2.56 s, 3H), 2 doublet methyls (δ_H 1.67, d, J = 6.1 Hz; δ_H 1.49, d, J = 6.5 Hz), and 5 aromatic protons [δ_H 8.27 (2H, d, J = 8.4 Hz); 7.41 (2H, t, J = 8.4 Hz); 7.55 (1H, t, J = 8.4 Hz)]. The ¹³C NMR and DEPT spectroscopic data (Table 3; Figure S2, Supplementary data) of 1 displayed the signals for six methyls (one methoxy group), eight methylenes, twenty methines (including five olefinic/aromatic and thirteen oxygenated), and seven quaternary carbons (including two olefinic/aromatic and three carbonyls). The aforementioned data showed a similarity to those of periplocoside A [13], except for the presence of an additional carbonyl (δ_C 211.5), benzoyl group, and sp³ methyl group (δ_C 12.1); however, the absence of a dioxygenated quaternary carbon (δ_C 115.7) and the C-18 ester carbonyl in 1 indicated that 1 was a typical pregnane glycoside with a benzoyl group and 2 sugar moieties. The changes were confirmed by the key HMBC correlations (Figure 2) from H-6 (δ_H 1.46, m; 1.41, m), H-9 (δ_H 3.29, d, J = 10.0 Hz), and H-7 (δ_H 2.05; 2.22) to C-8 (δ_C 211.5); from H-18 (δ_H 1.19, s) to C-12 (δ_C 81.0), C-13 (δ_C 55.8), and the hemiketal carbon C-14 (δ_C 115.7); and from H-11 (δ_H 5.06) to C-14. These HMBC correlations also suggested that the bond between C-8 and C-14 was cleaved to form two carbonyls through oxidative cleavage of the C-8 and C-14 vicinal diol. The position of benzoxy residue was assigned at C-12 by the HMBC correlation from H-12 (δ_H 6.59) to the carbonyl group of Bz (C-1′, δ_C 165.9). The relative configuration of the aglycone part of 1 was established by the analysis of key correlations in the NOESY spectrum (Figure 3). The NOESY correlations of H-17/H-12, of H-9/H-5/H-3/H-1α, and of Me-19/H-1β suggested that H-3, H-9, H-12, and H-17 were in α-configuration, while Me-18 and Me-19 were in β-configuration.

Compound 1 had 2 sugar units, which was revealed by the existence of 2 anomeric protons at δ_H 4.87 (d, J = 9.3 Hz) and 5.18 (d, J = 8.3 Hz), which corresponded to the carbon resonances at δ_C 97.6 and 103.2, respectively, in the ¹³C NMR spectrum. The sugar chain was linked at C-3 according to the key HMBC correlation of H-3 (3.80 m) to the anomeric carbon (δ_C 97.6). In addition, the ¹H NMR spectrum showed 2 methyl doublets at δ_H 1.67 (d, J = 6.1 Hz, 3H) and 1.49 (d, J = 6.5 Hz, 3H) and 1 methoxy group at δ_H 3.84, corresponding to the carbon signals at δ_C 18.3, 18.0, and 62.1, respectively. The ¹H-¹H COSY correlations of H-1′′′/H-2′′′/H-3′′′/H-4′′′/H-5′′′/Me-6′′′, together with the HMBC correlations of H-5′′′ (δ_H 3.68) to C-1′′′ (δ_C 97.6), enabled the assignment of the carbons of 1 sugar unit. The key NOESY correlations of H-1′′′/H-2′′′α (δ_H 2.57)/H-3′′′/H-5′′′ and of H-2′′′β (δ_H 2.03)/H-4 helped to assign the sugar as 3-O-demethyl-oleandropyranose. The ¹H-¹H COSY correlations of H-1′′′′/H-2′′′′/H-3′′′′/H-4′′′′/H-5′′′′/Me-6′′′′, along with the HMBC correlations from H-5′′′′ (δ_H 4.24) to C-1′′′′ (δ_C 103.2) and from the methoxy group (δ_H 3.84) to C-3′′′′, allowed the determination of the carbons of the other sugar unit, which was further determined as 6-deoxy-3-O-methyl-allopyranose by the diagnostic NOESY correlations of H-1′′′′/H-5′′′′,and of H-2′′′′/H-3′′′′/H-4′′′′/Me-6′′′′. The key HMBC correlations of H-1′′′′ to C-4′′′ revealed that the latter sugar unit was the terminal one, and the two sugar units were connected by the C-1′′′′ and C-4′′′. The absolute configurations of the sugar moieties were assigned by TLC analysis and comparing the optical rotation data of the hydrolysates with those of published data [14,15]. Two sugar units were finally identified as 6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-3-O-demethyl-oleandropyranose by comparison of the R_f data and optical rotations, respectively. Thus, the structure of compound 1 was defined to be marsdeoside A.

Compound 2 was isolated as a white amorphous powder. The absorption bands for the hydroxy group (3406 cm⁻¹), carbonyl group (1689 cm⁻¹), and double bond (1446 cm⁻¹) were shown in the IR spectrum. It had the molecular formula of C₄₄H₆₆O₁₆, as determined by the sodium-adduct ion peak at m/z 873.42303 ([M + Na]⁺, calculated for C₄₄H₆₆O₁₆Na, 873.42431) in the HRESIMS analysis. The 1D NMR spectroscopic data of 2 (Table 1, Table 2 and Table 3; Figures S8 and S9, Supplementary data) were similar to iloneoside (3-O-[6-deoxy-3-O-methyl-β-dallopyranosyl-(1→14)-β-D-oleandropyranosyl]-11,12-di-O-tigloyl-17β-marsdenin) [16], except for the species of the terminal sugar unit. By analysis of the 2D NMR data of 2, the presence of a glucose moiety was confirmed by the key ¹H-¹H COSY correlations (Figure 2) of H-1′′′′/H-2′′′′/H-3′′′′/H-4′′′′/H-5′′′′/H-6′′′′ and NOESY correlations (Figure 3) of H-5′′′′/H-1′′′′/H-3′′′′, and H-2′′′′/H-4′′′′. The key HMBC (Figure 2) correlation from H-1′′′′ to C-4′′′ of the oleandropyranosyl suggested a (1→4) glycosidic linkage. The absolute configurations of the sugars were determined as follows. Compound 2 was subjected to acidic hydrolysis, and the side chain sugars were identified as D-oleandropyranose (Ole) and D-glucose (Glc) by comparing the R_f values and optical rotations to the literature data [8,17]. Thus, the structure of compound 2 was defined to be marsdeoside B.

The molecular formula of compound 3 was determined to be C₄₈H₆₄O₁₆ by HRESIMS m/z 919.40680 ([M + Na]⁺, calculated for C₄₈H₆₄O₁₆Na, 919.40866). The absorption bands for the hydroxy group (3402 cm⁻¹), carbonyl group (1720 cm⁻¹), and double bond (1450 cm⁻¹) were shown in the IR spectrum. The 1D NMR spectroscopic data (Table 1, Table 2 and Table 3; Figures S15 and S16, Supplementary data) showed that 3 was also a pregnane glycoside with a bi-sugar moiety. With the careful comparison of NMR chemical shifts with compound 2 and the literature data, it was found that the bi-sugar moiety was the same as compound 2, while the aglycone moiety, including the absolute configuration, was identical with that of the compound 3-O-[6-deoxy-3-O-methyl-β-allopyanosyl-(1→4)-β-digitoxopyranoside]-11α,12β-di-O-benzoyl-17β-marsdenin-5,6-dihydrogen (Compound 3 in [18]). Further elucidation of the 2D NMR spectra confirmed the above assignments. By comparison of the R_f values and optical rotations of the acidic-hydrolyzed sugars of compound 3, the sugars were identified as D-oleandrose and D-glucose [8,13,17]. Thus, the structure of compound 3 was defined to be marsdeoside C.

Compound 4 had a molecular formula determined to be C₄₈H₆₆O₁₆ by HRESIMS m/z 897.42218 ([M + Na]⁺, calculated for C₄₆H₆₆O₁₆Na, 897.42431). The IR spectrum displayed absorption bands for the hydroxy group (3413 cm⁻¹), carbonyl group (1691 cm⁻¹), and double bonds (1450 cm⁻¹). The 1D NMR spectroscopic data (Table 1, Table 2 and Table 3; Figures S22 and S23, Supplementary data) showed that 4 was also a pregnane glycoside with a bi-sugar moiety, which was indicated by the anomeric protons at δ_H 5.16 (d, J = 7.8 Hz) and 4.87 (d, J = 9.5 Hz), corresponding to the carbon resonances at δ_C 97.4 and 104.4 in the 1D NMR spectrum. Comparing the chemical shifts with those of compound 3, it was found that the composition and connection of bi-sugar moieties were the same as 3. However, the modifying moieties of the aglycone part were different between compounds 3 and 4. In compound 4, the 12-O was attached to a benzoyl group, instead of being attached to a tigolyl group as in compound 3. This change was corroborated by the key HMBC correlation from H-12 (δ_H 5.62) to C-1′′ (δ_C 166.8) and the ¹H-¹H COSY correlations of H-3′′/H-4′′/H-5′′/H-6′′/H-7′′ (Figure 2). By comparison of the Rf and optical rotations of the sugars that hydrolyzed from 4 in an acidic condition, the sugars were identified as D-oleandrose and D-glucose [8,13,17]. Therefore, the structure of compound 4 was defined to be marsdeoside D.

The molecular formula of compound 5 was determined to be C₃₉H₆₀O₁₄ by HRESIMS m/z 775.38599 ([M + Na]⁺, calculated for C₃₉H₆₀O₁₄Na, 775.38753). The IR spectrum displayed absorption bands for the hydroxy group (3410 cm⁻¹), carbonyl group (1685 cm⁻¹), and double bonds (1446 cm⁻¹). The 1D NMR spectroscopic data (Table 1, Table 2 and Table 3; Figures S29 and S30, Supplementary data) showed that 5 was a pregnane glycoside with a bi-sugar moiety. Comparing the NMR data with those of compounds 1 and 2, it was found that compound 5 also harbored a 6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-3-O-demethyl-oleandropyranosyl sugar moiety, which was identical with that of 1. The aglycone part of 5 also exhibits a high similarity with its counterpart in compound 2, except for the absence of a tigolyl group of the 11-OH. The presence of exchangeable protons at δ_H 6.49 (d, J = 6.4 Hz) and the diagnostic HMBC correlations (Figure 2) from this exchangeable proton to C-9 (δ_C 50.4), C-11 (δ_C 68.5) and C-12 (δ_C 81.1) suggested that the 11-OH was free in 5 instead of substituted by a tigolyl group. The configurations of the aglycone and sugars were determined to be consistent with the corresponding parts of compounds 1 and 2. Thus, compound 5 was defined to be marsdeoside E.

Compound 6 had a molecular formula of C₃₅H₅₆O₁₂ that was determined by HRESIMS m/z 691.36763 ([M + Na]⁺, calculated for C₃₅H₅₆O₁₂Na, 691.36640). The IR spectrum displayed absorption bands for the hydroxy group (3446 cm⁻¹), carbonyl group (1693 cm⁻¹), and double bond (1548 cm⁻¹). The 1D NMR spectroscopic data (Table 1, Table 2 and Table 3; Figures S36 and S37, Supplementary data) showed that 6 was also a pregnane glycoside with a bi-sugar unit. The key 1H-1H COSY correlations (Figure 2) of H-1′′′/H-2′′′/H-3′′′/H-4′′′/H-5′′′/Me-6′′′ and H-1′′′′/H-2′′′′/H-3′′′′/H-4′′′′/H-5′′′′/Me-6′′′′, together with the HMBC correlations (Figure 2) from one methoxy to C-3′′′, from the other methoxy to C-3′′′′, and from H-1′′′′ to C-4′′′, suggested the presence of an oleandrose and a terminal 6-deoxy-3-O-methylallose, which was connected by a (1→4) glycosidic linkage.

Furthermore, the aglycone was further elucidated by analysis of the HSQC, HMBC, and ¹H-¹H COSY correlations and comparison of the NMR data with those of compounds 2–5. Except for the carbons that were already assigned to the bi-sugar moiety, only 21 carbons remained, which suggested the nonexistence of any modification groups of the aglycone part. Furthermore, a comparison of the chemical shifts of the remaining 21 carbons with those of the core steroidal skeleton of 5 revealed that the only difference was the oxygenated status of C-8. The key ¹H-¹H COSY correlations of H-7/H-8/H-9 demonstrated that C-8 was a methine instead of an oxygenated quaternary carbon as in 5. Analysis of the NOESY spectrum suggested the β orientation of H-8 by the key NOESY correlations of H-8/H-11/Me-18. The configurations of the other chiral carbons of 6 were identical with those of compound 5. The sugars were determined to be 6-deoxy-3-O-methyl-β-D-allopyranose and β-D-oleandropyranose by comparing the physical data with the authenticate samples. Thus, the structure of compound 6 was identified to be marsdeoside F.

Compound 7 was obtained as a white amorphous powder. It had a molecular formula of C₃₇H₅₈O₁₃, as determined by m/z 733.37496 ([M + Na]⁺, calculated for C₃₇H₅₈O₁₃Na, 733.37696). The 1D NMR spectra (Table 1, Table 2 and Table 3; Figures S43 and S44, Supplementary data) of 7 exhibited a high similarity to that of 6, except for the existence of a carbonyl (δ_C 170.4) and a methyl singlet (δ_H 1.98, s; δ_C 21.3) in 7, corresponding to an acetyl group. Analysis of the 2D NMR spectra enabled the assignment of the acetyl group at the 12-O position by the key HMBC correlation (Figure 2) of H-12 (δ_H 5.32) to C-1′ (δ_C 170.4). The pivotal NOESY correlations between Me-18 (δ_H 1.74)/H-17 (δ_H 3.57) indicated that the H-17 was in β orientation, which was different from compounds 1–6. Therefore, compound 7 was assigned to be marsdeoside G.

Compound 8 had a molecular formula of C₃₆H₅₆O₁₃ by HRESIMS analysis. The 1D NMR data (Table 1, Table 2 and Table 3; Figures S50 and S51, Supplementary data) of 8 were highly similar to those of compound 7. The differences between these two compounds were the sugar species and the location of the acetyl group. Elucidation of the 2D NMR spectra suggested that the sugar part of 8 was consistent with that of compounds 1 and 5. However, the key HMBC correlation (Figure 2) from H-11 (δ_H 5.68) to the acetyl carbonyl group at δ_C 170.9, C-9 (δ_C 48.5), C-10 (δ_C 39.7), and C-12 (δ_C 72.4) revealed that the acetoxy group was located at C-11 in 8. The key NOESY correlations between Me-18 (δ_H 1.79)/H-17 (δ_H 3.49) indicated that the H-17 was in β orientation. Therefore, compound 8 was identified as marsdeoside H.

Compound 9 had a molecular formula of C₃₅H₅₈O₁₂, as determined by HRESIMS with the sodium-adduct ion peak at m/z 693.38004 ([M + Na]⁺, calculated for C₃₅H₅₈O₁₂Na, 693.38205). The 1D NMR spectra (Table 1, Table 2 and Table 3; Figures S57 and S58, Supplementary data) of 9 were highly similar to those of 6, except for the absence of double-bond data in 9. The key ¹H-¹H COSY correlations (Figure 2) between H-4/H-5/H-6/H-7 suggested that C-5 was a methine and C-6 was a methylene in compound 9. The key NOESY correlations of H-12/H-9/H-5/H-3 suggested the α orientation of H-5. Further analysis of the 2D NMR data suggested that the configurations of the other chiral carbons were the same as those of compound 6. Thus, compound 9 was assigned to be marsdeoside I.

2.2. Inhibitory Activities of Compounds on NO Production by Lipopolysaccharide-Activated Macrophage (RAW264.7)

Based on the clinical applications and previous biological studies on the secondary metabolites of this plant, the isolates in this study were subjected to anti-inflammatory activity assays. Nine compounds were evaluated for their inhibitory activities on nitric oxide production by a lipopolysaccharide-activated macrophage (RAW264.7). The N^G-monomethyl-L-arginine (L-NMMA) was used as a positive control. As shown in Table 4, compounds 1, 8, and 9 showed anti-inflammatory activities, with IC₅₀ values of 37.5, 38.8, and 42.8 μM, respectively, which were comparable to the positive control.

3. Experimental Section

3.1. General Experimental Procedures

High-resolution electrospray ionization mass spectra (HRESIMS) were measured with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The instrumental parameters for HRESIMS analysis were as follows: capillary temperature 325 °C, spray voltage 3.80 kV, sheath gas flow rate 40 mL/min, auxiliary gas flow rate 20 mL/min. Medium pressure liquid chromatography (MPLC) was performed on an Interchim system equipped with a column packed with Chromatorex C₁₈ gel (40–75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan). UV spectra were recorded on a Hitachi UH5300 spectrophotometer (Hitachi, Tokyo, Japan). The NMR spectra (¹H, ¹³C, and 2D NMR) were measured on Bruker Avance III NMR instruments at 600 MHz for ¹H and 150 MHz for ¹³C NMR. IR spectra were obtained in a Tenor 27 spectrophotometer (Bio-Rad, Richmond, CA, USA) using KBr pellets. Column chromatography (CC) was executed on silica gel (80–100 mesh or 200−300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden), and Reverse-phase silica gel (20−45 μm, Fuji Silysia Chemical Ltd.). Medium-pressure liquid chromatography (MPLC) was applied to Biotage SP2 equipment, and columns were packed with reverse-phase silica gel (C₁₈). Preparative high-performance liquid chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with YMC-Pack ODS-A columns (S-5 μm, 250 × 10 mm) and a DAD detector (Agilent Technologies, Santa Clara, CA, USA). All fractions were monitored by thin-layer chromatography (TLC) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Preparative TLC (silica gel 60, Merck KGaA, 64271 Darmstadt) was used to purify the acid-hydrolyzed sugars. The spots were visualized by heating silica gel plates soaked with a vanillin–sulfuric acid color component solvent.

3.2. Plant Material

The stems of Marsdenia tenacissima were collected before flowering from Baoshan County, Yunnan Province, People’s Republic of China, in May 2021. The plant was identified by Dr. Hong-Lian Ai (Associate Professor of South-Central Minzu University, Wuhan, Hubei 430074, China). A voucher specimen (2021103FD) was deposited in the School of Pharmaceutical Sciences, South-Central Minzu University. The stems were dried and then stored at 4 °C until extraction.

3.3. Extraction and Isolation

The dried stems of Marsdenia tenacissima (1.3 kg) were mechanically crushed and percolated with EtOH/H₂O (95:5) at room temperature for cell rupture by water absorption. After filtration, the samples were extracted exhaustively with dichloromethane/methanol (1:1, v/v; 5 L × 4) at room temperature. The solvent was evaporated in vacuo to give a dark gum (89 g), which was dissolved in water (1 L) and then extracted with ethyl acetate (2 L × 4) to give ethyl acetate extract parts (36 g). The ethyl acetate extract parts were dissolved in dichloromethane and then placed on a silica gel column eluted with dichloromethane containing increasing amounts of methanol. Five fractions (BE-1/BE-2/BE-3/BE-4/BE-5) were collected. Among them, the last fraction was eluted with methanol. Fraction BE-3 (20.0 g) was subjected to ODS silica gel CC and eluted with MeOH−H₂O (10:90→100:1, v/v) to yield 11 fractions (Frs. 3-3-1→3-3-11). Fr. 3-3-2 was purified by preparative HPLC with CH₃CN−H₂O (20:80→40:60, v/v) to give 1 (5.0 mg, t_R = 6.5 min, 0.14 ‰ of yield) and 5 (20.0 mg, t_R = 21 min, 0.56 ‰ of yield). Fr. 3-3-3 was purified by semi-preparative HPLC with CH₃CN−H₂O (35:65→53:47, v/v) to give 2 (18 mg, 0.5 ‰ of yield), 4 (22 mg, 0.61 ‰ of yield), and 3 (16 mg, 0.44 ‰ of yield) at 22 min, 27 min, and 29 min, respectively. Fr. 3-3-10 was also treated by preparative HPLC (CH₃CN−H₂O, 20:50→50:50, v/v) and yielded 6 (4.3 mg, 0.12 ‰ of yield) and 7 (3.0 mg, 0.08 ‰ of yield) at 20 min and 17 min, respectively. Fr. 3-3-11 was purified by preparative HPLC with CH₃CN−H₂O (20:80→28:72, v/v) to give 8 (4.0 mg, 0.11 ‰ of yield) and 9 (6.5 mg, 0.18 ‰ of yield) at 10.5 min and 15 min, respectively.

3.3.1. Marsdeoside A (1)

White amorphous powder;

{[α]}_{D}^{26}

−96.6 (c = 0.09, MeOH); UV (MeOH) λ_max (log ε): 250 (3.10) nm; IR (KBr) ν_max 3411, 2935, 1691, 1549, 1441, 1305, 1164, and 1031 cm⁻¹; HRESIMS m/z 797.37292 ([M + Na]⁺, calculated for C₄₁H₅₈O₁₄Na, 797.37188). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.2. Marsdeoside B (2)

White amorphous powder;

{[α]}_{D}^{26}

−56.8 (c = 0.09, MeOH); UV (MeOH) λ_max (log ε): 250 (3.08) nm; IR (KBr) ν_max 3406, 2935, 1689, 1446, 1377, 1220, 1104, 1049, and 1037 cm⁻¹; HRESIMS m/z 873.42303 ([M + Na]⁺, calculated for C₄₄H₆₆O₁₆Na, 873.42431). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.3. Marsdeoside C (3)

White amorphous powder;

{[α]}_{D}^{22}

−27.0 (c = 0.12, MeOH); UV (MeOH) λ_max (log ε): 250 (3.27) nm; IR (KBr) ν_max 3402, 2935, 1720, 1450, 1387, 1276, 1095, and 1068 cm⁻¹; HRESIMS C₄₈H₆₄O₁₆ by HRESIMS m/z 919.40680 ([M + Na]⁺, calculated for C₄₈H₆₄O₁₆Na, 919.40866). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.4. Marsdeoside D (4)

White amorphous powder;

{[α]}_{D}^{20}

+98.3 (c = 0.11, MeOH); UV (MeOH) λ_max (log ε): 250 (3.23) nm; IR (KBr) ν_max 3413, 2931, 1691, 1450, 1373, 1164, 1130, and 1082 cm⁻¹; HRESIMS m/z 897.42218 ([M + Na]⁺, calculated for C₄₆H₆₆O₁₆Na, 897.42431). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.5. Marsdeoside E (5)

White amorphous powder;

{[α]}_{D}^{26}

+61.1 (c = 0.09, MeOH); UV (MeOH) λ_max (log ε): 250 (3.08) nm; IR (KBr) ν_max 3410, 2935, 1685, 1446, 1377, 1165, 1128, and 1064 cm⁻¹; HRESIMS m/z 775.38599 ([M + Na]⁺, calculated for C₃₉H₆₀O₁₄Na, 775.38753). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.6. Marsdeoside F (6)

White amorphous powder;

{[α]}_{D}^{26}

−35.6 (c = 0.11, MeOH); UV (MeOH) λ_max (log ε): 250 (0.74) nm; IR (KBr) ν_max 3446, 2935, 1693, 1548, 1377, 1165, 1126, and 1064 cm⁻¹; HRESIMS m/z 691.36763 ([M + Na]⁺, calculated for C₃₅H₅₆O₁₂Na, 691.36640). ¹H NMR and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.7. Marsdeoside G (7)

White amorphous powder;

{[α]}_{D}^{26}

+23.4 (c = 0.10, MeOH); UV (MeOH) λ_max (log ε): 250 (3.07) nm; IR (KBr) ν_max 3410, 2935, 1689, 1446, 1377, 1165, 1125, and 1067 cm⁻¹; HRESIMS m/z 733.37496 ([M + Na]⁺, calculated for C₃₇H₅₈O₁₃Na, 733.37696). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.8. Marsdeoside H (8)

White amorphous powder;

{[α]}_{D}^{27}

−88.5 (c = 0.11, MeOH); UV (MeOH) λ_max (log ε): 250 (3.07) nm; IR (KBr) ν_max 3421, 2947, 1651, 1492, 1377, 1165, and 1024 cm⁻¹; HRESIMS m/z 719.35931 ([M + Na]⁺, calculated for C₃₆H₅₆O₁₃Na, 719.36131). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.3.9. Marsdeoside I (9)

White amorphous powder;

{[α]}_{D}^{27}

−96.6 (c = 0.11, MeOH); UV (MeOH) λ_max (log ε): 250 (0.615) nm; IR (KBr) ν_max 3421, 2930, 1651, 1548, 1492, 1374, 1304, and 1163 cm⁻¹; HRESIMS m/z 693.38004 ([M + Na]⁺, calculated for C₃₅H₅₈O₁₂Na, 693.38205). ¹H and ¹³C NMR data displayed in Table 1 and Table 2.

3.4. Acidic Hydrolysis of Compounds 1–9

Compounds 1–9 (each 2.0 mg) were dissolved in 2 M HCl (1,4-dioxane/H₂O, 1:1 v/v, 1 mL). The solution was kept at 60 °C for 2 h and then was attenuated with H₂O (3 mL). The hydrolyzed mixture was extracted with CH₂Cl₂ (4 mL × 3). The sugars were detected by TLC and compared to the standard compounds. The four sugars were confirmed as glucose, oleandrose, 3-O-demethyl-oleandrose, and 6-deoxy-3-O-methyl-allose, respectively, on the basis of the R_f values. The sugars were purified by preparative TLC and were subjected to measurement of the specific optical rotation values. Moreover, as a result, 3-O-demethyl-oleandrose was detected in the glycosides 1, 5, and 8; 6-deoxy-3-O-methyl-allose was detected from 1, 5–9; glucose was detected from 2–4; and oleandrose was detected in 2–4, 6, 7, and 9. In addition, monosaccharides of compounds 1–9 were all considered to be D-form by comparing their optical rotation (OR) with those reported in the literature [8,14,17].

3.5. Anti-Inflammatory Activity Assays

The murine mononuclear macrophages RAW264.7 were seeded into 96-well plates and stimulated with 1 μg/mL lipopolysaccharide (LPS). At the same time, the compounds with different concentrations were added. The drug-free group and the L-NMMA-positive drug group were set approximately equal as a comparison. After the cells were cultured overnight, the medium was taken to detect the production of nitric oxide (NO), and the absorbance was measured at 570 nm. MTS was added to the remaining medium for cell viability assays to exclude the toxic effects of compounds on cells. The assays were carried out as a triplicate batch of experiments. The NO production inhibition rate (%) = [(OD_{570 nm} of non-drug treatment group − OD_{570 nm} of sample group)/OD_{570 nm} of non-drug treatment group] × 100%. IC₅₀ (50% concentration of inhibition) was calculated by the Reed & Muench method [19,20].

3.6. Determination of the Absolute Configuration of the Monosaccharides

The optical rotations of 6-deoxy-3-O-methyl-D-allose, D-oleandrose, 3-O-demethyl-D-oleandrose, and D-glucose in the literature were

{[α]}_{D}^{20}

+10.0,

{[α]}_{D}^{20}

−12.0,

{[a]}_{D}^{20}

+43.0, and

{[a]}_{D}^{20}

+48.0, respectively [8,14,17]. The optical rotations of 6-deoxy-3-O-methyl-allose and 3-O-demethyl-D-oleandrose were

{[α]}_{D}^{20}

+10.2 (c = 0.17, H₂O) and

{[a]}_{D}^{20}

+43.5 (c = 0.20, H₂O), respectively, in 1; the optical rotation of oleandrose

{[α]}_{D}^{20}

−12.3 and glucose

{[a]}_{D}^{20}

+49.3 (c = 0.20, H₂O) in 2; the optical rotation of oleandrose

{[α]}_{D}^{20}

−12.3 (c = 0.21, H₂O) and glucose

{[a]}_{D}^{20}

+48.3 (c = 0.20, H₂O) in 3; the optical rotation of oleandrose

{[α]}_{D}^{20}

−12.1(c = 0.19, H₂O) and glucose

{[a]}_{D}^{20}

+49.0 (c = 0.20, H₂O) in 4; the optical rotation of 6-deoxy-3-O-methyl-allose

{[α]}_{D}^{20}

+10.2 (c = 0.17, H₂O) and 3-O-demethyl-D-oleandrose

{[a]}_{D}^{20}

+49.0 (c = 0.20, H₂O) in 5; the optical rotation of 6-deoxy-3-O-methyl-allose

{[α]}_{D}^{20}

+10.2 (c = 0.17, H₂O) and oleandrose

{[α]}_{D}^{20}

−12.6 (c = 0.22, H₂O) in 6; the optical rotation of 6-deoxy-3-O-methyl-allose

{[α]}_{D}^{20}

+10.3 (c = 0.18, H₂O) and oleandrose

{[α]}_{D}^{20}

−12.2 (c = 0.22, H₂O) in 7; the optical rotation of 6-deoxy-3-O-methyl-allose

{[α]}_{D}^{20}

+10.3 (c = 0.18, H₂O) and 3-O-demethyl-D-oleandrose

{[a]}_{D}^{20}

+43.6 (c = 0.19, H₂O) in 8; and the optical rotation of 6-deoxy-3-O-methyl-allose

{[α]}_{D}^{20}

+10.2 (c = 0.17, H₂O) and oleandrose

{[α]}_{D}^{20}

−12.6 (c = 0.22, H₂O) in 9. Thus, monosaccharides of compounds 1–9 were all considered to be D-form by comparing their specific optical rotations with those reported in the literature [8,14,17].

4. Conclusions

This phytochemical study of the cultivated medicinal plant Marsdenia tenacissima led to the isolation and structural elucidation of nine new pregnane glycosides (Figure 1), including one new 8,14-seco-pregnane glycoside (1) harboring a new hemiketal formed at the C-11 and C-8 positions. All of the undescribed pregnane glycosides were evaluated for inhibitory activity against nitric oxide production by a lipopolysaccharide-stimulated macrophage (RAW264.7), and three compounds showed comparable inhibitory activity to the positive control in vitro (Table 4). The structures and in vitro anti-inflammatory activity of these nine new pregnane glycosides were reported for the first time. This study broadens the horizon of the structural diversity of preganane glycosides of Marsdenia tenacissima, but also provides new evidence for the clinical applications of the botanical drug of this plant. Folk and ethnic medicines are of great importance and are valuable reservoirs for lead compounds in the field of drug research and development. The deeper the understanding of the chemistry of a medicinal plant, the better the way the sustainable utilization of the plant resources will be adopted.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules28062705/s1; Figure S1–S6 NMR spectra of Marsdeoside A (1), Figure S7 HRESIMS spectrum of Marsdeoside A (1), Figure S8–S13 NMR spectra of Marsdeoside B (2), Figure S14 HRESIMS spectrum of Marsdeoside B (2), Figure S15–S20 NMR spectra of Marsdeoside C (3), Figure S21 HRESIMS spectrum of Marsdeoside C (4), Figure S22–27 NMR spectra of Marsdeoside D (4), Figure S28 HRESIMS spectrum of Marsdeoside D (4), Figure S29–34 NMR spectra of Marsdeoside E (5), Figure S35 HRESIMS spectrum of Marsdeoside E (5), Figure S36–41 NMR spectra of Marsdeoside F (6), Figure S42 HRESIMS spectrum of Marsdeoside F (6), Figure S43–48 NMR spectra of Marsdeoside G (7), Figure S49 HRESIMS spectrum of Marsdeoside G (7), Figure S50–55 NMR spectra of Marsdeoside H (8), Figure S56 HRESIMS spectrum of Marsdeoside H (8), Figure S57–62 NMR spectra of Marsdeoside H (9), Figure S63 HRESIMS spectrum of Marsdeoside H (9).

Author Contributions

H.-P.C. and J.-K.L. designed the experiment; Q.-Q.M. performed the isolation and identification of all the compounds and also wrote the manuscript; S.-Y.T. and Y.-Q.Z. contributed to the isolation of the compounds; Z.-H.L. helped to buy the plant material and gave suggestions on the isolation; X.-R.P. reviewed the manuscript; H.-P.C. and J.-K.L. provided comments and suggestions on structure elucidation and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (22177138).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data in this research were presented in the manuscript and Supplementary Material.

Acknowledgments

The authors thank the Bioactivity Screening Center, Kunming Institute of Botany, Chinese Academy of Sciences, for screening the bioactivity of the compounds.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors upon reasonable request.

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Figure 1. Structures of new compounds 1–9 from Marsdenia tenacissima.

Figure 2. Selected HMBC and 1H–1H COSY correlations of compound 1–9.

Figure 3. Selected NOESY correlations of compound 1–9.

Table 1. ¹H NMR Spectroscopic Data of the Aglycones of Compounds 1–9 in Pyridine-d₅ (600 MHz, J in Hz).

No.	1	2	3	4	5	6	7	8	9
1	2.51, overlapped 1.69, overlapped	2.47, dt (13.5, 3.5) 1.43, td (13.5, 3.5)	2.27, dt (13.5, 3.8) 1.36, td (13.5, 3.8)	2.24, dt (13.2, 4.0) 1.29, td (13.2, 4.3)	2.62, overlapped 2.63, overlapped	2.60, br. d (12.0) 2.46, overlapped	2.61, br. d (12.0) 2.47, overlapped	2.58, overlapped 2.40, br. t (12.0)	3.14, br. d (14.0) 1.46, br. t (14.0)
2	1.99, m 1.58, m	2.12, overlapped 1.92, overlapped	1.86, overlapped 1.65, ddd (13.5, 13.5, 13.5)	2.03, m 1.77, overlapped	2.13, m 1.99, overlapped	2.11, m 1.86, m	2.14, m 1.85, overlapped	2.17, overlapped 1.83, overlapped	2.10, overlapped 1.74, overlapped
3	3.80, m	3.89, m	3.91, m	3.94, m	3.93, m	3.86, m	3.87, m	3.83, m	3.91, m
4	1.73, m 1.22, br. t (12.2)	2.63, dd (13.0, 4.5) 2.56, t (13.0)	1.84, overlapped 1.53, overlapped	1.83, br. d (12.0) 1.53, overlapped	3.35, br. d (13.6) 1.44, td (13.6, 3.8)	3.25, br. d (14.3) 1.43, td (14.8, 3.0)	3.26, br. d (13.9) 1.48, td (14.3, 3.6)	2.16, overlapped 1.41, td (12.6, 3.6)	1.77, overlapped 1.39, overlapped
5	1.57, m		1.23, m	1.19, m					1.11, m
6	1.46, m 1.41, m	5.44, d (5.5)	1.96, ddd (13.0, 13.0, 13.0) 1.19, m	1.93, m 1.16, overlapped	5.41, d (5.1)	5.55, overlapped	5.57, d (5.6)	5.55, d (5.4)	2.50, br .d (12.1) 1.16, overlapped
7	2.22, m 2.05, br. d (12.0)	2.66, dd (18.0, 5.5) 2.39, br. d (18.0)	2.41, overlapped 1.56, overlapped	2.37, m 1.52, overlapped	2.62, m 2.29, m	2.67, br. d (17.0) 1.97, m	2.66, overlapped 1.98, overlapped	2.70, overlapped 2.07, overlapped	1.28, overlapped 1.21, overlapped
8						2.03, overlapped	2.14, overlapped	2.21, td (11.7, 4.7)	1.92, overlapped
9	3.29, d (10.0)	2.27, d (10.5)	2.11, d (11.0)	1.97, d (11.0)	1.99, overlapped	1.61, t (11.0)	1.73, t (9.7)	1.79, overlapped	1.31, overlapped
11	5.06, overlapped	6.47, br. t (10.5)	6.82, t (10.6)	6.63, t (10.7)	4.89, td (10.7, 6.4)	4.10, overlapped	4.19, overlapped	5.68, t (9.9)	3.96, t (9.7)
12	6.59, d (4.7)	5.50, d (10.5)	5.76, d (10.6)	5.62, d (10.0)	5.49, d (9.7)	3.56, overlapped	5.32, d (9.7)	3.90, d (9.6)	3.48, d (9.5)
15	2.21, m, 2H	2.29, overlapped2.09, m	2.47, overlapped2.17, m	2.40, overlapped 2.13, m	2.19, m 2.04, m	2.06, overlapped 1.94, overlapped	2.09, overlapped 1.86, overlapped	2.11, overlapped 1.95, overlapped	2.07, overlapped 1.96, overlapped
16	2.47, m 1.80, m	2.33, m 2.07, m	2.33, m 2.17, m	2.31, m 2.14, m	2.28, m 2.05, m	2.22, m 1.86, overlapped	2.68, overlapped 1.87, overlapped	2.71, overlapped 1.99, overlapped	2.26, m 1.86, m
17	3.25, br. t (8.0)	3.22, dd (9.0, 5.0)	3.37, dd (9.8, 5.7)	3.36, dd (9.3, 5.5)	2.21, overlapped	3.88, overlapped	3.57, overlapped	3.49 br. t (8.8)	3.83, dd (8.8, 5.2)
18	1.19, s, 3H	1.68, s, 3H	1.79, s, 3H	1.74, s, 3H	1.64, s, 3H	1.35, s	1.74, s, 3H	1.79, s, 3H	1.36, s, 3H
19	0.89, s, 3H	1.72, s, 3H	1.58, s, 3H	1.55, s, 3H	1.82, s, 3H	1.39, s	1.33, s, 3H	1.33, s, 3H	1.01, s, 3H
21	2.56, s, 3H	2.20, s, 3H	2.07, s, 3H	2.06, s, 3H	2.21, s, 3H	2.30, s	2.28, s, 3H	2.37, s, 3H	2.31, s, 3H
8-OH		5.30, s		4.90, s	4.77, s
11-OH					6.49, d (6.4)
14-OH	8.16, s	5.82, s	6.00, s	5.96, s	5.60, s

Table 2. ¹H NMR Spectroscopic Data of the Decorating Groups of Compounds 1–9 in Pyridine-d₅ (600 MHz, J in Hz).

	1	2	3	4	5	6	7	8	9
Substituents	11-Benzoyl	11-Tigloyl	11-Benzoyl	11-Tigloyl				11-Acetyl
1′
2′								1.96, s, 3H
3′	8.27, d (8.4)	7.01, q (7.2)	8.05, d (8.4)	6.84, q (7.1)
4′	7.41, t (8.4)	1.61, d (7.2), 3H	7.23, t (8.4)	1.39, d (7.1)
5′	7.55, t (8.4)	1.84, s, 3H	7.31, t (8.4)	1.55, s, 3H
6′	7.41, t (8.4)		7.23, t (8.4)
7′	8.27, d (8.4)		8.05, d (8.4)
		12-Tigloyl	12-Benzoyl	12-Benzoyl	12-Tigloyl		12-Acetyl
1′′
2′′							1.98, s, 3H
3′′		7.10, q (7.1)	8.10, d (8.4)	8.28, d (8.4)	7.03, q (7.1)
4′′		1.64, d (7.1), 3H	7.25, t (8.4)	7.42, t (8.4)	1.56, d (7.1)
5′′		1.91, s, 3H	7.36 t (8.4)	7.52, t (8.4)	1.85, s, 3H
6′′			7.25, t (8.4)	7.42, t (8.4)
7′′			8.10, d (8.4)	8.28, d (84)
Sugar moieties	3-O-deMe-D-Ole	D-Ole	D-Ole	D-Ole	3-O-deMe-D-Ole	D-Ole	D-Ole	3-O-deMe-D-Ole	D-Ole
1′′′	4.87, br. d (9.3)	4.81, br. d (9.9)	4.84, d (9.6)	4.87, d (9.5)	4.93, d (9.7)	4.81, d (9.5)	4.80, d (9.7)	4.90, d (9.6)	4.84, d (9.8)
2′′′	2.57, overlapped 2.03, overlapped	2.42, dd (12.8, 5.4) 1.75, overlapped	2.42, overlapped 1.75, overlapped	2.45, dd (12.0, 4.8) 1.77, overlapped	2.55, dd (12.2, 5.3) 2.02, overlapped	2.46, overlapped 1.79, overlapped	2.45, overlapped 1.80, overlapped	2.58, overlapped 2.04, overlapped	2.48, br. d (12.5) 1.82, overlapped
3′′′	4.07, m	3.65, m	3.67, m	3.68, m	4.03, ddd (13.6, 8.6, 5.3)	3.64, overlapped	3.64, overlapped	4.04, ddd (13.4, 8.5, 5.2)	3.66, overlapped
4′′′	3.42, t (9.0)	3.74, t (9.0)	3.74, t (9.0)	3.76, t (8.9)	3.39, t (9.6)	3.63, overlapped	3.65, overlapped	3.40, t (9.0)	3.64, overlapped
5′′′	3.68, dq (9.0, 6.5)	3.68, dq (15.0, 6.0)	3.73, overlapped	3.75, m	3.64, overlapped	3.61, overlapped	3.58, overlapped	3.66, overlapped	3.66, overlapped
6′′′	1.67, d (6.1), 3H	1.73, d (6.5), 3H	1.75, d (6.0), 3H	1.78, d (5.9), 3H	1.60, d (6.0), 3H	1.66, d (5.8), 3H	1.66, d (6.0), 3H	1.61, d (6.3), 3H	1.69, d (4.3), 3H
-OMe		3.52, s, 3H	3.51, s, 3H	3.52, s, 3H		3.53, s, 3H	3.54, s, 3H		3.56, s, 3H
Sugar moieties	6-deoxy-3-O-deMe-D-Allo	D-Glc	D-Glc	D-Glc	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo
1′′′′	5.18, d (8.3)	5.15, d (7.8)	5.15, d (7.8)	5.16, d (7.8)	5.15, d (8.0)	5.31, d (8.2)	5.33, d (8.1)	5.15, d (8.0)	5.34, d (8.2)
2′′′′	3.98, dd (8.3, 3.0)	4.03, br. t (7.0)	4.03, br. t (8.0)	4.04, br. t (7.7)	3.96, overlapped	3.89, m	3.91, m	3.97, dd (8.1, 3.0)	3.93, overlapped
3′′′′	4.10, t (3.0)	4.24, overlapped	4.24, overlapped	4.25, overlapped	4.09, t (3.0)	4.10, overlapped	4.10, t (2.7)	4.09, t (3.0)	4.10, m
4′′′′	3.64, m	4.24, overlapped	4.24, overlapped	4.24, overlapped	3.63, overlapped	3.63, overlapped	3.65, overlapped	3.64, overlapped	3.65, overlapped
5′′′′	4.24, dq (10.0, 6.5)	3.96, m	3.95, m	3.96, m	4.23, dq (9.7, 6.3)	4.17, m	4.19, overlapped	4.23, m	4.18, overlapped
6′′′′	1.49, d (6.5), 3H	4.54, br. d (11.0) 4.38, dd (11.0, 6.0)	4.54, d (11.7) 4.37, dd (11.4, 5.4)	4.54, d (11.4) 4.38, dd (11.4, 5.5)	1.49, d (6.0), 3H	1.55, d (6.2), 3H	1.56, d (6.2), 3H	1.49, d (6.0), 3H	1.56, d (6.3), 3H
-OMe	3.84, s	3.52, s, 3H			3.84, s, 3H	3.84, s, 3H	3.85, s, 3H	3.84, s, 3H	3.85, s, 3H

Table 3. ¹³C NMR Data of Compounds 1−9 (150 MHz, δ_H in ppm, J in Hz, pyridine-d₅).

No.	1	2	3	4	5	6	7	8	9
1	38.5, CH₂	40.3, CH₂	39.6, CH₂	39.4, CH₂	39.7, CH₂	40.1, CH₂	40.2, CH₂	40.2, CH₂	40.0, CH₂
2	29.4, CH₂	30.0, CH₂	29.7, CH₂	29.8, CH₂	30.0, CH₂	30.8, CH₂	30.8, CH₂	30.9, CH₂	30.8, CH₂
3	76.2, CH	77.7, CH	76.0, CH	76.2, CH	78.1, CH	78.1, CH₂	78.1, CH₂	77.8, CH	77.0, CH
4	34.1, CH₂	39.7, CH₂	35.0, CH₂	35.0, CH₂	40.8, CH₂	40.0, CH₂	40.1, CH₂	39.1, CH₂	35.9, CH₂
5	43.7, CH	139.6, C	45.6, CH	45.6, CH	140.5, C	140.8, C	141.2, C	140.5, C	45.6, CH
6	30.6, CH₂	118.8, CH	25.3, CH₂	25.3, CH₂	118.5, CH	122.6, CH	122.5, CH	122.9, CH	28.9, CH₂
7	42.5, CH₂	35.5, CH₂	35.6, CH₂	35.6, CH₂	35.6, CH₂	28.5, CH₂	27.9, CH₂	28.2, CH₂	30.1, CH₂
8	211.5, C	76.0, C	78.3, C	78.3, C	75.9, C	37.6, CH	38.2, CH	38.0, CH	40.4, CH
9	62.5, CH	49.2, CH	51.4, CH	51.3, CH	50.4, CH	49.9, CH	50.4, CH	48.5, CH	52.5, CH
10	42.7, C	39.2, C	38.4, C	38.4, C	39.5, C	39.6, C	40.0, C	39.7, C	38.3, C
11	76.5, CH	71.5, CH	72.0, CH	71.2, CH	68.5, CH	72.0, CH	71.0, CH	75.5, CH	72.3, CH
12	81.0, CH	78.3, CH	79.6, CH	79.7, CH	81.1, CH	78.5, CH	76.2, CH	72.4, CH	79.4, CH
13	55.8, C	55.5, C	55.7, C	55.6, C	55.8, C	55.9, C	55.3, C	57.2, C	56.0, C
14	115.7, C	85.5, C	85.5, C	85.5, C	85.6, C	85.1, C	85.6, C	85.5, C	84.9 C
15	36.8, CH₂	36.6, CH₂	36.2, CH₂	36.1, CH₂	36.6, CH₂	35.3, CH₂	31.9, CH₂	32.4, CH₂	34.8, CH₂
16	23.3, CH₂	24.2, CH₂	24.7, CH₂	24.7, CH₂	24.3, CH₂	24.6, CH₂	21.2, CH₂	21.5, CH₂	24.9, CH₂
17	58.1, CH	59.2, CH	59.4, CH	59.4, CH	59.6, CH	58.9, CH	61.4, CH	61.9, CH	59.1, CH
18	12.1, CH₃	13.5, CH₃	14.1, CH₃	14.0, CH₃	13.8, CH₃	11.2, CH₃	15.8, CH₃	4.9, CH₃	11.4, CH₃
19	12.5, CH₃	18.1, CH₃	13.4, CH₃	13.3, CH₃	17.4, CH₃	19.2, CH₃	19.2, CH₃	19.8, CH₃	12.7, CH₃
20	207.7, C	213.1, C	213.6, C	213.7, C	213.9, C	217.0, C	208.9 C	209.9, C	216.7, C
21	31.1, CH₃	31.2, CH₃	31.7, CH₃	31.6, CH₃	31.4, CH₃	32.8, CH₃	31.7, CH₃	32.0, CH₃	32.8, CH₃
	11-Bz	11-Tig	11-Bz	11-Tig				11-Ac
1′	165.9, C	166.8, C	165.9, C	166.9, C				170.9, C
2′	130.2, C	129.1, C	130.5, C	128.9, C				22.4, CH₃
3′(-7′)	129.9, CH	138.3, CH	129.8, CH	138.5, CH
4′(-6′)	128.9, CH	14.2, CH₃	128.4, CH	14.0, CH₃
5′	133.5, CH	12.0, CH₃	133.0, CH	11.6, CH₃
		12-Tig	12-Bz	12-Bz	12-Tig		12-Ac
1′′		167.9, C	166.8, C	166.8, C	168.3, C		170.4, C
2′′		128.4, C	129.9, C	130.3, C	129.2, C		21.3, CH₃
3′′(-7′′)		138.5, CH	129.8, CH	130.0, CH	137.2, CH
4′′(-6′′)		14.2, CH₃	128.6, CH	128.8, CH	14.0, CH₃
5′′		12.0, CH₃	133.3, CH	133.5, CH	12.2, CH₃
	3-O-deMe-D-Ole	D-Ole	D-Ole	D-Ole	3-O-deMe-D-Ole	D-Ole	D-Ole	3-O-deMe-D-Ole	D-Ole
1′′′	97.6, CH	97.9, CH	97.3, CH	97.4, CH	97.8, CH	98.1, CH	98.2, CH	98.5, CH	97.8, CH
2′′′	40.0, CH₂	37.5, CH₂	37.5, CH₂	37.5, CH2	40.1, CH₂	38.1, CH₂	38.2, CH₂	40.5, CH₂	38.2, CH₂
3′′′	70.1, CH	79.5, CH	79.5, CH	79.5, CH	70.1, CH	79.9, CH	79.9, CH	70.6, CH	80.0, CH
4′′′	88.6, CH	83.4, CH	83.4, CH	83.4, CH	88.6, CH	83.6, CH	83.6, CH	89.0, CH	83.7, CH
5′′′	71.2, CH	71.9, CH	71.9, CH	71.9, CH	71.1, CH	72.2, CH	72.3, CH	71.6, CH	72.4, CH
6′′′	18.3, CH₃	18.9, CH₃	18.9, CH₃	18.9, CH	18.2, CH₃	19.4, CH₃	19.4, CH₃	18.7, CH₃	19.5,CH₃
-OMe		57.0, CH₃	56.9, CH₃	57.0, CH₃		57.5, CH₃	57.5, CH₃		57.5, CH₃
	6-deoxy-3-O-deMe-D-Allo	D-Glc	D-Glc	D-Glc	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo	6-deoxy-3-O-deMe-D-Allo
1′′′′	103.2, CH	104.4, CH	104.4, CH	104.4, CH	103.2, CH	102.4, CH	102.5, CH	103.7, CH	102.4, CH
2′′′′	72.5, CH	75.6, CH	75.6, CH	75.6, CH	72.5, CH	73.5, CH	73.6, CH	73.0, CH	73.6, CH
3′′′′	83.8, CH	78.6, CH	78.6, CH	78.6, CH	83.8, CH	84.3, CH	84.4, CH	84.3, CH	84.4, CH
4′′′′	74.1, CH	71.8, CH	71.8, CH	71.8, CH	74.0, CH	74.9, CH	74.9, CH	74.5, CH	74.9, CH
5′′′′	71.1, CH	78.0, CH	78.0, CH	78.0, CH	71.1, CH	71.2, CH	71.3, CH	71.5, CH	71.3, CH
6′′′′	18.0, CH₃	62.9, CH₂	62.9, CH₂	62.9, CH₂	18.0, CH₃	18.9, CH₃	19.0, CH₃	18.5, CH₃	19.0, CH₃
-OMe	62.1, CH₃				62.0, CH₃	62.4, CH₃	62.4, CH₃	62.5, CH₃	62.4, CH₃

Table 4. Inhibitory activities of compounds 1, 8, and 9 on NO production by lipopolysaccharide-activated macrophage (RAW264.7).

Compounds	IC₅₀ (μM)
L-NMMA	39.30 ± 1.23
1	37.46 ± 1.91
8	38.80 ± 0.76
9	42.78 ± 1.43

L-NMMA (N^G-Monomethyl-L-Arginine, monoacetate salt) was used as positive control.

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Meng, Q.-Q.; Tong, S.-Y.; Peng, X.-R.; Zhao, Y.-Q.; Li, Z.-H.; Chen, H.-P.; Liu, J.-K. Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima. Molecules 2023, 28, 2705. https://doi.org/10.3390/molecules28062705

AMA Style

Meng Q-Q, Tong S-Y, Peng X-R, Zhao Y-Q, Li Z-H, Chen H-P, Liu J-K. Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima. Molecules. 2023; 28(6):2705. https://doi.org/10.3390/molecules28062705

Chicago/Turabian Style

Meng, Qian-Qian, Shun-Yao Tong, Xing-Rong Peng, Yu-Qing Zhao, Zheng-Hui Li, He-Ping Chen, and Ji-Kai Liu. 2023. "Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima" Molecules 28, no. 6: 2705. https://doi.org/10.3390/molecules28062705

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Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima

Abstract

1. Introduction

2. Results and Discussion

2.1. Structural Elucidation of Compounds 1–9

2.2. Inhibitory Activities of Compounds on NO Production by Lipopolysaccharide-Activated Macrophage (RAW264.7)

3. Experimental Section

3.1. General Experimental Procedures

3.2. Plant Material

3.3. Extraction and Isolation

3.3.1. Marsdeoside A (1)

3.3.2. Marsdeoside B (2)

3.3.3. Marsdeoside C (3)

3.3.4. Marsdeoside D (4)

3.3.5. Marsdeoside E (5)

3.3.6. Marsdeoside F (6)

3.3.7. Marsdeoside G (7)

3.3.8. Marsdeoside H (8)

3.3.9. Marsdeoside I (9)

3.4. Acidic Hydrolysis of Compounds 1–9

3.5. Anti-Inflammatory Activity Assays

3.6. Determination of the Absolute Configuration of the Monosaccharides

4. Conclusions

Supplementary Materials

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Acknowledgments

Conflicts of Interest

Sample Availability

References

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