Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima

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 Rf 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.


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.

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 Compound 1, obtained as white amorphous powders, had the molecular formula of C 41 H 58 O 14 , as determined by the sodium-adduct ion peak at m/z 797.37292 ([M + Na] + , calculated for C 41 H 58 O 14 Na,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 1 H NMR spectrum of 1 (Tables 1 and 2; Figure S1, Supplementary data) showed 3 singlet methyls (δ H 1. 19 13 C 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 sp 3 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 13 C 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 1 H 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 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-Odemethyl-oleandropyranose by comparison of the R f data and optical rotations, respectively. Thus, the structure of compound 1 was defined to be marsdeoside A.               [8,17]. Thus, the structure of compound 2 was defined to be marsdeoside B.  (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 Dglucose (Glc) by comparing the Rf 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 (Tables 1-3; Figures S15 and S16, Supplementary data) showed that 3 was also a pregnane glycoside with a bi- The molecular formula of compound 3 was determined to be C 48 16 Na, 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 (Tables 1-3; 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)-βdigitoxopyranoside]-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.

6-deoxy-3-O-deMe-D-Allo
Compound 4 had a molecular formula determined to be C 48 16 Na,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 (Tables 1-3; 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 1 H-1 H 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 C 39 14 Na,775.38753). The IR spec-trum 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 (Tables 1-3; 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.  13 Na,733.37696). The 1D NMR spectra (Tables 1-3; 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 C 36 H 56 O 13 by HRESIMS analysis. The 1D NMR data (Tables 1-3; 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 C 35 H 58 O 12 , as determined by HRESIMS with the sodium-adduct ion peak at m/z 693.38004 ([M + Na] + , calculated for C 35 H 58 O 12 Na,693.38205). The 1D NMR spectra (Tables 1-3; 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 1 H-1 H 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.

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 Gmonomethyl-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 IC 50 values of 37.5, 38.8, and 42.8 µM, respectively, which were comparable to the positive control. Table 4. Inhibitory activities of compounds 1, 8, and 9 on NO production by lipopolysaccharideactivated macrophage (RAW264.7).
spots were visualized by heating silica gel plates soaked with a vanillin-sulfuric acid color component solvent.

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.

Extraction and Isolation
The dried stems of Marsdenia tenacissima (1.3 kg) were mechanically crushed and percolated with EtOH/H 2 O (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.

Acidic Hydrolysis of Compounds 1-9
Compounds 1-9 (each 2.0 mg) were dissolved in 2 M HCl (1,4-dioxane/H 2 O, 1:1 v/v, 1 mL). The solution was kept at 60 • C for 2 h and then was attenuated with H 2 O (3 mL). The hydrolyzed mixture was extracted with CH 2 Cl 2 (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-demethyloleandrose 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].

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%. IC 50 (50% concentration of inhibition) was calculated by the Reed & Muench method [19,20]. 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].

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.