Polyoxypregnane Glycosides from Root of Marsdenia tenacissima and Inhibited Nitric Oxide Levels in LPS Stimulated RAW 264.7 Cells

Six new polyoxypregnane glycosides, marstenacisside F1–F3 (1–3), G1–G2 (4–5) and H1 (6), as well as 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-benzoyl-tenacigenin B (7), were isolated from the roots of Marsdenia tenacissima. Their structures were established by an extensive interpretation of their 1D and 2D NMR and HRESIMS data. Compounds 1–7 were tenacigenin B derivatives with an oligosaccharide chain at C-3. This was the first time that compound 7 had been isolated from the title plant and its 1H and 13C NMR data were reported. Compounds 4 and 5 were the first examples of C21 steroid glycoside bearing unique β-glucopyranosyl-(1→4)-β-glucopyranose sugar moiety. All the isolated compounds were evaluated for anti-inflammatory activity by inhibiting nitric oxide (NO) production in the lipopolysaccharide-induced RAW 264.7 cells. The results showed that marstenacisside F1 and F2 exhibited significant NO inhibitory activity with an inhibition rate of 48.19 ± 4.14% and 70.33 ± 5.39%, respectively, at 40 μM, approximately equal to the positive control (L-NMMA, 68.03 ± 0.72%).


Introduction
Marsdenia tenacissima (Roxb.) Moon (Asclepiadaceae), a perennial climber, is distributed mainly in the southwest of China and other parts of tropical and subtropical Asia. The stems and roots of M. tenacissima are traditional Chinese medicine and Dai herbal medicine, respectively. The dried stems of M. tenacissima, known as "tongguanteng", have been used in the treatment of asthma, cancer, and trachitis [1]. The roots of this plant, known as "Dai-Bai-Jie", have been widely used as a Dai herbal medicine by Dai people living in Laos, Myanmar and the Yunnan province of China due to the root's pharmacological functions of relieving pain, clearing heat, decreasing swelling and detoxification, etc. [2]. There have been many more chemical investigations on the stems than the roots of M. tenacissima. Previous phytochemical studies on the stems had revealed this plant as an extremely rich source of C 21 steroid glycosides [3][4][5][6][7][8][9][10][11][12][13][14]. Although "Dai-Bai-Jie" has been widely used as a Dai herbal medicine, few phytochemical studies on the roots of this plant have been reported so far [15][16][17]. These studies showed that the main chemical composition of roots was also the same as the stems, i.e., C 21 steroid glycosides. These compounds were only screened for anti-HIV activity and were necessary for anti-inflammatory activity, because the traditional usage for "Dai-Bai-Jie" was the treatment of inflammatory-associated diseases. Inflammation is a response of the organism to injury related to physical or chemical noxious stimuli or microbiological toxins, which is involved in multiple pathologies such as arthritis, asthma, multiple sclerosis, colitis, inflammatory bowel diseases, and atherosclerosis [18]. It can be speculated that the presence of key chemical constituents with effective Molecules 2023, 28, 886 2 of 12 anti-inflammatory activity had led to the extensive clinical application of "Dai-Bai-Jie" in traditional ethnomedicine, so we were interested in clarifying the relationship between the constituents and anti-inflammatory activity of this plant.
In order to search for more novel natural products, particularly those with potential antiinflammatory activity, from the roots of M. tenacissima, a systematic phytochemical study was carried out on their 95% ethanol extract. As a result, six new polyoxypregnane glycosides, named marstenacisside F1-F3 (1-3), G1-G2 (4)(5), and H1 (6), as well as 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-benzoyltenacigenin B (7), were isolated ( Figure 1). Compound 7 was isolated from the title plant, and its 1 H and 13 C NMR data were reported, for the first time. In the present paper, we describe the isolation and structure elucidation of these compounds, and we also evaluate the anti-inflammatory activity of the isolated compounds in terms of the inhibitory effect on NO production in LPS-induced RAW 264.7 cells.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13 bowel diseases, and atherosclerosis [18]. It can be speculated that the presence of key chemical constituents with effective anti-inflammatory activity had led to the extensive clinical application of "Dai-Bai-Jie" in traditional ethnomedicine, so we were interested in clarifying the relationship between the constituents and anti-inflammatory activity of this plant.
In order to search for more novel natural products, particularly those with potential anti-inflammatory activity, from the roots of M. tenacissima, a systematic phytochemical study was carried out on their 95% ethanol extract. As a result, six new polyoxypregnane glycosides, named marstenacisside F1-F3 (1-3), G1-G2 (4)(5), and H1 (6), as well as 3-Oβ-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-benzoyl-tenacigenin B (7), were isolated ( Figure 1). Compound 7 was isolated from the title plant, and its 1 H and 13 C NMR data were reported, for the first time. In the present paper, we describe the isolation and structure elucidation of these compounds, and we also evaluate the anti-inflammatory activity of the isolated compounds in terms of the inhibitory effect on NO production in LPS-induced RAW 264.7 cells.
Compound 2 Figure S8). The NMR data of 2 showed a pattern analogous to 1, except for an ester group (Figures S9 and S10). In the 13 C NMR spectrum of 2, there were signals of two benzoyl groups on the aglycone of 2.
Compound 3 showed a quasi-molecular ion peak at m/z 825.4040 [M + Na] + , in accordance with the molecular formula C 43 H 62 O 14 (calcd for C 43 H 62 NaO 14 , 825.4032) ( Figure S15). In the 1 H NMR spectrum of 3 ( Figure S16), due to the absence of an ester group at C-12, there was a higher-field shift signal at δ H 3.27 (1H, d, J = 9.7 Hz), compared to the δ H 5.35 (1H, d, J = 10.2 Hz) in the 1 H NMR spectrum of 2. Consequently, the aglycone of 3 was a monoester of tenacigenin B. The signals at δ H 7.09 and 6.75 (d, J = 8. 6 Hz, each 2H) and the 13 C NMR ( Figure S17) singlet at δ C 155.2 indicated that 3 contained a 4-hydroxyphenyl group. In the HMBC spectrum ( Figure S20), the aromatic resonances at δ C 125.3 and 130.7 were correlated with the signal at δ H 3.54 (m, 2H), and 7.09 correlated with the δ C 41.1. Moreover, the HMBC spectrum showed a correlation between δ H 3.54 and δ C 172.0. Therefore, a methylene group (δ H 3.54, δ C 41.1) was located between the 4-hydroxyphenyl moiety and the carbonyl group (δ C 172.0). Hence, 3 contained a (4hydroxyphenyl) acetyl (HPA) group. The HPA group was assigned at C-11 by HMBC correlation of the proton at δ H 5.05 (H-11) with the carbonyl carbon at δ C 172.0 of the HPA group. The glycosidation site was deduced from the HMBC cross peaks between δ H 4.58 (Ole-H-1) and δ C 76.6 (aglycone-C-3). Therefore, 3 was defined as 3-O-6-deoxy- (Figures S18-S21). The 1 H NMR data of the aglycone moiety of 4 were very close to those of 1, except for the presence of the signal for an acetyl group at δ H 1.90 (3H, s) and the absence of the protons signals for the benzoyl group. HMBC cross-peaks between δ H 5.76 (H-11) and δ C 167.2 (Tig-C-1), and between δ H 5.40 (H-12) and δc 170.7 (Ac-C-1) ( Figure S27), indicated that a tigloyl and acetyl group were located at C-11 and C-12, respectively. Accordingly, the aglycone of 4 was identified as 11α-O-tigloyl-12β-O-acetyl-tenacigenin B.
The 1 H and 13 CNMR data of the sugar moiety of 5 were well in agreement with those of 4. Accordingly, 5 had the same sugar moiety as 4.
Compound  Figure S29). The NMR data of 5 (Figures S30 and S31) showed a similar pattern to 4, except for an ester substitution.

NO Inhibitory Evaluations
Compounds 1-7 were screened for anti-inflammatory activity by inhibiting NO production in LPS-induced RAW 264.7 cells. Compounds 1 and 2 showed significant NO inhibitory activity with an inhibition rate of 48.19 ± 4.14% and 70.33 ± 5.39%, respectively, at 40 µM, approximately equal to the positive control (L-NMMA, 68.03 ± 0.72%) (Figure 4 and Table 4). The effects of compounds 1-7 on cell viability are shown in Figure S50. All compounds showed dose-dependent NO inhibitory activity. Only 1 and 2 showed significant NO inhibitory activity, and other compounds did not show the activity. The above facts may be related to the structure of compounds 1 and 2. There were tigloyl and/or benzoyl groups at C-11 and C-12 of 1 and 2, and the sugar moiety was pachybiose. Although 7 also had two benzoyl groups at C-11 and C-12, the sugar moiety was neocondurangotriose.

NO Inhibitory Evaluations
Compounds 1-7 were screened for anti-inflammatory activity by inhibiting NO production in LPS-induced RAW 264.7 cells. Compounds 1 and 2 showed significant NO inhibitory activity with an inhibition rate of 48.19 ± 4.14% and 70.33 ± 5.39%, respectively, at 40 μM, approximately equal to the positive control (L-NMMA, 68.03 ± 0.72%) ( Figure 4 and Table 4). The effects of compounds 1-7 on cell viability are shown in Figure S50. All compounds showed dose-dependent NO inhibitory activity. Only 1 and 2 showed significant NO inhibitory activity, and other compounds did not show the activity. The above facts may be related to the structure of compounds 1 and 2. There were tigloyl and/or benzoyl groups at C-11 and C-12 of 1 and 2, and the sugar moiety was pachybiose. Although 7 also had two benzoyl groups at C-11 and C-12, the sugar moiety was neocondurangotriose.

Plant Material
The roots of Marsdenia tenacissima were collected from Simao, Yunnan Province, China in January 2020. A voucher specimen (No. 20200101) was deposited in the authors' research group.

Extraction and Isolation
The dried powder roots of Marsdenia tenacissima (2.5 kg) were percolated with 95% ethanol at room temperature three times (3 days each time) and then concentrated under reduced pressure to give concentrated extract. The concentrated extract was efficiently partitioned with ethyl acetate (EtOAc min) to afford compounds 7 (11 mg, t R = 12.3 min), 1 (9 mg, t R = 16.6 min), and 2 (10 mg, t R = 20.7 min).  Table 1. For 1 H and 13 C NMR data of the sugar moiety of 1 see Tables 2 and 3. Marstenacisside  Table 1. For 1 H and 13 C NMR data of the sugar moiety of 2 see Tables 2 and 3 Table 1. For 1 H and 13 C NMR data of the sugar moiety of 3 see Tables 2 and 3 time, and its 1 H and 13 C NMR data were reported. The patterns of compounds 1-7 were consistent with those of compounds previously isolated from this plant. All isolates were evaluated for anti-inflammatory activity by inhibiting the production of NO stimulated by LPS in RAW 264.7 cells, with L-NMMA as a positive control. Among those compounds, compounds 1 and 2 exhibited significant NO inhibition at 40 µM.
Author Contributions: Z.N. designed the experiment, performed the isolation and identification of all the compounds, and also wrote this paper; P.G. contributed to the nitric oxide inhibition assay, data analysis, and wrote this section; Q.F. provided comments and suggestions on structure elucidation and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.