Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Ypsilandrosides U-Y, five new steroidal saponins from Ypsilandrathibetica

Ypsilandrosides U-Y, five new steroidal saponins from Ypsilandrathibetica 1 Introduction Ypsilandra (Melanthiaceae) is distributed in south- western China and Myanmar, which contains 5 species according to the updated classification of the Angio - *Correspondence: haiyangliu@mail.kib.ac.cn Wen‑ Tao Gao and Ling‑Ling Yu contributed equally to this work sperm Phylogeny Group IV [1]. Among them, Ypsilandra State Key Laboratory of Phytochemistry and Plant Resources thibetica has been used in folk medicine for treatment of in West China, and Yunnan Key Laboratory of Natural Medicinal scrofula, dysuria, edema, uterine bleeding, and traumatic Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China hemorrhage in China by the local people [2, 3]. Our Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 2 of 9 previous investigations discovered twenty eight new ste- four methyl groups, nine methylene groups (one oxygen- roidal glycosides including nineteen spirostanol saponins, ated), ten methine groups (one olefinic and three oxy - two furostanol saponins, three cholestanol saponins, two genated), and four quaternary carbons (one olefinic and pregnane glycosides, and two C -steroidal lactone gly- one ketal). The above NMR data suggested that com - cosides from this species [4–10], some of which showed pound 1 is a typical C-27 steroidal saponin and its agly- cytotoxicity [4, 5], antifungal [4, 6], antibacterial [6], anti- cone is heloniogen [11]. This deduction can be confirmed 1 1 HIV-1 activities [7], and so on. For further investigation by 2D-NMR spectra. The H‒ H COSY correlations on the chemical constituents of this herb, four new spi- revealed that the aglycone for 1 had four structural frag- rostanol saponins (1‒4) and one new cholestanol saponin ments as shown in (Fig. 2). Furthermore, the key HMBC (5) (Fig.  1) were obtained and structurally characterized. correlations (Fig.  2) from CH -18 (δ 0.89) to C-12 (δ 3 H C The current paper reports the isolation, structural eluci - 82.4)/C-13 (δ 44.9)/C-14 (δ 44.4)/C-17 (δ 53.1), from C C C dation, and the induced platelet aggregation activity of CH -19 (δ 0.91) to C-1 (δ 37.1)/C-5 (δ 141.1)/C-9 (δ 3 H C C C these isolates. 49.0)/C-10 (δ 36.9), from CH -21 (δ 1.38)/H-20 (δ C 3 H H 2.00)/H-23a (δ 1.77)/H-26a (δ 3.53) to C-22 (δ 109.3) H H C 2 Results and discussion were observed. In addition, the ROESY correlations of Compound 1 was isolated as an amorphous powder. H-12 (δ 3.88) with H-18 (δ 0.89) and H-20 (δ 2.00) H H H Its molecular formula was determined as C H O by indicated that the OH-12 was α-oriented (Fig. 3). 44 70 17 the positive-ion HRESI-MS at m/z 893.4500 [M + Na] For the sugar part, the pentose was inferred as 13 13 (calcd. for C H O Na, 893.4505) and C NMR data β-d-apiofuranoside by the C NMR signals at δc 108.1 44 70 17 (Table  2). The H NMR spectrum of 1 (Table  1) showed (d, C-1’), 78.4 (d, C-2’), 79.0 (s, C-3’), 74.8 (t, C-4’), and four methyl proton signals at δ 0.89 (s, CH -18), 0.91 72.9 (t, C-5’) with those of corresponding carbons of H 3 (s, CH -19), 1.38 (d, J = 7.0 Hz, CH -21), and 0.67 (d, α- and β-d-apiofuranoside and α- and β-l-apiofuranoside 3 3 J = 5.4 Hz, CH -27), one olefinic proton signal at δ 5.18 [12, 13]. And the two hexose units were assigned to be 3 H (o, H-6), while three anomeric protons at δ 5.64 (d, a l-rhamnopyranosyl and a d-glucopyranosyl by their J = 3.2 Hz, H-1’), 5.30 (br s, H-1’’), and 4.86 (d, J = 7.8 Hz, NMR data, the acid hydrolysis of 1, and the HPLC anal- H-1’’’), which suggested that 1 was a glycoside with three ysis (retention time) of their L-cysteine methyl esters monosaccharide moieties. The C NMR spectra dis- followed by conversion into O-tolyl isothiocyanate deriv- played 44 carbon signals, of which 17 were assigned to atives and the authentic samples’ derivatives. And the those of one pentose and two hexose units, whereas other β-configuration of glucopyranosyl was revealed by the 27 ones were assigned to the aglycone moiety, including coupling constant ( J > 7.0 Hz) [14], while the anomeric 1,2 OH OH OH 21 O HO O 25 18 O HO OH 22 H HO OH 17 23 H O HO OH O HO H H 1 9 H HO O H H OH H H HO O HO 7 HO O 3 O O 12 3 OH O HO HO OH OH O HO HO HO OH OH HO OH HO 22' H 16' OH OH 5 7 H H H HO 6 CHO O O O HO 4 5 HO O OH HO HO HO HO O OH HO OH O HO HO HO OH OH HO OH Fig. 1 Chemical structures of saponins 1‒5 G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 3 of 9 Table 1 H NMR spectroscopic data of compounds 1–5 (δ in ppm, J in Hz, C D N) 5 5 a a a a b Position 1 2 3 4 5 1a 1.73 (o) 1.75 (d, 3.6) 1.73 (o) 1.70 (o) 1.73 (m) 1b 1.07 (d, 3.8) 0.97 (s) 1.00 (s) 1.15 (o) 0.95 (m) 2a 1.99 (m) 2.07 (o) 2.21 (m) 2.18 (m) 2.06 (d) 2b 1.62 (m) 1.72 (d, 6.3) 1.86 (m) 1.89 (m) 1.86 (d) 3 3.58 (o) 3.69 (m) 3.95 (o) 4.03 (m) 3.87 (m) 4a 2.45 (m) 2.60 (o) 2.72 (o) 4.02 (m) 2.81 (o) 4b 2.29 (m) 2.45 (t, 12.3) 1.82 (o) 2.47 (m) 2.74 (o) 6 5.18 (o) 5.26 (o) 5.22 (d, 5.0) 10.22 (s) 5.35 (o) 7a 1.76 (o) 1.91 (o) 1.83 (m) 1.95 (o) 7b 1.73 (o) 1.60 (o) 1.47 (m) 1.58 (o) 8 1.44 (m) 1.51 (m) 1.49 (m) 2.67 (m) 1.61 (m) 9 1.87 (d, 3.8) 0.95 (d, 20.9) 0.91 (m) 1.03 (m) 0.98 (m) 11a 2.22 (m) 1.56 (m) 1.48 (m) 1.32 (o) 1.57 (2H, m) 11b 1.57 (m) 1.47 (m) 1.03 (o) 12a 3.88 (br, s) 2.18 (m) 2.12 (m) 1.69 (m) 2.40 (m) 12b 1.91 (m) 1.83 (m) 1.03 (m) 1.60 (m) 14 1.56 (o) 2.04 (m) 2.03 (m) 1.36 (m) 1.50 (m) 15a 1.94 (o) 2.17 (m) 2.21 (m) 2.65 (m) 2.61 (m) 15b 1.44 (o) 1.51 (m) 1.56 (m) 2.01 (m) 2.41 (m) 16 4.42 (m) 4.43 (t, 6.9) 4.58 (t, 7.2) 4.59 (m) 17 3.31 (dd, 8.6, 6.1) 1.80 (dd, 8.5, 6.1) 18 0.89 (s) 0.93 (s) 1.14 (s) 0.88 (s) 0.94 (s) 19 0.91 (s) 0.97 (s) 1.00 (s) 0.83 (s) 1.10 (s) 20 2.00 (m) 2.20 (q) 3.39 (q, 7.2) 1.98 (m) 21 1.38 (d, 7.0) 1.18 (d, 7.1) 1.31 (d, 7.2) 1.13 (d, 7.0) 2.34 (s) 23a 1.77 (o) 1.92 (m) 4.00 (m) 1.67 (m) 2.76 (m) 23b 1.38 (o) 1.52 (m) 1.58 (m) 2.72 (m) 24a 1.67 (m) 2.21 (m) 2.29 (o) 1.58 (m) 1.95 (m) 24b 1.28 (m) 1.86 (m) 2.21 (o) 1.24 (m) 1.51 (m) 25 1.55 (d, 6.2) 1.87 (m) 2.29 (m) 1.59 (o) 1.92 (m) 26a 3.53 (o) 4.12 (o) 3.99 (m) 4.90 (br s) 3.79 (m) 26b 3.46 (o) 3.92 (m) 3.90 (m) 3.54 (br s) 3.73 (m) 27a 0.67 (d, 5.4) 4.12 (o) 3.73 (m) 0.71 (d, 4.7z) 1.14 (d, 6.6) 27b 3.94 (m) 3.68 (m) 16’ 3‑Api 3‑Api 3‑ Glc 3‑ Glc 7.12 (o) 22’ 7.07 (o) 3‑ Glc 1’ 5.64 (d, 3.2) 5.72 (o) 4.92 (d, 7.1) 5.02 (d, 7.3) 4.96 (o) 2’ 4.63 (o) 4.83 (m) 4.18 (o) 4.23 (m) 4.22 (m) 3’ 4.18 (o) 4.20 (m) 4.22 (m) 4’a 4.51 (m) 4.48 (d, 9.3) 4.19 (o) 4.40 (m) 4.41 (m) 4’b 4.25 (m) 4.24 (d, 9.3) 3.89 (o) 3.64 (m) 3.61 (m) 5’a 4.62 (o) 4.13 (2H, o) 5’b 4.43 (o) 6’a 4.74 (d, 5.5) 4.21 (o) 4.19 (o) 6’b 4.28 (d, 5.5) 4.05 (o) 4.05 (o) 5’‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 1’’ 5.30 (br s) 5.85 (br s) 6.31 (br s) 6.44 (br s) 6.41 (br s) 2’’ 3.92 (m) 4.56 (o) 4.76 (o) 4.86 (m) 4.87 (m) 3’’ 4.08 (m) 4.70 (br, s) 4.58 (o) 4.62 (m) 4.67 (m) Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 4 of 9 Table 1 (continued) a a a a b Position 1 2 3 4 5 4’’ 3.63 (m) 4.31 (o) 4.32 (o 4.36 (m) 4.38 (m) 5’’ 4.24 (m) 4.49 (m) 4.94 (m) 4.93 (m) 4.97 (m) 6’’ 1.55 (d, 6.2) 1.72 (d, 6.3) 1.75 (d, 6.1) 1.59 (d, 6.1) 1.60 (o) 12‑ Glc 6’‑ Glc 4′‑Rha 4′‑Rha 1’’’ 4.86 (d, 7.8) 5.04 (d, 8.0) 5.82 (br s) 5.84 (br s) 2’’’ 4.06 (m) 4.01 (o) 4.54 (m) 4.52 (m) 3’’’ 4.23 (m) 4.18 (o) 4.54 (m) 4.54 (m) 4’’’ 4.25 (m) 4.12 (o) 4.44 (m) 4.45 (m) 5’’’ 3.97 (m) 4.18 (o) 4.92 (m) 4.94 (m) 6’’’a 4.53 (o) 4.49 (d, 11.5) 1.59 (d, 6.1) 1.59 (o) 6’’’b 4.39 (d, 5.0) 4.33 (o) 4′′‑Rha 4′′‑Rha 1’’’’ 6.28 (br s) 6.29 (br s) 2’’’’ 4.90 (m) 4.90 (m) 3’’’’ 4.54 (m) 4.52 (m) 4’’’’ 4.33 (m) 4.31 (m) 5’’’’ 4.94 (m) 4.37 (m) 6’’’’ 1.73 (d, 6.2) 1.78 (d, 6.1) s singlet, d doublet, t triplet, q quartet, br broad, m multiplet, o overlapped a b Measured at 500 MHz. Measured at 600 MHz 1 1 Fig. 2 H‒ H COSY and Key HMBC correlations of 1‒5 configuration of rhamnopyranosyl was identified as aglycone was established from the following HMBC cor- α-orientated on the basis of the chemical shift values of rletions: H-1’ (δ 5.64) of Api with C-3 (δ 77.5) of the H C C-3’’ (δ 72.9) and C-5’’ (δ 70.6) with those of corre- aglycone, H-1’’ (δ 5.30) of the Rha with C-5’ (δ 72.9) of C C H C sponding carbons of methyl α- and β-rhamnopyranoside Api, and H-1’’’ (δ 4.86) of the Glc with C-12 (δ 82.4) H C [15]. The sequence of the sugar chain at C-3 of the of the aglycone (Fig.  2). Thus, the structure of 1 was G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 5 of 9 Me H 18 Me Me H Me 19 H H 19 Me H 20 O Me H 17 Me H 12 CH OH H 2 1 1 O HO 3 O H O 3 H HO O Glc H H H H Fig. 3 Key ROESY correlations for the aglycone moieties of 1 and 3 elucidated as 12-O-β-d-glucopyranosy-(25R)-spirost-5- 6.31) to C-2’ (δ 77.5), and from H-1’’’ (δ 5.04) to C-6’ C H en-3β,12β-diol-3-O-α-l -rhamnopyranosyl-(1 → 5)-β-d - (δ 69.9)  (Fig.  2). Consequently, the structure of 3 was apiofuranoside, and named ypsilandroside U. established as (23S,25S)-spirost-5-en-3β,17α,23,27- Compound 2 was isolated as an amorphous powder t e tra ol-3- O - β - d -g lu c opy rano s yl-(1 → 6)-[ α - l - with a molecular formula of C H O determined by rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside, and 38 60 13 the positive-ion HRESI-MS at m/z 747.3921 [M + Na] , named ypsilandroside W. (calcd. for C H O Na, 747.3926) and C NMR data Compound 4 possessed a molecular formula C H O 38 60 13 51 80 21 (Table  2). Its NMR spectra suggested that 2 is a spiros- determined by the HRESI-MS at m/z 1051.5077 tane saponin with a disaccharide chain. Comparison [M + Na] , (calcd. for C H O Na, 1051.5084) and 51 80 21 1 13 13 of the H and C NMR data of 2 (Tables  1 and 2) with C NMR data (Table  2). The UV spectrum of 4 showed those of ypsiparoside C obtained from the same genus absorption maxima at 254.5  nm, suggesting the pres- [16] revealed that they shared the same aglycone. The ence of a conjugated enal system. When comparing its 1 13 two monosaccharides and their absolute configura - H and C NMR data (Tables  1 and 2) with those of tions were determined as β-d-apiose and α-l-rhamnose ypsilandroside H [10], it was suggested that they shared by the same methods with compound 1. The HMBC the same sugar sequence and the similar aglycone, correlations from H-1’ (δ 5.72) to C-3 (δ 77.7), and except for the compound 4 has no hydroxyl substituent H C from H-1’’ (δ 5.85) of the rhamnopyransyl to C-2’ (δ at the C-17. The above deduction could be verified by H C 82.4) established the sequence for 3-O-sugar chain as the HMBC correlations from H-21 (δ 1.13) and H-18 1 1 O-α-l -rhamnopyranosyl-(1 → 2)-β-d -apiofuranoside (δ 0.88) to C-17 (δ 62.4) and H‒ H COSY correla- H C (Fig.  2). Therefore, the structure of 2 was deter - tions between H-16 (δ 4.59) and H-17 (δ 1.80) (Fig. 2). H H mined as (25R)-spirost-5-en-3β,17α,27-triol-3-O-α- The HMBC correlations from H-1’ (δ 5.02) to C-3 l-rhamnopyranosyl-(1 → 2)-β-d-apiofuranoside, and (δ 77.7), from H-1’’ (δ 6.44) to C-2’ (δ 77.9), from C H C named ypsilandroside V. H-1’’’ (δ 5.82) to C-4’ (δ 77.7), and from H-1’’’’ (δ H C H Compound 3 was isolated as an amorphous pow- 6.28) to C-4’’’ (δ 80.4) confirmed that compound 3 der and had a molecular formula of C H O as had the same sequence of 3-O-sugar chain as that of 45 72 20 determined by the positive-ion HRESI-MS data (m/z ypsilandroside H  (Fig.  2). Thus, the structure of 4 was 955.4505 [M + Na] , calcd. for C H O Na, 955.4509) elucidated as (25R)-B-nor(7)-6-carboxaldehyde-spirost- 45 72 20 and C NMR data (Table  2). Inspection of the NMR 5(7)-en-3β-ol-3-O-α-l -rhamnopyranosyl-(1 → 4)-α-l - spectra (Tables  1 and 2) of 3 revealed that it possessed r h a m n o p y r a n o s y l - ( 1 → 4 ) - [ α - l - r h a m n o p y r a n o - a spirotanol skeleton with a trisaccharide chain consist- syl-(1 → 2)]-β-d-glucopyranoside, and named ing of one rhamnopyranosyl and two glucopyranosyls. ypsilandroside X. 1 13 Comparing its H and C NMR data (Tables  1 and 2) The molecular formula of compound 5 was deter- with those of trillitschonide S6 [17] indicated that they mined as C H O by the HRESI-MS at m/z 1045.5352 53 82 19 + 13 shared the same aglycone. The α-orientations of OH-23 [M + Na] (calcd. for C H O Na, 1045.5343) and C 53 82 19 and CH OH-25 were supported by the ROESY correla- NMR data (Table  2). Its NMR spectra indicated that tions between H-23 (δ 4.00) and H-20 (δ 3.39)/H- compound 5 was a cholestane tetraglycosides contain- H H 1 13 25 (δ 2.29)  (Fig.  3). The absolute configurations and ing an aromatic ring. Analysis of the H and C NMR the anomeric configurations of monosaccharides were data (Tables  1 and 2) of 5 suggested that it was similar determined by the same methods with the above com- to that of parispseudoside A [18], and the major dif- pounds. The sequence of the sugar chain at C-3 of the ference was the absence of a glucopyranosyl group aglycone was established by the HMBC correlations at OH-26 site. With the assistance of HSQC experi- 1 13 from H-1’ (δ 4.92) to C-3 (δ 76.8), from H-1’’ (δ ment, H and C NMR data (Tables  1 and 2) showed H C H Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 6 of 9 Table 2 C NMR spectroscopic data of compounds 1–5 (δ in Table 2 (continued) ppm, C D N) a a a a b 5 5 Position 1 2 3 4 5 a a a a b Position 1 2 3 4 5 63.1 (t) 62.7 (t) 18.6 (q) 18.8 (q) 6′′′ 1 37.1 (t) 37.6 (t) 37.6 (t) 36.3 (t) 37.2 (t) 4′′‑Rha 4′′‑Rha 2 30.2 (t) 30.3 (t) 30.3 (t) 29.9 (t) 30.0 (t) 1′′′′ 103.4 (d) 103.2 (d) 3 77.5 (d) 77.7 (d) 76.8 (d) 77.7 (d) 77.8 (d) 2′′′′ 72.7 (d) 72.8 (d) 4 39.3 (t) 39.3 (t) 39.1 (t) 30.7 (t) 38.9 (t) 3′′′′ 72.9 (d) 72.4 (d) 5 141.1 (s) 140.8 (s) 140.9 (s) 169.5 (s) 140.9 (s) 74.0 (d) 74.1 (d) 4′′′′ 6 121.6 (d) 121.9 (d) 121.7 (d) 189.3 (d) 121.7 (d) 5′′′′ 70.5 (d) 70.3 (d) 7 32.0 (t) 32.4 (t) 32.4 (t) 139.6 (s) 31.9 (t) 18.9 (q) 18.6 (q) 6′′′′ 8 31.8 (d) 32.3 (d) 32.3 (d) 45.8 (d) 30.8 (d) a b Measured at 125 MHz. Measured at 150 MHz 9 49.0 (d) 50.2 (d) 50.1 (d) 60.4 (d) 50.4 (d) 10 36.9 (s) 37.1 (s) 37.1 (s) 46.5 (s) 37.0 (s) four anomeric protons at δ 4.96 (o, H-1’), 6.41 (br s, 11 27.6 (t) 20.9 (t) 20.9 (t) 20.8 (t) 21.2 (t) H-1’’), 5.84 (br s, H-1’’’), and 6.29 (s, H-1’’’’) and their 12 82.4 (d) 32.1 (t) 32.4 (t) 40.1 (t) 36.8 (t) corresponding anomeric carbons at δ 100.2 (C-1’), 13 44.9 (s) 45.1 (s) 45.8 (s) 43.3 (s) 47.1 (s) 102.1 (C-1’’), 102.1 (C-1’’’), and 103.2 (C-1’’’’). The 14 44.4 (d) 53.0 (d) 53.1 (d) 54.3 (d) 57.6 (d) sequence of sugar units was consistent with that of 15 32.1 (t) 31.8 (t) 31.9 (t) 35.3 (t) 32.3 (t) compound 4 by HMBC experiment (Fig. 2). As a result, 16 81.0 (d) 90.2 (d) 90.8 (d) 81.3 (d) 140.6 (s) the structure of 5 was assigned as homo-aro-cholest-5- 17 53.1 (d) 90.1 (s) 90.1 (s) 62.4 (d) 151.8 (s) en-3β,26-diol-3-O-α-l -rhamnopyranosyl-(1 → 4)-α-l - 18 17.0 (q) 17.2 (q) 17.4 (q) 16.8 (q) 16.4 (q) r hamnopy rano s yl-(1 → 4)-[ α - l - 19 19.3 (q) 19.5 (q) 19.4 (q) 15.5 (q) 19.2 (q) rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside, and 20 42.2 (d) 45.3 (d) 38.8 (d) 41.9 (d) 131.1 (s) named ypsilandroside Y. 21 15.3 (q) 9.6 (q) 9.4 (q) 15.1 (q) 14.6 (q) Because the whole plants of Y. thibetica has been 22 109.3 (s) 110.5 (s) 112.7 (s) 109.2 (s) 139.9 (s) used in folk medicine for treatment of uterine bleeding 23 32.0 (t) 27.5 (t) 68.1(d) 31.9 (t) 31.4 (t) and traumatic hemorrhage in China, the isolated com- 24 29.4 (t) 21.2 (t) 33.1 (t) 29.3 (t) 35.4 (t) pounds (1–5) were evaluated for their induced platelet 25 30.6 (d) 36.1 (d) 40.4 (d) 30.7 (d) 36.6 (d) aggregation activity and ADP (adenosine diphosphate) 26 66.8 (t) 60.6 (t) 63.1 (t) 66.9 (t) 67.3 (t) was used as a positive control. Unfortunately, the 27 17.4 (q) 61.4 (t) 64.0 (t) 17.4 (q) 17.2 (q) results showed all isolated saponins did not exhibit the 16’ 122.8 (d) 22’ 127.3 (d) inducing platelet aggregation activity at the tested con- 3‑Api 3‑Api 3‑ Glc 3‑ Glc 3‑ Glc centration of 100 μM. 1′ 108.1 (d) 107.0 (d) 100.7 (d) 100.9 (d) 100.2 (d) 78.4 (d) 82.4 (d) 77.5 (d) 77.9 (d) 77.9 (d) 2′ 3′ 79.0 (s) 80.5 (s) 79.5 (d) 77.4 (d) 77.6 (d)3 Experimental section 4′ 74.8 (t) 74.9 (t) 71.6 (d) 77.7 (d) 77.6 (d) 3.1 General experimental procedures 72.9 (t) 65.9 (t) 78.4 (d) 77.2 (d) 76.9 (d) Optical rotations were measured by a JASCO P-1020 5′ polarimeter (Jasco Corp., Japan). UV spectra were 6′ 69.9 (t) 61.3 (t) 61.1 (t) recorded on a Shimadzu UV2401 PC spectrophotom- 5’‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 2′‑Rha eter (Shimadzu Corp., Japan). HRESI-MS was recorded 1′′ 102.8 (d) 102.0 (d) 102.0 (d) 101.9 (d) 102.1 (d) on an Agilent 1290 UPLC/6540 Q-TOF mass spectrom- 2′′ 72.4 (d) 72.7 (d) 72.6 (d) 72.5 (d) 72.6 (d) eter (Agilent Corp., USA). The NMR experiments were 3′′ 72.9 (d) 72.0 (d) 72.8 (d) 72.9 (d) 72.8 (d) performed on Bruker AVANCE III 500, Avance III-600, 4′′ 74.3 (d) 74.0 (d) 74.2 (d) 74.2 (d) 74.0 (d) and AV 800 spectrometers (Bruker Corp., Switzerland). 70.6 (d) 70.3 (d) 69.5 (d) 69.5 (d) 69.5 (d) 5′′ Silica gel (200–300 mesh, Qingdao Marine Chemical Co., 6′′ 18.6 (q) 18.7 (q) 18.7 (q) 18.5 (q) 18.3 (q) Ltd., People’s Republic of China), RP-18 (50  μm, Merck, 12‑ Glc 6’‑ Glc 4′‑Rha 4′‑Rha Germany), and Sephadex LH-20 (Pharmacia, Stockholm, 1′′′ 106.6 (d) 105.5 (d) 102.3 (d) 102.1 (d) Sweden) were used for column chromatography (CC). 2′′′ 75.6 (d) 75.2 (d) 72.9 (d) 72.8 (d) An Agilent 1260 system (Agilent Corp., America) with a 78.8 (d) 78.4 (d) 73.3 (d) 73.2 (d) 3′′′ Zorbax SB-C18 column (5  μm, 9.4 × 250  mm) was used 4′′′ 71.9 (d) 71.6 (d) 80.4 (d) 80.3 (d) for HPLC separation. TLC was carried out on silica gel 78.4 (d) 78.4 (d) 68.4 (d) 68.2 (d) 5′′′ G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 7 of 9 HSGF plates (Qingdao Marine Chemical Co., China) ([M + Na] , calcd. for C H O Na, 747.3926) (Addi- 254 38 60 13 or RP-18 F (Merck, Darmstadt, Germany). tional file 1). 3.2 Plant material 3.4.3 Y psilandroside W (3) 20.5 1 The whole plant materials of Y. thibetica were collected Amorphous solid; [α] ‒125.67 (c 0.12, MeOH); H in August 2010 from Zhaotong City, Yunnan Provence, (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) 5 5 China, and identified by Prof. Xin-Qi Chen, Institute of NMR data, see Tables  1 and 2; HRESIMS m/z 955.4505 Botany, Chinese Academy of Sciences, Beijing. A voucher [M + Na] (calcd. for C H O Na, 955.4509) (Addi- 45 72 20 specimen was deposited at the State Key Laboratory of tional file 1). Phytochemistry and Plant Resources in West China, Kun- ming Institute of Botany, Chinese Academy of Sciences. 3.4.4 Y psilandroside X (4) 18.6 Amorphous solid; [α] ‒106.40 (c 0.15, MeOH); UV 3.3 Extraction and isolation (MeOH) λ (log ε) 202.5 (3.9), 254.5 (3.9) nm; H max The dried whole plants of Y. thibetica (110  kg) were (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) 5 5 crushed and extracted three times with 70% EtOH under NMR data, see Tables 1 and 2; HRESIMS m/z 1051.5077 reflux for a 3 h, 2 h and 2 h. Then, the combined extract [M + Na] (calcd. for C H O Na, 1051.5084) (Addi- 51 80 21 was concentrated under reduced pressure. The crude tional file 1). extract (30  kg) was passed through YWD-3F macropo- rous resin and eluted successively with H O, 40% EtOH, 3.4.5 Y psilandroside Y (5) 18.6 75% EtOH, and 95% EtOH, respectively. Evaporated 75% Amorphous solid; [α] ‒48.18 (c 0.11, MeOH); UV EtOH fraction (crude saponin-rich mixture, 10  kg) was (MeOH) λ (log ε) 203 (4.5) nm; H (600  MHz, pyr- max subjected to a silica gel column chromatography (CHCl – idine-d ) and C (150  MHz, pyridine-d ) NMR data, 3 5 5 MeOH, 20:1 → 8:2, v/v) to give eleven fractions (Fr. A–Fr. see Tables  1 and 2; HRESIMS m/z 1045.5352 [M + Na] K). Fr. C (560 g) was subjected to a silica gel column chro- (calcd. for C H O Na, 1045.5343) (Additional file 1). 53 82 19 matography (CHCl –MeOH, 20:1 → 1:1, v/v) to give 14 fractions (Fr. C-1–Fr. C-14). Fr. C-11 (80 mg) was submit- 3.5 Acid hydrolysis of compounds 1–5 and determination ted to Sephadex LH-20 (MeOH) and chromatographically of the absolute configuration of the sugars by HPLC separated on an RP-18 column eluted with MeOH–H O Compounds 1‒5 (1.0  mg each) in 6  M C F COOH 2 3 (40:60 → 70:30, v/v) and purified by preparative HPLC (1,4-dioxane-H O 1:1, 1.0  mL) were heated at 99 ℃ (MeCN–H O, 40:60 → 50:50, v/v) to afford saponin for 2  h, respectively. The reaction mixture was diluted 2 (t = 12.8  min, 10  mg). Fr. C-13 (45  g) was submitted with H O (1.0  mL) and then extracted with EtOAc R 2 to Sephadex LH-20 (MeOH) to give three subfractions (3 × 2.0  mL). Next, each aqueous layer was evaporated (C-13–1–C-13–3). Subsequently, Fr. C-13–1 (150  mg) to dryness using rotary evaporation. Each dried residue was further purified by preparative HPLC (MeCN–H O, was dissolved in pyridine (1.0 mL) mixed with l-cysteine 25:75 → 35:65, v/v) to afford saponins 5 (t = 10.8  min, methyl ester hydrochloride (1.0  mg) (Aldrich, Japan) 7  mg) and 4 (t = 11.9  min, 12  mg), whereas saponins 3 and heated at 60 °C for 1 h. Then, O-tolyl isothiocyanate (t = 11.1  min, 10  mg) and 1 (t = 14.8  min, 9  mg) were (5.0 μL) (Tokyo Chemical Industry Co., Ltd., Japan) was R R obtained from Fr. C-13–3 (208 mg) by preparative HPLC added to the mixture, this being heated at 60 °C for 1 h. (MeCN–H O, 30:70 → 45:55, v/v). Each reaction mixture was directly analyzed by reversed phase HPLC following the above procedure. Each reac- 3.4 P hysical and spectroscopic data of new glycosides tion mixture was directly analyzed by analytical HPLC on 3.4.1 Ypsilandroside U (1) a Poroshell 120 SB-C18 column (100 × 4.6  mm, 2.7  μm, 18.6 1 Amorphous solid; [α] ‒55.80 (c 0.20, MeOH); H Agilent) using an elution of C H CN‒H O (20:75 → 40:60, 3 2 (500  MHz, pyridine d ) and C (125  MHz, pyridine d ) v/v) at a flow rate of 0.6 mL/min. As a result, the sugars 5 5 NMR data, see Tables  1 and 2; HRESIMS m/z 893.4500 in the test compounds were identified as d-glucose and [M + Na] (calcd. for C H O Na, 893.4505) (Addi- l-rhamnose, respectively, by comparing their molecu - 44 70 17 tional file 1). lar weight and retention time with the standards (t 13.90 min for d-glucose; t 17.72 min for l-rhamnose). 3.4.2 Ypsilandroside V (2) 18.6 1 Amorphous solid; [α] ‒190.00 (c 0.12, MeOH); H 3.6 Platelet aggregation assays (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) Turbidometric measurements of platelet aggregation of 5 5 NMR data, see Tables  1 and 2; HRESIMS m/z 747.3921 the samples were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown, Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 8 of 9 PA, USA) according to Born’s method [19, 20]. Rabbit of compound 4 in pyridine‑d . Fig. S26. HMBC spectrum of compound 4 platelet aggregation study was completed within 3.0 h of in pyridine‑d . Fig. S27. ROESY spectrum of compound 4 in pyridine‑d . 5 5 Fig. S28. HRESI (+) MS spectrum of compound 4. Fig. S29. UV spectrum preparation of platelet-rich plasma (PRP). Immediately of compound 4. Fig. S30. H NMR spectrum (600 MHz) of compound 5 after preparation of PRP, 250  μL was incubated in each in pyridine‑d . Fig. S31. C NMR spectrum (150 MHz) of compound 5 in 1 1 test tube at 37  °C for 5.0  min and then 2.5  μL of com- pyridine‑d . Fig. S32. H– H COSY spectrum of compound 5 in pyridine‑ d . Fig. S33. HSQC spectrum of compound 5 in pyridine‑d . Fig. S34. pounds (100  μM) were individually added. The changes 5 5 HMBC spectrum of compound 5 in pyridine‑d . Fig. S35. ROESY spectrum in absorbance as a result of platelet aggregation were of compound 5 in pyridine‑d . Fig. S36. HRESI (+) MS spectrum of com‑ recorded. The extent of aggregation was estimated by the pound 5. Fig. S37. UV spectrum of compound 5. percentage of maximum increase in light transmittance, with the buffer representing 100% transmittance. ADP Acknowledgements (adenosine diphosphate) was used as a positive control This work was financially supported by the National Natural Science Foundation of China (U1802287 and 32000280), the Ten Thousand Talents with a 59.5 ± 6.1% maximal platelet aggregation rate at a Plan of Yunnan Province for Industrial Technology Leading Talents, and the concentration of 10  μM. 1% DMSO was used as a blank State Key Laboratory of Phytochemistry and Plant Resources in West China control with a 2.7 ± 0.6% maximal platelet aggregation. (P2019‑ZZ02). Data counting and analysis was done on SPSS 16.0, with Author contributions experimental results expressed as mean ± standard error. All authors read and approved the final manuscript. Declarations 4 Conclusion Phytochemical reinvestigation on the whole plants Competing interests of Y. thibetica obtained four new spirostanol glyco- The authors declare that there are no conflicts of interest associated with this work. sides, named ypsilandrosides U-X (1–4), and one new cholestanol glycoside, named ypsilandroside Y (5). Their Author details structures have been illustrated by extensive spectro- College of Traditional Chinese Medicine, Yunnan University of Chinese Medicine, Kunming 650500, China. State Key Laboratory of Phytochemistry scopic data and chemical methods. Among them, com- and Plant Resources in West China, and Yunnan Key Laboratory of Natural pound 4 is a rare spirostanol glycoside which possesses a Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sci‑ novel 5(6 → 7) abeo-steroidal aglycone, while compound ences, Kunming 650201, China. University of Chinese Academy of Sciences, Beijing 100049, China. 1 is a first spirostanol bisdesmoside attached to C-3 and C-12, respectively, obtained from the Ypsilandra species. Received: 21 February 2022 Accepted: 28 March 2022 This investigation enriched the cognition of the chemical constituents in Y. thibetica. Unfortunately, the bioassay results showed the five new saponins have no the activity of inducing platelet aggregation. References 1. Li D‑Z. The families and genera of Chinese vascular plants, vol. 1. Beijing: China Science Press; 2020. p. 354–5. Supplementary Information 2. College JNM. Dictionary of traditional Chinese materia medica. Shanghai: The online version contains supplementary material available at https:// doi. China Shanghai Scientific and Technological Press; 1977. p. 1841. org/ 10. 1007/ s13659‑ 022‑ 00337‑0. 3. Yunnan Food and Drug Administration. The Yunnan Chinese materia medica standards, Yi Nationality Medicine (III), Kunming: China Shanghai Additional file 1: Fig. S1. H NMR spectrum (500 MHz) of compound 1 Yunnan Scientific and Technological Press; 2005. p. 5–6. in pyridine‑d . Fig. S2. C NMR spectrum (125 MHz) of compound 1 in 4. Xie B‑B, Liu H‑ Y, Ni W, Chen C‑ X. Ypsilandrosides C‑ G, five new spirostanol 1 1 pyridine‑d . Fig. S3. H– H COSY spectrum of compound 1 in pyridine‑d . saponins from Ypsilandra thibetica. Steroids. 2009;74:950–5. 5 5 Fig. S4. HSQC spectrum of compound 1 in pyridine‑d . Fig. S5. HMBC 5. Liu H‑ Y, Chen C‑ X, Lu Y, Yang J‑ Y, Ni W. Steroidal and pregnane glycosides spectrum of compound 1 in pyridine‑d . Fig. S6. ROESY spectrum of from Ypsilandra thibetica. Nat Prod Bioprospect. 2012;2:11–5. compound 1 in pyridine‑d . Fig. S7. HRESI (+) MS spectrum of compound 6. Xie B‑B, Liu H‑ Y, Ni W, Chen C‑ X, Lu Y, Wu L, Zheng Q‑ T. Five new steroidal 1. Fig. S8. H NMR spectrum (500 MHz) of compound 2 in pyridine‑d . compounds from Ypsilandra thibetica. Chem Biodivers. 2006;3:1211–8. Fig. S9. C NMR spectrum (125 MHz) of compound 2 in pyridine‑d . 7. Xie B‑B, Chen C‑ X, Guo Y‑H, Li Y ‑ Y, Liu Y‑ J, Ni W, Yang L‑M, Gong N‑B, 1 1 Fig. S10. H– H COSY spectrum of compound 2 in pyridine‑d . Fig. S11. Zheng Y‑ T, Wang R‑R, Lu Y, Liu H‑ Y. New 23‑spirocholestane derivatives HSQC spectrum of compound 2 in pyridine‑d . Fig. S12. HMBC spectrum from Ypsilandra thibetica. Planta Med. 2013;79:1063–7. of compound 2 in pyridine‑d . Fig. S13. ROESY spectrum of compound 8. Lu Y, Xie B‑B, Chen C‑ X, Ni W, Hua Y, Liu H‑ Y. Ypsilactosides A and B, two 2 in pyridine‑ d5. Fig. S14. HRESI (+) MS spectrum of compound 2. Fig. new C22‑steroidal lactone glycosides from Ypsilandra thibetica. Helv Chim S15. H NMR spectrum (500 MHz) of compound 3 in pyridine‑d . Fig. S16. Acta. 2011;94:92–7. C NMR spectrum (125 MHz) of compound 3 in pyridine‑d . Fig. S17. 9. Si Y‑A, Yan H, Ni W, Liu Z ‑H, Lu T ‑ X, Chen C‑ X, Liu H‑ Y. Two new steroidal 1 1 H– H COSY spectrum of compound 3 in pyridine‑d . Fig. S18. HSQC saponins from Ypsilandra thibetica. Nat Prod Bioprospect. 2014;4:315–8. spectrum of compound 3 in pyridine‑d . Fig. S19. HMBC spectrum of 10. Lu Y, Chen C‑ X, Ni W, Hua Y, Liu H‑ Y. Spirostanol tetraglycosides from compound 3 in pyridine‑d . Fig. S20. ROESY spectrum of compound 3 Ypsilandra thibetica. Steroids. 2010;75:982–7. in pyridine‑d . Fig. S21. HRESI (+) MS spectrum of compound 3. Fig. S22 11. Nakano K, Murakami K, Takaishi Y, Tomimatsu T, Nohara T. Studies on the 1 13 H NMR spectrum (500 MHz) of compound 4 in pyridine‑d .Fig. S23. C constituents of Heloniopsis orientalis ( Thunb.) C. Tanaka. Chem Pharm 1 1 NMR spectrum (125 MHz) of compound 4 in pyridine‑d . Fig. S24. H– H Bull. 1989;37:116–8. COSY spectrum of compound 4 in pyridine‑d . Fig. S25. HSQC spectrum 5 G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 9 of 9 12. Snyder J‑R, Serianni A‑S. DL ‑Apiose substituted with stable isotpoes‑ synthesis, NMR‑spectral analysis, and furanose anomerization. Carbohydr Res. 1987;166:85–99. 13. Kitagawa I, Sakagami M, Hashiuchi F, Zhou J‑L, Yoshikawa M, Ren J. Apioglycyrrhizin and araboglycyrrhizin, 2 new sweet oleanene‑type trit ‑ erpene oligoglycosides from the root of Glycyrrhiza inflata. Chem Pharm Bull. 1989;37:551–3. 14. Agrawal P‑K. NMR‑spectroscopy in the structural elucidation of oligosac‑ charides and glycosides. Phytochemistry. 1992;31:3307–30. 15. Kasai M‑ O‑R, Asakawa J, Mizutani K, Tanaka O. C NMR study of α‑ and β‑anomeric pairs of D ‑mannopyranosides and L ‑rhamnopyranosides. Tetrahedron. 1979;35:1427–32. 16. Lu T‑ X, Shu T, Qin X‑ J, Ni W, Ji Y‑H, Chen Q ‑R, Khanf A, Zhao Q, Liu H‑ Y. Spi‑ rostanol saponins from Ypsilandra parviflora induce platelet aggregation. Steroids. 2017;123:55–60. 17. Yang Y‑ J, Pang X, Wang B, Yang J, Chen X‑ J, Sun X‑ G, Li Q, Zhang J, Guo B‑L, Ma B‑P. Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells. Steroids. 2020;156: 108587. 18. Xiao C‑M, Huang J, Zhong X ‑M, Tan X ‑ Y, Deng P‑ C. Two new homo‑aro ‑ cholestane glycosides and a new cholestane glycoside from the roots and rhizomes of Paris polyphylla var. pseudothibetica. Helv Chim Acta. 2009;92:2587–95. 19. Born G‑ V‑R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194:927–9. 20. Born G‑ V‑R, Cross M ‑ J. The aggregation of blood platelets. J Physiol. 1963;168:178–95. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Natural Products and Bioprospecting Springer Journals

Ypsilandrosides U-Y, five new steroidal saponins from Ypsilandrathibetica

Loading next page...
 
/lp/springer-journals/ypsilandrosides-u-y-five-new-steroidal-saponins-from-apmfXAjxy6

References (20)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
ISSN
2192-2195
eISSN
2192-2209
DOI
10.1007/s13659-022-00337-0
Publisher site
See Article on Publisher Site

Abstract

1 Introduction Ypsilandra (Melanthiaceae) is distributed in south- western China and Myanmar, which contains 5 species according to the updated classification of the Angio - *Correspondence: haiyangliu@mail.kib.ac.cn Wen‑ Tao Gao and Ling‑Ling Yu contributed equally to this work sperm Phylogeny Group IV [1]. Among them, Ypsilandra State Key Laboratory of Phytochemistry and Plant Resources thibetica has been used in folk medicine for treatment of in West China, and Yunnan Key Laboratory of Natural Medicinal scrofula, dysuria, edema, uterine bleeding, and traumatic Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China hemorrhage in China by the local people [2, 3]. Our Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 2 of 9 previous investigations discovered twenty eight new ste- four methyl groups, nine methylene groups (one oxygen- roidal glycosides including nineteen spirostanol saponins, ated), ten methine groups (one olefinic and three oxy - two furostanol saponins, three cholestanol saponins, two genated), and four quaternary carbons (one olefinic and pregnane glycosides, and two C -steroidal lactone gly- one ketal). The above NMR data suggested that com - cosides from this species [4–10], some of which showed pound 1 is a typical C-27 steroidal saponin and its agly- cytotoxicity [4, 5], antifungal [4, 6], antibacterial [6], anti- cone is heloniogen [11]. This deduction can be confirmed 1 1 HIV-1 activities [7], and so on. For further investigation by 2D-NMR spectra. The H‒ H COSY correlations on the chemical constituents of this herb, four new spi- revealed that the aglycone for 1 had four structural frag- rostanol saponins (1‒4) and one new cholestanol saponin ments as shown in (Fig. 2). Furthermore, the key HMBC (5) (Fig.  1) were obtained and structurally characterized. correlations (Fig.  2) from CH -18 (δ 0.89) to C-12 (δ 3 H C The current paper reports the isolation, structural eluci - 82.4)/C-13 (δ 44.9)/C-14 (δ 44.4)/C-17 (δ 53.1), from C C C dation, and the induced platelet aggregation activity of CH -19 (δ 0.91) to C-1 (δ 37.1)/C-5 (δ 141.1)/C-9 (δ 3 H C C C these isolates. 49.0)/C-10 (δ 36.9), from CH -21 (δ 1.38)/H-20 (δ C 3 H H 2.00)/H-23a (δ 1.77)/H-26a (δ 3.53) to C-22 (δ 109.3) H H C 2 Results and discussion were observed. In addition, the ROESY correlations of Compound 1 was isolated as an amorphous powder. H-12 (δ 3.88) with H-18 (δ 0.89) and H-20 (δ 2.00) H H H Its molecular formula was determined as C H O by indicated that the OH-12 was α-oriented (Fig. 3). 44 70 17 the positive-ion HRESI-MS at m/z 893.4500 [M + Na] For the sugar part, the pentose was inferred as 13 13 (calcd. for C H O Na, 893.4505) and C NMR data β-d-apiofuranoside by the C NMR signals at δc 108.1 44 70 17 (Table  2). The H NMR spectrum of 1 (Table  1) showed (d, C-1’), 78.4 (d, C-2’), 79.0 (s, C-3’), 74.8 (t, C-4’), and four methyl proton signals at δ 0.89 (s, CH -18), 0.91 72.9 (t, C-5’) with those of corresponding carbons of H 3 (s, CH -19), 1.38 (d, J = 7.0 Hz, CH -21), and 0.67 (d, α- and β-d-apiofuranoside and α- and β-l-apiofuranoside 3 3 J = 5.4 Hz, CH -27), one olefinic proton signal at δ 5.18 [12, 13]. And the two hexose units were assigned to be 3 H (o, H-6), while three anomeric protons at δ 5.64 (d, a l-rhamnopyranosyl and a d-glucopyranosyl by their J = 3.2 Hz, H-1’), 5.30 (br s, H-1’’), and 4.86 (d, J = 7.8 Hz, NMR data, the acid hydrolysis of 1, and the HPLC anal- H-1’’’), which suggested that 1 was a glycoside with three ysis (retention time) of their L-cysteine methyl esters monosaccharide moieties. The C NMR spectra dis- followed by conversion into O-tolyl isothiocyanate deriv- played 44 carbon signals, of which 17 were assigned to atives and the authentic samples’ derivatives. And the those of one pentose and two hexose units, whereas other β-configuration of glucopyranosyl was revealed by the 27 ones were assigned to the aglycone moiety, including coupling constant ( J > 7.0 Hz) [14], while the anomeric 1,2 OH OH OH 21 O HO O 25 18 O HO OH 22 H HO OH 17 23 H O HO OH O HO H H 1 9 H HO O H H OH H H HO O HO 7 HO O 3 O O 12 3 OH O HO HO OH OH O HO HO HO OH OH HO OH HO 22' H 16' OH OH 5 7 H H H HO 6 CHO O O O HO 4 5 HO O OH HO HO HO HO O OH HO OH O HO HO HO OH OH HO OH Fig. 1 Chemical structures of saponins 1‒5 G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 3 of 9 Table 1 H NMR spectroscopic data of compounds 1–5 (δ in ppm, J in Hz, C D N) 5 5 a a a a b Position 1 2 3 4 5 1a 1.73 (o) 1.75 (d, 3.6) 1.73 (o) 1.70 (o) 1.73 (m) 1b 1.07 (d, 3.8) 0.97 (s) 1.00 (s) 1.15 (o) 0.95 (m) 2a 1.99 (m) 2.07 (o) 2.21 (m) 2.18 (m) 2.06 (d) 2b 1.62 (m) 1.72 (d, 6.3) 1.86 (m) 1.89 (m) 1.86 (d) 3 3.58 (o) 3.69 (m) 3.95 (o) 4.03 (m) 3.87 (m) 4a 2.45 (m) 2.60 (o) 2.72 (o) 4.02 (m) 2.81 (o) 4b 2.29 (m) 2.45 (t, 12.3) 1.82 (o) 2.47 (m) 2.74 (o) 6 5.18 (o) 5.26 (o) 5.22 (d, 5.0) 10.22 (s) 5.35 (o) 7a 1.76 (o) 1.91 (o) 1.83 (m) 1.95 (o) 7b 1.73 (o) 1.60 (o) 1.47 (m) 1.58 (o) 8 1.44 (m) 1.51 (m) 1.49 (m) 2.67 (m) 1.61 (m) 9 1.87 (d, 3.8) 0.95 (d, 20.9) 0.91 (m) 1.03 (m) 0.98 (m) 11a 2.22 (m) 1.56 (m) 1.48 (m) 1.32 (o) 1.57 (2H, m) 11b 1.57 (m) 1.47 (m) 1.03 (o) 12a 3.88 (br, s) 2.18 (m) 2.12 (m) 1.69 (m) 2.40 (m) 12b 1.91 (m) 1.83 (m) 1.03 (m) 1.60 (m) 14 1.56 (o) 2.04 (m) 2.03 (m) 1.36 (m) 1.50 (m) 15a 1.94 (o) 2.17 (m) 2.21 (m) 2.65 (m) 2.61 (m) 15b 1.44 (o) 1.51 (m) 1.56 (m) 2.01 (m) 2.41 (m) 16 4.42 (m) 4.43 (t, 6.9) 4.58 (t, 7.2) 4.59 (m) 17 3.31 (dd, 8.6, 6.1) 1.80 (dd, 8.5, 6.1) 18 0.89 (s) 0.93 (s) 1.14 (s) 0.88 (s) 0.94 (s) 19 0.91 (s) 0.97 (s) 1.00 (s) 0.83 (s) 1.10 (s) 20 2.00 (m) 2.20 (q) 3.39 (q, 7.2) 1.98 (m) 21 1.38 (d, 7.0) 1.18 (d, 7.1) 1.31 (d, 7.2) 1.13 (d, 7.0) 2.34 (s) 23a 1.77 (o) 1.92 (m) 4.00 (m) 1.67 (m) 2.76 (m) 23b 1.38 (o) 1.52 (m) 1.58 (m) 2.72 (m) 24a 1.67 (m) 2.21 (m) 2.29 (o) 1.58 (m) 1.95 (m) 24b 1.28 (m) 1.86 (m) 2.21 (o) 1.24 (m) 1.51 (m) 25 1.55 (d, 6.2) 1.87 (m) 2.29 (m) 1.59 (o) 1.92 (m) 26a 3.53 (o) 4.12 (o) 3.99 (m) 4.90 (br s) 3.79 (m) 26b 3.46 (o) 3.92 (m) 3.90 (m) 3.54 (br s) 3.73 (m) 27a 0.67 (d, 5.4) 4.12 (o) 3.73 (m) 0.71 (d, 4.7z) 1.14 (d, 6.6) 27b 3.94 (m) 3.68 (m) 16’ 3‑Api 3‑Api 3‑ Glc 3‑ Glc 7.12 (o) 22’ 7.07 (o) 3‑ Glc 1’ 5.64 (d, 3.2) 5.72 (o) 4.92 (d, 7.1) 5.02 (d, 7.3) 4.96 (o) 2’ 4.63 (o) 4.83 (m) 4.18 (o) 4.23 (m) 4.22 (m) 3’ 4.18 (o) 4.20 (m) 4.22 (m) 4’a 4.51 (m) 4.48 (d, 9.3) 4.19 (o) 4.40 (m) 4.41 (m) 4’b 4.25 (m) 4.24 (d, 9.3) 3.89 (o) 3.64 (m) 3.61 (m) 5’a 4.62 (o) 4.13 (2H, o) 5’b 4.43 (o) 6’a 4.74 (d, 5.5) 4.21 (o) 4.19 (o) 6’b 4.28 (d, 5.5) 4.05 (o) 4.05 (o) 5’‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 1’’ 5.30 (br s) 5.85 (br s) 6.31 (br s) 6.44 (br s) 6.41 (br s) 2’’ 3.92 (m) 4.56 (o) 4.76 (o) 4.86 (m) 4.87 (m) 3’’ 4.08 (m) 4.70 (br, s) 4.58 (o) 4.62 (m) 4.67 (m) Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 4 of 9 Table 1 (continued) a a a a b Position 1 2 3 4 5 4’’ 3.63 (m) 4.31 (o) 4.32 (o 4.36 (m) 4.38 (m) 5’’ 4.24 (m) 4.49 (m) 4.94 (m) 4.93 (m) 4.97 (m) 6’’ 1.55 (d, 6.2) 1.72 (d, 6.3) 1.75 (d, 6.1) 1.59 (d, 6.1) 1.60 (o) 12‑ Glc 6’‑ Glc 4′‑Rha 4′‑Rha 1’’’ 4.86 (d, 7.8) 5.04 (d, 8.0) 5.82 (br s) 5.84 (br s) 2’’’ 4.06 (m) 4.01 (o) 4.54 (m) 4.52 (m) 3’’’ 4.23 (m) 4.18 (o) 4.54 (m) 4.54 (m) 4’’’ 4.25 (m) 4.12 (o) 4.44 (m) 4.45 (m) 5’’’ 3.97 (m) 4.18 (o) 4.92 (m) 4.94 (m) 6’’’a 4.53 (o) 4.49 (d, 11.5) 1.59 (d, 6.1) 1.59 (o) 6’’’b 4.39 (d, 5.0) 4.33 (o) 4′′‑Rha 4′′‑Rha 1’’’’ 6.28 (br s) 6.29 (br s) 2’’’’ 4.90 (m) 4.90 (m) 3’’’’ 4.54 (m) 4.52 (m) 4’’’’ 4.33 (m) 4.31 (m) 5’’’’ 4.94 (m) 4.37 (m) 6’’’’ 1.73 (d, 6.2) 1.78 (d, 6.1) s singlet, d doublet, t triplet, q quartet, br broad, m multiplet, o overlapped a b Measured at 500 MHz. Measured at 600 MHz 1 1 Fig. 2 H‒ H COSY and Key HMBC correlations of 1‒5 configuration of rhamnopyranosyl was identified as aglycone was established from the following HMBC cor- α-orientated on the basis of the chemical shift values of rletions: H-1’ (δ 5.64) of Api with C-3 (δ 77.5) of the H C C-3’’ (δ 72.9) and C-5’’ (δ 70.6) with those of corre- aglycone, H-1’’ (δ 5.30) of the Rha with C-5’ (δ 72.9) of C C H C sponding carbons of methyl α- and β-rhamnopyranoside Api, and H-1’’’ (δ 4.86) of the Glc with C-12 (δ 82.4) H C [15]. The sequence of the sugar chain at C-3 of the of the aglycone (Fig.  2). Thus, the structure of 1 was G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 5 of 9 Me H 18 Me Me H Me 19 H H 19 Me H 20 O Me H 17 Me H 12 CH OH H 2 1 1 O HO 3 O H O 3 H HO O Glc H H H H Fig. 3 Key ROESY correlations for the aglycone moieties of 1 and 3 elucidated as 12-O-β-d-glucopyranosy-(25R)-spirost-5- 6.31) to C-2’ (δ 77.5), and from H-1’’’ (δ 5.04) to C-6’ C H en-3β,12β-diol-3-O-α-l -rhamnopyranosyl-(1 → 5)-β-d - (δ 69.9)  (Fig.  2). Consequently, the structure of 3 was apiofuranoside, and named ypsilandroside U. established as (23S,25S)-spirost-5-en-3β,17α,23,27- Compound 2 was isolated as an amorphous powder t e tra ol-3- O - β - d -g lu c opy rano s yl-(1 → 6)-[ α - l - with a molecular formula of C H O determined by rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside, and 38 60 13 the positive-ion HRESI-MS at m/z 747.3921 [M + Na] , named ypsilandroside W. (calcd. for C H O Na, 747.3926) and C NMR data Compound 4 possessed a molecular formula C H O 38 60 13 51 80 21 (Table  2). Its NMR spectra suggested that 2 is a spiros- determined by the HRESI-MS at m/z 1051.5077 tane saponin with a disaccharide chain. Comparison [M + Na] , (calcd. for C H O Na, 1051.5084) and 51 80 21 1 13 13 of the H and C NMR data of 2 (Tables  1 and 2) with C NMR data (Table  2). The UV spectrum of 4 showed those of ypsiparoside C obtained from the same genus absorption maxima at 254.5  nm, suggesting the pres- [16] revealed that they shared the same aglycone. The ence of a conjugated enal system. When comparing its 1 13 two monosaccharides and their absolute configura - H and C NMR data (Tables  1 and 2) with those of tions were determined as β-d-apiose and α-l-rhamnose ypsilandroside H [10], it was suggested that they shared by the same methods with compound 1. The HMBC the same sugar sequence and the similar aglycone, correlations from H-1’ (δ 5.72) to C-3 (δ 77.7), and except for the compound 4 has no hydroxyl substituent H C from H-1’’ (δ 5.85) of the rhamnopyransyl to C-2’ (δ at the C-17. The above deduction could be verified by H C 82.4) established the sequence for 3-O-sugar chain as the HMBC correlations from H-21 (δ 1.13) and H-18 1 1 O-α-l -rhamnopyranosyl-(1 → 2)-β-d -apiofuranoside (δ 0.88) to C-17 (δ 62.4) and H‒ H COSY correla- H C (Fig.  2). Therefore, the structure of 2 was deter - tions between H-16 (δ 4.59) and H-17 (δ 1.80) (Fig. 2). H H mined as (25R)-spirost-5-en-3β,17α,27-triol-3-O-α- The HMBC correlations from H-1’ (δ 5.02) to C-3 l-rhamnopyranosyl-(1 → 2)-β-d-apiofuranoside, and (δ 77.7), from H-1’’ (δ 6.44) to C-2’ (δ 77.9), from C H C named ypsilandroside V. H-1’’’ (δ 5.82) to C-4’ (δ 77.7), and from H-1’’’’ (δ H C H Compound 3 was isolated as an amorphous pow- 6.28) to C-4’’’ (δ 80.4) confirmed that compound 3 der and had a molecular formula of C H O as had the same sequence of 3-O-sugar chain as that of 45 72 20 determined by the positive-ion HRESI-MS data (m/z ypsilandroside H  (Fig.  2). Thus, the structure of 4 was 955.4505 [M + Na] , calcd. for C H O Na, 955.4509) elucidated as (25R)-B-nor(7)-6-carboxaldehyde-spirost- 45 72 20 and C NMR data (Table  2). Inspection of the NMR 5(7)-en-3β-ol-3-O-α-l -rhamnopyranosyl-(1 → 4)-α-l - spectra (Tables  1 and 2) of 3 revealed that it possessed r h a m n o p y r a n o s y l - ( 1 → 4 ) - [ α - l - r h a m n o p y r a n o - a spirotanol skeleton with a trisaccharide chain consist- syl-(1 → 2)]-β-d-glucopyranoside, and named ing of one rhamnopyranosyl and two glucopyranosyls. ypsilandroside X. 1 13 Comparing its H and C NMR data (Tables  1 and 2) The molecular formula of compound 5 was deter- with those of trillitschonide S6 [17] indicated that they mined as C H O by the HRESI-MS at m/z 1045.5352 53 82 19 + 13 shared the same aglycone. The α-orientations of OH-23 [M + Na] (calcd. for C H O Na, 1045.5343) and C 53 82 19 and CH OH-25 were supported by the ROESY correla- NMR data (Table  2). Its NMR spectra indicated that tions between H-23 (δ 4.00) and H-20 (δ 3.39)/H- compound 5 was a cholestane tetraglycosides contain- H H 1 13 25 (δ 2.29)  (Fig.  3). The absolute configurations and ing an aromatic ring. Analysis of the H and C NMR the anomeric configurations of monosaccharides were data (Tables  1 and 2) of 5 suggested that it was similar determined by the same methods with the above com- to that of parispseudoside A [18], and the major dif- pounds. The sequence of the sugar chain at C-3 of the ference was the absence of a glucopyranosyl group aglycone was established by the HMBC correlations at OH-26 site. With the assistance of HSQC experi- 1 13 from H-1’ (δ 4.92) to C-3 (δ 76.8), from H-1’’ (δ ment, H and C NMR data (Tables  1 and 2) showed H C H Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 6 of 9 Table 2 C NMR spectroscopic data of compounds 1–5 (δ in Table 2 (continued) ppm, C D N) a a a a b 5 5 Position 1 2 3 4 5 a a a a b Position 1 2 3 4 5 63.1 (t) 62.7 (t) 18.6 (q) 18.8 (q) 6′′′ 1 37.1 (t) 37.6 (t) 37.6 (t) 36.3 (t) 37.2 (t) 4′′‑Rha 4′′‑Rha 2 30.2 (t) 30.3 (t) 30.3 (t) 29.9 (t) 30.0 (t) 1′′′′ 103.4 (d) 103.2 (d) 3 77.5 (d) 77.7 (d) 76.8 (d) 77.7 (d) 77.8 (d) 2′′′′ 72.7 (d) 72.8 (d) 4 39.3 (t) 39.3 (t) 39.1 (t) 30.7 (t) 38.9 (t) 3′′′′ 72.9 (d) 72.4 (d) 5 141.1 (s) 140.8 (s) 140.9 (s) 169.5 (s) 140.9 (s) 74.0 (d) 74.1 (d) 4′′′′ 6 121.6 (d) 121.9 (d) 121.7 (d) 189.3 (d) 121.7 (d) 5′′′′ 70.5 (d) 70.3 (d) 7 32.0 (t) 32.4 (t) 32.4 (t) 139.6 (s) 31.9 (t) 18.9 (q) 18.6 (q) 6′′′′ 8 31.8 (d) 32.3 (d) 32.3 (d) 45.8 (d) 30.8 (d) a b Measured at 125 MHz. Measured at 150 MHz 9 49.0 (d) 50.2 (d) 50.1 (d) 60.4 (d) 50.4 (d) 10 36.9 (s) 37.1 (s) 37.1 (s) 46.5 (s) 37.0 (s) four anomeric protons at δ 4.96 (o, H-1’), 6.41 (br s, 11 27.6 (t) 20.9 (t) 20.9 (t) 20.8 (t) 21.2 (t) H-1’’), 5.84 (br s, H-1’’’), and 6.29 (s, H-1’’’’) and their 12 82.4 (d) 32.1 (t) 32.4 (t) 40.1 (t) 36.8 (t) corresponding anomeric carbons at δ 100.2 (C-1’), 13 44.9 (s) 45.1 (s) 45.8 (s) 43.3 (s) 47.1 (s) 102.1 (C-1’’), 102.1 (C-1’’’), and 103.2 (C-1’’’’). The 14 44.4 (d) 53.0 (d) 53.1 (d) 54.3 (d) 57.6 (d) sequence of sugar units was consistent with that of 15 32.1 (t) 31.8 (t) 31.9 (t) 35.3 (t) 32.3 (t) compound 4 by HMBC experiment (Fig. 2). As a result, 16 81.0 (d) 90.2 (d) 90.8 (d) 81.3 (d) 140.6 (s) the structure of 5 was assigned as homo-aro-cholest-5- 17 53.1 (d) 90.1 (s) 90.1 (s) 62.4 (d) 151.8 (s) en-3β,26-diol-3-O-α-l -rhamnopyranosyl-(1 → 4)-α-l - 18 17.0 (q) 17.2 (q) 17.4 (q) 16.8 (q) 16.4 (q) r hamnopy rano s yl-(1 → 4)-[ α - l - 19 19.3 (q) 19.5 (q) 19.4 (q) 15.5 (q) 19.2 (q) rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside, and 20 42.2 (d) 45.3 (d) 38.8 (d) 41.9 (d) 131.1 (s) named ypsilandroside Y. 21 15.3 (q) 9.6 (q) 9.4 (q) 15.1 (q) 14.6 (q) Because the whole plants of Y. thibetica has been 22 109.3 (s) 110.5 (s) 112.7 (s) 109.2 (s) 139.9 (s) used in folk medicine for treatment of uterine bleeding 23 32.0 (t) 27.5 (t) 68.1(d) 31.9 (t) 31.4 (t) and traumatic hemorrhage in China, the isolated com- 24 29.4 (t) 21.2 (t) 33.1 (t) 29.3 (t) 35.4 (t) pounds (1–5) were evaluated for their induced platelet 25 30.6 (d) 36.1 (d) 40.4 (d) 30.7 (d) 36.6 (d) aggregation activity and ADP (adenosine diphosphate) 26 66.8 (t) 60.6 (t) 63.1 (t) 66.9 (t) 67.3 (t) was used as a positive control. Unfortunately, the 27 17.4 (q) 61.4 (t) 64.0 (t) 17.4 (q) 17.2 (q) results showed all isolated saponins did not exhibit the 16’ 122.8 (d) 22’ 127.3 (d) inducing platelet aggregation activity at the tested con- 3‑Api 3‑Api 3‑ Glc 3‑ Glc 3‑ Glc centration of 100 μM. 1′ 108.1 (d) 107.0 (d) 100.7 (d) 100.9 (d) 100.2 (d) 78.4 (d) 82.4 (d) 77.5 (d) 77.9 (d) 77.9 (d) 2′ 3′ 79.0 (s) 80.5 (s) 79.5 (d) 77.4 (d) 77.6 (d)3 Experimental section 4′ 74.8 (t) 74.9 (t) 71.6 (d) 77.7 (d) 77.6 (d) 3.1 General experimental procedures 72.9 (t) 65.9 (t) 78.4 (d) 77.2 (d) 76.9 (d) Optical rotations were measured by a JASCO P-1020 5′ polarimeter (Jasco Corp., Japan). UV spectra were 6′ 69.9 (t) 61.3 (t) 61.1 (t) recorded on a Shimadzu UV2401 PC spectrophotom- 5’‑Rha 2′‑Rha 2′‑Rha 2′‑Rha 2′‑Rha eter (Shimadzu Corp., Japan). HRESI-MS was recorded 1′′ 102.8 (d) 102.0 (d) 102.0 (d) 101.9 (d) 102.1 (d) on an Agilent 1290 UPLC/6540 Q-TOF mass spectrom- 2′′ 72.4 (d) 72.7 (d) 72.6 (d) 72.5 (d) 72.6 (d) eter (Agilent Corp., USA). The NMR experiments were 3′′ 72.9 (d) 72.0 (d) 72.8 (d) 72.9 (d) 72.8 (d) performed on Bruker AVANCE III 500, Avance III-600, 4′′ 74.3 (d) 74.0 (d) 74.2 (d) 74.2 (d) 74.0 (d) and AV 800 spectrometers (Bruker Corp., Switzerland). 70.6 (d) 70.3 (d) 69.5 (d) 69.5 (d) 69.5 (d) 5′′ Silica gel (200–300 mesh, Qingdao Marine Chemical Co., 6′′ 18.6 (q) 18.7 (q) 18.7 (q) 18.5 (q) 18.3 (q) Ltd., People’s Republic of China), RP-18 (50  μm, Merck, 12‑ Glc 6’‑ Glc 4′‑Rha 4′‑Rha Germany), and Sephadex LH-20 (Pharmacia, Stockholm, 1′′′ 106.6 (d) 105.5 (d) 102.3 (d) 102.1 (d) Sweden) were used for column chromatography (CC). 2′′′ 75.6 (d) 75.2 (d) 72.9 (d) 72.8 (d) An Agilent 1260 system (Agilent Corp., America) with a 78.8 (d) 78.4 (d) 73.3 (d) 73.2 (d) 3′′′ Zorbax SB-C18 column (5  μm, 9.4 × 250  mm) was used 4′′′ 71.9 (d) 71.6 (d) 80.4 (d) 80.3 (d) for HPLC separation. TLC was carried out on silica gel 78.4 (d) 78.4 (d) 68.4 (d) 68.2 (d) 5′′′ G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 7 of 9 HSGF plates (Qingdao Marine Chemical Co., China) ([M + Na] , calcd. for C H O Na, 747.3926) (Addi- 254 38 60 13 or RP-18 F (Merck, Darmstadt, Germany). tional file 1). 3.2 Plant material 3.4.3 Y psilandroside W (3) 20.5 1 The whole plant materials of Y. thibetica were collected Amorphous solid; [α] ‒125.67 (c 0.12, MeOH); H in August 2010 from Zhaotong City, Yunnan Provence, (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) 5 5 China, and identified by Prof. Xin-Qi Chen, Institute of NMR data, see Tables  1 and 2; HRESIMS m/z 955.4505 Botany, Chinese Academy of Sciences, Beijing. A voucher [M + Na] (calcd. for C H O Na, 955.4509) (Addi- 45 72 20 specimen was deposited at the State Key Laboratory of tional file 1). Phytochemistry and Plant Resources in West China, Kun- ming Institute of Botany, Chinese Academy of Sciences. 3.4.4 Y psilandroside X (4) 18.6 Amorphous solid; [α] ‒106.40 (c 0.15, MeOH); UV 3.3 Extraction and isolation (MeOH) λ (log ε) 202.5 (3.9), 254.5 (3.9) nm; H max The dried whole plants of Y. thibetica (110  kg) were (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) 5 5 crushed and extracted three times with 70% EtOH under NMR data, see Tables 1 and 2; HRESIMS m/z 1051.5077 reflux for a 3 h, 2 h and 2 h. Then, the combined extract [M + Na] (calcd. for C H O Na, 1051.5084) (Addi- 51 80 21 was concentrated under reduced pressure. The crude tional file 1). extract (30  kg) was passed through YWD-3F macropo- rous resin and eluted successively with H O, 40% EtOH, 3.4.5 Y psilandroside Y (5) 18.6 75% EtOH, and 95% EtOH, respectively. Evaporated 75% Amorphous solid; [α] ‒48.18 (c 0.11, MeOH); UV EtOH fraction (crude saponin-rich mixture, 10  kg) was (MeOH) λ (log ε) 203 (4.5) nm; H (600  MHz, pyr- max subjected to a silica gel column chromatography (CHCl – idine-d ) and C (150  MHz, pyridine-d ) NMR data, 3 5 5 MeOH, 20:1 → 8:2, v/v) to give eleven fractions (Fr. A–Fr. see Tables  1 and 2; HRESIMS m/z 1045.5352 [M + Na] K). Fr. C (560 g) was subjected to a silica gel column chro- (calcd. for C H O Na, 1045.5343) (Additional file 1). 53 82 19 matography (CHCl –MeOH, 20:1 → 1:1, v/v) to give 14 fractions (Fr. C-1–Fr. C-14). Fr. C-11 (80 mg) was submit- 3.5 Acid hydrolysis of compounds 1–5 and determination ted to Sephadex LH-20 (MeOH) and chromatographically of the absolute configuration of the sugars by HPLC separated on an RP-18 column eluted with MeOH–H O Compounds 1‒5 (1.0  mg each) in 6  M C F COOH 2 3 (40:60 → 70:30, v/v) and purified by preparative HPLC (1,4-dioxane-H O 1:1, 1.0  mL) were heated at 99 ℃ (MeCN–H O, 40:60 → 50:50, v/v) to afford saponin for 2  h, respectively. The reaction mixture was diluted 2 (t = 12.8  min, 10  mg). Fr. C-13 (45  g) was submitted with H O (1.0  mL) and then extracted with EtOAc R 2 to Sephadex LH-20 (MeOH) to give three subfractions (3 × 2.0  mL). Next, each aqueous layer was evaporated (C-13–1–C-13–3). Subsequently, Fr. C-13–1 (150  mg) to dryness using rotary evaporation. Each dried residue was further purified by preparative HPLC (MeCN–H O, was dissolved in pyridine (1.0 mL) mixed with l-cysteine 25:75 → 35:65, v/v) to afford saponins 5 (t = 10.8  min, methyl ester hydrochloride (1.0  mg) (Aldrich, Japan) 7  mg) and 4 (t = 11.9  min, 12  mg), whereas saponins 3 and heated at 60 °C for 1 h. Then, O-tolyl isothiocyanate (t = 11.1  min, 10  mg) and 1 (t = 14.8  min, 9  mg) were (5.0 μL) (Tokyo Chemical Industry Co., Ltd., Japan) was R R obtained from Fr. C-13–3 (208 mg) by preparative HPLC added to the mixture, this being heated at 60 °C for 1 h. (MeCN–H O, 30:70 → 45:55, v/v). Each reaction mixture was directly analyzed by reversed phase HPLC following the above procedure. Each reac- 3.4 P hysical and spectroscopic data of new glycosides tion mixture was directly analyzed by analytical HPLC on 3.4.1 Ypsilandroside U (1) a Poroshell 120 SB-C18 column (100 × 4.6  mm, 2.7  μm, 18.6 1 Amorphous solid; [α] ‒55.80 (c 0.20, MeOH); H Agilent) using an elution of C H CN‒H O (20:75 → 40:60, 3 2 (500  MHz, pyridine d ) and C (125  MHz, pyridine d ) v/v) at a flow rate of 0.6 mL/min. As a result, the sugars 5 5 NMR data, see Tables  1 and 2; HRESIMS m/z 893.4500 in the test compounds were identified as d-glucose and [M + Na] (calcd. for C H O Na, 893.4505) (Addi- l-rhamnose, respectively, by comparing their molecu - 44 70 17 tional file 1). lar weight and retention time with the standards (t 13.90 min for d-glucose; t 17.72 min for l-rhamnose). 3.4.2 Ypsilandroside V (2) 18.6 1 Amorphous solid; [α] ‒190.00 (c 0.12, MeOH); H 3.6 Platelet aggregation assays (500  MHz, pyridine-d ) and C (125  MHz, pyridine-d ) Turbidometric measurements of platelet aggregation of 5 5 NMR data, see Tables  1 and 2; HRESIMS m/z 747.3921 the samples were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown, Gao et al. Natural Products and Bioprospecting (2022) 12:17 Page 8 of 9 PA, USA) according to Born’s method [19, 20]. Rabbit of compound 4 in pyridine‑d . Fig. S26. HMBC spectrum of compound 4 platelet aggregation study was completed within 3.0 h of in pyridine‑d . Fig. S27. ROESY spectrum of compound 4 in pyridine‑d . 5 5 Fig. S28. HRESI (+) MS spectrum of compound 4. Fig. S29. UV spectrum preparation of platelet-rich plasma (PRP). Immediately of compound 4. Fig. S30. H NMR spectrum (600 MHz) of compound 5 after preparation of PRP, 250  μL was incubated in each in pyridine‑d . Fig. S31. C NMR spectrum (150 MHz) of compound 5 in 1 1 test tube at 37  °C for 5.0  min and then 2.5  μL of com- pyridine‑d . Fig. S32. H– H COSY spectrum of compound 5 in pyridine‑ d . Fig. S33. HSQC spectrum of compound 5 in pyridine‑d . Fig. S34. pounds (100  μM) were individually added. The changes 5 5 HMBC spectrum of compound 5 in pyridine‑d . Fig. S35. ROESY spectrum in absorbance as a result of platelet aggregation were of compound 5 in pyridine‑d . Fig. S36. HRESI (+) MS spectrum of com‑ recorded. The extent of aggregation was estimated by the pound 5. Fig. S37. UV spectrum of compound 5. percentage of maximum increase in light transmittance, with the buffer representing 100% transmittance. ADP Acknowledgements (adenosine diphosphate) was used as a positive control This work was financially supported by the National Natural Science Foundation of China (U1802287 and 32000280), the Ten Thousand Talents with a 59.5 ± 6.1% maximal platelet aggregation rate at a Plan of Yunnan Province for Industrial Technology Leading Talents, and the concentration of 10  μM. 1% DMSO was used as a blank State Key Laboratory of Phytochemistry and Plant Resources in West China control with a 2.7 ± 0.6% maximal platelet aggregation. (P2019‑ZZ02). Data counting and analysis was done on SPSS 16.0, with Author contributions experimental results expressed as mean ± standard error. All authors read and approved the final manuscript. Declarations 4 Conclusion Phytochemical reinvestigation on the whole plants Competing interests of Y. thibetica obtained four new spirostanol glyco- The authors declare that there are no conflicts of interest associated with this work. sides, named ypsilandrosides U-X (1–4), and one new cholestanol glycoside, named ypsilandroside Y (5). Their Author details structures have been illustrated by extensive spectro- College of Traditional Chinese Medicine, Yunnan University of Chinese Medicine, Kunming 650500, China. State Key Laboratory of Phytochemistry scopic data and chemical methods. Among them, com- and Plant Resources in West China, and Yunnan Key Laboratory of Natural pound 4 is a rare spirostanol glycoside which possesses a Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sci‑ novel 5(6 → 7) abeo-steroidal aglycone, while compound ences, Kunming 650201, China. University of Chinese Academy of Sciences, Beijing 100049, China. 1 is a first spirostanol bisdesmoside attached to C-3 and C-12, respectively, obtained from the Ypsilandra species. Received: 21 February 2022 Accepted: 28 March 2022 This investigation enriched the cognition of the chemical constituents in Y. thibetica. Unfortunately, the bioassay results showed the five new saponins have no the activity of inducing platelet aggregation. References 1. Li D‑Z. The families and genera of Chinese vascular plants, vol. 1. Beijing: China Science Press; 2020. p. 354–5. Supplementary Information 2. College JNM. Dictionary of traditional Chinese materia medica. Shanghai: The online version contains supplementary material available at https:// doi. China Shanghai Scientific and Technological Press; 1977. p. 1841. org/ 10. 1007/ s13659‑ 022‑ 00337‑0. 3. Yunnan Food and Drug Administration. The Yunnan Chinese materia medica standards, Yi Nationality Medicine (III), Kunming: China Shanghai Additional file 1: Fig. S1. H NMR spectrum (500 MHz) of compound 1 Yunnan Scientific and Technological Press; 2005. p. 5–6. in pyridine‑d . Fig. S2. C NMR spectrum (125 MHz) of compound 1 in 4. Xie B‑B, Liu H‑ Y, Ni W, Chen C‑ X. Ypsilandrosides C‑ G, five new spirostanol 1 1 pyridine‑d . Fig. S3. H– H COSY spectrum of compound 1 in pyridine‑d . saponins from Ypsilandra thibetica. Steroids. 2009;74:950–5. 5 5 Fig. S4. HSQC spectrum of compound 1 in pyridine‑d . Fig. S5. HMBC 5. Liu H‑ Y, Chen C‑ X, Lu Y, Yang J‑ Y, Ni W. Steroidal and pregnane glycosides spectrum of compound 1 in pyridine‑d . Fig. S6. ROESY spectrum of from Ypsilandra thibetica. Nat Prod Bioprospect. 2012;2:11–5. compound 1 in pyridine‑d . Fig. S7. HRESI (+) MS spectrum of compound 6. Xie B‑B, Liu H‑ Y, Ni W, Chen C‑ X, Lu Y, Wu L, Zheng Q‑ T. Five new steroidal 1. Fig. S8. H NMR spectrum (500 MHz) of compound 2 in pyridine‑d . compounds from Ypsilandra thibetica. Chem Biodivers. 2006;3:1211–8. Fig. S9. C NMR spectrum (125 MHz) of compound 2 in pyridine‑d . 7. Xie B‑B, Chen C‑ X, Guo Y‑H, Li Y ‑ Y, Liu Y‑ J, Ni W, Yang L‑M, Gong N‑B, 1 1 Fig. S10. H– H COSY spectrum of compound 2 in pyridine‑d . Fig. S11. Zheng Y‑ T, Wang R‑R, Lu Y, Liu H‑ Y. New 23‑spirocholestane derivatives HSQC spectrum of compound 2 in pyridine‑d . Fig. S12. HMBC spectrum from Ypsilandra thibetica. Planta Med. 2013;79:1063–7. of compound 2 in pyridine‑d . Fig. S13. ROESY spectrum of compound 8. Lu Y, Xie B‑B, Chen C‑ X, Ni W, Hua Y, Liu H‑ Y. Ypsilactosides A and B, two 2 in pyridine‑ d5. Fig. S14. HRESI (+) MS spectrum of compound 2. Fig. new C22‑steroidal lactone glycosides from Ypsilandra thibetica. Helv Chim S15. H NMR spectrum (500 MHz) of compound 3 in pyridine‑d . Fig. S16. Acta. 2011;94:92–7. C NMR spectrum (125 MHz) of compound 3 in pyridine‑d . Fig. S17. 9. Si Y‑A, Yan H, Ni W, Liu Z ‑H, Lu T ‑ X, Chen C‑ X, Liu H‑ Y. Two new steroidal 1 1 H– H COSY spectrum of compound 3 in pyridine‑d . Fig. S18. HSQC saponins from Ypsilandra thibetica. Nat Prod Bioprospect. 2014;4:315–8. spectrum of compound 3 in pyridine‑d . Fig. S19. HMBC spectrum of 10. Lu Y, Chen C‑ X, Ni W, Hua Y, Liu H‑ Y. Spirostanol tetraglycosides from compound 3 in pyridine‑d . Fig. S20. ROESY spectrum of compound 3 Ypsilandra thibetica. Steroids. 2010;75:982–7. in pyridine‑d . Fig. S21. HRESI (+) MS spectrum of compound 3. Fig. S22 11. Nakano K, Murakami K, Takaishi Y, Tomimatsu T, Nohara T. Studies on the 1 13 H NMR spectrum (500 MHz) of compound 4 in pyridine‑d .Fig. S23. C constituents of Heloniopsis orientalis ( Thunb.) C. Tanaka. Chem Pharm 1 1 NMR spectrum (125 MHz) of compound 4 in pyridine‑d . Fig. S24. H– H Bull. 1989;37:116–8. COSY spectrum of compound 4 in pyridine‑d . Fig. S25. HSQC spectrum 5 G ao et al. Natural Products and Bioprospecting (2022) 12:17 Page 9 of 9 12. Snyder J‑R, Serianni A‑S. DL ‑Apiose substituted with stable isotpoes‑ synthesis, NMR‑spectral analysis, and furanose anomerization. Carbohydr Res. 1987;166:85–99. 13. Kitagawa I, Sakagami M, Hashiuchi F, Zhou J‑L, Yoshikawa M, Ren J. Apioglycyrrhizin and araboglycyrrhizin, 2 new sweet oleanene‑type trit ‑ erpene oligoglycosides from the root of Glycyrrhiza inflata. Chem Pharm Bull. 1989;37:551–3. 14. Agrawal P‑K. NMR‑spectroscopy in the structural elucidation of oligosac‑ charides and glycosides. Phytochemistry. 1992;31:3307–30. 15. Kasai M‑ O‑R, Asakawa J, Mizutani K, Tanaka O. C NMR study of α‑ and β‑anomeric pairs of D ‑mannopyranosides and L ‑rhamnopyranosides. Tetrahedron. 1979;35:1427–32. 16. Lu T‑ X, Shu T, Qin X‑ J, Ni W, Ji Y‑H, Chen Q ‑R, Khanf A, Zhao Q, Liu H‑ Y. Spi‑ rostanol saponins from Ypsilandra parviflora induce platelet aggregation. Steroids. 2017;123:55–60. 17. Yang Y‑ J, Pang X, Wang B, Yang J, Chen X‑ J, Sun X‑ G, Li Q, Zhang J, Guo B‑L, Ma B‑P. Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells. Steroids. 2020;156: 108587. 18. Xiao C‑M, Huang J, Zhong X ‑M, Tan X ‑ Y, Deng P‑ C. Two new homo‑aro ‑ cholestane glycosides and a new cholestane glycoside from the roots and rhizomes of Paris polyphylla var. pseudothibetica. Helv Chim Acta. 2009;92:2587–95. 19. Born G‑ V‑R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194:927–9. 20. Born G‑ V‑R, Cross M ‑ J. The aggregation of blood platelets. J Physiol. 1963;168:178–95. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations.

Journal

Natural Products and BioprospectingSpringer Journals

Published: Dec 1, 2022

Keywords: Ypsilandrathibetica; Melanthiaceae; Ypsilandrosides U-Y; Spirostanol saponins; Cholestanol saponins

There are no references for this article.