Ypsilandrosides U-Y,five new steroidal saponins from Ypsilandra thibetica

Wen?Tao Gao,Ling?Ling Yu,Jing Xie,Long?Gao Xiao,Shi?Juan Zhang,Wen?Yi Ma,Huan Yan and Hai?Yang Liu*

?Wen?Tao Gao and Ling?Ling Yu contributed equally to this work

2 State Key Laboratory of Phytochemistry and Plant Resources in West China,and Yunnan Key Laboratory of Natural Medicinal Chemistry,Kunming Institute of Botany,Chinese Academy of Sciences,Kunming 650201,China

Abstract Phytochemical reinvestigation on the whole plants of Ypsilandra thibetica obtained four new spirostanol glycosides,named ypsilandrosides U?X (1—4),and one new cholestanol glycoside,named ypsilandroside Y (5).Their structures have been established by extensive spectroscopic data and chemical methods.Among them,compound 4 is a rare spirostanol glycoside which possesses a novel 5(6→7) abeo?steroidal aglycone,while compound 1 is a first spiro?stanol bisdesmoside attached to C?3 and C?12,respectively,isolated from the genus Ypsilandra.The induced platelet aggregation activity of the isolates was tested.

Keywords: Ypsilandra thibetica,Melanthiaceae,Ypsilandrosides U?Y,Spirostanol saponins,Cholestanol saponins

1 Introduction

Ypsilandra(Melanthiaceae) is distributed in southwestern China and Myanmar,which contains 5 species according to the updated classification of the Angiosperm Phylogeny Group IV [1].Among them,Ypsilandra thibeticahas been used in folk medicine for treatment of scrofula,dysuria,edema,uterine bleeding,and traumatic hemorrhage in China by the local people [2,3].Our previous investigations discovered twenty eight new steroidal glycosides including nineteen spirostanol saponins,two furostanol saponins,three cholestanol saponins,two pregnane glycosides,and two C22-steroidal lactone glycosides from this species [4–10],some of which showed cytotoxicity [4,5],antifungal [4,6],antibacterial [6],anti-HIV-1 activities [7],and so on.For further investigation on the chemical constituents of this herb,four new spirostanol saponins (1?4) and one new cholestanol saponin(5) (Fig.1) were obtained and structurally characterized.The current paper reports the isolation,structural elucidation,and the induced platelet aggregation activity of these isolates.

Fig.1 Chemical structures of saponins 1?5

2 Results and discussion

Compound 1 was isolated as an amorphous powder.Its molecular formula was determined as C44H70O17by the positive-ion HRESI-MS atm/z893.4500 [M + Na]+(calcd.for C44H70O17Na,893.4505) and13C NMR data(Table 2).The1H NMR spectrum of 1 (Table 1) showed four methyl proton signals atδH0.89 (s,CH3-18),0.91(s,CH3-19),1.38 (d,J=7.0 Hz,CH3-21),and 0.67 (d,J=5.4 Hz,CH3-27),one olefinic proton signal atδH5.18(o,H-6),while three anomeric protons atδH5.64 (d,J=3.2 Hz,H-1’),5.30 (br s,H-1’’),and 4.86 (d,J=7.8 Hz,H-1’’),which suggested that 1 was a glycoside with three monosaccharide moieties.The13C NMR spectra displayed 44 carbon signals,of which 17 were assigned to those of one pentose and two hexose units,whereas other 27 ones were assigned to the aglycone moiety,including four methyl groups,nine methylene groups (one oxygenated),ten methine groups (one olefinic and three oxygenated),and four quaternary carbons (one olefinic and one ketal).The above NMR data suggested that compound 1 is a typical C-27 steroidal saponin and its aglycone is heloniogen [11].This deduction can be confirmed by 2D-NMR spectra.The1H?1H COSY correlations revealed that the aglycone for 1 had four structural fragments as shown in (Fig.2).Furthermore,the key HMBC correlations (Fig.2) from CH3-18 (δH0.89) to C-12 (δC82.4)/C-13 (δC44.9)/C-14 (δC44.4)/C-17 (δC53.1),from CH3-19 (δH0.91) to C-1 (δC37.1)/C-5 (δC141.1)/C-9 (δC49.0)/C-10 (δC36.9),from CH3-21 (δH1.38)/H-20 (δH2.00)/H-23a (δH1.77)/H-26a (δH3.53) to C-22 (δC109.3)were observed.In addition,the ROESY correlations of H-12 (δH3.88) with H-18 (δH0.89) and H-20 (δH2.00)indicated that the OH-12 wasα-oriented (Fig.3).

Fig.2 1H?1H COSY and Key HMBC correlations of 1?5

Fig.3 Key ROESY correlations for the aglycone moieties of 1 and 3

Table 1 1H NMR spectroscopic data of compounds 1—5 (δ in ppm,J in Hz,C5D5N)

Table 1(continued)

For the sugar part,the pentose was inferred asβ-D-apiofuranoside by the13C NMR signals atδc 108.1(d,C-1’),78.4 (d,C-2’),79.0 (s,C-3’),74.8 (t,C-4’),and 72.9 (t,C-5’) with those of corresponding carbons ofα-andβ-D-apiofuranoside andα-andβ-L-apiofuranoside[12,13].And the two hexose units were assigned to be a L-rhamnopyranosyl and a D-glucopyranosyl by their NMR data,the acid hydrolysis of 1,and the HPLC analysis (retention time) of their L-cysteine methyl esters followed by conversion intoO-tolyl isothiocyanate derivatives and the authentic samples’ derivatives.And theβ-configuration of glucopyranosyl was revealed by the coupling constant (3J1,2>7.0 Hz) [14],while the anomeric configuration of rhamnopyranosyl was identified asα-orientated on the basis of the chemical shift values of C-3’’(δC72.9) and C-5’(δC70.6) with those of corresponding carbons of methylα-andβ-rhamnopyranoside[15].The sequence of the sugar chain at C-3 of the aglycone was established from the following HMBC corrletions: H-1’(δH5.64) of Api with C-3 (δC77.5) of the aglycone,H-1’’(δH5.30) of the Rha with C-5’(δC72.9) of Api,and H-1’’(δH4.86) of the Glc with C-12 (δC82.4)of the aglycone (Fig.2).Thus,the structure of 1 was

elucidated as 12-O-β-D-glucopyranosy-(25R)-spirost-5-en-3β,12β-diol-3-O-α-L-rhamnopyranosyl-(1→5)-β-Dapiofuranoside,and named ypsilandroside U.

Compound 2 was isolated as an amorphous powder with a molecular formula of C38H60O13determined by the positive-ion HRESI-MS atm/z747.3921 [M + Na]+,(calcd.for C38H60O13Na,747.3926) and13C NMR data(Table 2).Its NMR spectra suggested that 2 is a spirostane saponin with a disaccharide chain.Comparison of the1H and13C NMR data of 2 (Tables 1 and 2) with those of ypsiparoside C obtained from the same genus[16] revealed that they shared the same aglycone.The two monosaccharides and their absolute configurations were determined asβ-D-apiose andα-L-rhamnose by the same methods with compound 1.The HMBC correlations from H-1’(δH5.72) to C-3 (δC77.7),and from H-1’(δH5.85) of the rhamnopyransyl to C-2’(δC82.4) established the sequence for 3-O-sugar chain asO-α-L-rhamnopyranosyl-(1→2)-β-D-apiofuranoside(Fig.2).Therefore,the structure of 2 was determined as (25R)-spirost-5-en-3β,17α,27-triol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-apiofuranoside,and named ypsilandroside V.

Compound 3 was isolated as an amorphous powder and had a molecular formula of C45H72O20as determined by the positive-ion HRESI-MS data (m/z955.4505 [M + Na]+,calcd.for C45H72O20Na,955.4509)and13C NMR data (Table 2).Inspection of the NMR spectra (Tables 1 and 2) of 3 revealed that it possessed a spirotanol skeleton with a trisaccharide chain consisting of one rhamnopyranosyl and two glucopyranosyls.Comparing its1H and13C NMR data (Tables 1 and 2)with those of trillitschonide S6 [17] indicated that they shared the same aglycone.Theα-orientations of OH-23 and CH2OH-25 were supported by the ROESY correlations between H-23 (δH4.00) and H-20 (δH3.39)/H-25 (δH2.29) (Fig.3).The absolute configurations and the anomeric configurations of monosaccharides were determined by the same methods with the above compounds.The sequence of the sugar chain at C-3 of the aglycone was established by the HMBC correlations from H-1’(δH4.92) to C-3 (δC76.8),from H-1’(δH6.31) to C-2’(δC77.5),and from H-1’(δH5.04) to C-6’(δC69.9) (Fig.2).Consequently,the structure of 3 was established as (23S,25S)-spirost-5-en-3β,17α,23,27-tetraol-3-O-β-D-glucopyranosyl-(1→6)-[α-Lrhamnopyranosyl-(1→2)]-β-D-glucopyranoside,and named ypsilandroside W.

Compound 4 possessed a molecular formula C51H80O21determined by the HRESI-MS atm/z1051.5077[M + Na]+,(calcd.for C51H80O21Na,1051.5084) and13C NMR data (Table 2).The UV spectrum of 4 showed absorption maxima at 254.5 nm,suggesting the presence of a conjugated enal system.When comparing its1H and13C NMR data (Tables 1 and 2) with those of ypsilandroside H [10],it was suggested that they shared the same sugar sequence and the similar aglycone,except for the compound 4 has no hydroxyl substituent at the C-17.The above deduction could be verified by the HMBC correlations from H-21 (δH1.13) and H-18(δH0.88) to C-17 (δC62.4) and1H?1H COSY correlations between H-16 (δH4.59) and H-17 (δH1.80) (Fig.2).The HMBC correlations from H-1’(δH5.02) to C-3(δC77.7),from H-1’(δH6.44) to C-2’(δC77.9),from H-1’(δH5.82) to C-4’(δC77.7),and from H-1’’(δH6.28) to C-4’(δC80.4) confirmed that compound 3 had the same sequence of 3-O-sugar chain as that of ypsilandroside H (Fig.2).Thus,the structure of 4 was elucidated as (25R)-B-nor(7)-6-carboxaldehyde-spirost-5(7)-en-3β-ol-3-O-α-L-rhamnopyranosyl-(1→4)-α-Lrhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside,and named ypsilandroside X.

The molecular formula of compound 5 was determined as C53H82O19by the HRESI-MS atm/z1045.5352[M + Na]+(calcd.for C53H82O19Na,1045.5343) and13C NMR data (Table 2).Its NMR spectra indicated that compound 5 was a cholestane tetraglycosides containing an aromatic ring.Analysis of the1H and13C NMR data (Tables 1 and 2) of 5 suggested that it was similar to that of parispseudoside A [18],and the major difference was the absence of a glucopyranosyl group at OH-26 site.With the assistance of HSQC experiment,1H and13C NMR data (Tables 1 and 2) showed four anomeric protons atδH4.96 (o,H-1’),6.41 (br s,H-1’’),5.84 (br s,H-1’’’),and 6.29 (s,H-1’’’’) and their corresponding anomeric carbons atδC100.2 (C-1’),102.1 (C-1’’),102.1 (C-1’’’),and 103.2 (C-1’’’’).The sequence of sugar units was consistent with that of compound 4 by HMBC experiment (Fig.2).As a result,the structure of 5 was assigned as homo-aro-cholest-5-en-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1→4)-α-Lrhamnopyranosyl-(1→4 )-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside,and named ypsilandroside Y.

Table 2 13C NMR spectroscopic data of compounds 1—5 (δ in ppm,C5D5N)

Table 2(continued)

Because the whole plants ofY.thibeticahas been used in folk medicine for treatment of uterine bleeding and traumatic hemorrhage in China,the isolated compounds (1–5) were evaluated for their induced platelet aggregation activity and ADP (adenosine diphosphate)was used as a positive control.Unfortunately,the results showed all isolated saponins did not exhibit the inducing platelet aggregation activity at the tested concentration of 100 μM.

3 Experimental section

3.1 General experimental procedures

Optical rotations were measured by a JASCO P-1020 polarimeter (Jasco Corp.,Japan).UV spectra were recorded on a Shimadzu UV2401 PC spectrophotometer (Shimadzu Corp.,Japan).HRESI-MS was recorded on an Agilent 1290 UPLC/6540 Q-TOF mass spectrometer (Agilent Corp.,USA).The NMR experiments were performed on Bruker AVANCE III 500,Avance III-600,and AV 800 spectrometers (Bruker Corp.,Switzerland).Silica gel (200–300 mesh,Qingdao Marine Chemical Co.,Ltd.,People’s Republic of China),RP-18 (50 μm,Merck,Germany),and Sephadex LH-20 (Pharmacia,Stockholm,Sweden) were used for column chromatography (CC).An Agilent 1260 system (Agilent Corp.,America) with a Zorbax SB-C18 column (5 μm,9.4 × 250 mm) was used for HPLC separation.TLC was carried out on silica gel HSGF254plates (Qingdao Marine Chemical Co.,China)or RP-18 F254(Merck,Darmstadt,Germany).

3.2 Plant material

The whole plant materials ofY.thibeticawere collected in August 2010 from Zhaotong City,Yunnan Provence,China,and identified by Prof.Xin-Qi Chen,Institute of Botany,Chinese Academy of Sciences,Beijing.A voucher specimen was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany,Chinese Academy of Sciences.

3.3 Extraction and isolation

The dried whole plants ofY.thibetica(110 kg) were crushed and extracted three times with 70% EtOH under reflux for a 3 h,2 h and 2 h.Then,the combined extract was concentrated under reduced pressure.The crude extract (30 kg) was passed through YWD-3F macroporous resin and eluted successively with H2O,40% EtOH,75% EtOH,and 95% EtOH,respectively.Evaporated 75%EtOH fraction (crude saponin-rich mixture,10 kg) was subjected to a silica gel column chromatography (CHCl3–MeOH,20:1→8:2,v/v) to give eleven fractions (Fr.A–Fr.K).Fr.C (560 g) was subjected to a silica gel column chromatography (CHCl3–MeOH,20:1→1:1,v/v) to give 14 fractions (Fr.C-1–Fr.C-14).Fr.C-11 (80 mg) was submitted to Sephadex LH-20 (MeOH) and chromatographically separated on an RP-18 column eluted with MeOH–H2O(40:60→70:30,v/v) and purified by preparative HPLC(MeCN–H2O,40:60→50:50,v/v) to afford saponin 2 (tR=12.8 min,10 mg).Fr.C-13 (45 g) was submitted to Sephadex LH-20 (MeOH) to give three subfractions(C-13–1–C-13–3).Subsequently,Fr.C-13–1 (150 mg)was further purified by preparative HPLC (MeCN–H2O,25:75→35:65,v/v) to afford saponins 5 (tR=10.8 min,7 mg) and 4 (tR=11.9 min,12 mg),whereas saponins 3(tR=11.1 min,10 mg) and 1 (tR=14.8 min,9 mg) were obtained from Fr.C-13–3 (208 mg) by preparative HPLC(MeCN–H2O,30:70→45:55,v/v).

3.4 Physical and spectroscopic data of new glycosides

3.4.1 Ypsilandroside U (1)

Amorphous solid;[α]1D8.6?55.80 (c0.20,MeOH);1H(500 MHz,pyridined5) and13C (125 MHz,pyridined5)NMR data,see Tables 1 and 2;HRESIMSm/z893.4500[M + Na]+(calcd.for C44H70O17Na,893.4505) (Additional file 1).

3.4.2 Ypsilandroside V (2)

Amorphous solid;[α]1D8.6?190.00 (c0.12,MeOH);1H(500 MHz,pyridine-d5) and13C (125 MHz,pyridine-d5)NMR data,see Tables 1 and 2;HRESIMSm/z747.3921([M + Na]+,calcd.for C38H60O13Na,747.3926) (Additional file 1).

3.4.3 Ypsilandroside W (3)

Amorphous solid;[α]2D0.5?125.67 (c0.12,MeOH);1H(500 MHz,pyridine-d5) and13C (125 MHz,pyridine-d5)NMR data,see Tables 1 and 2;HRESIMSm/z955.4505[M + Na]+(calcd.for C45H72O20Na,955.4509) (Additional file 1).

3.4.4 Ypsilandroside X (4)

Amorphous solid;[α]18.6D?106.40 (c0.15,MeOH);UV(MeOH) λmax(logε) 202.5 (3.9),254.5 (3.9) nm;1H(500 MHz,pyridine-d5) and13C (125 MHz,pyridine-d5)NMR data,see Tables 1 and 2;HRESIMSm/z1051.5077[M + Na]+(calcd.for C51H80O21Na,1051.5084) (Additional file 1).

3.4.5 Ypsilandroside Y (5)

Amorphous solid;[α]18.6D?48.18 (c0.11,MeOH);UV(MeOH) λmax(logε) 203 (4.5) nm;1H (600 MHz,pyridine-d5) and13C (150 MHz,pyridine-d5) NMR data,see Tables 1 and 2;HRESIMSm/z1045.5352 [M + Na]+(calcd.for C53H82O19Na,1045.5343) (Additional file 1).

3.5 Acid hydrolysis of compounds 1–5 and determination of the absolute configuration of the sugars by HPLC

Compounds 1?5 (1.0 mg each) in 6 M CF3COOH(1,4-dioxane-H2O 1:1,1.0 mL) were heated at 99 ℃for 2 h,respectively.The reaction mixture was diluted with H2O (1.0 mL) and then extracted with EtOAc(3 × 2.0 mL).Next,each aqueous layer was evaporated to dryness using rotary evaporation.Each dried residue was dissolved in pyridine (1.0 mL) mixed with L-cysteine methyl ester hydrochloride (1.0 mg) (Aldrich,Japan)and heated at 60 °C for 1 h.Then,O-tolyl isothiocyanate(5.0 μL) (Tokyo Chemical Industry Co.,Ltd.,Japan) was added to the mixture,this being heated at 60 °C for 1 h.Each reaction mixture was directly analyzed by reversed phase HPLC following the above procedure.Each reaction mixture was directly analyzed by analytical HPLC on a Poroshell 120 SB-C18 column (100 × 4.6 mm,2.7 μm,Agilent) using an elution of CH3CN?H2O (20:75→40:60,v/v) at a flow rate of 0.6 mL/min.As a result,the sugars in the test compounds were identified as D-glucose and L-rhamnose,respectively,by comparing their molecular weight and retention time with the standards (tR13.90 min for D-glucose;tR17.72 min for L-rhamnose).

3.6 Platelet aggregation assays

Turbidometric measurements of platelet aggregation of the samples were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation,Havertown,PA,USA) according to Born’s method [19,20].Rabbit platelet aggregation study was completed within 3.0 h of preparation of platelet-rich plasma (PRP).Immediately after preparation of PRP,250 μL was incubated in each test tube at 37 °C for 5.0 min and then 2.5 μL of compounds (100 μM) were individually added.The changes in absorbance as a result of platelet aggregation were recorded.The extent of aggregation was estimated by the percentage of maximum increase in light transmittance,with the buffer representing 100% transmittance.ADP(adenosine diphosphate) was used as a positive control with a 59.5 ± 6.1% maximal platelet aggregation rate at a concentration of 10 μM.1% DMSO was used as a blank control with a 2.7 ± 0.6% maximal platelet aggregation.Data counting and analysis was done on SPSS 16.0,with experimental results expressed as mean ± standard error.

4 Conclusion

Phytochemical reinvestigation on the whole plants ofY.thibeticaobtained four new spirostanol glycosides,named ypsilandrosides U-X (1–4),and one new cholestanol glycoside,named ypsilandroside Y (5).Their structures have been illustrated by extensive spectroscopic data and chemical methods.Among them,compound 4 is a rare spirostanol glycoside which possesses a novel 5(6→7) abeo-steroidal aglycone,while compound 1 is a first spirostanol bisdesmoside attached to C-3 and C-12,respectively,obtained from theYpsilandraspecies.This investigation enriched the cognition of the chemical constituents inY.thibetica.Unfortunately,the bioassay results showed the five new saponins have no the activity of inducing platelet aggregation.

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10.1007/ s13659?022?00337?0.

Additional file 1: Fig.S1.1H NMR spectrum (500 MHz) of compound1in pyridine?d5.Fig.S2.13C NMR spectrum (125 MHz) of compound1in pyridine?d5.Fig.S3.1H—1H COSY spectrum of compound1in pyridine?d5.Fig.S4.HSQC spectrum of compound1in pyridine?d5.Fig.S5.HMBC spectrum of compound1in pyridine?d5.Fig.S6.ROESY spectrum of compound1in pyridine?d5.Fig.S7.HRESI (+) MS spectrum of compound1.Fig.S8.1H NMR spectrum (500 MHz) of compound2in pyridine?d5.Fig.S9.13C NMR spectrum (125 MHz) of compound2in pyridine?d5.Fig.S10.1H—1H COSY spectrum of compound2in pyridine?d5.Fig.S11.HSQC spectrum of compound2in pyridine?d5.Fig.S12.HMBC spectrum of compound2in pyridine?d5.Fig.S13.ROESY spectrum of compound2in pyridine?d5.Fig.S14.HRESI (+) MS spectrum of compound2.Fig.S15.1H NMR spectrum (500 MHz) of compound3in pyridine?d5.Fig.S16.13C NMR spectrum (125 MHz) of compound3in pyridine?d5.Fig.S17.1H—1H COSY spectrum of compound3in pyridine?d5.Fig.S18.HSQC spectrum of compound3in pyridine?d5.Fig.S19.HMBC spectrum of compound3in pyridine?d5.Fig.S20.ROESY spectrum of compound3in pyridine?d5.Fig.S21.HRESI (+) MS spectrum of compound3.Fig.S221H NMR spectrum (500 MHz) of compound4in pyridine?d5.Fig.S23.13C NMR spectrum (125 MHz) of compound4in pyridine?d5.Fig.S24.1H—1H COSY spectrum of compound4in pyridine?d5.Fig.S25.HSQC spectrum of compound4in pyridine?d5.Fig.S26.HMBC spectrum of compound4in pyridine?d5.Fig.S27.ROESY spectrum of compound4in pyridine?d5.Fig.S28.HRESI (+) MS spectrum of compound4.Fig.S29.UV spectrum of compound4.Fig.S30.1H NMR spectrum (600 MHz) of compound5in pyridine?d5.Fig.S31.13C NMR spectrum (150 MHz) of compound5in pyridine?d5.Fig.S32.1H—1H COSY spectrum of compound5in pyridine?d5.Fig.S33.HSQC spectrum of compound5in pyridine?d5.Fig.S34.HMBC spectrum of compound5in pyridine?d5.Fig.S35.ROESY spectrum of compound5in pyridine?d5.Fig.S36.HRESI (+) MS spectrum of com?pound5.Fig.S37.UV spectrum of compound5.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (U1802287 and 32000280),the Ten Thousand Talents Plan of Yunnan Province for Industrial Technology Leading Talents,and the State Key Laboratory of Phytochemistry and Plant Resources in West China(P2019?ZZ02).

Author contributions

All authors read and approved the final manuscript.

Declarations

Competing interests

The authors declare that there are no conflicts of interest associated with this work.

Author details

1College of Traditional Chinese Medicine,Yunnan University of Chinese Medicine,Kunming 650500,China.2State Key Laboratory of Phytochemistry and Plant Resources in West China,and Yunnan Key Laboratory of Natural Medicinal Chemistry,Kunming Institute of Botany,Chinese Academy of Sci?ences,Kunming 650201,China.3University of Chinese Academy of Sciences,Beijing 100049,China.