Antiproliferative Triterpenoid Saponins from Leptaulus citroides Baill.from the Madagascar Rain Forest

Qingxi Su.Peggy J.Brodie.Yixi Liu.James S.Miller.Naina M.Andrianjafy.Rabodo Antsiferana.Vincent E.Rasamison.David G.I.Kingston

ORIGINAL ARTICLE

Antiproliferative Triterpenoid Saponins from Leptaulus citroides Baill.from the Madagascar Rain Forest

Qingxi Su.Peggy J.Brodie.Yixi Liu.James S.Miller.Naina M.Andrianjafy.Rabodo Antsiferana.Vincent E.Rasamison.David G.I.Kingston

?The Author(s)2016.This article is published with open access at Springerlink.com

Bioassay-guided fractionation of EtOH extracts obtained from the roots and wood of the Madagascan plantLeptaulus citroidesBaill.(Cardiopteridaceae)led to the isolation of ethyl esters of three new triterpenoid saponins(1–3)and the known sesquiterpenoid cinnamosmolide(4).The structures of1–3were elucidated by extensive 1D and 2D NMR experiments and mass spectrometry.Compounds1,2,and4showed moderate cytotoxicity against the A2780 human ovarian cancer cell line with IC50values of 2.8,10.2 and 2.0 μM,respectively.

Graphical Abstract

Antiproliferative activity ·Triterpenoid saponins·Leptaulus citroides(Cardiopteridacea)

1 Introduction

In our continuing search for bioactive natural products from Madagascar rainforests as part of the International Cooperative Biodiversity Group(ICBG)project,we have focused on the isolation of antiproliferative natural products from plant extracts collected from Madagascar forests[2,3].As part of the research,we selected the EtOH extracts of the roots and wood of the plantLeptaulus citroides(Cardiopteridaceae)for investigation of antiproliferative natural products,since no previous chemical work has been reported on this genus.Bioassay-guided fractionation of the roots and wood extracts yielded ethyl esters of three new triterpenoid saponins(1–3)and the known sesquiterpenoid cinnamosmolide(4)[4].Here we report the structure elucidation of compounds1–3and their antiproliferative activities.

2 Results and Discussion

Ethyl leptauloside A(1)was obtained as an amorphous white powder.Its HRESIMS revealed a sodiated quasimolecular ion peak atm/z1223.5806[M+Na]+,corresponding to the molecular formula C59H92O25.Compound1was assigned as an olean-12-ene triterpene derivative based on its 1D and 2D NMR spectra.The1H NMR spectrum of the aglycone part of compound1displayed characteristic signals of six singlet methyl groups[δH0.91,0.95,1.03,1.05,1.16 and 1.50,(each 3H,all s,29,26,25,30,24,27-H3)],an olefin group(δH5.35,t,J=3.5 Hz,H-12)and an aldehyde group(δH9.44,s).The position of the olefin group was confirmed by HMBC correlations of H3-27(δH1.50,s)to C-13(δC144.1),and H-12 to C-11(δC24.6)[5].The aglycone moiety was oxygenated at C-3,16,22,and 28,based on the HMBC correlations between H3-24(δH1.16,s)and C-3(δC86.3);H-22/H3-29/H3-30(δH5.44,dd,J=12.1,5.6 Hz/0.91 s/1.05 s)and C-21(δC42.1);between H-16/H-22(δH4.11,brs/δH5.44,dd,J=12.1,5.6 Hz)and C-17(δC45.3);and between H2-28(δH3.05,d,J=10.9 Hz/3.25 m)and C-22(δC73.8).The aldehyde group[(δH/δC):9.44,s/210.6]was located at C-4 based on the HMBC correlation between the aldehyde proton and C-24(δC10.8),in addition to comparison of 1D NMR data of1with previously reported data of similar compounds[6].The 1D NMR and HSQC spectra of compound1showed the characteristic chemical shifts and coupling patterns of an angeloyl group,with one olefinic proton[δH6.07(1H,qq,J=7.3,1.5 Hz,H-3′′′′′)],two methyl groups[δH1.98(3H,dq,J=7.3,1.5 Hz,H3-4′′′′′)and 1.90(3H,m,H3-5′′′′′)],a carboxyl carbon(δC168.8,C-1′′′′′)and twosp2carbons(δC138.1 and 130.0,C-3′′′′′and C-2′′′′′),in agreement with the NMR data of related compounds[5–7].The angeloyl group was assigned to C-22 based on the HMBC correlation between H-22 and the angeloyl carbonyl carbon.The spectroscopic data of the aglycone of1showed good agreement with the data of similar compounds reported previously[6–10].The relative configurations of the aglycone and tetrasaccharide moiety were determined from coupling constants and ROESY correlations.The aldehyde group at C-4 was assigned the α-equatorial orientation on the basis of ROESY correlations of H3-24(δH1.16,s)to the β-axially oriented H3-25(δH1.03,s)and of H-23(δH9.44,s)to H-3(δH3.87,m)and H-5(δH1.35,m),both of which were βaxial(Fig.1).H-16(δH4.11,brs)was assigned a β-equatorial orientation based on its appearance as a broad singlet,indicating small coupling constants,while H-22(δH5.44,dd,J=12.3,5.6 Hz)had the β-axial orientation based on its coupling constants;these assignments were confirmed by comparison with the chemical shifts and coupling constants of related protons of apodytines A-C[8].The relative configurations of the aglycone were supported by comparison of its1H and13C NMR data with those of assamsaponin A[10]and camelliasaponin B1[7].

The structure of the sugarmoiety of1was elucidated on the bases of1H-1HCOSY,TOCSY,ROESY,HSQC,andHMBC spectra.Four anomericprotons[δH4.97(1H,d,J=7.2 Hz),4.92(1H,d,J=7.1 Hz),4.51(1H,d,J=7.6 Hz)and 4.45(1H,d,J=7.5 Hz)]correlated with carbons at δC102.7,102.0,107.5 and 104.8 were observedin the HSQC spectrum of1,in dicating the presence of four sugarun its.The four sugar units were identified as β-glucuronopyranosyl(GlcA-1′-6′),β-galactopyranosyl(Gal-1′′-6′′), β-xylopyranosyl(Xyl-1′′′-5′′′),and β-xylopyranosyl(Xyl-1′′′′-5′′′′),by comparison of their13C NMR data with those of apodytine B[8],isotheasaponin B1[11],and assamsaponin A[10].HMBC correlations of H-1′′(δH4.97,d)to C-2′(δC77.8),H-1′′′(δH4.92,d)toC-3′(δC83.6),and H-1′′′′(δH4.51,d)toC-2′′′(δC85.1)indicated the connectivity of the four sugar units(Fig.2).The HMBC correlation of H-1′(δH4.45,d,J=7.5 Hz)to C-3(δC86.3),indicated that the tetrasaccharide moiety was connected to the aglycone at C-3.An ethyl ester group was present at C-5′(δC76.5)based on the COSY correlations of H2-7′(δH4.22,q,J=7.1 Hz)to H3-8′(δH1.28,t,J=7.1 Hz)and HMBC correlations of H2-7′(δH4.22,q,J=7.1 Hz)and H-5′(δH3.84,d,J=8.2 Hz)to a carboxyl carbon C-6′(δC170.3).These facts led to the assignment of ethyl leptauloside A (1)as 3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosiduronate}-(3β,4α,16α,22α)-16,28-dihydroxy-22-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl.

The structures and absolute configurations of the sugar moieties of1were confirmed by the method described by Tanaka and Kouno[12].Compound1was subjected to acid hydrolysis and the resulting mixture was derivatized by treatment withL-cysteine methyl ester ando-tolylisothiocyanate.Comparison of the HPLC retention times of the resultingo-tolylthiocarbamoyl-thiazolidine derivatives of the sugar units with those of standards prepared from L-cysteine methyl ester and D and L-glucose,D and L-galactose,D and L-xylose,D and L-arabinose and D-glucuronic acid,and from D-cysteine methyl ester andD-glucuronic acid,confirmed the structures of the carbohydrate units as D-galactose,two D-xyloses,and D-glucuronic acid.

Ethyl esters of glucuronopyranosyl derivatives are unlikely to be natural products,so the ethyl ester was probably formed as the plant material was extracted with EtOH and the solvent was evaporated.It was regrettably not feasible to collect fresh plant material and extract it with a different solvent to confirm this hypothesis.

Ethyl leptauloside B(2)was obtained as an amorphous white powder.The quasi-molecular ion peak atm/z1223.5781[M+Na]+corresponded to a molecular formula of C59H92O25.Further comparison of 1D NMR data of2suggested it is an isomer of1with the same sugar moiety and a similar aglycone to that of1.The only difference between1and2was the position of the angeloyl and hydroxyl groups.The angeloyl group was assigned to C-16 in2,as indicated by the deshielded signal of H-16(δH5.63,brs),and the hydroxyl group was assigned to C-22 due to the more shielded signal of H-22(δH4.06,dd,J=12.3,5.6 Hz)compared to that of compound1[8,11].The aglycone of2was therefore elucidated as identical to that of theasaponin G2[13].Compound2contained the same sugar moieties as1as shown by the essentially identical 1D NMR data of the two compounds.The structure of2was further confirmed by comparison of HSQC,HMBC and ROESY data with those of1.Thus,the structure of ethyl leptauloside B(2)was determined as 3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][-β-D-xylopyrano syl-(1 → 2)-β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosi duronate}-(3β,4α,16α,22α)-22,28-dihydroxy-16-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl(Fig.1).

Ethyl leptauloside C(3)was obtained as an amorphous white powder.The quasi-molecular ion peak atm/z1301.5982[M+Na]+corresponded to a molecular formula of C64H94O26.Comparison of1H and13C NMR data with those of2indicated that they were closely related except that signals of the angeloyl group were replaced by those of ap-methoxycinnamoyl group in the spectra of3.Thus the1H NMR spectrum of3showed signals of apara-substituted benzene ring at δH7.54(2H,d,J=8.8 Hz)and 6.98(2H,d,J=8.8 Hz),two olefinic protons withE-configuration δH7.75(1H,d,J=16.0 Hz)and δH6.35(1H,d,J=16.0 Hz)and a methoxy group δH3.84(3H,s).These signals suggested the occurrence of a 4-methoxycinnamoyl group,and this was confirmed by HMBC correlations between the methoxy group(δH3.84,s,3H)with C7,and H-acyl-3(δH7.75,d,J=16.0 Hz)with C-acyl-4(δC128.2).Comparisons of chemical shifts and coupling constants of H-16 and H-22 in2and3suggested the 4-methoxycinnamoyl group was attached to C-16[8,11].Thus,the structure of ethyl leptauloside C(3)was determined as 3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][-β-D-xylopyranosyl-(1 → 2)-β-D-xylo pyranosyl-(1 → 3)]-β-D-glucopyranosiduronate}-(3β,4α,6α,22α)-22,28-dihydroxy-16-[(E-4-methoxycinnamoyl)oxy]-23-oxoolean-12-en-3-yl(Fig.1).

Compound4was isolated as a white solid.Its structure was assigned as shown based on comparison of its spectroscopic data with those reported in the literature[4].Cinnamosmolide(4)has also been reported to display antifungal and α-glucosidase inhibitory activities.[14,15].

Compounds1,2,3and4were evaluated for antiproliferative activity against the A2780 human ovarian cancer cell line.Compounds1,2and4showed IC50values of 2.8,10.2,and 2.0 μM respectively,while compound3was inactive (IC50＞20 μg/mL,inhibiting 14% ofcells growth at a concentration of 20 μg/mL)in this assay.Previous studies suggested that acylation with angeloyl groups at C-21 and C-22 can affect the biological activities of oleanane triterpenoid saponins[5,7,16,17].These results support the importance of an angeloylated 22-hydroxyl group for antiproliferative activity.The reduced activity of compound3compared to2is possibly due to the bulkiness of the 4-coumaroyl group that acylates the C-16 hydroxyl group.

3 Conclusions

Ethyl esters of the three new triterpenoid saponins leptaulosides A,B and C(1–3)and the known sesquiterpenoid cinnamosmolide(4)were isolated fromLeptaulus citroides.Compounds1,2and4showed moderate antiproliferative activity against the A2780 human ovarian cancer cell line in the A2780 assay.Ethyl leptauloside C(3)contained an aglycone moiety with an uncommon C-16 4-methoxycinnamate group;camelliagenin A cinnamate is one of the few examples of C-16 cinnamates of oleanane triterpenes[18].

4 Experimental Section

4.1 General Experimental Procedures

IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers,respectively.1D and 2D NMR spectra were recorded on a Bruker Avance 500 spectrometer in CD3OD;chemical shifts are given in δ(ppm),and coupling constants are reported in Hz.Mass spectra were obtained on an Agilent 6220 LC-TOF–MS in the positive ion mode.Optical rotations were recorded on a JASCO P-2000 polarimeter.Open column chromatography was performed using Sephadex LH-20 and silica gel(40–63 μm,Silicycle Co.USA).HPLC was performed on a Shimadzu LC-10AT instrument with a semipreparative C18(Phenomenex Luna column,5 μm,250 X10 mm),a Shimadzu SPD M10A diode array detector,and a SCL-10A system controller.All isolated compounds were purified to 95%purity or better,as judged by HPLC(both UV and ELSD detection)before determining bioactivity.

4.2 Antiproliferative Bioassays

Antiproliferative activities were determined at Virginia Tech against the drug-sensitive A2780 human ovarian cancer cell line as previously described[19,20].

4.3 Plant Material

Leptaulus citroidesBaill.(Cardiopteridaceae)(vernacular name Tabonaka)were collected by N.M.Andrianjafy and coworkers at an elevation of about 600 m from a 10 m tall tree.Collection was made on a slope near the town of Ambodimangavalo in the district of Vavatenina,on the d’Ihofika river near the Andran of antsona camp,at coordinates 17°39′07′S 048°58′14′E(-17.6519400,48.9705500).Dupl icate voucher specimens(Andrianjafy 323)were deposited at the Centre National d’Application des Recherches Pharmaceutiques(CNARP),the Herbarium of the Department of Forestry and Fishery Research(TEF),and the Missouri Botanical Garden,St.Louis,Missouri(MO).

4.4 Extraction and Isolation

A ground sample ofL.citroidesbark(420 g)was extracted withEtOH(1000 mL)at room temperature to yield23.8 gof crude EtOH extract designated MG 1619,and a 5 g portion of this extract was made available to Virginia Tech for bioassay-guided isolation.Similar treatment of the wood(412 g)(1900 mL)yielded 9.8 g of extract designated MG 1620,and a 1.7 g portion of this extract was made available to Virginia Tech for bioassay-guided isolation.

4.5 Isolation of Bioactive Constituents

The EtOH extract of the root ofL.citroides(MG 1619,3 g,IC50=20 μg/mL)was suspended in aqueous MeOH(MeOH–H2O,9:1,100 mL)and extracted with hexane(5×100 mL portions).The aqueous fraction was then diluted to 60%MeOH and further extracted with CH2Cl2(5×100 mL portions)to give a CH2Cl2fraction(477 mg)with an IC50value of 11 μg/mL.This fraction was further subjected to size exclusion open column chromatography on Sephadex LH-20(I.D.×L 3×50 cm)eluted with 1:1 CH2Cl2:MeOH to yield four fractions,of which the most active fraction F3(168 mg)exhibited an IC50of 7.9 μg/mL.Fraction F3 was applied to a silica gel column(I.D.×L 3×50 cm,40–63 μm)and eluted with CHCl3:MeOH:H2O,15:6:1 to give five fractions based on TLC profile.Fractions F3-3(22.4 mg,IC50=2.5 μg/mL)and F3-4(32.0 mg,IC50=9.9 μg/mL)were combined and further separated by HPLC on a semipreparative C18 column(Phenomenex Luna column,5 μm,25×1 cm)with elution by a solvent gradient from CH3OH:H2O,50:50to60:40from0to10 min,to70:30 from 20 to 30 min,to 100:0 from 30 to 35 min,ending with 100%CH3OH from 35 to 45 min.This process gave crude compounds1(3.4 mg,tR22 min)and2(3.0 mg,tR23 min),and compound3(3.0 mg,tR26 min).Compounds1and2were each purified by HPLC on a semipreparative C18 column(Phenomenex Luna column,5 μm,25×1 cm)eluted with a same solvent gradient from CH3CN:H2O,30:70 to 40:60 from 0 to 10 min,to 50:50 from 10 to 40 min,ending with 100%CH3CN from 40 to 45 min to give purified compounds1(3.0 mg,tR27 min)and2(2.8 mg,tR40 min).

The EtOH extract of the wood ofL.citroides(MG 1620,1.5 g,IC50=20 μg/mL)was subjected to liquid–liquid partition using same procedures described above.The active dichloromethane fraction(97 mg,IC50=3.5 μg/mL)was subjected to Sephadex LH-20(I.D.×L 3×50 cm)chromatography to give four fractions.The active fraction F-4b(57.5 mg,IC50=2.1 μg/mL)was then subjected to open silica gel column(I.D.×L 3×50 cm,40-63 μm)eluted with CHCl3-MeOH,20:1 to give four fractions.Fraction F-4b-2(29.2 mg,IC50=1.4 μg/mL)was further separated by HPLC on a semipreparative C18 column(Phenomenex Lunacolumn,5 μm,25×1 cm)eluted by a solvent gradient from CH3CN:H2O,70:30to90:10 from0to30 min,to100:0 from 30 to 40 min,ending with 100%CH3CN from 40 to 45 min.This process gave compound4(7.6 mg,tR32 min).

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4.6 Ethyl leptauloside A(1)3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][-β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosiduronate}-(3β,4α,16α,22α)-16,28-dihydroxy-22-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl

4.7 Ethyl leptauloside B(2)3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][-β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosiduronate}-(3β,4α,16α,22α)-22,28-dihydroxy-16-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl

4.8 Ethyl leptauloside C(3)3-O-{ethyl[β-D-galactopyranosyl-(1 → 2)][-β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosiduronate}-(3β,4α,16α,22α)-22,28-dihydroxy-16-[(E-4-methoxycinnamoyl)oxy]-23-oxoolean-12-en-3-yl.

4.9 Hydrolysis of Ethyl Leptauloside A(1)and Absolute Configurations of Its Carbohydrate Moieties.

Authentic methyl2-(polyhydroxyalkyl)-3-(o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylates were prepared from D-andL-galactose,D-andL-glucose,D-andL-xylose,D-andL-galactose,D-andL-galactose,andD-glucuronic acid by reaction withL-cysteine methyl ester ando-tolylisothiocyanate as described[12].SinceL-glucuronic acidwas not available,the enantiomeric (o-tolylthiocarbamoyl)-thiazolidine-4(S)-carboxylate of D-glucuronic acid was prepared by reaction with D-cysteinemethyl ester ando-tolylisothiocyanate.Compound1(2.0 mg)was treated with 3 MHCl for 4 hat100°C,andthe solution was then neutralized with sodium bicarbonate and extracted thrice with EtOAc.The aqueous fraction was evaporated to dryness under reduced pressure.The resulting mixture of carbohydrates(0.7 mg)was then dissolved in 0.5 mL pyridine,0.9 mg ofL-cysteine methyl ester was added,and the mixture was heated at 60°C for 1 h.o-Tolylisothiocyanate(0.9 mg)was then added to the mixture,which was againheatedat 60°Cfor1 h.There action mixture was directly analyzed by reverse-phase HPLC on a Phenomenex Luna column(5 μm,25×1 cm),eluted with isocratic 0.1%formic acid in CH3CN/H2O(15:85)at a flow rate of 2.5 mL/min for 5 min,followed by 0.1%formic acid in CH3CN/H2O(25:75)for 30 min,and a wash with 100%CH3CN for 10 min.The resulting chromatogram contained three major peaks with retention times of 25.29,29.12,and 31.25 min,identical to those of the derivatives of D-xylose,D-galactose andD-glucuronic acid.Co-injection of each of the derivatives obtained by hydrolysis of1with its corresponding synthetic counterpart confirmed the identity of the compounds.The peak corresponding to the D-xylose derivative was approximately twice as large as that for theD-galactose derivative,consistent with the presence of two D-xylose units in1.

AcknowledgmentsThis project was supported by the Fogarty International Center,the National Cancer Institute,the National Institute of Allergy and Infectious Diseases,the National Institute of Mental Health,the National Institute on Drug Abuse,the National Heart Lung and Blood Institute,the National Center for Complementary and Alternative Medicine,the Office of Dietary Supplements,the National Institute of General Medical Sciences,the Biological Sciences Directorate of the National Science Foundation,and the Office of Biological and Environmental Research of the U.S.Department of Energy under Cooperative Agreement U01 TW00313 with the International Cooperative Biodiversity Groups.This project was also supported by the National Research Initiative of the Cooperative State Research,Education and Extension Service,USDA,Grant#2008-35621-04732.These supports are gratefully acknowledged.Work at Virginia Tech was supported by the National Science Foundation under Grant CHE-0722638 for the purchase of the Agilent 6220 mass spectrometer.We thank Mr.B.Bebout and Mr.Mehdi Ashraf-Khorassani for recording the mass spectra.Fieldwork essential for this project was conducted under a collaborative agreement between the Missouri Botanical Garden and the Parc Botanique et Zoologique de Tsimbazaza and a multilateral agreement between the ICBG partners,including the Centre National d’Application des Recherches Pharmaceutiques.We thank J.Razafitsalama,S.Randrianasolo,A.Rakotondrafara,L.Randrianjanaka,R.Mananjara,and C.Razanadrainy for assistance with plant collection,and we gratefully acknowledge courtesies extended by the Government of Madagascar(Ministe`re des Eaux et Fore?ts).

Compliance with Ethical StandardsThe botanical collection in Madagascar was carried out under a benefit-sharing agreement between all the parties involved,and under the authority of plant collection and extract export permits issued by the government of Madagascar.

Conflict of InterestThe authors declare no conflict of interest.

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12 October 2015/Accepted:19 December 2015/Published online:8 January 2016

Biodiversity Conservation and Drug Discovery in Madagascar,Part 67.For Part 66,see[1].

Electronic supplementary material The online version of this article(

10.1007/s13659-015-0083-1)contains supplementary material,which is available to authorized users.

Q.Su·P.J.Brodie·Y.Liu·D.G.I.Kingston(?)

Department of Chemistry,Virginia Tech Center for Drug

Discovery,M/C 0212,Virginia Tech,Blacksburg VA,

24061,USA

e-mail:dkingston@vt.edu

J.S.Miller

Missouri Botanical Garden,P.O.Box 299,St.Louis,

MO 63166-0299,USA

N.M.Andrianjafy

Missouri Botanical Garden,B.P 3391,101 Antananarivo,

Madagascar

R.Antsiferana·V.E.Rasamison

Centre National d’Application des Recherches Pharmaceutiques,

B.P 702,101 Antananarivo,Madagascar