Formosins A–F:Diterpenoids with Anti-microbial Activities from Excoecaria formosana

Bing-Dong Lin.Bin Zhou.Lei Dong.Yan Wu.Jian-Min Yue

ORIGINAL ARTICLE

Formosins A–F:Diterpenoids with Anti-microbial Activities from Excoecaria formosana

Bing-Dong Lin.Bin Zhou.Lei Dong.Yan Wu.Jian-Min Yue

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

Three new halimane-type diterpenoids formosins A–C(1–3),and three clerodane-type diterpenoids formosins D–F(4–6),were isolated from the twigs ofExcoecaria formosana.Their structures were assigned on the basis of spectroscopic data analysis.Compounds1and4showed moderate anti-microbial activities againstBacillus subtilis(MIC=50 and 50 μg/mL,respectively).Compound6exhibited moderate anti-microbial activities against two strains ofHelicobacter pylori(Hp-SS1 and ATCC 43504)with MIC values of 50 and 50 μg/mL,respectively.

Graphical abstract

Excoecaria formosana·Halimane-type·Clerodane-type·Diterpenoid·Anti-microbial

1 Introduction

The genusExcoecaria(Euphorbiaceae)compromising 40 species,are widely distributed in Africa and East Asia[1].Several plants in this genus have been used in folk medicine to treat psoriasis,dermatitis and pruritus[2–4].The characteristic of this plant genus is the poisonous milk latex,which causes skin blister[5].Chemical investigations on this plantgenus have led to the isolation of structurally diverse compounds with significant biological activities including antitumor promoting,anti-ulcer,and anti-microbial activities[6–10].In the current study,three new halimane-type diterpenoids formosins A–C(1–3),and three clerodane-type diterpenoids formosins D–F(4–6),were isolated from the twigs ofExcoecaria formosana.Presented herein are the isolation,structural characterization,and biological evaluation of these compounds.

Table 11H NMR data for compounds 1–6 in CDCl3at 400 MHz

2 Results and Discussion

Compound1,a white powder,gave a molecular formula C20H28O3as determined by the(+)-HRESIMS ion atm/z339.1937[M+Na]+(calcd 339.1931)requiring seven degrees of unsaturation.The IR absorptions(3000–2800 cm-1,broad band)and(1695 cm-1)showed the presence of a carboxylic group.The diagnostic NMR data(Tables 1,2)suggested the presence of aβ-substituted furan ring(δH6.27,7.22,and 7.34),a persubstituted double bond,and a carboxylic group(δC181.5).These functionalities accounted for five out of the seven indices of hydrogen deficiency,requiring the presence of two additional rings in the structure of1.The aforementioned data suggested that compound1is a halimane-type diterpenoid and is structurally related to crotohalimaneic acid[11].The planar structure of1was deduced by 2D NMR spectra.In the HMBC spectrum(Fig.1),two tertiary methyls(δH1.00,and 1.04,each 3H,s)correlating with C-3,C-4 and C-5 were attached to C-4.The multiple HMBC correlations of H-3,H-7,andH3-19/C-5(δC141.3);H-1,H-8,andH-11/C-10(δC125.2);and H3-17/C-7,C-8,and C-9 indicated the presence of a typical Δ5(10)double bond and 8-Me(C-17).Theβsubstituted furan ring and carboxylic group were located at C-12andC-9 by the HMBC correlations of H-12/C-13,C-14 and C-16;and H-8 and H-11/C-20,respectively.There lative configuration of1was established by the ROESY experiment(Fig.1).The ROESY cross-peaks of H3-18/H-2β,H-2β/H-1β,H-1β/H-11,and H-12/H-8 indicated that they are co-facial and randomly assigned in aβ-configuration.In consequence,H3-19 and H3-17 wereα-oriented by the ROESY correlations of H3-19/H-6αand H3-17/H-7α.The structure of1(formosin A)was herein elucidated as shown.

Table 213C NMR data for compounds 1–6 in CDCl3at 100 MHz

Compound2had the molecular formula of C20H28O4,as determined by the13C NMR data and the(+)-HRESIMS ion atm/z355.1839[M+Na]+(calcd 355.1880),which is 16 mass units more than that of1.Comparison of its NMR spectroscopic data(Tables 1,2)with those of1revealed they are structural analogues with the obvious difference being the presence of anα,β-unsaturated-γ-lactone moiety instead of theβ-substituted furan ring.It was confirmed by the NMR data(δH4.77 and 7.17;δC70.2,134.5,143.9,and 174.3),as well as the key HMBC correlations from H-12 to C-13,C-14,and C-16(Figure S13,Supporting Information).Thus,the structure of2(formosin B)was determined as shown.

Compound3displayed a molecular formula of C21H30O5as established by the(+)-HRESIMS atm/z385.1991[M+Na]+(calcd 385.1991)and the13C NMR data.Analysis of the NMR data(Tables 1,2)of3showed many similarities to those of2.The only difference was the presence of an additional methoxy group(δH3.49,s,3H),which was located at C-15 to form the acetal motif,which was confirmed by the downfield shifted C-15(ΔδC32.3)as compared to that of2.Compound3was obtained as a pair of inseparable C-15 epimers,which exhibited several pairs of very close carbon resonances in the13C NMR spectrum(Figure S19,Supplementary Material).Therefore,the structure of3(formosin C)with its relative con figuration was con firmed as depicted by the HMBC and ROESY spectra(Figures S21 and S22,Supplementary Material).

Compound4was obtained as a white powder with a molecular formula of C21H30O4as established by the13C NMR data and the(+)-HRESIMS ion atm/z369.1970[M+Na]+(calcd 369.2036),demanding seven degrees of unsaturation.The IR absorption bands(3000–2800 cm-1,broad band;and 1695 cm-1)showed the presence of a carboxylic group.The characteristic NMR signals for aβfuran ring,a trisubstituted double bond,a methoxyl and a carboxylic groups were observed from the1H and13C NMR spectroscopic data analysis(Tables 1,2).Comprehensive analysis of the NMR spectra of4revealed its structure is highly related with that of junceic acid[12]with a clerodane-type diterpenoid backbone.The only difference was the presence of an additional methoxyl group in4,which was placed at C-2 by the HMBC correlation(Fig.2)fromto C-2(δC74.3).The carboxylic group was attached to C-9 via the HMBC correlations from H-8,H-10 and H-11 to C-20(δC182.6).The key HMBC cross-peaks from H3-18 to C-3(δC120.6),C-4(δC149.2)and C-5,and from H3-19 to C-4 revealed the presence of Δ3double bond.The relative con figuration of4was established by the ROESY experiment(Fig.2).The ROESY correlations of H-10/H-6βand H-10/H-8 indicated that H-8 and H-10 are co-facial and randomly assigned in aβ-configuration.Consequently,H3-19 and H-2 were thus assigned to beα-directed by the ROESY correlations of H3-19/H-1α,H3-19/H-7α,and H-2/H-1α.Therefore,the structure of4(formosin D)was established as depicted.

Compound5possessed a molecular formula C20H28O4based on the13C NMR data and the(+)-HRESIMS ion atm/z355.1885[M+Na]+(calcd 355.1888),which is 14 mass units less than that of4.Detailed analysis of the NMR data(Tables 1,2)of5revealed that its structure is closely related with that of4with the only difference being the absence of the methyl etherification,which is consistent with the molecular formula.The structure of5(formosin E)with the relative configuration was further confirmed by HMBC and ROESY spectra(Figures S39 and S40,Supplementary Material).

Compound6,named formosin F,exhibited a sodiated molecular ion atm/z353.1724 [M+Na]+(calcd 353.1723)in the(+)-HRESIMS,consistent with a molecular formula of C20H26O4,which was supported by the13C NMR data.Comparison of the NMR data(Tables 1,2)of6with those of5revealed they are structural analogues.The main difference was the presence of a keto group at C-2 in6instead of the hydroxy group in the latter,which was confirmed by the HMBC correlations(Figure S42,Supporting Information)from H-3 and H-10 to C-2(δC199.9).The structure of6was thus determined as shown.

The new isolates were tested for anti-microbial activities against a panel of microbesin vitroby the microdilution method[13,14].Compounds1and4exhibited moderate activity againstBacillus subtilis ATCC 6633with MIC values of 50 and 50 μg/mL,respectively,where magnolol was used as the positive control(MIC=12.5 μg/mL).Compound6showed moderate antibacterial activities against two strainsHelicobacter pylori(Hp-SS1 or ATCC 43504)with MIC values of 50 and 50 μg/mL,respectively,and metronidazole was used as the positive control(MIC=0.312 and 128 μg/mL,respectively).

3 Experimental Section

3.1 General Experimental Procedures

Optical rotations were determined on a Perkin-Elmer 341 polarimeter.UV spectra were recorded on a Shimadzu UV-2550 spectrophotometer.IR spectra were acquired on a Perkin-Elmer 577 spectrometer.NMR spectra were measured on a Bruker AM-400 spectrometer with TMS as internal standard.ESIMS and HRESIMS were performed on a Bruker Daltonics Esquire 3000 plus and a Waters-Micromass Q-TQF Ultima Global mass spectrometer,respectively.Semi-preparative HPLC was performed on a Waters 1525 binary pump system with a Waters 2489 detector(210 nm)and equipped with a YMC-Pack ODS-A(250 × 10 mm,S-5 μm).Silica gel(200–300 mesh,Qingdao Haiyang Chemical Co.,Ltd.),C18reversed-phase(RP-18)silica gel(20–45 μm,Fuji Silysia Chemical Ltd.),CHP20P MCI gel(75–150 μm,Mitsubishi Chemical Corporation),and Sephadex LH-20 gel(Amersham Biosciences)were used for column chromatography(CC).Precoated silica gel GF254plates(Qingdao Haiyang Chemical Co.,Ltd.)were used for TLC detection.All solvents used for CC were of analytical grade(Shanghai Chemical Reagents Co.,Ltd.),and solvents used for HPLC were of HPLC grade(J&K Scientific Ltd.).

3.2 Plant Material

ThetwigsofE.formosanawere collected from Sanya city of Hainan Province,the People’s Republic of China,and authenticated by Prof.S.-M.Huang,Department of Biology,Hainan University.A voucher specimen has been deposited in Shanghai Institute of Materia Medica,Chinese Academy of Sciences(accession number:SMEF-2006-1Y).

3.3 Extraction and Isolation

The air-dried,powdered twigs ofE.formosana(6.0 kg)was extracted three times with 95%EtOH at room temperature to give a crude extract(290 g),which was partitioned between EtOAc and H2O.The EtOAc-soluble fraction(85 g)was subjected to passage over an MCI gel column(MeOH/H2O,3:7–9:1)to afford fractions A–G.Fraction C(25.7 g)was separated over a silica gel column eluted with gradient mixtures of petroleum ether–acetone(35:1–1:1,v/v)to afford major fractions C1-C6.Fraction C3(3.4 g)was separated on a reversed-phase C18silica gel column(MeOH/H2O,55–100%)to yield three major portions(C3a–C3c),and each of those was purified by a semi-preparative HPLC(60%CH3CN in H2O,3 mL/min)to yield compounds1(20 mg),2(10 mg),and6(100 mg),respectively.Fraction C4(515 mg)was purified by a semipreparative HPLC(55%CH3CN in H2O,3 mL/min)to give compound4(15 mg).Fraction C6(1.5 g)was separated on a column of Sephadex LH-20,and then purified by a semi-preparative HPLC(50%CH3CN in H2O,3 mL/min)to yield compound5(8 mg).Fraction E(11.4 g)was chromatographed on a silica gel column eluted with petroleum ether-ethyl acetate(25:1–1:4,v/v)to afford subfractions E1–E4.Fraction E2(217 mg)was separated on a reversed-phase column containing C18silica gel(MeOH/H2O,70–100%)to yield three fractions E2a–E2c.Fraction E2b(35 mg)was separated by a semi-preparative HPLC(70%CH3CN in H2O,3 mL/min)to yield compound3(8 mg).

3.4 Formosin A(1)

3.5 Formosin B(2)

3.6 Formosin C(3)

3.7 Formosin D(4)

3.8 Formosin E(5)

3.9 Formosin F(6)

3.10 Antimicrobial Activity Assay

The in vitro antibacterial activities againstBacillus subtilisATCC 6633 were tested by applying the protocols described in our previous research[13].

Antibacterial tests againstHelicobacter pyloristrains(Hp-SS1 or ATCC 43504 strain)were carried out in vitro according to the protocols described previously[14].

AcknowledgmentsFinancial support from the National Natural Science Foundation(Grant No.U1302222;81321092)of the People’s Republic of China is gratefully acknowledged.We thank Prof.S.-M.Huang of Hainan University for the identification of the plant material.

Compliance with Ethical Standards

Conflict of InterestThe authors declare no conflict of interest.

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16 December 2015/Accepted:4 January 2016/Published online:27 January 2016

Bing-Dong Lin and Bin Zhou have contributed equally to this work.Electronic supplementary material The online version of this article(

10.1007/s13659-016-0086-6)contains supplementary material,which is available to authorized users.

B.-D.Lin·B.Zhou·L.Dong·Y.Wu·J.-M.Yue(✉)

State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,555 Zuchongzhi Road,Shanghai 201203,People’s Republic of China

e-mail:jmyue@simm.ac.cn