1-s2.0-S0960894X15001687-main

Chemical constituents from Kandelia candel with their inhibitory effects on pro-in?ammatory cytokines production in LPS-stimulated bone marrow-derived dendritic cells

(BMDCs)

Le Duc Dat a ,b ,Nguyen Phuong Thao a ,b ,Bui Huu Tai a ,b ,Bui Thi Thuy Luyen b ,Sohyun Kim c ,Jung Eun Koo c ,Young Sang Koh c ,Nguyen The Cuong d ,Nguyen Van Thanh a ,Nguyen Xuan Cuong a ,Nguyen Hoai Nam a ,Phan Van Kiem a ,Chau Van Minh a ,?,Young Ho Kim b ,?

Institute of Marine Biochemistry,Vietnam Academy of Science and Technology (VAST),18Hoang Quoc Viet,Nghido,Caugiay,Hanoi,Vietnam b

College of Pharmacy,Chungnam National University,Daejeon 305-764,South Korea c

School of Medicine,Brain Korea 21PLUS Program,and Institute of Medical Science,Jeju National University,Jeju 690-756,South Korea d

Institute of Ecology and Biological Resources,VAST,18Hoang Quoc Viet,Nghido,Caugiay,Hanoi,Vietnam

a r t i c l e i n f o Article history:

Received 12November 2014Revised 5February 2015Accepted 20February 2015

Available online 27February 2015Keywords:

Kandelia candel Rhizpphoraceae Kandelside

Anti-in?ammatory activity

a b s t r a c t

Chemical investigation of Kandelia candel resulted in the isolation of 19compounds (1–19),including one new sesquiterpene glycoside,kandelside (1),three megastigman glycoside compounds (7–9),16known phenolic compounds (2–6and 10–19).Structures of the isolated compounds were elucidated based on spectral data comparison with reported values.Isolated compounds were also evaluated for their inhibitory effects on the production of pro-in?ammatory cytokines interleukin (IL)-12p40,IL-6,and tumor necrosis factor a (TNF-a )in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells.Among these compounds,compound 9exhibited strong inhibitory activity against IL-6production (IC 50=0.07±0.05l M)and moderate inhibitory activity against TNF-a production (IC 50=49.86±1.02l M),but exhibited no activity on https://www.360docs.net/doc/845284764.html,pounds 5and 6signi?cantly inhibited IL-12p40,IL-6,and TNF-a production with IC 50values of 11.68±0.38,44.52±1.08,and 28.73±0.96l M,respectively.

Mangroves are a diverse group of trees that grow in intertidal tropical and subtropical forests.In mangrove species,phenolics are abundant components that prevent damage from herbivores 1,2and exhibit a diversity of other biological activities of historic and potential importance to humans.3Mangrove extracts have been used for diverse medicinal purposes and are known to exhibit antibacterial,antiherpetic,and antihelminthic activities.4

Kandelia candel (Rhizophoraceae)is most widely distributed in the Asian coastline.According to previous study,the hypocotyls of K.candel have high levels of phenolic compounds.5Phenolics are also important components in the leaf extract of K.candel.The bioactivity of phenolic compounds were screened for their antioxidant activities.Total phenolic content in the leaves of K.candel was about 130.32mg/g,evaluated with the pharmaco-logical effect for anti-oxidant activity.6While the anti-in?ammatory activity was not investigated at the moment.

Many studies have shown that in?ammation is part of a com-plex biological response of vascular tissue toward harmful exoge-nous stimuli 7and is mediated by a variety of soluble factors,including a group of secreted polypeptides known as cytokines,which play a key role in the modulation of immune responses.BMDCs play a key role in the interface between the innate and acquired immune systems.8Activated BMDCs perform crucial functions in immune and in?ammatory responses via the patho-gen-associated molecular patterns (PAMPs)-stimulated production of pro-in?ammatory cytokines such as IL-12p40,IL-6and TNF-a .This study describes the isolation and structure elucidation of 19compounds (see Fig.1)were isolated from K.candel ,as well as an evaluation of their in vitro anti-in?ammatory effects.

The methanolic extract of K.candel were partitioned into frac-tions and isolated by multiple chromatographic steps over silica gel,Sephadex LH-20,and YMC RP-18column chromatography (CC)9to yield compounds 1–19.10

Kandelside (1)11was obtained as a white amorphous powder.According to high-resolution electron spray ionization mass spec-troscopy (HR-ESI-MS),a basic ion peak at m /z 439.2315[M+Na]+

https://www.360docs.net/doc/845284764.html,/10.1016/j.bmcl.2015.02.048

?Corresponding authors.Tel.:+84437917053;fax:+84437917054(C.V.M.);tel.:+82428215933;fax:+82428236566(Y.H.K.).

E-mail addresses:cvminh@vast.vn (C.V.Minh),yhk@cnu.ac.kr (Y.H.Kim).

(calcd.for C21H36O8Na,439.2410)con?rmed its molecular struc-ture of C21H36O8.The infrared(IR)spectrum of compound1 showed absorption due to a hydroxyl group(3373cmà1).The 13C-nuclear magnetic resonance(NMR)spectra of1illustrated sig-nals of21carbon atoms,including four methyls,four methylenes, ten methines,and three quaternary carbons,which were identi?ed by DEPT-135experiments(see Table1).Signals representing one anomeric carbon(d C102.4,C-10),six oxymethines(d C88.0,78.5, 78.5,78.4,75.0,and71.9),one oxygenated quaternary(d C73.9, C-12),and one oxymethylene(d C63.1,C-60)were also observed. The1H NMR spectrum of1showed an anomeric proton signal at d H4.20(1H,d,J=7.8Hz,H-10).Therefore,the glucose unit was sug-gested to be connected to aglycon in a b-glycosidic linkage. Moreover,the1H NMR spectrum showed four methyl groups at d H0.95(3H,s,H-11),d H1.05(3H,s,H-10),d H1.10(3H,s,H-14), and d H1.12(3H,s,H-13).The1H and13C NMR of compound1were similar to those of2a,12-dihydroxycopacamphan-15-one2-O-b-D-glucopyranoside,12except for the replacement of a carbonyl group in2a,12-dihydroxycopacamphan-15-one2-O-b-D-glucopy-ranoside by oxymethine,and a glucose moiety attach to C-8in1. The structure of1was further con?rmed by heteronuclear multiple bond correlation(HMBC)and heteronuclear single quantum coher-ence(HSQC)experiments.The placement of the oxymethine car-bon at C-15(d C88.0)was determined by HMBC correlation signals between H-4(d H1.52),H-5(d H1.39),H-6(d H1.76),H-11 (d H0.95),and C-15(d C88.0),con?rmed with HSQC correlation between H-15(d H3.44,br s)and C-15(d C88.0).The correlations between H-4(d H1.52),H-13(d H1.12),H-14(d H1.10)and C-12(d C 73.9),as well as H-11(d H0.95),H-6(d H1.76)and C-8(d C78.5),were also observed in HMBC spectra,which was con?rmed with HSQC correlation between H-8(d H4.28,dd,J=3.0,7.8Hz)and C-8(d C 78.5).According to the signal correlation between H glc-10(d H,d, J=7.8Hz)and C-8(d C78.5),the glucose moiety was determined to connect to C-8of aglycon.In addition,the hydrolysis of1was deter-mined as D-glucoside.13Detailed analyses of other HMBC correla-tions(see Fig.2)clearly identi?ed the planar structure of compound1as8,12,15-trihydroxycopacamphan-8-O-b-D-glucopyranoside.

The relative con?guration of three chiral centers(C-1,C-4,and C-9)in1were assigned to the same as those of2a,12-dihydroxyco-pacamphan-15-one2-O-b-D-glucopyranoside,12which was eluci-dated by X-ray diffraction analysis,and showed that CH3

-10, Figure1.The structures of isolated compounds(1–19)from K.candel.

CH3-11were in cis orientation(see Fig.3).Moreover,the nuclear overhauser effect(NOE)spectra of1revealed correlation signals between H-6(d H1.76,m)and H-8(d H4.28,dd,J=3.0,7.8Hz), H-10(d H1.05,s);H-11(d H0.95,s)and H-8(d H4.28,dd,J=3.0, 7.8Hz),H-10(d H1.05,s),H-15(d H3.44,br s).Therefore,compound1was?nally established as8a,12,15a-trihydroxycopacamphan-8-O-b-D-glucopyranoside,and subsequently termed kandelside.

In addition,eighteen known compounds was identi?ed as protocatechuic acid(2),14caffeic acid(3),15chlorogenic acid(4),16 threo and erythro-1-C-syringyl-glycerol(5and6),17blumenol C glucoside(7),18(3R,9S)-megastigman-5-ene-3,9-diol3-O-b-D-glu-copyranoside(8),19corchoionoside C(9),20syringaresinol-b-D glu-coside(10),21isorhamnetin3-O-b-D-glucopyranoside(11),22 isorhamnetin3-O-[a-rhamnopyranosyl-(1-6)-b-glucopyranoside] (12),22kaempferol3-neohesperidoside(13),23quercetin-3-O-ruti-noside(14),23quercetin-3-O-glucoside(15),24cathechin(16),25epi-catechin(17),25kaemferol-3-O-rhamnoside(18),26engeletin(19),27 in comparison with reported values(see Fig.1).To our knowledge, compounds(7–10)are reported from this species for the ?rst time.

We then evaluated the effects of all isolated compounds(1–19) in the pro-in?ammatory response of BMDCs(see Supplementary data).First,a colorimetric MTT assay was used to con?rm that these compounds had little or no effect on cell viability28(data not shown).Upon treatment with LPS,dendritic cells(DCs) secreted pro-in?ammatory cytokines,including IL-12p40,IL-6, and TNF-a.

In our experiments,DCs were incubated in48-well plates at a density of2?105cells/mL,treated for1h with tested compounds at concentrations of2.0,10.0,25.0,and50.0l M,and stimulated with LPS(10.0ng/mL)(see Fig.4).Supernatants were harvested 16h after stimulation.SB203580,an inhibitor of cytokine suppres-sive binding protein/p38kinase,served as a positive control. SB203580inhibited IL-12p40,IL-6,and TNF-a production with half maximal inhibitory concentrations(IC50)of 5.00±0.10, 3.50±0.10,and7.20±0.20l M,respectively.Among the isolated compounds,a mixture of compounds(5+6)inhibited IL-12p40, IL-6,and TNF-a production in LPS-stimulated BMDCs with IC50val-ues of11.68±0.38,44.52±1.08,and28.73±0.96l M,respectively. Compound9potently exhibited signi?cant inhibitory activity on IL-6,with an IC50value of0.07±0.05l M.In addition,(9)also mod-erately inhibited TNF-a production with an IC50value of 49.86±1.02l M.Other compounds were weak and/or inactive on IL-12p40,IL-6,and TNF-a production(IC50>50l M).Further stud-ies are required to elucidate the anti-in?ammatory effects of the active compounds identi?ed in the present study.

Figure3.Key NOESY correlations of1.

Table1

The NMR(CD3OD)spectrosopic data of1

Position d H b(mult.,J in Hz)d C c

1—50.1

2 1.42(1H,m)a30.9

1.17(1H,m)

3 1.42(1H,m)a22.1

1.28(1H,m)

4 1.52(1H,m)55.0

5 1.39(1H,m)49.9

6 1.76(1H,m)a43.3

7 1.87(1H,m)35.8

1.73(1H,m)

8 4.28(1H,dd,3.0,7.8)78.5

9—54.1

10 1.05(3H,s)22.1

110.95(3H,s)10.3

12—73.9

13 1.12(3H,s)28.1

14 1.10(3H,s)26.1

15 3.44(1H,br s)88.0

10 4.20(1H,d,7.8)102.4

20 3.08(1H,t,7.8)75.0

Figure2.Key HMBC correlations of1.

1414L.D.Dat et al./Bioorg.Med.Chem.Lett.25(2015)1412–1416

Acknowledgments

This study was?nally supported by a Grant from the Vietnam Academy of Science&Technology(code:VAST04.09/15-16)and by a Grant from the Priority Research Center Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology(2009-0093815), Republic of Korea.

L.D.Dat et al./Bioorg.Med.Chem.Lett.25(2015)1412–14161415

Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.360docs.net/doc/845284764.html,/10.1016/j.bmcl.2015.02. 048.

References and notes

1.Feller,I.C.;Whigham,D.F.;O’Neill,J.P.;McKee,K.L.Ecology1999,80,2193.

2.Hernes,P.J.;Benner,R.;Cowie,G.L.;Goni,M.A.;Bergamaschi,B.A.;Hedges,J.

I.Geochim.Cosmochim.Acta2001,65,3109.

3.Mainoya,J.;Mesaki,S.;Banyikwa,F.F.United Nat.Univ.Tokyo,Jpn.1986,87.

4.Bandarnayake,W.M.Wetlands Ecol.Manage.2002,10,421.

5.Wei,S.D.;Zhou,H.C.;Lin,Y.M.Int.J.Mol.Sci.2010,11,4080.

6.Zhang,L.L.;Lin,Y.M.;Zhou,H.C.;Wei,S.D.;Chen,J.H.Molecules2010,15,420.

7.Nathan,C.Nature2002,420,846.

8.Efron,P.;Tsujimoto,H.;Bahjat,F.;Ungaro,R.;Debernardis,J.;Tannahill,C.;

Baker,H.;Edwards,C.;Moldawer,L.J.Endotoxin.Res.2005,11,145.

9.General procedures:Optical rotations were determined on a JASCO P-2000

polarimeter(Hachioji,Tokyo,Japan).IR spectra were obtained on a Bruker

TENSOR37FT-IR spectrometer(Bruker Optics,Ettlingen,Germany).The high-

resolution electrospray ionization mass spectra(HR-ESI-MS)were obtained

using an Agilent6530Accurate-Mass Q-TOF LC/MS system.The1H NMR

(600MHz)and13C NMR(150MHz)spectra were recorded on a Bruker AM600

FT-NMR spectrometer(Bruker,Billerica,MA,USA)and TMS was used as an

internal standard.Column chromatography CC was performed on silica gel

(Kieselgel60,70–230mesh and230–400mesh,Merck,Germany)and YMC RP-

18resins(30–50l m,Fuji Silisa Chemical Ltd.,Kasugai,Aichi,Japan).Thin layer chromatography(TLC)used pre-coated silica gel60F254(1.05554.0001,Merck,

Germany)and RP-18F254S plates(1.15685.0001,Merck,Darmstadt,Germany)

and compounds were visualized by spraying with aqueous10%H2SO4and

heating for3–5min.

10.K.candel samples were collected in July2014at Ha Long,Quang Ninh province,

Vietnam,and identi?ed by Dr.Nguyen The Cuong,Institute of Ecology and

Biological Resources,VAST,Vietnam.A voucher specimen(CB01C)was

deposited at the Herbarium of Institute of Ecology and Biological Resources,

VAST,Vietnam.Fresh leaves and stems of K.candel were dried under shinny for

dried samples.The dried of K.candel(3.0kg)were well grinded and extracted

three times with MeOH at room temperature for6h each to give a MeOH

residue(400g)after removal of the solvent under reduced pressure.This

residue was supended in water(2L)and partitioned in turn with CH2Cl2

(3?2L)and EtOAc(3?2L)to furnish corresponding extracts:CH2Cl2(B,

112g)and EtOAc(C,180g)and water layer(D,2L).The EtOAc fraction(C)was

crudely separated by silica gel CC using gradient concentration of methanol in

dichloromethane(from50%to100%).Fractions were pooled after TLC analysis

to give four fractions(C1–C4).Fraction C1(10g)was divided in to seven

subfractions(C1.1–C1.7)by silica gel CC using stepwise elution with EtOAc/

methanol(30:1–10:1,v/v)and further puri?ed by silica gel CC,YMC RP-18,and

Sephadex LH-20afford to get15(7mg),8(6mg),16(7mg)from subfraction

C1.1(1.0g).Next,fraction C2(1.2g)afforded2(6mg),3(7mg),17(9mg),and

18(8mg)after subjecting it to Sephadex LH-20and YMC RP-18CC with

methanol/water(1:1,v/v).The water layer was removed the solvent and added

to Dianion HP-20CC using concentration of methanol in water(from0%to

100%)to get?ve fractions(D1–D5).Compounds4(6mg)and5(7mg)were

obtained from fraction D1(0.5g)after subjecting it to silica gel CC with dichloromethane-methanol(4-1,v/v),followed by YMC RP-18CC with methanol/water(1-1,v/v).Next,fraction D2(2.5g)was separated by YMC RP-18CC eluting with methanol-water(2-1,v/v)and puri?ed by Sephadex LH-20CC,followed by silica gel CC eluting with dichloromethane/methanol(8-1, v/v),silica gel CC eluting with EtOAc/methanol(9-1,v/v)afforded1(5mg),6 (7mg),7(8mg),13(10mg),and14(10mg).Continued separating fraction D4

(1.8g)by YMC RP-18CC eluting with acetone/water(1-2,v/v),followed silica

gel CC with dichloromethane-methanol(4-1,v/v)and further puri?cation by Sephadex LH-20to get9(6mg),10(11mg),11(12mg),and12(9mg).

11.Physical and spectroscopic data of new compound:Kacdelside(1):a white

morphous powder;a D29à106.08(c0.08,MeOH);IR(KBr)m max3373,1637, 1073,and1036,cmà1;1H NMR(CD3OD,600MHz)and13C-NMR(CD3OD, 150MHz)are given in Table1;HR-ESI-MS m/z439.2315[M+Na]+(calcd.for C21H32O8Na,439.2410).

12.Zhao,C.;Liu,Q.;Halaweish,F.;Shao,B.;Ye,Y.;Zhao,W.J.Nat.Prod.2003,66,

1140.

13.Hydrolysis of1was ditermined as followings:compound1(1.5mg)was

dissolved in 1.0N HCl(dioxane/H2O,1:1,v/v, 1.0ml)and then heated to 80°C in a water bath for3h.The acidic solution was neutralized with Ag2CO3 and reduced solvent out under N2gas overnight.After extraction with CHCl3, the aqueous layer was concentrated to dryness by N2gas.The residue was dissolved in0.1ml of dry pyridine,and solution was added L-cysteine methyl ester hydrochloride in pyridine(0.06M,0.1mL).The reaction mixture was heated at60°C for2h,then0.1mL trimethylsilylimadazole solution was added,and heated at60°C for1.5h.The dried product was partitioned with n-hexane and H2O(0.1mL,each).The n-hexane partition was analyzed by gas chromatography(GC).The retention time of persilylated glucose was found to be14.11min,respectively,compared with the standard solution prepared by the same reaction from standard monosaccharides(retention times of perilylate D-glucose,and L-glucose were14.11min,and14.26,respectively).

14.Flamini,G.;Antognoli,E.;Morelli,L.Phytochemistry2001,57,559.

15.Nakazawa,T.;Ohsawa,K.J.Nat.Prod.1998,61,993.

16.Ting,H.;Huiliang,L.;Qiaoyan,Z.;Hanchen,Z.;Luping,https://www.360docs.net/doc/845284764.html,pd.

2006,42,567.

17.Otsuka,H.;Takeuchi,M.;Inoshiri,S.;Sato,T.;Yamasaki,K.Phytochemistry

1989,28,883.

18.Matsunami,K.;Otsuka,H.;Takeda,Y.Chem.Pharm.Bull.2010,58,438.

19.Nakanishi,T.;Iida,N.;Inatomi,Y.;Murata,H.;Inada,A.;Murata,J.;Lang,F.;

Iinuma,M.;Tanaka,T.;Samagami,Y.Chem.Pharm.Bull.2005,53,783.

20.Yajima,A.;Oono,Y.;Nakagawa,R.;Nukada,T.;Yabuta,G.Bioorg.Med.Chem.

2009,17,189.

21.Shahat,A.A.;Abdel-Azim,N.S.;Pieters,L.;Vlietinck,A.J.Fitoterapia2004,75,

771.

22.Beck,M.;H?berlein,H.Phytochemistry1999,50,329.

23.Oliveira,D.M.;Siqueira,E.P.;Nunes,Y.R.F.;Cota,B.B.Revista Brasileira Farm.

Brazilian J.Pharm.2013,23,614.

24.Kazuma,K.;Noda,N.;Suzuki,M.Phytochemistry2003,62,229.

25.Mendoza-Wilson,A.;Glossman-Mitnik,D.J.Mol.Struct.Theochem.2006,761,

97.

26.Takehiko,F.;Koichi,N.;Ikuko,K.;Yoshikuni,W.;Nobou,S.;Koichi,T.;Hidejix,

I.Chem.Pharm.Bull.1988,36,1180.

27.Huang,H.;Cheng,Z.;Shi,H.;Xin,W.;Wang,T.;Yu,L.J.Agric.Food Chem.2011,

59,4562.

28.Carmichael,J.;DeGraff,W.G.;Gazdar,A.F.;Minna,J.D.;Mitchell,J.B.Cancer

Res.1987,47,943.

1416L.D.Dat et al./Bioorg.Med.Chem.Lett.25(2015)1412–1416