張利康，陳海霞 ，2*，焦 健

1天津大學(xué)藥物科學(xué)與技術(shù)學(xué)院;2天津市現(xiàn)代藥物傳遞及功能高效化重點(diǎn)實(shí)驗(yàn)室 天津 300072;3河北醫(yī)科大學(xué) 石家莊 050000

Introduction

The fruits of Schisandra chinensis(Wuweizi in Chinese)are used as functional foods and traditional Chinese medicine(TCM)for thousands of years.The fruits have multiple functions on treating impairment of body fluids with thirst，diabetes and wasting-thirst，spermatorrhea，protracted diarrhea enuresis，spontaneous sweating，frequent urination，shortness of breath，night sweating and feeble pulse，nocturnal emission，chronic cough and dyspnea［1］.S.chinensis extract has high antioxidant activity［2］.Components of Schisandra Chinensis mainly include organic acid，polysaccharide，lignin，amino acid，triterpenoid，sesquiterpene，vitamin and volatile oil etc.［3］.In this study，organic acids were isolated from Schisandra chinensis，and their structures were determined mainly by NMR experiments.Among those，1，5-Dimethyl citrate(1)，1-Monomethyl citrate(2)were isolated from Schisandra chinensis for the first time.

Materials and Methods

General Experimental Procedures

1H NMR spectra were recordedon a Bruker AM-400 NMR(Bruker，Bremerhaven，Germany).Silica gel for column chromatography was obtained from Qingdao Haiyang Chemical Company(Qingdao，China).Sephadex LH-20 was purchased from GE Healthcare.

Plant Material

Schisandra chinensis was collected in November 2009 from natural habitat in Kuandian County(Liaoning，China)and authenticated by associated Professor Haixia Chen，School of Pharmaceutical Science and Technology，Tianjin University，Tianjin，where a voucher specimen has been deposited.

Extraction and Isolation

Schizandra Chinensis(2.3 Kg)was macerated and repeatedly extracted with H2O-EtOH(15∶85).The combined extracts were partitioned between Petroleum ether and H2O and the latter fraction was repartitioned between H2O and Ethyl acetate.

P.E.extract layer was separated by silica gel column chromatography eluted with P.E.∶EtOAc(6∶1，0∶1，v/v)to give Fourteen fractions.the seventh fraction was furtuer purified on a silica gel column eluted with P.E.:Acetone(90∶10，v/v)and P.E.∶EtOAc(6∶1，0∶1，v/v)to yield Sitosterol(39 mg).

EtOAc extract layer was separated with silica gel columnchromatographyeluted with P.E.∶EtOAc(80∶20，0∶100，v/v)and finally MeOH to give ten fractions.The third fraction was eluted with P.E.∶EtOAc(40∶60，v/v)and further with CH2Cl2∶MeOH(98∶2，96∶4，v/v)to yield 5-hydroxymethyl-2-furancarboxaldehyde(35 mg).The fifth fraction was purified onto a silica gel column chromatography eluted with CH2Cl2∶MeOH(98∶2，96∶4，94∶6，0∶1，v/v)and furtuer eluted with P.E.∶EtOAc∶Acid(2∶3∶0.5，v/v)to afford protocatechuic acid(301.7 mg).The seventh fraction was eluted with CH2Cl2∶MeOH(40∶60，v/v)and EtOAc to afford Daucosterol(20 mg).The eighth fraction was loaded onto a silica gel column chromatography eluted with CH2Cl2∶EtOAc(5∶5，7∶3，8∶2，v/v)and loaded onto Sephadex LH-20(MeOH)to yield 1-Monomethyl citrate(22.94 mg).The ninth fraction was recrystallizated and loaded onto a silica gel column chromatography(CH2Cl2∶MeOH，100∶0，96∶4 and P.E.∶EtOAc，1∶1，v/v)to yield Citric acid(21.4 mg)and 1，5-Dimethyl citrate(150 mg).

Structure Identification

1，5 -dimethyl citrate(1)

Colorless solid(MeOH).1H NMR(CD3OD，400 MHz):δ3.70(each 3H，s，1-OCH3，5-OCH3)，2.95 and 2.84(each 2H，AB system，d，2-CH2， 4-CH2).These data accounted for all the NMR resonances for 1 are consistent with literature values［4］.

1-monomethyl citrate(2)

Colorless solid(MeOH).1H NMR(CD3OD，400 MHz):δ3.80(3H，s，1-CH3)，2.77(1H，d)，2.81(1H，d)，2.90(1H，d)，2.95(1H，d).These data accounted for all the NMR resonances for 2 are consistent with literature values［5］.

Citric acid(3)

Colorless solid(MeOH).1H NMR(CD3OD，400 MHz):δ2.83(2H，d，J=16.0 Hz，H-2a，4a)，2.94(2H，d，J=16.0 Hz，H-2b，4b).These data accounted for all the NMR resonances for 3 are consistent with literature values［6］.

Protocatechuic acid(4)

Yellow needle crystalm (Methanol).1H NMR(CD3OD，400 MHz):δ7.49(1 H，d，H-2)，7.47(1 H，dd，H-6)，6.85(1 H，d，H-5).These data accounted for all the NMR resonances for 4 are consistent with literature values［7］.

5-hydroxymethyl-2-furancarboxaldehyde(5)

Pale yellow liquid.1H NMR(CDCl3，400 MHz):δ4.69(d，2H，7-H)，6.50(t，1H，4-H)，7.20(t，1H，3-H)，9.23(s，1H，6-H).These data accounted for all the NMR resonances for 5 are consistent with literature values［7］.

β-sitosterol(6)

Colorlessneedle crystal(Chloroform).1H NMR(CDCl3，400 MHz):δ 0.98(3H，s，Me-19)，0.90(3H，d，J=6.5 Hz，Me-26)，0.83(3H，d，J=7.7Hz，Me-21)，0.81(3H，overlap，Me-29)，0.79(3H，d，J=6.8 Hz，Me-27)，0.65(3H，S，Me-18)，3.47(1H，m，C3-OH)，5.32(1H，t).These data accounted for all the NMR resonances for 6 are consistent with literature values［8］.

Daucosterol(7)

White powder(MeOH).1HNMR(DMSO，400 MHz):δ3.45(m，1H，3-CH)，5.31(d，1H，6-CH)，4.20(d，1H，1'-CH)，2.89(dt，1H，2'-CH)，3.11(dt，1H，3'-CH)，3.00(dt，1H，4'-CH)，3.05(dd，1H，5'-CH)，3.64(ddd，1H，6'a-CH)，3.40(dd，1H，6'b-CH)，0.64(s，3H，18-CH3)，0.94(s，3H，19-CH3)，0.89(d，3H，21-CH3)，0.78(d，3H，26-CH3)，0.80(d，3H，27-CH3)，0.81(d，3H，29-CH3)，4.83(d，1H，2'-OH)，4.84(d，1H，3'-OH)，4.82(d，1H，4'-OH)，4.39(t，1H，6'-OH).These data accounted for all the NMR resonances for 7 are consistent with literature values［9］.

1 Lu Y，Chen DF.Analysis of Schisandra chinensis and Schisandra sphenanthera.J Chromatography A，2009，1216:1980-1990.

2 Seung Sik Lee，et al.Effects of irradiation on decolourisation and biological activity in Schizandra chinensis extracts.Food Chem，2011，125:214-220.

3 Wang ZS，et al.Comparative studies on the chemical composition and antioxidant activities of Schisandra chinensis and Schisandra sphenanthera fruits.J Med Plants Res，2011，5:1207-1216.

4 Tang TJ，Wu FE.Studies on the chemical constituents of Embelia Laeta.Nat Prod Res Dev，2004，16:129-130.

5 Richard J，et al.Warrinner.On the interactions of alkyl 2-hydroxycarboxylic acids with alkoxysilanes 2.Complexation and esterification of di-and tricarboxylic acids.Dalton Trans，2009:1460-1470.

6 Wang LW.Studies on the Chemical Constituents in the Fruit of Schisandra Chinensis.J Hangzhou Teachers College(Natural Science Edition)，2007，6:363-365.

7 Dai HF，et al.Studies on the chemical constituents of Schisandra chinensis.Nat Prod Res Dev，2000，13:24-26.

8 Guan YL，et al.Chemical Study on Schisandra chinensis.Chin Tradit Herb Drugs，2006，37:185-187.

9 Shaheen Faizi，et al.Spectral Assignments and Reference Data Magn Reson Chem，2001，39:399-405.