Chemical Constituents of Ceratocarpus arenarius L．

LIU Shan-shan，SUN Wen，YANG Hong-bing* ，SUN Wan-fuXinjiang Bingtuan Key Laboratory for Green Processing of Chemical Engineering，School of Chemistry and Chemical Engineering，Shihezi University，Shihezi 83003，China;Analysis and Research Center，Xinjiang University，Urumqi 830046，China

Introduction

Xinjiang is the largest distribution area of Chenopodiaceae plants in China due to its complex geographical environment and unique climatic conditions.Plants of Chenopodiaceae family are closely related to human life.For example，Beta vulgaris is the raw material for sugar manufacture;Chenopodium ambrosioides and Salsola collina are pharmaceutical raw materials;Anabasis aphylla is raw material for agricultural insecticide manufacture.

Ceratocarpus arenarius L.is an annual herb belonging to genus Ceratocarpus of Chenopodiaceae and is only distributed in Xinjiang in China.Current studies on C．arenarius L.mainly focused on ecology［1，2］，researches on its chemical composition had not been reported.

Experimental

General

Melting point was determined on XT4-100B microelting point apparatus and uncorrected.1H NMR(400 MHz)and13C NMR(100 MHz)spectra were recorded on a Bruker-DMX 400 spectrometer in CDCl3，DMSO-d6and CD3OD，with TMS as an internal standard and reported in ppm(δ).Column chromatograph was performed with silica gel(200-300 mesh，Zhigao Huangwu Chemical Co.，China)and Sephadex LH-20(GE healthcare Bio-science AB).TLC on silica gel GF254was detected with I2and 5%H2SO4-EtOH solution.

Plant material

C．arenarius L.sample was collected from Manasi in Xinjiang，in September 2009，and was identified by Professor Yan Ping of Shihezi Unverisity.A voucher specimen had been kept in the laboratory for future reference.All plant materials were dried at room temperature and divided into small pieces before extraction.

Extraction and isolation

The air-dried aerial parts of C．arenarius L.(10 kg)were exhaustively extracted with alcohol at room temperature.The extract was evaporated under reduced pressure.The EtOH extract(1501.01 g)was then extracted with petroleum ether，ethyl acetate，chloroform and n-butanol.

The ethyl acetate extract(300 g)was subjected to a silica gel column chromatography using different solvent systems and yielded 8 fractions.Fractions 1-3 were not further purified because of low polarity.Compound 1(8.9 mg)was obtained from fraction 5 after subjecting to column chromatography eluted with petroleum ether-acetone(1∶0;6∶1)as white powder.Fraction 6 was subjected to column chromatography using petroleum ether-acetone(1∶0;3∶1)and Sephadex LH-20 column eluting with CHCl3-MeOH(1∶1)，compound 2(35.8 mg)was obtained as white needle.Fraction 7 was subjected to silica gel column chromatography to give compound 3(30 mg).Compound 6(25 mg)was obtained from fraction 8 eluted with n-hexane-EtOAc(6∶1).Compounds 4(15 mg)and 5(20 mg)were obtained from fraction 4 after subjecting to repeated column chromatography using petroleum ether-acetone(1∶0;10∶1;5∶1).

Chloroform extract(12.06 g)was subjected to column chromatography on silica gel，six fractions were obtained by gradient elution with EtOAc-MeOH(1∶0;80∶1-20∶1).Fraction 1 was further separated by silica gel column chromatography and eluted with n-hexane-EtOAc(9∶1)to give compound 7(16 mg)as yellow powder.Similarly，Compound 8(30.1 mg)was isolated from fraction 2 eluting with CHCl3-MeOH(7∶3)as white needle.Compound 9(20 mg)was obtained from fraction 3 after subjecting to column chromatography eluted with CHCl3-MeOH(1∶0;80∶1)as colorless oil.Repeated column chromatography of fractions 4-6 eluting with CHCl3-MeOH(1∶0;15∶1;9∶1)afforded compound 10(11 mg)and compound 11(14 mg).

Structural identification

Compound 1White powder(EtOAc)，mp 120-123oC;1H NMR(400 MHz，CDC13)δ:5.69(1H，br.s，H-6)，3.68(1H，tt，J=5.2，10.8 Hz，H-3)，2.52(1H，ddd，J=2.0，5.1，12.9 Hz，H-4α)，2.40(1H，ddt，J=2.0，11.0，13.2 Hz，H-4β)，2.24(1H，t，J=12.0 Hz，H-8)，2.05(1H，dt，J=4.2，12.5 Hz，H-12β)，1.20(3H，s，H-19)，0.94(3H，d，J=7.0 Hz，H-21)，0.87(3H，d，J=6.5 Hz，H-27)，0.82(3H，t，J=7.0 Hz，H-29)，0.68(3H，s，H-18);13C NMR(100 MHz，CDC13)δ:202.4(C-7)，165.2(C-5)，126.1(C-6)，70.5(C-3)，54.7(C-17)，49.9(C-9，14)，45.8(C-24)，45.4(C-8)，43.1(C-13)，41.8(C-14)，38.7(C-12)，38.3(C-10)，36.3(C-1)，36.1(C-20)，33.9(C-22)，31.2(C-2)，29.1(C-25)，28.5(C-16)，26.3(C-15)，26.0(C-23)，23.0(C-28)，21.2(C-11)，19.8(C-27)，19.0(C-26)，18.9(C-21)，17.3(C-19)，12.0(C-18，29).The spectral data were matched with literature［3］，hence it was identified as 7-Oxo-β-sitosterol.

Compound 2White needle(EtOAc)，mp 137-139oC;TLC and IR spectrum were identical to those of authentic sample;1H NMR(400 MHz，CDCl3)δ:5.33(1H，br.d，J=5.2 Hz，H-6)，3.50(1H，m，H-3)，0.99(3H，s，H-19)，0.90(3H，d，J=6.6 Hz，H-21)，0.84(3H，t，J=7.5 Hz，H-29)，0.80(3H，d，J=6.9 Hz，H-27)，0.78(3H，d，J=6.9 Hz，H-26)，0.69(3H，s，H-18);13C NMR(100 MHz，CDCl3)δ:140.8(C-5)，121.7(C-6)，71.8(C-3)，56.8(C-14)，56.0(C-17)，50.1(C-9)，45.8(C-24)，42.3(C-13)，42.2(C-4)，39.8(C-12)，37.2(C-1)，36.5(C-10)，36.1(C-20)，33.9(C-22)，31.9(C-8)，31.9(C-7)，31.7(C-2)，29.1(C-23)，28.2(C-16)，26.1(C-25)，24.3(C-15)，23.1(C-28)，21.2(C-11)，19.8(C-27)，19.4(C-19)，19.0(C-21)，18.8(C-26)，11.9(C-18)，11.8(C-29).The data were in accordance with β-sitosterol［4，5］.

Compound 3White amorphous powder(EtOAc)，mp 264-266oC;Liebermann-Burchard reaction showed blue-green result;1H NMR(400 MHz，DMSO-d6)δ:5.33(2H，br.d，J=5.2 Hz，H-22，23)，5.16(1H，m，H-6)，4.90(1H，d，J=4.8 Hz，H-1')，4.43(1H，t，J=5.6 Hz，H-4')，0.95(3H，s，H-19)，0.90(3H，d，J=6.4 Hz，H-21)，0.83(3H，dd，J=2.8，7.2 Hz，H-29)，0.78(3H，d，J=4.0 Hz，H-27)，0.66(3H，d，J=8.0 Hz，H-26)，0.62(3H，s，H-18);13C NMR(100 MHz，DMSO-d6)δ:140.7(C-5)，138.3(C-22)，129.0(C-23)，121.4(C-6)，100.8(C-1')，77.1(C-3)，76.9(C-3')，76.7(C-5')，73.7(C-2')，70.3(C-4')，61.3(C-6')，56.4(C-14)，55.6(C-17)，49.8(C-9)，45.4(C-24)，41.9(C-13)，39.1(C-12)，38.5(C-4)，37.0(C-1)，36.4(C-10)，35.7(C-20)，31.6(C-7)，31.3(C-8)，29.4(C-2)，28.9(C-25)，27.7(C-16)，24.0(C-15)，22.8(C-28)，20.8(C-11)，19.9(C-26)，19.3(C-19)，19.1(C-27)，18.8(C-21)，12.0(C-29)，11.9(C-18).Compound 3 was identified as stigmasteryl-3-O-β-D-glucopyranoside by a comparison of its spectral data and physical properties with those reported［6，7］.

Compound 4Colorless solid(EtOAc);1H NMR(400 MHz，CDCl3)δ:3.64(2H，t，J=13.2 Hz，H-1)，1.57(2H，t，J=6.4 Hz，H-2)，1.38-1.19(26H，br.s，H-3 ～ H-14)，0.89(3H，t，J=8.4 Hz，H-16);13C NMR(100 MHz，CDCl3)δ:63.1(C-1)，32.8(C-2)，31.9(C-14)，29.7(C-5，13)，29.7(C-6 ～C-12)，29.4(C-4)，25.8(C-3)，22.7(C-15)，14.1(C-16).The spectral data were matched with the reported［8］，hence it was identified as n-hexadecanol.

Compound 5White needle crystal(EtOAc);1H NMR(400 MHz，CDCl3)δ:5.72(1H，br.s，H-4)，1.18(3H，s，H-19)，0.91(3H，d，J=6.4 Hz，H-21)，0.84(3H，t，J=7.2 Hz，H-29)，0.81(3H，d，J=7.2 Hz，H-26)，0.73(3H，d，J=6.6 Hz，H-27)，0.71(3H，s，H-18);13C NMR(400 MHz，CDC13)δ:199.7(C-3)，171.8(C-5)，123.8(C-4)，56.0(C-17)，55.9(C-14)，53.8(C-9)，45.8(C-24)，42.4(C-13)，39.6(C-12)，38.6(C-10)，36.1(C-20)，35.7(C-8)，35.6(C-1)，34.0(C-22)，33.9(C-2)，33.0(C-6)，32.1(C-7)，29.7(C-25)，29.1(C-16)，28.2(C-23)，26.1(C-15)，24.2(C-28)，23.1(C-11)，21.0(C-26)，19.8(C-27)，19.0(C-19)，18.7(C-21)，17.4(C-29)，11.9(C-18).The spectral data were matched with literature［9］，hence it was identified as［24S］stigmast-4-en-3-one.

Compound 6Black-green solid(EtOAc);1H NMR(400 MHz，CDCl3)δ:9.50(1H，s，H-10)，9.35(1H，s，H-5)，8.56(1H，s，H-20)，7.97(1H，dd，J=11.6，18.0 Hz，H-31)，6.30(1H，d，J=1.6 Hz，H-32(E))，6.27(1H，s，H-132)，6.19(1H，d，J=1.2 Hz，H-32(Z))，4.40(1H，m，H-18)，4.23(1H，m，H-17)，4.05(2H，m，H-174)，3.89(3H，s，H-134)，3.69(3H，s，H-121)，3.66(2H，q，J=5.6 Hz，H-81)，3.40(3H，s，H-21)，3.21(3H，s，H-71)，1.82(3H，d，J=7.2 Hz，H-181)，1.69(3H，t，J=7.6 Hz，H-82)，1.11(3H，t，J=7.2 Hz，H-175);13C NMR(100 MHz，CDCl3)δ:189.6(C-131)，172.9(C-133)，172.1(C-173)，169.6(C-19)，161.2(C-16)，155.6(C-6)，151.0(C-9)，149.6(C-14)，145.2(C-8)，142.1(C-1)，137.9(C-11)，136.5(C-3)，136.3(C-4)，136.1(C-7)，131.9(C-2)，129.0(C-13)，129.0(C-12)，129.0(C-31)，122.8(C-32)，105.1(C-15)，104.4(C-10)，97.5(C-5)，93.1(C-20)，64.7(C-132)，60.5(C-174)，52.9(C-134)，51.1(C-17)，50.1(C-18)，31.1(C-172)，29.8(C-171)，23(C-181)，19.4(C-81)，17.4(C-82)，14.0(C-175)，12.1(C-21)，12.1(C-121)，11.2(C-71).Compound 8 was identified as 173–ethoxyphaeophorbidea by comparison of its spectral data and physical properties with those reported［10，11］.

Compound 7Yellow powder(chloroform)，mp:291-292oC;1H NMR(400 MHz，DMSO-d6)δ:12.96(1H，s，H-5)，10.84(1H，s，H-7)，9.28(1H，s，H-4')，7.32(2H，s，H-2'，6')，6.96(1H，s，H-3)，6.56(1H，d，J=1.6 Hz，H-8)，6.21(1H，d，J=2.0 Hz，H-6)，3.89(6H，s，2 × OMe);13C NMR(100 MHz，DMSO-d6)δ:181.7(C-4)，164.0(C-2)，163.5(C-7)，161.3(C-5)，157.2(C-9)，148.1(C-3'，5')，139.8(C-4')，120.3(C-1')，104.3(C-2'，6')，103.6(C-10)，103.5(C-3)，98.7(C-6)，94.1(C-8)，56.3(C-OMe).The data were in accordance with tricin［12，13］.

Compound 8White needle(chloroform)，mp:87-89oC;IR(KBr)cm-1:3340，3230，3020，1650，1590，1550，1510，1460，1380，1360，1280，1160，1120，1030，980，805;1H NMR(400 MHz，CD3OD)δ:7.43(1H，d，J=15.6 Hz，H-7')，7.06(1H，br.s，-NH-)，7.05(1H，d，J=2.0 Hz，H-2')，7.00(2H，d，J=8.0 Hz，H-2，6)，6.76(1H，dd，J=2.0，8.1 Hz，H-6')，6.69(1H，d，J=8.2 Hz，H-5')，6.43(2H，d，J=8.6 Hz，H-3，5)，6.40(1H，d，J=15.6 Hz，H-8')，3.81(3H，s，3'-OMe)，3.45(2H，t，J=7.2 Hz，H-8)，2.72(2H，t，J=7.5 Hz，H-7).The data were in accordance with those of reported［14，15］，hence compound 8 was identified as moupinamide.

Compound 9Colorless oil(chloroform);GC-MS(70 eV)m/z:41，57，83，113，131，149，167，279，390;1H NMR(400 MHz，CDCl3)δ:7.69(2H，dd，J=3.2，5.6 Hz，H-3，6)，7.50(2H，dd，J=3.2，5.6 Hz，H-4，5)，4.21(4H，m，H-2'，2'')，1.67(2H，m，H-3'，3'')，1.35(16H，m，H-4'，5'，6'，8'，4''，5''，6''，8'')，0.89(12H，m，H-7'，7''，9'，9'');13C NMR(100 MHz，CDCl3)δ:167.8(C-1'，1'')，132.5(C-1，2)，130.9(C-4，5)，128.8(C-3，6)，68.2(C-2'，2'')，38.8(C-3'，3'')，30.4(C-5'，5'')，28.9(C-4'，4'')，23.7(C-8'，8'')，23.0(C-6'，6'')，14.1(C-9'，9'')，10.9(C-7'，7'').Compound 9 was identified as 1，2-benzenedicarboxylic acid，bis(2-ethylhexyl)ester by comparison of its spectral data with literature［16］.

Compound 10Pale yellow oil jelly(chloroform);1H NMR(400 MHz，CDCl3)δ:5.41(5H，m，H-6，9'，10'，12'，13')，4.17(1H，m，H-3)，2.33(2H，t，J=7.5 Hz，H-11')，2.01(2H，t，J=14.0 Hz，H-2')，2.00(4H，m，H-8'，14')，1.62(16H，m，H-3'-7'，15'-17')，1.04(3H，s，H-19)，1.02(3H，s，H-21)，0.89(3H，t，J=6.3 Hz，H-18')，0.88(3H，m，H-29)，0.85(3H，s，H-26)，0.84(3H，s，H-27)，0.69(3H，s，H-18);13C NMR(100 MHz，CDCl3)δ:173.5(C-1')，146.3(C-5)，138.3(C-10')，129.3(C-12')，128.9(C-13')，125.4(C-9')，123.8(C-6)，73.4(C-3)，65.4(C-14)，56.0(C-17)，51.5(C-9)，49.4(C-24)，42.8(C-13)，41.7(C-12)，39.2(C-4)，37.5(C-1)，37.1(C-10)，37.0(C-20)，34.2(C-2')，34.0(C-22)，32.0(C-7)，31.9(C-8)，31.9(C-6')，31.6(C-16')，29.7(C-7')，29.7(C-15')，29.5(C-4')，29.5(C-5')，29.4(C-25)，28.8(C-16)，28.0(C-2)，27.7(C-14')，27.4(C-8')，26.4(C-23)，25.9(C-11')，25.5(C-3')，24.9(C-15)，23.1(C-28)，22.7(C-17')，21.2(C-11)，21.0(C-27)，19.6(C-19)，19.2(C-21)，19.0(C-26)，14.1(C-18')，12.3(C-29)，12.0(C-18).Compound 10 was identified as β-sitosteryl linoleate by comparison of its spectral data with literature［17］.

Compound 11Amorphous solid(chloroform);TLC reaction detected glucose;1H NMR(400 MHz，DMSO-d6)δ:6.66(2H，s，H-2'，6')，6.60(2H，s，H-2″，6″)，5.80(1H，dd，J=4.0，7.0 Hz，Glc-1)，4.90(2H，t，J=4.5 Hz，H-2，6)，4.24(7H，m，H-4 or H-8，Glc-2，3，4，6)，4.05(2H，m，H-8 or H-4)，3.93(1H，m，Glc-5)，3.82(6H，m，2 ×OMe)，3.79(6H，m，2 × OMe)，3.11(2H，m，H-1，5);13C NMR(100 MHz，DMSO-d6)δ:152.8(C-3'，C-5')，148.1(C-3″，C-5″)，137.3(C-1')，135.0(C-4″)，133.8(C-4')，131.5(C-1″)，104.3(C-G1)，103.8(C-2'，6')，102.8(C-2″，6″)，85.5(C-6)，85.3(C-2)，77.4(C-G5)，76.7(C-G3)，74.3(C-G2)，71.4(C-4，8)，71.3(C-G4)，61.1(C-G6)，56.6(C-1)，53.8(C-5).From the analysis of NMR spectra and by comparison with reported spectral data［18，19］，compound 11 was identified as syringaresinol mono-β-D-glucoside.

Reference

1 Gao R，Wei Y，Yan C.Amphicarpy and seed germination behavior of Ceratocarpus arenarius L..Chin J Ecol，2008，27:23-27.

2 Tian ZP，Lu JH，Yang QL，et al.The Anatomical structure of Ceratocarpes arenarius and its adaptation to three pieces of environmrnt.J Shihezi Univ，2008，26:668-671.

3 Ma XL，Lin WB，Zhang GL.Chemical constituents of Osmanthus yunnanensis．Nat Prod Res Dev，2009，21:593-599.

4 Li WH，Chang ST，Chang SC，et al.Isolation of antibacterial diterpenoids from Cryptomeria japonica bark.Nat Prod Res，2008，22:1085-1093.

5 Li C，Bu PB，Yue DK，et al.Chemical constituents from roots of Ficus hirta.China J Chin Mat Med，2006，31:131-133.

6 Lou FC，Ma QY，Du FL.Phytochemical study of Lysimachia foenumgraecum I.J China Pharm Univ，1989，20:37-39.

7 Wang JR，Peng SL，Wang MK，et al.Chemical constituents of Anemone tomentosa root.Acta Bot Sin，1999，41:107-110.

8 Xu SH，Yang K，Guo SH，et al.Studies on chemical constituents from Acropora pulchra.Nat Prod Res Dev，2003，15:109-112.

9 Cao JQ，Wang YN，Zhou YZ，et al.Isolation and identification of the chemical constituents from Blumea riparia DC．(Ⅱ).Chin J Med Chem，2008，18:449-451.

10 Wang LN，Xu BX，Lin HR，et al.Study on the chemical constituents of Geum japonicum var.chinense.Lishizhen med materia medica res，2009，20，798-799.

11 Jin PF，Deng ZW，Pei YH，et al.Two phaeophytin type analogues from marine sponge Dysidea sp．．Chin Chem Lett，2005，16，209-211.

12 Wang JY，Chen D，Liang LJ，et al.Chemical constituents from flowers of Chrysanthemum indicum.China J Chin Mat Med，2010，35:718-721.

13 Hoang TL，Do TH，Chau TAM，et al.Constituents from the stem barks of Canarium bengalense with cytoprotective activity against hydrogen peroxide-induced hepatotoxicity.Arch Pharm Res，2012，35(1):87-92.

14 Qian TX，Yang SL，Xu LZ.Study on chemical constituents of Phaeanthus saccopetaloides(I).Nat prod Res Dev，1997，9(2):32-34.

15 Li Y，Qiu C，Zhang DM，et al.Studies on chemical constituents from branch of Trema angustifolia.China J Chin Mat Med，2004，29:235-237.

16 Su K，Gong M，Zhou J，et al.Chemical constituents from Nauclea Officinalis leaves.J Shehezi Univ，2010，28:257-259.

17 Wang R，Chou GX，Zhu EY，et al.Studies on chemical constituents of Paeonia veitchii L..Chin Pharm J，2007，42:661-663.

18 Lami N，Kadota S，Kikuchi T，et al.Constituents of the roots of Boerhaavia diffua L.III.Identification of Ca2+channel antagonistic compound from the methanol extract.Chem Pharm Bull，1991，39，1551-1555.

19 Song YB，Cheng WM，Qu GX，et al.Chemical constituents of Sinomenium acutym.J Shenyang Pharm Univ，2007，24，79-81.