Chemical Constituents from the Leaves of Trigonostemon flavidus

TANG Gui-hua，ZHANG Yu，HE Hong-ping* ，HAO Xiao-jiang*

1State Key Laboratory of Phytochemistry and Plant Resources in West China，Kunming Institute of Botany，Chinese Academy of Sciences，Kunming 650201，Yunnan，China;2University of Chinese Academy of Sciences，Beijing 100049，China

Introduction

Trigonostemon flavidus Gagnepain，also known as Trigonostemon heterophyllus Merrill，is an evergreen shrub belonging to the Euphorbiaceae family，and is only distributed in Sanya city，Hainan Province in China［1］.Previous research on this plant have revealed the occurrence of modified daphnane diterpenoids［2］，3，4-seco-diterpenoids［2，3］，degraded diterpenoids［4］，and phenylpropanoids［5］.As our ongoing work，we have carried out a phytochemical investigation on the leaves of T．flavidus collected in Hainan Province.Herein，we report the identification of nine known compounds(Fig.1)，including two flavonoids，robustic acid(1)and1-［6-hydroxy-2-methoxy-2″，2″-dimethylpyrano-(5″，6″:3，4)］-2-(4'-methoxyphenyl)-1，2-ethanedione(2)，one triterpene，3β-ursolic acid(3)，four degraded sesquiterpenoids，(6S，7E)-6-hydroxy-4，7-megastigmadien-3，9-dione(4)，3β-hydroxy-5，6-epoxy-7-megastigmen-9-one(5)，(3R，6R，7E)-3-hydroxy-4，7-megastigmadien-9-one(6)and loliolide(7)，and two phenylpropanoids，methyl P-coumarate(8)and methyl sinapate(9).Compounds 1-7 and 9 were obtained from Trigonostemon for the first time.

Experimental section

Experimental instruments

1H and13C NMR spectra were recorded on Bruker AM-400 and Bruker DRX-500 spectrometers using TMS as an internal standard.ESI-MS analyses were carried out on an API Qstar Pulsar 1 instrument.Silica gel(80-100 and 300-400 mesh，Qingdao Makall Group Co.，Ltd.)，MCI gel CHP 20P(75-150 μm，Mitsubishi Chemical Corporation，Tokyo)，C8silica gel(20-45 μm，Fuji Silysia Chemical Ltd.)，and SephadexLH-20(GE Healthcare Bio-Xciences AB)were used for column chromatography，and silica gel GF254(Qingdao)was used for preparative TLC in the form of precoated plates.TLC spots were visualized under UV light and by dipping into 5%H2SO4in EtOH followed by heating.

Plant material

The leaves of T．flavidus were collected from Sanya city，Hainan Province，the People’s Republic of China，in December 2010.The plant was identified by one of the authors(Gui-hua Tang)，and a voucher specimen(H20101201)was deposited at State Key Laboratory of Phytochemistry and Plant Resources in West China，Kunming Institute of Botany.

Extraction and isolation

The air-dried leaves of T．flavidus(13.0 kg)were powdered and extracted with MeOH for three times(4，3，and 3 h，respectively)under reflux.After evaporating the solution under reduced pressure，the crude residue was suspended in water and then partitioned successively with petroleum ether(PE)and EtOAc to give two corresponding portions.The PE extract(340.0 g)was subjected to column chromatography(CC)over silica gel(80-100 mesh)using PE-Me2CO(100∶1→0∶1)to yield five fractions(A-E).Fr.B(36.5 g)was subjected to CC over MCI gel CHP 20P(80%-90%MeOH)and then over C8silica gel eluting with a gradient of increasing MeOH in H2O(35%-95%)to gain seven fractions(B1-B7).Subsequently，Fr.B1was chromatographed on a Sephadex LH-20 column(MeOH)and a silica gel column(300-400 mesh，PEMe2CO，10∶1)followed by a Sephadex LH-20 column(Me2CO)to obtain 4(48.4 mg).After recrystallization of 1(85.8 mg)from Fr.B2，the stock was chromatographed on a Sephadex LH-20 column(MeOH)and a silica gel column(300-400 mesh，PE-Me2CO，6∶1)to afford 3(16.5 mg).Fr.B3was subjected to CC over Sephadex LH-20 column(CHCl3-MeOH，1∶1)to gain two subfractions(B31-B32).Subfraction B31was purified by a silica gel column(300-400 mesh，PEMe2CO，12∶1)and a Sephadex LH-20column(MeOH)to get 7(8.8 mg).Subfraction B32was subjected to CC over a silica gel column(300-400 mesh，PE-Me2CO，12∶1)and a Sephadex LH-20 column(Me2CO)to obtain 5(9.7 mg).Fr.B4was chromatographed on a Sephadex LH-20 column(MeOH)and a silica gel column(PE-Me2CO，6∶1)to yield 6(10.1 mg)，purified by preparative TLC(CHCl3-Me2CO，3∶1).Fr.B5was purified by a silica gel column(300-400 mesh，PE-EtOAc，6∶1)to get 8(7.5 mg)and 9(6.0 mg).Fr.B6was subjected to CC over a Sephadex LH-20 column(MeOH)followed by a silica gel column(300-400 mesh，PE-Me2CO，10∶1)to obtain 2(5.4 mg).

Structure identification

Robustic acid(1)White crystal;C22H20O6ESI-MS(positive)m/z 403 ［M+Na］+;1H NMR(CDCl3，400 MHz)δ:7.47(2H，d，J=8.8 Hz，H-2'，6')，6.96(2H，d，J=8.8 Hz，H-3'，5')，6.64(1H，s，H-8)，6.51(1H，d，J=10.0 Hz，H-4″)，5.78(1H，d，J=10.0 Hz，H-3″)，3.98(3H，s，5-OCH3)，3.83(3H，s，4(-OCH3)，1.49(6H，s，2″-CH3× 2);13C NMR(CDCl3，100 MHz)δ:162.6(s，C-4)，160.2(s，C-2)，158.9(s，C-7)，157.1(s，C-4')，153.7(d，C-5)，152.1(s，C-9)，131.7(d，C-2'，6')，131.4(d，C-3″)，123.3(s，C-1')，115.0(d，C-4″)，113.5(d，C-3'，5')，110.7(s，C-6)，103.8(s，C-3)，101.8(s，C-10)，101.7(d，C-8)，77.6(s，C-2″)，64.4(q，5-OCH3)，55.2(q，4'-OCH3)，28.0(q，2″-CH3×2).Its NMR and MS data were identical with those reported in the literature［6，7］.

1-［6-Hydroxy-2-methoxy-2″，2″-dimethylpyrano-(5″，6″:3，4)］-2-(4'-methoxyphenyl)-1，2-ethanedione(2) Yellow oil;C21H20O6ESI-MS(positive)m/z 391 ［M+Na］+;1H NMR(CDCl3，400 MHz)δ:7.87(2H，d，J=8.7 Hz，H-2'，6')，6.97(2H，d，J=8.7 Hz，H-3'，5')，6.31(1H，d，J=10.0 Hz，H-4″)，6.24(1H，s，H-5)，5.58(1H，d，J=10.0 Hz，H-3″)，3.88(3H，s，4'-OCH3)，3.52(3H，s，2-OCH3)，1.44(6H，s，2″-CH3×2);13C NMR(CDCl3，100 MHz)δ:197.9(s，C-7)，189.4(s，C-8)，166.2(s，C-6)，164.2(s，C-4')，163.3(s，C-4)，159.2(s，C-2)，131.5(d，C-2'，6')，128.2(d，C-3″)，125.9(s，C-1')，116.1(d，C-4″)，114.2(d，C-3'，5')，107.2(s，C-1)，106.4(s，C-3)，100.9(d，C-5)，78.0(s，C-2″)，55.5(q，4(-OCH3)，63.0(q，2-OCH3)，28.1(q，2″-CH3×2).The NMR and MS data were in consistent with those reported in the literature［7］.

3β-Ursolic acid(3) White amorphous powder;C30H48O3ESI-MS(positive)m/z 479［M+Na］+;1H NMR(C5D5N，500 MHz)δ:5.49(1H，br.s，H-12)，3.45(1H，dd，J=10.0 and 6.1 Hz，H-3)，1.24(3H，s，H-23)，1.22(3H，s，H-27)，1.05(3H，s，H-26)，1.02(3H，s，H-24)，0.99(3H，d，J=6.3 Hz，H-29)，0.94(3H，d，J=6.1 Hz，H-30)，0.88(3H，s，H-25);13C NMR(C5D5N，100 MHz)δ:180.0(s，C-28)，139.3(s，C-13)，125.7(d，C-12)，78.2(d，C-3)，55.9(d，C-5)，53.6(d，C-18)，48.1(d，C-9)，48.1(s，C-17)，42.6(s，C-14)，40.0(s，C-8)，39.54(s，C-4)，39.45(d，C-19，20)，39.4(t，C-1)，37.5(s，C-10)，37.3(t，C-22)，33.6(t，C-7)，31.2(t，C-21)，28.9(q，C-23)，28.7(t，C-15)，28.2(t，C-2)，25.0(t，C-16)，24.0(q，C-27)，23.7(t，C-11)，21.5(q，C-30)，18.8(t，C-6)，17.6(q，C-29)，17.5(q，C-26)，16.6(q，C-24)，15.7(q，C-25).Its NMR and MS data were in accordance with those reported in the literature［8］.

(6S，7E)-6-Hydroxy-4，7-megastigmadien-3，9-dione(4)Colorless oil;C13H18O3ESI-MS(positive)m/z 245 ［M+Na］+;1H NMR(CDCl3，400 MHz)δ:6.83(1H，d，J=15.7 Hz，H-7)，6.45(1H，d，J=15.7 Hz，H-8)，5.94(1H，s，H-4)，2.58(1H，d，J=17.0 Hz，H-2a)，2.32(1H，d，J=17.0 Hz，H-2b)，2.29(3H，s，H-10)，1.88(3H，s，H-13)，1.09(3H，s，H-11)，1.01(3H，s，H-12);13C NMR(CDCl3，100 MHz)δ:197.7(s，C-9)，197.3(s，C-3)，160.9(s，C-5)，145.3(d，C-7)，130.3(d，C-8)，127.6(d，C-4)，79.2(s，C-6)，49.5(t，C-2)，41.5(s，C-1)，28.3(q，C-10)，24.3(q，C-12)，22.9(q，C-11)，18.7(q，C-13).Its NMR and MS data were in consistent with those reported in the literature［9］.

3(-Hydroxy-5，6-epoxy-7-megastigmen-9-one(5)Colorless oil;C13H20O3ESI-MS(positive)m/z 247［M+Na］+;1H NMR(CDCl3，400 MHz)δ:7.00(1H，d，J=15.6 Hz，H-7)，6.27(1H，d，J=15.6 Hz，H-8)，3.88(1H，m，H-3)，2.37(1H，dd，J=14.4 and 4.9 Hz，H-4a)，2.27(3H，s，H-10)，1.75(1H，dd，J=14.4 and 4.4 Hz，H-2a)，1.63(1H，dd，J=14.4 and 8.8 Hz，H-4b)，1.25(1H，dd，J=14.4 and 9.0 Hz，H-2b)，1.18(6H，s，H-12，13)，0.96(3H，s，H-11);13C NMR(CDCl3，100 MHz)δ:197.5(s，C-9)，142.5(d，C-7)，132.5(d，C-8)，69.4(s，C-6)，67.3(s，C-5)，63.9(d，C-3)，46.6(t，C-2)，40.5(d，C-4)，35.1(s，C-1)，29.3(q，C-12)，28.2(q，C-10)，24.9(q，C-11)，19.8(q，C-13).Its NMR and MS data were identical with those reported in the literature［10］.

(3R，6R，7E)-3-Hydroxy-4，7-megastigmadien-9-one(6)Colorless oil;C13H20O2ESI-MS(positive)m/z 231［M+Na］+;1H NMR(CD3OD，400 MHz)δ:6.67(1H，dd，J=15.8 and 10.3 Hz，H-7)，6.13(1H，d，J=15.8 Hz，H-8)，5.60(1H，br.s，H-4)，4.22(1H，br.s，H-3)，2.58(1H，d，J=10.3 Hz，H-6)，2.27(3H，s，H-10)，1.79(1H，dd，J=13.2 and 5.4 Hz，H-2a)，1.62(3H，s，H-13)，1.39(1H，dd，J=13.2 and 7.1 Hz，H-2b)，1.01(3H，s，H-11)，0.90(3H，s，H-12);13C NMR(CD3OD，100 MHz)δ:200.8(s，C-9)，149.8(d，C-7)，135.9(s，C-5)，134.7(d，C-8)，127.3(d，C-4)，65.9(d，C-3)，55.5(d，C-6)，45.0(t，C-2)，35.0(s，C-1)，29.8(q，C-11)，27.1(q，C-10)，24.5(q，C-12)，22.8(q，C-13).Its NMR and MS data were in accordance with those reported in the literature［11］.

Loliolide(7)Colorless oil;C11H16O3ESI-MS(positive)m/z 219 ［M+Na］+;1H NMR(CDCl3，400 MHz)δ:5.68(1H，s，H-7)，4，32(1H，br.s，H-3)，2.47(1H，dd，J=14.0 and 2.2 Hz，H-4a)，1.97(1H，dd，J=14.0 and 2.5 Hz，H-2a)，1.74(1H，dd，J=14.5 and 3.6 Hz，H-4b)，1.51(1H，dd，J=14.5 and 3.6 Hz，H-2b)，1.77(3H，s，H-11)，1.46(3H，s，H-9)，1.26(3H，s，H-10);13C NMR(CDCl3，100 MHz)δ:182.7(s，C-6)，172.9(d，C-8)，112.8(d，C-7)，86.8(s，C-5)，66.7(d，C-3)，47.2(t，C-2)，45.5(t，C-4)，35.0(s，C-1)，30.6(q，C-9)，26.9(q，C-11)，26.4(q，C-10).Its NMR and MS data were identical with those reported in the literature［12］.

Methyl P-coumarate(8) White amorphous powder;C10H10O3ESI-MS(positive)m/z 201［M +Na］+;1H NMR(CD3OD，400 MHz)δ:7.59(1H，d，J=16.0 Hz，H-7)，7.45(2H，d，J=8.6 Hz，H-2，6)，6.79(2H，d，J=8.6 Hz，H-3，5)，6.32(1H，d，J=16.0 Hz，H-8)，3.75(3H，s，9-OCH3).The NMR and MS data were in accordance with those reported in the literature［13］.

Methyl sinapate(9)White amorphous powder;C12H14O5ESI-MS(positive)m/z 261［M+Na］+;1H NMR(CD3OD，400 MHz)δ:7.59(1H，d，J=15.9 Hz，H-7)，6.89(2H，s，H-2，6)，6.38(1H，d，J=15.9 Hz，H-8)，3.87(6H，s，3，5-OCH3)，3.76(3H，s，9-OCH3);13C NMR(CD3OD，100 MHz)δ:169.6(s，C-9)，149.4(s，C-3，5)，147.0(d，C-7)，139.6(s，C-4)，126.6(s，C-1)，115.6(d，C-8)，106.8(d，C-2，6)，56.8(q，3，5-OCH3)，52.0(q，9-OCH3).The NMR and MS data were in accordance with those reported in the literature［14］.

Acknowledgment We are grateful to all members of the analytical group for recording spectra in the State Key Laboratory of Phytochemistry and Plant Resources in West China，Kunming Institute of Botany，CAS.This work was supported by the National Natural Science Foundation of China(21072199)，the Natural Science Funding of Yunnan Province(2009CD112)，and Foundation of Chinese Academy of Sciences to Dr.H.P.He，the Yong Academic and Technical Leader Raising Foundation of Yunnan Province(2010CI047).

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