王春輝 ，魏攀蕾，嚴(yán)詩(shī)楷，金慧子 ，張衛(wèi)東，2

1上海交通大學(xué)藥學(xué)院，上海 200240;2 第二軍醫(yī)大學(xué)藥學(xué)院，上海 200433

Introduction

Inula wissmanniana，a suffrutescent plant belonging to the Asteraceae family，mainly distributed in the south of Yunnan province of China，growing at 1200-1650 m above sea level［1］.So far，no chemical constituents have been reported from I.wissmanniana.In this study，we isolated and identified sixteen compounds from an ethyl acetate (EtOAc)extract of the aerial parts of this plant，including luteolin (1)，3-O-methylquercetin(2)，5，6，4'-trihydroxy-3，7-dimethoxyflavone (3)，artemetin (4)，taxifolin (5)，dihydrokaempferol (6)，3，4-di-O-caffeoyl quinic acid (7)，3，5-di-O-caffeoylquinic acid (8)，C-veratroylglycol (9)，2，3-dihydroxy-1-(4-hydroxy-3，5-dimethoxyphenyl)-1-propanone (10)，caffeic acid (11)，dibutylphthalate (12)，3，4-dihydroxybenzoic acid (13)，3-hydroxy-4-methoxybenzoic acid (14)，p-hydroxybenzoic acid (15)and vanillin (16).All the compounds were isolated from this plant for the first time.

Experimental

General procedures

The normal phase silica gel (100-200，200-300 mesh，Yantai，China)，MCI gel (CHP20P 75-150 μm，Mitsubishi Chemical Co.，Japan)，and Sephadex LH-20(GE Healthcare Bio-Sciences AB，Sweden)were used for column chromatography，and precoated silica HSGF254plates were used for TLC (Yantai，China).HPLC were performed with SHIMADZU LC 2010AHT，Agilent Technologies 1200 series，semipreparative HPLC was obtained on a SHIMADZU LC-6AD series using a Zorbax-SB-C18(5 μm，9.4 × 250 nm).The ESI-MS were measured on an Agilent 1100 series mass spectrometer.1H and13C NMR spectra were measured on a Bruker DRX-400 spectrometer (400 MHz for1H NMR and 100 MHz for13C NMR).Chemical shift (δ)were given in ppm relative to TMS as internal reference and coupling constants (J)in Hz.

Plant material

The aerial parts of I.wissmanniana were collected from Pingbian county of Yunnan Province，China，in August 2010 and identified by Prof.Zhang Han-Ming，Department of Pharmacognosy，School of Pharmacy，Second Military Medical University.A voucher specimen has been deposited at School of Pharmacy (NO.201008DNYEJ)Shanghai Jiao Tong University.

Extraction and isolation

The air-dried and powdered aerial parts of I.wissmanniana (30.0 kg)were extracted with 95% EtOH for three times at room temperature，the extracts were combined and concentrated to yield a residue (631.4 g).The residue was suspended in H2O (6.0 L)and then partitioned successively with petroleum ether (12.0 L×5)，CH2Cl2(12.0 L×5)，EtOAc (12.0 L×5)and n-butanol (12.0 L×5)，giving 133.2 g，179.6 g，28.1 g and 35.2 g，respectively.The EtOAc fraction was chromatographed on a silica gel column eluting with CH2Cl2-MeOH (100 ∶1 to 0 ∶100)gradient to obtain six fractions (Fr.1-Fr.6).Six fractions were all applied to MCI gel column chromatography (MeOHH2O，9∶1).Fr.2 (2.1 g)was further subjected to a silica gel column and eluted with CH2Cl2-MeOH (100∶1 to 20 ∶1)to give compound 4 (20.1 mg).Fr.3(3.1 g)was separated by Sephadex LH-20 (MeOH)to give 4 subfractions.The subfraction 2 was submitted to preparative HPLC (RP18，210 nm，CH3CN-H2OHCOOH，17 ∶83 ∶0.1)，yielding compounds 9 (3.8 mg，tR=13.4 min)，10 (1.6 mg，tR=14.3 min)，14(50.8 mg，tR=25.4 min)and 15 (23.0 mg，tR=35.5 min).The subfraction 4 was submitted to preparative HPLC (RP18，210 nm，MeOH-H2O，80 ∶20)to obtain 12 (9.0 mg，tR=24.0 min).Fr.4 (3.7 g)was subjected to Sephadex LH-20 (MeOH)to give 5 subfractions.The subfraction 3 was separated by preparative HPLC (RP18，210 nm，CH3CN-H2O-HCOOH，17∶83∶0.1)to give compounds 3 (11.1 mg，tR=72.1 min)，11 (29.0 mg，tR=19.3 min)，13 (17.1 mg，tR=12.5 min)and 16 (21.6 mg，tR=41.8 min)，the subfraction 4 was separated by preparative HPLC(RP18，210 nm，CH3CN-H2O，20 ∶80)to yield compounds 1 (102.2 mg，tR=51.3 min)，2 (5.0 mg，tR=97.2 min)，5 (58.4 mg，tR=27.0 min)and 6(13.3 mg，tR=35.2 min).Compounds 7 (37.7 mg，tR=60.5 min)and 8 (25.8 mg，tR=78.6 min)were obtained after the purification of Fr.6 (5.2 g)by Sephadex LH-20 (MeOH) and preparative HPLC(RP18，210 nm，CH3CN-H2O-HCOOH，20∶80∶0.1).

Structure identification

Luteolin (1)C15H10O6，yellow amorphous powder，ESI-MS (positive)m/z 309 ［M+Na］+，ESI-MS(negative)m/z 285 ［M-H］-;1H NMR (400 MHz，DMSO-d6)δ:12.94 (1H，s，5-OH)，7.40 (1H，brs，H-6')，7.38 (1H，s，H-2')，6.87 (1H，d，J=8.1 Hz，H-5')，6.64 (1H，s，H-3)，6.43 (1H，d，J=1.4 Hz，H-8)，6.17 (1H，d，J=1.4 Hz，H-6);13C NMR(100 MHz，DMSO-d6)δ:181.7 (C-4)，164.1 (C-7)，163.9 (C-2)，161.5 (C-9)，157.3 (C-5)，149.7(C-4')，145.8 (C-3')，121.5 (C-1')，119.0 (C-6')，116.1 (C-5')113.4 (C-2')，103.7 (C-10)，102.9 (C-3)，98.9 (C-6)，93.9 (C-8).The NMR and MS data were in accordance with those reported in the literature［2］，and identified 1 as luteolin.

3-O-Methylquercetin (2)C16H12O7，yellow amorphous powder，ESI-MS (positive)m/z 339 ［M +Na］+，ESI-MS (negative)m/z 315［M-H］-;1H NMR(400 MHz，CD3OD)δ:7.61 (1H，s，H-2')，7.52(1H，d，J=8.5 Hz，H-6')，6.89 (1H，d，J=8.5 Hz，H-5')，6.39 (1H，s，H-8)，6.19 (1H，brs，H-6)，3.77 (3H，s，3-OCH3);13C NMR (100 MHz，CD3OD)δ:180.0 (C-4)，166.0 (C-7)，163.1 (C-5)，158.4(C-9)，158.0 (C-2)，150.0 (C-4')，146.5 (C-3')，139.5 (C-3)，122.9 (C-1')，122.3 (C-6')，116.5(C-2')，116.4 (C-5')，105.9 (C-10)，99.8 (C-6)，94.6 (C-8)，60.5 (3-OCH3).The NMR and MS data were in accordance with those reported in the literature［3］，and identified 2 as 3-O-methylquercetin.

5，6，4'-Trihydroxy-3，7-dimethoxyflavone (3)C17H14O7，yellow needle crystals，ESI-MS (positive)m/z 353［M+Na］+，ESI-MS (negative)m/z 329 ［MH］-;1H NMR (400 MHz，DMSO-d6)δ:12.38 (1H，brs，5-OH)，7.96 (2H，d，J=8.6 Hz，H-2'，6')，6.94(2H，d，J=8.6 Hz，H-3'，5')，6.85 (1H，s，H-8)，3.90 (3H，s，7-OCH3)，3.79 (3H，s，3-OCH3);13C NMR (100 MHz，DMSO-d6)δ:178.1 (C-4)，160.3(C-4')，155.7 (C-2)，154.5 (C-7)，148.9 (C-9)，145.7 (C-5)，137.5 (C-3)，130.0 (C-2'，6')，129.6(C-6)，120.7 (C-1')，115.7 (C-3'，5')，105.6 (C-10)，90.9 (C-8)，59.7 (3-OCH3)，56.3 (7-OCH3).The NMR and MS data were in accordance with those reported in the literature［4］，and identified 3 as 5，6，4'-trihydroxy-3，7-dimethoxyflavone.

Artemetin (4)C20H20O8，yellow amorphous powder，ESI-MS (positive)m/z 411 ［M +Na］+，ESI-MS(negative)m/z 387 ［M-H］-;1H NMR (400 MHz，DMSO-d6)δ:12.6 (1H，brs，5-OH)，7.74 (1H，dd，J=8.6，1.8 Hz，H-6')，7.67 (1H，d，J=1.8 Hz，H-2')，7.16 (1H，d，J=8.6 Hz，H-5')，6.94 (1H，s，H-8)，3.94，3.83，3.70 (each 3H，s，3 ×-OCH3)，3.87 (6H，s，2 ×-OCH3);13C NMR (400 MHz，DMSO-d6)δ:178.3 (C-4)，158.7 (C-7)，155.5 (C-9)，151.8 (C-2)，151.6 (C-5)，151.3 (C-4')，148.5(C-3')，138.1 (C-3)，131.6 (C-6)，122.1 (C-1')，122.0 (C-6')，111.6 (C-5')，111.3 (C-2')，105.6(C-10)，91.5 (C-8)，60.0，59.8，56.5，55.7，55.6(6 ×-OCH3).The NMR and MS data were in accordance with those reported in the literature［5］，and identified 4 as artemetin.

Taxifolin (5)C15H12O7，yellow amorphous powder，ESI-MS (positive)m/z 327 ［M+Na］+，ESI-MS(negative)m/z 303 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:6.97 (1H，s，H-2')，6.84 (1H，d，J=8.0 Hz，H-5')，6.79 (1H，d，J=8.0 Hz，H-6')，5.91(1H，s，H-8)，5.87 (1H，s，H-6)，4.89 (1H，d，J=11.4 Hz，H-2)，4.49 (1H，d，J=11.4 Hz，H-3);13C NMR (100 MHz，CD3OD)δ:198.2 (C-4)，168.6(C-7)，164.4 (C-5)，164.3 (C-9)，147.0 (C-4')，146.2 (C-3')，129.8 (C-1')，120.9 (C-6')，116.1(C-2')，115.9 (C-5')，101.8 (C-10)，97.3 (C-6)，96.3 (C-8)，84.9 (C-2)，73.5 (C-3).The NMR and MS data were in accordance with those reported in the literature［6］，and identified 5 as toxifolin.

Dihydrokaempferol (6)C15H12O6，yellow needle crystals，ESI-MS (positive)m/z 311［M+Na］+，ESIMS (negative)m/z 287 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.34 (2H，d，J=8.6 Hz，H-2'，6')，6.83 (2H，d，J=8.6 Hz，H-3'，5')，5.92 (1H，s，H-8)，5.88 (1H，s，H-6)，4.97 (1H，d，J=11.4 Hz，H-2)，4.53 (1H，d，J=11.4 Hz，H-3);13C NMR(100 MHz，CD3OD)δ:198.5 (C-4)，168.8 (C-5)，164.6 (C-9)，159.2 (C-4')，130.4 (C-2'，6')，129.3 (C-1')，116.2 (C-3'，5')，101.9 (C-10)，97.4 (C-6)，96.3 (C-8)，85.0 (C-2)，73.7 (C-3).The NMR and MS data were in accordance with those reported in the literature［7］，and identified 6 as dihydrokaempferol.

3，4-di-O-Caffeoyl quinic acid (7) C25H24O12，yellow amorphous powder，ESI-MS (negative)m/z 515［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.62 (1H，d，J=15.0 Hz，H-7')，7.53 (1H，d，J=15.0 Hz，H-7″)，7.03 (2H，s，H-2'，2″)，6.91 (2H，d，J=7.0 Hz，H-6'，6″)，6.77 (2H，d，J=8.1 Hz，H-5'，5″)，6.30 (1H，d，J=14.0 Hz，H-8″)，6.21 (1H，d，J=14.0 Hz，H-8')，5.67 (1H，m，H-3)，4.32 (1H，m，H-5)，3.73 (1H，m，H-4)，2.00?2.31 (4H，m，H-2，6);13C NMR (100 MHz，CD3OD)δ:168.5 (C-9')，168.3 (C-9″)，149.5 (C-4'，4″)，147.7 (C-7'，7″)，147.5 (C-3″)，146.6 (C-3')，127.7 (C-1″)，127.6(C-1')，123.1 (C-6'，6″)，116.4 (C-5'，5″)，115.2(C-8'，8″)，114.6 (C-2'，2″)，75.9 (C-4)，69.6 (C-3)，69.1(C-5)，39.5 (C-6)，38.3 (C-2).The NMR and MS data were in accordance with those reported in the literature［8］，and identified 7 as 3，4-di-O-caffeoyl quinic acid.

3，5-di-O-Caffeoyl quinic acid (8) C25H24O12，yellow amorphous powder，ESI-MS (negative)m/z 515［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.63 (1H，d，J=15.6 Hz，H-7″)，7.57 (1H，t，J=15.6 Hz，H-7')，7.06 (2H，brs，H-2'，2″)，6.96 (2H，d，J=8.0 Hz，H-6'，6″)，6.77 (2H，d，J=8.0 Hz，H-5'，5″)，6.35 (1H，d，J=15.8 Hz，H-8″)，6.26 (1H，d，J=15.8 Hz，H-8')，5.42 (2H，brs，H-3，5)，3.96 (1H，brs，H-4)，2.02-2.23 (4H，m，H-2，6);13C NMR(100 MHz，CD3OD)δ:168.9 (C-9″)，168.4 (C-9')，149.6 (C-4″)，149.5 (C-4')，147.9 (C-3″)，147.1(C-7″)，146.8 (C-3'，7')，127.9 (C-1″)，127.8 (C-1')，123.1 (C-6″)，123.0 (C-6')，116.5 (C-5″，8″)，115.6 (C-5')，115.3 (C-2″)，115.2 (C-2')，115.1(C-8')，74.7 (C-1)，72.5 (C-3)，72.1 (C-5)，70.6(C-4)，38.2 (C-2)，36.3 (C-6).The NMR and MS data were in accordance with those reported in the literature［9］，and identified 8 as 3，5-di-O-caffeoyl quinic acid.

C-Veratroylglycol (9)C10H12O5，brown amorphous powder，ESI-MS (positive)m/z 235［M+Na］+，ESIMS (negative)m/z 211 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.59 (1H，s，H-2)，7.57 (1H，d，J=8.5 Hz，H-6)，6.87 (1H，d，J=8.5 Hz，H-5)，5.11 (1H，t，J=4.8 Hz，H-8)，3.88 (3H，s，3-OCH3)，3.86 (1H，m，H-9α)，3.72 (1H，m，H-9β);13C NMR (100 MHz，CD3OD)δ:199.7 (C-7)，153.9 (C-4)，149.3 (C-3)，128.0 (C-1)，125.1 (C-6)，115.9 (C-5)，112.5 (C-2)，75.5 (C-8)，66.2(C-9)，56.5 (3-OCH3).The NMR and MS data were in accordance with those reported in the literature［10］，and identified 9 as C-veratroylglycol.

2，3-Dihydroxy-1-(4-hydroxy-3，5-dimethoxyphenyl)-1-propanone (10)C11H14O6，white amorphous powder，ESI-MS (positive)m/z 265［M +Na］+，ESIMS (negative)m/z 241 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.34 (2H，s，H-2'，6')，5.11 (1H，dd，J=5.0，4.0 Hz，H-2)，3.90 (6H，s，3'，5'-OCH3)，3.85 (1H，dd，J=11.6，4.0 Hz，H-3α)，3.73 (1H，dd，J=11.6，5.0 Hz，H-3β);13C NMR(100 MHz，CD3OD)δ:199.6 (C-1)，149.5 (C-3'，5')，107.9 (C-2'，6')，75.6 (C-2)，66.3 (C-3)，57.0 (3'，5'-OCH3).The NMR and MS data were in accordance with those reported in the literature［11］，and identified 10 as 2，3-dihydroxy-1-(4-hydroxy-3，5-dimethoxyphenyl)-1-propanone.

Caffeic acid (11)C9H8O4，brown amorphous powder，ESI-MS (positive)m/z 203 ［M +Na］+，ESI-MS(negative)m/z 179 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.52 (1H，d，J=15.6 Hz，H-1')，7.03(1H，s，H-2)，6.91 (1H，d，J=8.1 Hz，H-6)，6.76(1H，d，J=8.1 Hz，H-5)，6.21 (1H，d，J=15.6 Hz，H-2');13C NMR (100 MHz，CD3OD)δ:171.4(C-3')，149.4 (C-4)，147.0 (C-3)，146.7 (C-2)，127.8 (C-1)，122.8 (C-5)，116.5 (C-6)，115.7 (C-1')，115.1 (C-2').The NMR and MS data were in accordance with those reported in the literature［12］，and identified 11 as caffeic acid.

Dibutylphthalate (12)C16H22O4，pink oil，ESI-MS(positive)m/z 301 ［M +Na］+;1H NMR (CD3OD，400 MHz)δ:7.71 (2H，dd，J=5.7，3.3 Hz，H-3，6)，7.60 (2H，dd，J=5.7，3.3 Hz，H-4，5)，4.28(4H，t，J=6.6 Hz，H-8，8')，1.73 (4H，m，J=6.6 Hz，H-9，9')，1.44 (4H，m，J=7.6 Hz，H-10，10')，0.97 (6H，t，J=7.6 Hz，H-11，11');13C NMR(CD3OD，100 MHz)δ:169.3 (C-7，7')，133.6 (C-1，2)，132.3 (C-4，5)，129.9 (C-3，6)，66.7 (C-8，8')，31.7 (C-9，9')，20.2 (C-10，10')，14.0 (C-11，11').The NMR and MS data were in accordance with those reported in the literature［13］，and identified 12 as dibutylphthalate.

3，4-Dihydroxybenzoic acid (13) C7H6O4，white needle crystals，ESI-MS (positive)m/z 177 ［M +Na］+，ESI-MS (negative)m/z 153［M-H］-;1H NMR(400 MHz，CD3OD)δ:7.43 (1H，s，H-2)，7.41(1H，d，J=10.0 Hz，H-6)，6.78 (1H，d，J=10.0 Hz，H-5);13C NMR (100 MHz，CD3OD)δ:170.2 (C=O)，151.5 (C-4)，146.0 (C-3)，123.9 (C-2)，117.7 (C-6)，115.8 (C-5).The NMR and MS data were in accordance with those reported in the literature［14］，and identified 13 as 3，4-dihydroxybenzoic acid.

3-Hydroxy-4-methoxybenzoic acid (14)C8H8O4，white needle crystals，ESI-MS (positive)m/z 191［M+Na］+，ESI-MS (negative)m/z 167 ［M-H］-;1H NMR (400 MHz，CD3OD)δ:7.56 (1H，brs，H-2，)，7.55 (1H，brs，H-6)，6.83 (1H，d，J=8.3 Hz，H-5)，3.89 (3H，s，4-OCH3);13C NMR (100 MHz，CD3OD)δ:152.5 (C-4)，148.7 (C-3)，125.3 (C-6)，115.8 (C-2)，113.9 (C-5)，56.4 (4-OCH3).The NMR and MS data were in accordance with those reported in the literature［15］，and identified 14 as 3-hydroxy-4-methoxybenzoic acid.

p-Hydroxybenzoic acid (15)C7H6O3，white amorphous powder，ESI-MS (negative) m/z 137 ［MH］-;1H NMR (400 MHz，CD3OD)δ:7.86 (2H，d，J=8.0 Hz，H-2，6)，6.80 (2H，d，J=8.0 Hz，H-3，5);13C NMR (100 MHz，CD3OD)δ:170.8 (1-COOH)，163.6 (C-4)，133.3 (C-2，6)，123.5 (C-1)，116.3 (C-3，5).The NMR and MS data were in accordance with those reported in the literature［16］，and identified 15 as p-hydroxybenzoic acid.

Vanillin (16)C8H8O3，white needle crystal，ESI-MS(negative)m/z 151 ［M-H］-;1H NMR (CD3OD，400 MHz)δ:9.63 (1H，s，1-CHO)，7.38 (2H，brs，H-2，6)，6.83 (1H，d，J=8.0 Hz，H-5)，3.87 (3H，s，3-OCH3);13C NMR (CD3OD，400 MHz)δ:190.7 (1-CHO)，151.7 (C-4)，147.2 (C-3)，129.9 (C-1)，127.3 (C-6)，114.4 (C-5)，108.9 (C-2)，56.1 (3-OCH3).The NMR and MS data were in accordance with those reported in the literature［17］，and identified 16 as vanillin.

1 Flora of China Editorial Committee.Chinese Academy of Sciences.Flora of China，Beijing:Science Press，2005.75:272-273.

2 Liang YH，Ye M，Han J，et al.Lignans and flavonoids from rhizome of Drynaria fortuner.Chin Tradit Herb Drugs，2011，42:25-30.

3 Mu LH，Zhang DM.Studies on chemical constituents of Cercis Chinensis.China J Chin Mater Med，2006，31:1795-1797.

4 Liu BL，Zhang T，Zhang XQ，et al.Chemical constituents of Laggera pterodont.China J Chin Mater Med，2010，35:602-606.

5 Song WX，Ji TF，Si YK，et al.Studies on chemical constituents in herb from Artemisia rupestris.China J Chin Mater Med，2006，31:1790-1792.

6 Wu ZY，Li RT.Chemical constituents from the roots of Rhododendron spiciferum.Nat Prod Res Dev，2011，23:253-257.

7 Liang WJ，Ma QY，Jiang HZ，et al.Chemical constituents from hulls of Nephelium lappaceum.China J Chin Mater Med，2011，42:1271-1275.

8 Zhang HY，Wei XY，Huang L.Study on four phenolic acids in Kouyanqing Liquid extraction.Modern Chin Med，2012，14:20-32.

9 Li J，Yu DQ.Chemical constituents from herbs of Erigeron breviscapus.China J Chin Mater Med，2011，36:1458-1462.

10 Li LY，Seeram NP.Maple syrup phytochemicals include lignans，coumarins，a stilbene，and other previously unreported antioxidant phenolic compounds.J Agric Food Chem，2010，58:11673-11679.

11 Lee TH，Kuo YC，Wang GJ，et al.Five new phenolics from the roots of Ficus beecheyane.J Nat Prod，2002，65:1497-1500

12 Qin LH，Guo XY，F(xiàn)an M，et al.Anti-anoxic constituents from Mesona chinensis Benth.J Shenyang Pharm Univer，2006，23:633-636.

13 Qu XY，Gu QQ，Cui CB，et al.Structural identification and antitumor activity of secondary metabolites of marine-derived actinomycete.China J Mar Drugs，2004，23:1-4.

14 Qiu YK，Dou DQ，Pei YP，et al.Chemical constituents of Opuntia dillenii.J China Pharm Univer，2005，36:213-215.

15 Duan YH，Dai Y，Gao H，et al.Studies on chemical constituents of Sarcandra glabra.Chin Tradit Herb Drugs，2010，41:29-32.

16 Chen YS，Lin XY，Zhong LJ，et al.Chemical constituents of Vitex trifolia L..Nat Prod Res Dev(天然產(chǎn)物研究與開(kāi)發(fā))，2011，23:1011-1013，1048.

17 Fu CH，Zhang YM，Tang NH，et al.Chemical constituents of Keteleeria evelyniana.Nat Prod Res Dev，2008，20:257-261，277.