Chemical constituents of Incarvillea delavayi

CHEN Hong，WANG Qi，WANG Gui-rong，F(xiàn)AN Yi-wen，SHEN Yun-heng

1Department of Pharmacognosy，Jilin Agricultural University，Changchun 130118，China;2School of Pharmacy，Second Military Medical University，Shanghai 200433，China

Introduction

Incarvillea delavayi Bur.et.Fronch，a member of the genus Incarvillea(Bignoniace)，is mainly distributed in Yunan and Sichuan provinces，China.In Chinese folk medicine，the roots of this plant are used to treat megrim，anemia and to relieve fatigue［1，2］.The presence of monoterpenoid alkaloids［3，4］，iridoid［5］and sesquiterpene［6］in this plant were previously reported.In this study，the isolation and structural identification of 12 known compounds from the ethanol extract of the whole plants of I．delavayi are presented．This is the first report of the isolation of these 12 compounds from the genus Incarvillea or I．delavayi.

Experimental

Plant materials

The whole plants of I．delavayi were collected in Eryuan county of Yunnan province，China，in September，2010.It was identified by Prof.Han Ming Zhang，the Second Military Medical University.The voucher specimen(No.201009011)of this herb was kept in the School of Pharmacy，the Second Military Medical University.

Instruments

NMR spectral data were recorded on Bruker DRX 400 and 500 MHz NMR spectrometers.Chemical shifts were recorded as δ values.The ESI-MS data were acquired on an Agilent-1100-LC/MSD-Trap-XCT mass spectrometer(Agilent，USA).

TLC and Column chromatography

TLC was done on precoated silica gel 254 plates(Huanghai，0.15-0.20 mm thick for TLC analysis，0.40-0.50 mm thick for preparative TLC).Column chromatography was performed using silica gel(200-300 mesh and 100-200 mesh)(Huiyou Silica Gel Development Co.Ltd，Yantai，P.R.China)，RP-C18(GHODS AQ 12S50，Japan)and Sephadex LH-20(40-70 μm)(Amersham Pharmacia Biotech Ab，Uppsala，Sweden).

Extraction and isolation

The dried whole plants of I．delavayi(18 kg)were extracted with 90%ethanol for three times.The extract was combined and evaporated.The concentrated residue was dissolved in 2% HCl(2 L)，and filtered through buchner filter(Jiangsu，China).The filtrate was extracted with ethyl acetate by liquid-liquid extraction to yield the ethyl acetate extract.The aqueous layer was adjusted to pH 10-11 with NH4OH，and then extracted with CHCl3to afford a total alkaloid fraction.The remained aqueous layer was adjusted to pH 7 with 2%HCl，and subsequently extracted with ethyl acetate.The obtained ethyl acetate extract was mixed with the previous ethyl acetate extract to yield a total ethyl acetate fraction.The total alkaloid fraction(80 g)was separated into 5 fractions(A-E)by a neutral alumina column chromatography eluting with a gradient of petroleum ether(PE)-acetone(100∶1-0∶1).Fraction A was further purified by column chromatography over neutral alumina，Sephadex LH-20(CHCl3∶CH3OH 1∶1)and preparative TLC，yielding compounds 1(23.6 mg)and 2(120.6 mg).Compound 6(5.5 mg)was obtained from fraction B through similar procedures.Repeated column chromatography of fraction C over Sephadex LH-20(CHCl3∶CH3OH 1∶1)led to compound 3(300.6 mg).Fraction D was subjected to neutral alumina column chromatography(PE∶acetone 7∶1)，followed by Sephadex LH-20(CHCl3∶CH3OH 1∶1)and preparative TLC(PE∶actetone 5∶1)，affording compound 4(115.0 mg)and 7(259.8 mg).Similarly，compound 5(5.6 mg)was isolated from fraction E.Ethyl acetate fraction was separated over silica gel column eluting with increasing concentrations of acetone in petroleum ether to give three fractions F-H.By repeated column chromatography over silica gel(PE∶CHCl3∶CH3OH 25∶2∶1)and Sephadex LH-20(CHCl3∶CH3OH 1∶1)，compounds 8(3.8 mg)and 11(7.2 mg)were isolated from fraction F.Fraction G was separated over silica gel column chromatography(PE∶acetone 10∶1)，and Sephadex LH-20 column chromatography(CHCl3∶CH3OH 1∶1)to yield compound 12(12.3 mg).Fraction H was subjected to medium pressure liquid chromatography (MPLC)(CH3OH∶H2O 0∶100-100∶0)，and further purified by repeated silica gel column chromatography(PE∶ethyl acetate 5∶1-3∶1)to yield compounds 9(29.8 mg)and 10(102.3 mg).

Structural identification

Compound 1C11H17NO，yellow oil;ESI-MS(positive)m/z 180.1［M+H］+，202.0［M+Na］+;1H NMR(400 MHz，CDCl3-d1)δ:3.24(1H，m，H-1a)，1.74(1H，t，J=11.4 Hz，H-1b)，3.01(1H，m，H-3a)，1.74(1H，t，J=11.4 Hz，H-3b)，2.72(1H，m，H-4)，5.83(1H，s，H-6)，1.94(1H，m，H-8)，2.58(1H，m，H-9)，1.17(3H，d，J=6.0 Hz，H-10)，1.14(3H，d，J=5.2 Hz，H-11)，2.32(3H，s，NCH3);13C NMR(100 MHz，CDCl3-d1)δ:63.3(C-1)，62.1(C-3)，35.2(C-4)，183.6(C-5)，124.4(C-6)，210.6(C-7)，45.2(C-8)，49.7(C-9)，15.1(C-10)，15.0(C-11)，45.6(C-12).The NMR data was consistent with the data reported in literature［7］.Hence，compound 1 was identified as tecomine.

Compound 2C11H19NO，yellow oil;ESI-MS(positive)m/z 182.1［M+H］+，204.0［M+Na］+;1H NMR(400 MHz，CDCl3-d1)δ:2.88(1H，m，H-1a)，2.10(1H，m，H-1b)，2.70(1H，m，H-3a)，1.57(1H，t，J=11.2 Hz，H-3b)，1.44(1H，m，H-4)，1.63(1H，m，H-5)，2.27(2H，m，H-6)，2.45(1H，m，H-8)，1.81(1H，m，H-9)，0.81(3H，d，J=6.4 Hz，H-10)，1.01(3H，d，J=6.8 Hz，H-11)，2.21(1H，s，NCH3);13C NMR(100 MHz，CDCl3-d1)δ:55.6(C-1)，62.9(C-3)，32.6(C-4)，39.2(C-5)，43.2(C-6)，221.3(C-7)，43.7(C-8)，44.8(C-9)，12.8(C-10)，17.7(C-11)，46.6(NCH3).The NMR data was consistent with the data reported in literature［8］.Hence，compound 2 was identified as epidihydrotecomanine.

Compound 3C9H16O2，yellow oil;ESI-MS(positive)m/z 179.1 ［M+Na］+，1H NMR(400 MHz，CDCl3)δ:1.81(3H，s，H-1)，2.07(3H，s，2-CH3)，5.95(1H，s，H-3)，2.49(2H，s，H-5)，1.15(6H，s，6-CH3，H-7)，4.19(1H，s，6-OH);13C NMR(100 MHz，CDCl3)δ:21.0(C-1)，157.2(C-2)，27.9(2-CH3)，124.7(C-3)，202.3(C-4)，54.2(C-5)，70.0(C-6)，29.5(2C，C-7，6-CH3).The NMR data was consistentwith the data reported in literature［9］.Hence，compound 3 was identified as 2，6-dimethyl-6-hydroxy-2-heptene-4-one.

Compound 4C11H21NO，yellow oil;ESI-MS(positive)m/z 184.2［M+H］+，206.3［M+Na］+;1H NMR(400 MHz，CDCl3-d1)δ:2.52(1H，m，H-1a)，1.59(1H，m，H-1b)，2.45(1H，m，H-3a)，1.63(1H，m，H-3b)，2.03(1H，m，H-4)，1.87(1H，m，H-5)，1.39(2H，m，H-6)，1.78(1H，m，H-7a)，1.08(1H，m，H-7b)，1.66(1H，m，H-8)，2.03(1H，m，H-9)，0.79(3H，d，J=8.0 Hz，H-10)，3.31(2H，d，J=4.0 Hz，H-11)，2.16(3H，s，NCH3);13C NMR(100 MHz，CDCl3-d1)δ:57.4(C-1)，58.0(C-3)，30.7(C-4)，41.2(C-5)，22.1(C-6)，26.0(C-7)，45.1(C-8)，40.8(C-9)，17.6(C-10)，66.3(C-11)，46.2(NCH3).The NMR data was consistent with the data reported in literature［10］.Hence，compound 4 was identified as marine B.

Compound 5C9H11NO，yellow oil;ESI-MS(positive)m/z 172.2 ［M+Na］+;1H NMR(400 MHz，C5D5N-d5)δ:7.56(1H，d，J=5.6 Hz，H-2)，7.16(1H，d，J=5.6 Hz，H-5)，8.72(1H，t，J=5.6 Hz，H-6)，3.46(1H，m，H-7)，2.43(1H，m，H-8a)，2.03(1H，m，H-8b)，5.53(1H，d，J=5.9 Hz，H-9)，1.17(3H，d，J=7.2 Hz，H-10);13C NMR(100 MHz，C5D5N-d5)δ:148.8(C-2)，144.4(C-3)，155.3(C-4)，120.5(C-5)，146.9(C-6)，36.1(C-7)，45.2(C-8)，74.2(C-9)，21.0(C-10).The NMR data was consistent with the data reported in literature［11］.Hence，compound 5 was identified as(3R，5S)-3-methyl-cyclopentyl［1，2-c］pyridine-5-ol.

Compound 6C12H16O5，yellow oil;ESI-MS(positive)m/z 263.1 ［M + Na］+，503.1 ［2M +Na］+;1H NMR(400 MHz，CDCl3-d1)δ:7.28(2H，s，H-2，H-6)，4.35(2H，q，J = 7.1Hz，-OCH2CH3)，1.38(3H，t，J=7.1 Hz，-OCH2CH3)，3.89(9H，d，J=3.5 Hz，-OCH3× 3);13C NMR(100 MHz，CDCl3-d1)δ:125.7(C-1)，106.9(C-2，C-6)，153.1(C-3，C-5)，142.3(C-4)，166.5(C-7)，61.4(-OCH2CH3)，14.6(-OCH2CH3)，56.4(3-OCH3，5-OCH3)，61.1(4-OCH3).The NMR data was consistent with the data reported in literature［12］.Hence，compound 6 was identified as Ethyl 3，4，5-trimethoxybenzoate.

Compound 7C8H14O3，yellow oil;ESI-MS(negative)m/z 157.1 ［M-H］-;1H NMR(400 MHz，CDCl3-d1)δ:2.05(2H，m，H-2a，H-6a)，1.68(2H，m，H-2b，H-6b)，2.67(2H，m，H-3a，H-5a)，2.17(2H，m，H-3b，H-5b)，1.75(2H，t，J=5.8 Hz，H-7)，3.88(2H，t，J=5.8 Hz，H-8);13C NMR(100 MHz，CDCl3-d1)δ:70.6(C-1)，36.9(C-2，C-6)，37.1(C-3，C-5)，213.3(C-4)，41.8(C-7)，59.5(C-8).The NMR data was consistent with the data reported in literature［13］.Hence，compound 7 was identified as cleroindicin B.

Compound 8C11H14O5，yellow oil;ESI-MS(positive)m/z 249.2 ［M + Na］+，475.1 ［2M +Na］+;1H NMR(500 MHz，CDCl3-d1)δ:7.28(2H，s，H-2，H-6)，3.88(3H，s，-COOCH3)，3.89(9H，s，-OCH3×3);13C NMR(125 MHz，CDCl3-d1)δ:125.4(C-1)，107.0(C-2，C-6)，153.1(C-3，C-5)，142.3(C-4)，167.0(C-7)，52.5(-COOCH3)，56.4(3-OCH3，5-OCH3)，61.1(4-OCH3).The NMR data was consistent with the data reported in literature［14］.Hence，compound 8 was identified as 3，4，5-trimethoxy-benzoic acid methyl ester.

Compound 9C10H14O2，yellow oil;ESI-MS(positive)m/z 189.2 ［M+Na］+;1H NMR(400 MHz，C5D5N-d5)δ:7.33(2H，d，J=8.6 Hz，H-2，H-6)，6.99(2H，d，J=8.6 Hz，H-3，H-5)，3.03(2H，t，J=7.0 Hz，H-7)，3.92(2H，q，J=7.0 Hz，H-8)，4.09(2H，t，J=7.0 Hz，-OCH2CH3)，1.28(3H，t，J=7.0 Hz，-OCH2CH3);13C NMR(C5D5N，100 MHz)δ:132.6(C-1)，130.8(C-2，C-6)，115.1(C-3，C-5)，158.2(C-4)，39.9(C-7)，63.8(C-8)，64.1(-OCH2CH3)，15.3(-OCH2CH3).By the above NMR data，the structure of compound 9 can beidentified as 2-(4'-ethoxy-phenyl)-ethanol，a known simple compound［15］.However，no NMR data of this compound are available in published literatures，although we searched a lot of literatures and databases.Herein，we reported the NMR data of 9 for the first time.

Compound 10C8H8O2，yellow oil;ESI-MS(negative)m/z 135.2 ［M-H］-;1H NMR(400 MHz，MeOD-d4)δ:4.44(2H，t，J=8.6，H-2)，3.10(2H，t，J=8.6 Hz，H-3)，6.51(1H，s，H-5)，6.66(1H，dd，J=7.8 Hz，H-7)，6.50(1H，d，J=7.8 Hz，H-8);13C NMR(100 MHz，CD3OD)δ:72.2(C-2)，31.2(C-3)，129.3(C-4)，114.9(C-5)，152.3(C-6)，113.2(C-7)，110.0(C-8)，154.7(C-9).The NMR data was consistent with the data reported in literature［16］.Hence，compound 10 was identified as 2，3-dihydro-benzofuran-6-ol.

Compound 11C29H48O，white needle crystal(CHCl3);EI-MS m/z 412［M］+;TLC behavior was identical to that of authentic β-sitosterone.

Compound 12C32H50O4，white needle crystal(CHCl3);ESI-MS m/z 521.6［M+Na］+;1H NMR(500 MHz，CDCl3)δ:4.47(1H，t，J=7.8 Hz，H-3)，5.24(1H，s，H-12)，0.82，0.83，0.87，0.90，0.91，1.10，1.22(7 × 3H，s，7 ×-CH3)，2.02(3H，s，-COCH3);13C NMR(125 MHz，CDCl3)δ:37.9(C-1)，28.2(C-2)，81.1(C-3)，38.2(C-4)，55.5(C-5)，18.4(C-6)，32.7(C-7)，39.5(C-8)，47.7(C-9)，37.2(C-10)，23.0(C-11)，122.7(C-12)，143.8(C-13)，41.7(C-14)，27.8(C-15)，23.6(C-16)，46.7(C-17)，41.1(C-18)，46.0(C-19)，30.7(C-20)，34.0(C-21)，32.6(C-22)，29.9(C-23)，15.3(C-24)，16.9(C-25)，17.3(C-26)，26.0(C-27)，183.1(C-28)，33.3(C-29)，23.7(C-30)，171.3(-COCH3)，21.5(-COCH3).The NMR data was consistent with the data reported in literature［17］.Hence，compound 12 was identified as 3β-acetyl-oleanolic acid.

Reference

1 Chinese herbal medicine，Volume seventh.Shanghai:Shanghai Science and Technology Press，1991，426-437.

2 Wang WC.Flora republicae popularis sinicae，Beijing Science and Technologypress，1990，44．

3 Motoyoki N，Katsmi K，Junei K，et al．Antinociceptive substances from Lncarvillea delavayi．Phytochem，2000，53:253．

4 Motoyoki N，Katsmi K，Junei K，et al．Two novel actinidinetype alkaloids from lncarvillea delavayi．Chem Pharm Bull，2000，48:1826．

5 Lu T，Zhang WD，Pei YH，et al．A new iridoid from Incarvillea delavayi．Chin Chem Lett，2007，18:1512-1515．

6 Chen YQ，Zhang WD，Kong LY，et al．Delavayol，a novel sesquiterpene from Incarvillea delavayi Bureau et Franchet．Nat Prod Res Dev，2010，24:915．

7 Costantino L，Raimondi L，Pirisino R，et al．Isolation and pharmacological activities of the Tecoma stans alkaloids．Farmaco，2003，58:781-785．

8 Cid MM，Pombo-Villar E．Enantioselective synthesis of 3-Azabicyclo［4．3．0］nonane Alkaloids．HeIv Chim Acta，1993，76:1591-1607．

9 Liu YF，Yang XW，Wu B．Chemical constituents of the flower buds of Tussilago farfara．J Chin Pharm Sci，2007，16:288-293．

10 Xing AT，Tian JM，Liu CM，et al．Three new monoterpene alkaloids and a new caffeic acid ester from Incarvillea mairei var．Multifoliolata．HeIv Chim Acta，2010，93:718-723．

11 Lopez JL，Pusset J，San Feliciano A．Plantes de nouvelle-caledonie 115．a(chǎn)lcaloides monoterpeniques de coelospermum billardieri．J Nat Prod，1988，51:829-835．

12 Yoshino T，Imori S，Togo H．Effecient esterification of carboxylic acids and phosphonic acids with trialkyl orthoacetate in ionic liquid．Tetrahedron，2006，62:1309-1307．

13 Tian J，Zhao QS，Zhang HJ．et al．New cleroindicins from Clerodendrum indicum．J Nat Prod，1997，60:766-769．

14 Castillo-Avila G，Margarita，Karlina GS，et al．Antioxidants from the leaf extract of Byrsonima bucidaefolia．Nat Prod Commun，2009，4:83-86．

15 Ramsden HE，Balint AE，Whitford WR，et al．Arylmagnesium chlorides．Preparations and characterizations．J Org Chem，1957，22:1202-1206．

16 Benbow JW，Katoch-Rouse R．A biomimetic approach to dihydrobenzofuran synthesis．J Org Chem，2001，66:4965-4972．

17 Tian MQ，Dai HF，Li XM，et al．Chemical constituents of marine medicinal mangrove plant Sonneratia caseolaris．CJOL，2009，27:288-296