药学学报  2015, Vol. 50 Issue (4): 475-479   PDF    
引用本文
LIU Yan, WANG Zhi-yao, HE Wen-jun, TAN Ning-hua, YIN Zhi-qi. Chemical constituents from stems and leaves of Micromelum integerrimum[J]. Acta Pharm Sin, 2015, 50(4): 475-479.  
刘燕, 王志尧, 贺文军, 谭宁华, 殷志琦. 小芸木茎和叶中化学成分研究[J]. 药学学报, 2015, 50(4): 475-479.  
小芸木茎和叶中化学成分研究
刘燕1,2, 王志尧1,2,3, 贺文军2, 谭宁华2 , 殷志琦1     
1. 中国药科大学天然药物化学教研室, 天然药物活性组分与药效国家重点实验室, 江苏 南京 210009;
2. 中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室, 云南 昆明 650201;
3. 河南省科学院天然产物重点实验室, 河南 郑州 450002
收稿日期: 2014-10-30;修回日期: 2014-12-05.
基金项目：Project supported by the National Natural Science Foundation of China (31470428, 21202147, 81001379);National New Drug Innovation Major Project of China (2011ZX09307-002-02);Natural Science Foundation of Yunnan Province (2012GA003).
* 通讯作者：Tel/Fax: 86-25-83271447,E-mail: chyzq2005@126.com;
Tel/Fax: 86-871-65223800,E-mail: nhtan@mail.kib.ac.cn
摘要：通过硅胶、凝胶、RP-18 和HPLC 等一系列色谱方法对小芸木茎和叶中化学成分进行研究,得到8 个化合物,包括两个新化合物(1、2)和6 个已知化合物(3～8).通过一维、二维核磁共振以及高分辨质谱等波谱数据对这些化合物进行结构鉴定,其中化合物microintegerrin C(1)为一个新的苯环衍生物,化合物microintegerrinD(2)为一个新的降倍半萜,化合物3～8 为首次从该植物中分离得到.
关键词： 小芸木     芸香科     苯环衍生物     降倍半萜     microintegerrins C-D    
Chemical constituents from stems and leaves of Micromelum integerrimum
LIU Yan1,2, WANG Zhi-yao1,2,3, HE Wen-jun2, TAN Ning-hua2 , YIN Zhi-qi1     
1. Department of Natural Medicinal Chemistry and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
2. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China;
3. Key Laboratory of Natural Products, Henan Academy of Sciences, Zhengzhou 450002, China
Abstract: A new benzene derivative microintegerrin C (1) and a new norsesquiterpenoid microintegerrin D (2), along with six known compounds (3-8), were isolated and identified from stems and leaves of Micromelum integerrimum by various chromatographies such as silica gel, Sephadex LH-20, RP-18 column chromatography and HPLC. Their structures were mainly identified based on the spectral data analysis such as 1D-, 2D-NMR and HR-EI-MS. All known compounds were isolated from this plant for the first time.
Key words: Micromelum integerrimum     Rutaceae     benzene derivative     norsesquiterpenoid     microintegerrins C-D    

Micromelum integerrimum (Buch.-Ham. ex DC.) Wight & Arn. ex M. Roem. (Rutaceae) is distributed mainly in China[1]. The leaves and barks have been used for the treatment of cold and trauma,and the roots for stomach pain[1]. Previous chemical investigations on this genus afforded a number of structurally inter­esting coumarins,alkaloids and other compounds[2,3,4,5,6,7]. Although coumarins,alkaloids and phenylpropanoidshave also been isolated from M. integerrimum,some coumarins showed cytotoxicity[3,4,5,8]. As part of our continuous investigation on the chemical and biological constituents of M. integerrimum,chemical investigation on the stems and leaves of M. integerrimum leads to the isolation of two new compounds (1,2) and six known compounds (3-8).

Results and discussion

Two new compounds,a benzene derivative (1) and a norsesquiterpenoid (2),together with six known compounds (3-8)were isolated and determined as microintegerrin C (1),microintegerrin D (2),2-methoxy- 4-(2-propenyl)-phenyl-β-D-glucoside (3)[9],erigesideⅡ (4)[10],acantrifoside E (5)[11],benzyl-β-D-glucoside (6)[12],5,7-dihydroxyl-3,8,4'-trimethoxylflavone (7)[13] and kaempferol 3-O-β-D-glucoside (8)[14] (Figure 1),based on the spectral data analysis such as 1D-,2D- NMR and HR-EI-MS.

Compound 1 was isolated as yellow oil. The molecular formula of C20H26O9 was determined on the basis of its HR-EI-MS molecular ion peak (m/z 410.156 7 [M]+),in combination with an analysis of the 13C NMR spectrum (DEPT),corresponding to eight degrees of unsaturation. The IR spectrum showed absorption bands of hydroxyl (3405 cm-1) and carbonyl groups (1 680 cm-1). The 13C NMR spectrum (Table 1) displayed 20 carbon signals: two CH3 (δC 18.5,14.5),three CH2 (δC 74.6,62.8,62.5),eleven CH (δC 134.4,130.6,130.6,129.7,129.7,122.3,103.3,78.2,78.1,75.2,71.7) and four C (δC 170.0,168.1,139.6,131.6).

Table 1 1H,13C NMR data of 1 and 2 at 600 and 150 MHz,in CD3OD,respectively (J in Hz)

The 1H NMR,COSY and HMBC spectra (Table 1,Figure 2) displayed the following moiety signals: one mono-substituted benzoyl [δC 168.1,134.4,131.6,130.6,130.6,129.7,129.7; δH 8.01 (2H,dd,J = 7.8,1.2 Hz),7.60 (1H,m),7.48 (2H,t,J = 7.8 Hz)],one glucosyl [δC 103.3,78.2,78.1,75.2,71.7,62.8; δH 4.28 (1H,d,J = 7.8 Hz),3.86 (1H,dd,J = 12.0,1.8 Hz),3.66 (1H,dd,J = 12.0,5.4 Hz),3.35 (1H,t,J = 9.0 Hz),3.25 (3H,overlapped)],one acetyl [δC 170.0,18.5; δH 2.21 (3H,s)],and one [-OCH2C(CH3)=CHCH2O-] unit [δC 139.6,122.3,74.6,62.5,14.5; δH 5.83 (1H,dt,J = 7.2,1.2 Hz),4.90 (2H,d,J = 7.2 Hz),4.31 (1H,d,J = 12.6 Hz),4.11 (1H,d,J = 12.6 Hz),1.84 (3H,s)]. The β-configuration of the glucose was determined from the coupling constant (7.8 Hz) of the anomeric proton signal in the 1H NMR spectrum[15]. Further analysis of the HMBC spectrum (Figure 2) revealed the following connections: the correlation of H-6''/C-7'' indicated that the acetyl was linked to C-6'' of the β-D-glucopyranosyl unit; the correlation of H-1/C-1'' indicated that the β-D- glucopyranosyl moiety was linked to C-1; the correlation of H-4/C-7' indicated that the [-OCH2C(CH3)=CHCH2O-] unit was linked to C-7'. Thus,the structure of 1 is elucidated and named as microintegerrin C (Figure 1).

Figure 1 Structures of compounds 1-8

Compound 2 was obtained as yellow oil. The HR-EI-MS revealed an ion peak at m/z 412.207 0 [M]+,corresponding to the molecular formula C21H32O8 (Calcd. 412.209 7). Comparison of the 1D- and 2D- NMR data of 2 with those of 4-oxo-β-ionol β-D- glucopyranoside[15] suggested their structures were closely related,except for an additional acetyl group [δC 170.0,18.5; δH 2.21 (3H,s)]. In the HMBC spectrum (Figure 2),the correlation of H-6'a/C-7' indicated the acetyl was connected with C-6' of the β-D-glucopyranose. Therefore,compound 2 was elucidated and named as microintegerrin D (Figure 1).

Figure 2 Key HMBC correlations of compounds 1 and 2
Experimental General experiment procedures

Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were recorded using a Shimadzu UV-2401A spectrophometer. CD spectra were recorded on a Chirascan Circular Dichroism spectrometer. IR spectra were obtained on a Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were performed on the Bruker AV-400 (1H: 400 MHz,13C: 100 MHz),Bruker AVANCE III-500 (1H: 500 MHz,13C: 125 MHz) or AV-600 (1H: 600 MHz,13C: 150 MHz) spectrometer with TMS as the internal standard. Mass spectra were measured on a VG Auto Spec-3000 or API-Qstar-Pulsar instrument. Column chromatography was performed using silica gel (100-200 and 200-300 mesh,Qingdao marine Chemical Inc.,China),Sephadex LH-20 (Amersham Biosciences,Sweden),MCI (CHP-20P,Mitsubishi,Japan) or Lichroprep RP-18 (40-63 mm,Merck,Darmstadt,Germany). Fractions were monitored by TLC (GF254,Qingdao Marine Chemical Inc.,China). Spots were first visualized under UV light (254 and 365 nm),followed by spraying with 5% H2SO4 in EtOH and then heating. Analytical or semi-preparative HPLC was performed on Agilent 1100 with Eclipse XDB-C18 (Agilnent,9.4 mm × 250 mm). Preparative HPLC was performed on an Agilent 1100 apparatus with a diode- array detector and a Sun FireTM Pre C18 OBDTM (Waters,19 mm × 250 mm,5 μm) column.

Plant material

The stems and leaves of M. integerrimum were collected in Xishuangbanna,Yunnan Province,China,in September 2011,and authenticated by Prof. Hua Peng,Kunming Institute of Botany,Chinese Academyof Sciences,where a voucher specimen (KUN No. 0182256) was deposited.

Extraction and isolation

Air-dried stems and leaves of M. integerrimum (29.0 kg) were extracted with methanol at 70 ℃ under reflux for four times. The extract was concentrated in vacuum to give a residue (4.5 kg),which was suspended in water,and then parti­tioned successively with petroleum ether,EtOAc,and n-BuOH. The EtOAc extract (530.0 g) and n-BuOH extract (600.0 g) were subjected separately to silica gel (100-200 mesh) column,and eluted with CHCl3- MeOH gradient (1∶0,30∶1,15∶1,9∶1,8∶2,7∶3,1∶1,0∶1,v/v) to give 26 fractions (Fr. A1 to Fr. A26) and 19 fractions (Fr. B1 to Fr. B19) separately,monitored by TLC. Fr. A18 (21.0 g) was further separated to obtain 2 (20.6 mg),3 (64.8 mg) and 6 (49.4 mg) by MPLC and preparative HPLC with the eluent of MeOH/H2O and CH3CN/H2O. Fr. A24 (10.0 g) was subjected to silica gel column (200-300 mesh) and eluted with a CH3Cl/MeOH gradient to afford 6 fractions (Fr. A24-1 to Fr. A24-6). Fr. A24-3 and Fr. A24-6 were chromatographed through sephadex LH-20 eluted with MeOH and semi-preparative HPLC with the eluent of CH3CN/H2O to yield 1 (5.9 mg) and 8 (16.0 mg). Fr. B3 (30.0 g) was further purified by means of MPLC on RP-18 eluting with MeOH/H2O,followed by semi-preparative HPLC with the eluent of CH3CN/H2O to give the pure 7 (696.1 mg). Fr. B7 was further chromatographed through MCI column with the eluent of MeOH/H2O,semi-preparative and preparative HPLC with the eluent of CH3CN/H2O to yield 4 (0.6 mg) and 5 (183.6 mg).

Microintegerrin C (1)

Yellow oil; C20H26O9; [α]17.9 D-22.4 (c 0.16,MeOH); positive ESI-MS m/z : 449 [M+K]+; HR-EI-MS: m/z 410.156 7 [M]+ (Cacld. For C20H26O9: 410.157 7); IR (KBr): 3 405,2 924,1 680,1 452,1 430,1 384,1 278,1 205,1 137,1 074,1 027,839,802 and 720 cm-1; UV (MeOH) λmax (log ε): 201 (4.2),226 (4.0),and 273 (3.4) nm; CD (MeOH): 218 (Δε -0.01) nm; 1H and 13C NMR spectral data,see Table 1.

Microintegerrin D (2)

Yellow oil; C21H32O8; [α]23.3 D-30.8 (c 0.21,MeOH); EI-MS m/z: 412 [M]+; HR-EI-MS m/z 412.207 0 [M]+ (Calcd. for C21H32O8: 412.209 7); IR (KBr): 3 383,2 969,2 934,1 677,1 514,1 429,1 383,1 204,1 187,1 138,1 076,1 037,840,802 and 723 cm-1; UV (MeOH) λmax (log ε): 203 (3.9),261 (3.5) nm; CD (MeOH): 265 (Δε -0.50) nm; 1H and 13C NMR spectral data,see Table 1.

2-Methoxy-4-(2-propenyl)-phenyl-β-D-glucopyra- noside (3)

White solid; C16H22O7; positive ESI-MS m/z 349 [M+Na]+; 1H NMR (CD3OD,600 MHz) δ: 7.08 (1H,d,J = 7.8 Hz,H-6),6.83 (1H,d,J = 1.8 Hz,H-3),6.72 (1H,dd,J = 7.8,1.8 Hz,H-5),5.95 (1H,m,H-8),5.06 (1H,dd,J = 16.8,1.8 Hz,H-9b),5.03 (1H,dd,J = 9.6,1.8 Hz,H-9a),4.86 (1H,d,J = 7.8 Hz,H-1'),3.87 (1H,d,J = 10.8 Hz,H-6'a),3.84 (3H,s,2-OCH3),3.69 (1H,m,H-6'b),3.31-3.50 (6H,m,H-2'-5',7); 13C NMR (CD3OD,150 MHz) δ: 150.8 (C,C-2),146.4 (C,C-1),139.2 (CH,C-8),136.5 (C,C-4),122.2 (CH,C-5),118.2 (CH,C-6),116.0 (CH2,C-9),114.1 (CH,C-3),103.1 (CH,C-1'),78.3 (CH,C-3'),77.9 (CH,C-5'),75.0 (CH,C-2'),71.4 (CH,C-4'),62.6 (CH2,C-6'),56.8 (3-OCH3),40.9 (CH2,C-7).

ErigesideⅡ (4)

Needle (CH3CN-H2O); C17H24O8; positive ESI-MS m/z 379 [M+Na]+,735 [2M+Na]+; 1H NMR (CDCl3, 500 MHz) δ: 6.41 (2H,s,H-3,5),5.89 (1H,m,H-8),5.08 (2H,m,H-9),4.53 (1H,d,J = 7.7 Hz,H-1'),3.81 (6H,s,OCH3 × 2),3.25-3.74 (6H,m,H-2'-6'); 13C NMR (CDCl3,100 MHz) δ: 152.4 (C,C-2,6),137.5 (C,C-1),136.7 (CH,C-8),130.4 (C,C-4),116.3 (CH2,C-9),105.7 (CH,C-3,5),103.0 (CH,C-1'),76.2 (CH,C-3'),76.1 (CH,C-5'),74.1 (CH,C-2') ,69.5 (CH,C-4'),61.6 (CH2,C-6'),56.2 (CH3,OCH3×2),40.4 (CH2,C-7).

Acantrifoside E (5)

White powder; C17H24O8; positive ESI-MS m/z 379 [M+Na]+,735 [2M+Na]+; 1H NMR (DMSO-d6,500 MHz) δ: 6.66 (2H,s,H-3,5),6.31 (1H,d,J = 16.1 Hz,H-7),6.23 (1H,m,H-8),4.87 (1H,d,J = 7.1 Hz,H-1'),3.74 (6H,s,2-OCH3,6-OCH3),3.00-3.56 (6H,m,H-2'-6'),1.81 (3H,d,J = 6.2 Hz,H-9); 13C NMR (DMSO-d6, 100 MHz) δ: 152.7 (C,C-2,6),133.5 (C,C-1),133.2 (C,C-4),130.7 (CH,C-7),125.0 (CH,C-8),104.0 (CH,C-3,5),102.6 (CH,C-1'),77.2 (CH,C-3'),76.5 (CH,C-5'),74.2 (CH,C-2'),69.9 (CH,H-4'),60.9 (CH2,C-6'),56.3 (CH3,3,5-OCH3),18.2 (CH3,C-9).

Benzyl β-D-glucopyranoside (6)

White solid; C13H18O6; positive ESI-MS m/z 293 [M+Na]+,563 [2M+Na]+; 1H NMR (CD3OD,400 MHz) δ: 7.44 (2H,d,J = 7.2 Hz,H-2,6),7.35 (2H,t,J = 7.2 Hz,H-3,5),7.29 (1H,t,J = 7.2 Hz,H-4),4.95 (1H,d,J = 12.0 Hz,H-7b),4.69 (1H,d,J = 12.0 Hz,H-7a),4.38 (1H,d,J = 7.7 Hz,H-1'),3.92 (1H,d,J = 11.9 Hz,H-6'a),3.72 (1H,dd,J = 11.9,5.3 Hz,H-6'b),3.26-3.38 (4H,m,H-2'-5'); 13C NMR (CD3OD,100 MHz) δ: 139.1 (C,C-1),129.3 (CH,C-3,5),129.2 (CH,C-2,6),128.7 (CH,C-4),103.3 (CH,C-1'),78.1 (CH,C-3') ,78.0 (CH,C-5'),75.1 (CH,C-2'),71.7 (CH2,C-7),71.7 (CH,C-4'),62.8 (CH2,C-6').

5,7-Dihydroxyl-3,8,4'-trimethoxylflavone (7)

Yellow needle (CHCl3-MeOH); C18H16O7; positive ESI- MS m/z 711 [2M+Na]+; 1H NMR (CDCl3,400 MHz) δ: 12.45 (1H,s,OH),8.13 (2H,d,J = 8.8 Hz,H-2',6'),7.06 (2H,d,J = 8.8 Hz,H-3',5'),6.43 (1H,s,H-6),4.10 (3H,s,OCH3),3.92 (3H,s,OCH3),3.87 (3H,s,OCH3); 13C NMR (CDCl3,100 MHz) δ: 178.9 (C,C-4),161.7 (C,C-4'),157.3 (C,C-7),155.6 (C,C-5),155.1 (C,C-3),148.0 (C,C-9),138.7 (C,C-2),130.0 (CH,C-2',6'),126.8 (C,C-8),122.7 (C,C-1'),114.2 (CH,C-3',5'),105.5 (C,C-10),98.5 (CH,C-6),61.9 (C,3-OCH3),60.2 (C,8-OCH3),55.4 (C,4'-OCH3),

Kaempferol 3-O-β-D-glucopyranoside (8)

Yellow needle (MeOH); C21H20O11: negative ESI-MS m/z 447 [M-H]+,895[2M-H]+; 1H NMR (DMSO-d6,500 MHz) δ: 12.62 (1H,s,OH),10.97 (1H,s,OH),10.26 (1H,s,OH),8.04 (2H,d,J = 8.7 Hz,H-2',H-6'),6.88 (2H,d,J = 8.7 Hz,H-3',H-5'),6.44 (1H,s,H-8),6.21 (1H,s,H-6),5.46 (1H,d,J = 7.5 Hz,H-1''),3.08-3.57 (6H,m,H-2''-6''); 13C NMR (DMSO-d6,150 MHz) δ: 177.5 (C,C-4),164.1 (C,C-7),161.2 (C,C-5),160.0 (C,C-4'),156.4 (C,C-2),156.2 (C,C-9),133.1 (C,C-3),130.9 (CH,C-2',6'),120.9 (C,C-1'),115.1 (CH,C-3',5'),104.0 (C,C-10),100.8 (CH,C-1''),98.7 (CH,C-6),93.6 (CH,C-8),77.5 (CH,C-3''),76.4 (CH,C-5''),74.2 (CH,C-2''),69.9 (CH,C-4''),60.8 (CH2,C-6').

Acknowledgments: The authors thank the analytical group from the State Key Laboratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany,Chinese Academy of Sciences,for measuring the IR,UV,CD,NMR,[α] and mass spectra.

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