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Phenylpropanoid glycosides and triterpenoid of Pedicularis kansuensis Maxim
Fitoterapia 82 (2011) 854–860
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Phenylpropanoid glycosides and triterpenoid of Pedicularis kansuensis Maxim Bei-bei Zhang a, Keli Shi a, Zhi-xin Liao a, b,⁎, Yuan Dai a, Zhi-hong Zou a a b
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China
a r t i c l e
i n f o
Article history: Received 3 March 2011 Accepted in revised form 7 April 2011 Available online 4 May 2011
Keywords: Pedicularis kansuensis Maxim Triterpenoid Phenylpropanoid glycoside Anti-tumor activity
a b s t r a c t Phytochemical investigation of the ethanol extract of Pedicularis kansuensis Maxim. led to the isolation of one new triterpenoid and two new phenylpropanoid glycosides, along with ten known compounds. Their structures were established by extensive 1D and 2D NMR, as well as other spectrum analysis. Biological evaluation of the three new compounds against Hela cell and Hep-6 cell with MIC values ranging from 9 to 20 μg/ml. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The genus Pedicularis L. (Scrophulariceae) is comprised of 500 species of plants, 340 of which are found in China [1]. As important herb medicines, nearly sixteen species of Scrophulariceae were used in Tibetan medicine system, such as Pedicularis muscicola, Pedicularis oliveriana, Pedicularis kansuensis Maxim. and Pedicularis rhinanthoides, which were described to exert relieving heat and toxic activities [2]. Especially, the aerial parts of P. kansuensis Maxim. have been used as herbal medicines for treatment of edema and boils [2]. P. kansuensis Maxim. is distributed abundantly in Gansu, Qinghai, west of Sichan province and east of Tibet Autonomous Region of China. However, there were few studies on the constituents of P. kansuensis Maxim. Previous phytochemical studies on other Pedicularis L. plants led to identification of alkaloids [3], iridoid glycosides [4] and phenylpropanoid glycosides [5,6]. Among the identified components, phenylpropanoid glycosides were proved to have significant activity [7]. In order to find biologically active components from Chinese traditional folk medicines, the ethanol extracts of P. kansuensis Maxim. was investigated. We described herein ⁎ Corresponding author. Tel.: + 86 25 52090620; fax: + 86 25 52090618. E-mail address: [email protected] (Z. Liao). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.04.005
the isolation and structural elucidation of one new triterpenoid: 1,2,3,16,19,20-hexahydroxyolean-12-en-28-oic acid (1), two new phenylpropanoid glycosides: 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)-3-(3,4-dihydroxyphenyl)-2-prope-noate]-Glucopyranoside (2) and 1-(2,3, 4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)-3-(3,4dihydroxyphenyl)-2-propenoate]-6-[(2E)-3-(3,4-dihydroxyphenyl)-2-prope-noate]-Glucopyranoside (3) (Fig. 1), as well as ten known compounds Acteoside (4), Echinacoside (5), Leucoseepyoside A (6), Pedicularioside (7), Luteolin (8), Orientin (9), Apigenin (10), Acacetin (11), 1-Hydroxy-3,5,8trimethoxy-xanthone (12) and β-sitosterol-3-O-β-D-glucoside (13). Their structures were mainly determined by application of spectroscopic methods. Compounds 1–7 showed anti-tumor activities in vitro through biological assessing. Especially, the three new compounds showed strong activity against Hela cell and Hep-6 cell.
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 spectropolarimeter. IR spectra were recorded on an Avatar
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Fig. 1. Chemical structures of Compounds 1–3.
360-ESP spectrophotometer. 1H and 13C NMR spectra were recorded on a DRX-300 and DRX-500 spectrometer. HR-ESI-MS was carried out on a Bruker APEX 7.0 TESLA FT-MS apparatus. Column chromatography (CC) was performed using silica gel (200–300 mesh). MCI GEL CHP20p (75–150 μ) and Sephdex LH-20 were used for CC. TLC was carried out on precoated silica gel plates. Spots were visualized by UV light or by spraying with H2SO4–EtOH. 2.2. Plant material The dried aerial parts of P. kansuensis Maxim. were collected in July, 2005 in the Beishan National Forest Park of Qinghai Province, China. It was identified by Prof. Shi-long
Chen of Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 05-01-02) was deposited at the laboratory of Zhixin Liao, Southeast University, Nanjing, China.
2.3. Extraction and isolation The dried and powdered plant (3.0 kg) of P. kansuensis Maxim. was percolated three times with 95% ethanol at room temperature. The filtrates were combined and evaporated to dryness in vacuum. The residue (153 g) was suspended in H2O (1.5 L), extracted with petroleum ether (3× 500 ml), ethyl acetate (3× 500 ml), and n-BuOH (3× 500 ml), successively.
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The petroleum ether extract (25.7 g) was subjected to a silica gel column eluting with petroleum ether–ethyl acetate (40:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield 1 (21 mg). The ethyl acetate fraction (39.0 g) was fractionated by column chromatography over silica gel eluting with petroleum ether followed by increasing concentration of ethyl acetate to yield 4 fractions (Fr.1–Fr.4). Fr.1 was firstly subjected to petroleum ether–ethyl acetate (30:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield 10 (16.0 mg) and 11 (15.0 mg). Fr.2 was subjected to a silica gel column eluting with petroleum ether–ethyl acetate (15:1) to yield 8 (60.0 mg), 9 (23.0 mg) and 12 (15.0 mg). Fr.3 was further purified on silica gel (200–300 mesh) with petroleum ether–ethyl acetate (10:1–1:4) to give 13 (22.0 mg), 2 (14.0 mg) and 6 (19.0 mg). Also the n-BuOH extract (46.0 g) was fractionated by column chromatography over silica gel eluting with chloroform followed by increasing concentration of methanol to yield 2 fractions (Fr.1–Fr.2). Fr.1 was firstly subjected to chloroform–methanol (40:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield Compounds 3 (12 mg), 4 (21 mg) and 5 (28 mg). Fr.2 was subjected to a silica gel column eluting with chloroform–methanol (20:1) to yield 7 (21 mg). 1,2,3,16,19,20-hexahydroxyolean-12-en-28-oic acid (1): white powder. [α]D20 = + 3.0[α]D20 = + 3.0(c = 0.01, MeOH), IR (KBr): 3434, 2935, 2867, 1642, 1464, 1380, and 1210. 1H and 13C NMR (DMSO) see (Table 1). HR-ESI-MS: 536.0602 ([M–H] −, C30H47O8−; calc. 535.3271). 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose)-4-[(2E)3-(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside (2): light viscous liquid. [α]20D = + 6.9(c = 0.01, MeOH), IR (KBr): 3365, 2939, 1696, 1630, 1446, 1372, 1159, and 1024. 1H and 13C NMR (DMSO) see (Table 2). HR-ESI-MS: 663.1666 + ([M + Na] +, C29H36Na1O16 ; calc. 663.1901). 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)3-(3,4-dihydroxyphenyl)-2-propenoate]-6-[(2E)-3-(3,4dihydroxyphenyl)-2-propenoate]-Glucopyranoside (3): light viscous liquid. [α]20D = + 9.7(c = 0.01, MeOH), IR (KBr): 3391, 2935, 1701, 1635, 1448, 1377, 1159, and 1034. 1H and 13C NMR (DMSO) see (Table 3). HR-ESI-MS: 809.2269 + ([M + Na] +, C38H42 Na1O18 ; calc. 809.2269). 2.4. Anti-tumor activity experiments Anti-tumor activities of Compounds 1–7 against Hela cell and Hep-6 cell were tested. The microbial cells were suspended in Columbia agar containing 5% sheep blood to form a final density of 5 × 10 − 5– 10 − 6 cfu/ml and incubated at 36–37 °C for 3 days, in 5% O2, 10% CO2, and 85% N2 atmosphere with the respective compounds. Compounds 1–7 were dissolved in DMSO. The blank controls of microbial culture were incubated with limited DMSO under the same conditions. DMSO was not toxic at a limited amount under the experimental conditions (Table 4). 3. Results and discussion The petroleum ether soluble fraction, ethyl acetate soluble fraction and n-BuOH soluble fraction of the EtOH–H2O (95:5) extract of the whole parts of P. kansuensis Maxim. were
Table 1 1 H, 13C NMR and HSQC spectral data of 1. Position
1
1 2 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19 20 21a 21b 22a 22b 23 24 25 26 27 28 29 30
4.70 (1H, d, 5.0 Hz) 4.26 (1H, d, 5.0 Hz) 3.72 (1H, s)
H NMR
1.34 1.54 1.25 1.51 1.30
(1H, m) (1H, d, 4.0 Hz) (1H, d, 4.0 Hz) (1H, d, 4.5 Hz) (1H, d, 4.5 Hz)
1.41 (1H, m) 1.90 (1H, m) 1.86 (1H, m) 5.18 (1H, s)
1.51 (1H, d, 4.5 Hz) 1.25 (1H, d, 4.5 Hz) 3.02 (1H, t, 4.5 Hz) 2.39 (1H, s)
1.62 (1H, d, 4.0 Hz) 1.47 (1H, d, 4.0 Hz) 1.70 (1H, d, 4.0 Hz) 1.54 (1H, d, 4.0 Hz) 0.92 (3H, s) 0.70 (3H, s) 0.87 (3H, s) 0.72 (3H, s) 0.87 (3H, s) 11.80 (1H, s) 1.09 (3H, s) 0.85 (3H, s)
13
C NMR
71.6 78.9 80.7 38.3 53.1 18.0 32.6 41.3 46.6 38.1 25.9 126.7 138.5 41.0 28.2 78.3 46.8 54.8 76.8 79.1 26.3 37.2 28.0 15.9 15.0 16.2 25.1 178.8 26.9 16.5
HSQC 4.70 (71.6) 4.26 (78.9) 3.72 (80.7) 1.34 1.54 1.25 1.51 1.30
(53.1) (18.0) (18.0) (32.6) (32.6)
1.41 (46.6) 1.90 (25.9) 1.86 (25.9) 5.18 (126.7)
1.51 (28.2) 1.25 (28.2) 3.02 (78.3) 2.39 (54.8)
1.62 1.47 1.70 1.54 0.92 0.70 0.87 0.72 0.87 11.80 1.09 0.85
(26.3) (26.3) (37.2) (37.2) (28.0) (15.9) (15.0) (16.2) (25.1) (178.8) (26.9) (16.5)
1 H, 13C NMR data and HSQC of 1 (at 500 and 125 MHz, in DMSO at 27°; δ in ppm).
submitted to multiple chromatographic steps to afford Compounds 1–13. Compound 1, was obtained as white powder, [α]20D= +3.0 (c =0.01,MeOH). The negative HR-ESI-MS of Compound 1 showed a molecular ion peak at m/z 536.0602 [M–H]−. When considered in conjunction with its 13C NMR data, it indicated a molecular formula of C30H48O8. The assignment of 1H and 13C NMR spectroscopic data of 1 was based on HSQC, HMBC and ROESY spectra. In the 1H NMR spectrum of 1 showed seven methyl signals at δ 0.70, 0.72, 0.85, 0.87 × 2, 0.92, and 1.09, an olefinic proton at δ 5.18 (s, 1H), and a carboxylic acid proton at δ 11.80 (s, 1H), together with a singlet at δ 2.39 (1H, s), which is a characteristic signal for the H-18 of an ursane type with 19-Osubstitution [8]. Furthermore, the 13C NMR spectrum of 1 showed seven methyl signals at δ 15.0, 15.9, 16.2, 16.5, 25.1, 26.9 and 28.0, also a double bond across C-12/C-13 with a pair of olefinic carbon signals at δ 126.7 and 138.5 [8]. In addition, in the 1H NMR spectrum: four oxygen bearing methine protons at δ 3.02 (d, J = 4.5 Hz, 1H), 3.72 (s, 1H), 4.26 (d, J = 5.0 Hz, 1H) and 4.70 (d, J = 5.0 Hz, 1H), corresponding to four oxygen bearing methine carbons at δ 71.6, 78.3, 78.9 and 80.7 could be observed respectively. And two oxygen bearing quartery carbons at δ 76.8 and 79.1 could also be
B. Zhang et al. / Fitoterapia 82 (2011) 854–860 Table 2 1 H, 13C NMR and HSQC spectral data of 2. Structures
Position
Phenethyl alcohol 1 groups 2 3 4 5 6 α β Caffeoyl groups 1′ 2′ 3′ 4′ 5′ 6′ α′ β′ 7′ Glc 1″ 2″ 3″ 4″ 5″ 6″a 6″b Rha 1″′ 2″′ 3″′ 4″′ 5″′ 6″′
1
H NMR
6.58 6.48 2.66 3.38
(1H, d, 7.6 Hz) (1H, d, 7.6 Hz) (2H, m) (2H, m)
7.01 (1H, s)
6.73 (1H, d, 7.6 Hz) 6.94 (1H, d, 7.6 Hz) 6.20 (1H, d, 15.6 Hz) 7.43 (1H, d, 15.6 Hz) 4.32 3.30 3.80 4.68 3.43 3.53 3.45 4.98 3.90 3.49 3.18 3.46 1.03
857
Table 3 1 H, 13C NMR and HSQC spectral data of 3. 13 C NMR
129.2 128.4 144.8 143.4 115.7 119.6 70.1 34.8 125.5 113.5 148.4 145.5 115.4 121.5 145.6 116.2 165.8 102.1 74.3 79.3 68.7 74.4 60.6
HSQC
6.58 (115.7) 6.48 (119.6) 2.66 (70.1) 3.38 (34.8) 7.01 (113.5)
6.73 (115.4) 6.94 (121.5) 6.20 (145.6) 7.43 (116.2)
(1H, d, 7.8 Hz) 4.32 (102.1) (1H, m) 3.30 (74.3) (1H, m) 3.80 (79.3) (1H, m) 4.68 (68.7) (1H, m) 3.43 (74.4) (1H, m) 3.53 (60.6) (1H, m) 3.45 (60.6) (1H, brs) 101.1 4.98 (101.1) (1H, s) 70.3 3.90 (70.3) (1H, m) 70.4 3.49 (70.4) (1H, m) 71.6 3.18 (71.6) (1H, m) 69.0 3.46 (69.0) (3H, t, 6.9 Hz) 18.2 1.03 (18.2)
1 H and 13C NMR data and HSQC of 2 (at 300 and 75 MHz, in DMSO at 27°; δ in ppm).
observed in the 13C NMR spectrum. Thus, the structure of 1 was elucidated as a polyhydroxyolean-12-ene triterpenoid derivative. In the HMBC spectrum of 1 (Fig. 2), the correlations of H-3, H-5 with C-1, of H-2 with C-4, and of H-1 with C-3, revealed the presence of three hydroxyl groups located at C-1, C-2 and C-3. Correlations of H-18 with C-12, revealed the presence of a double bond across C-12/C-13. Correlations of H-18 with C-16, C-19 and C-20, demonstrated that three hydroxyl groups were attached to C-16, C-19 and C-20. Correlations of H-18 with C-28, revealed a carbonyl group located at C-17. The relative configuration of 1 was assigned on the basis of coupling constants and a ROESY (Fig. 3) experiment. Compound 1 was determined to be 1,2,3,16,19,20hexahydroxyolean-12-en-28-oic acid. Compound 2, was obtained as light viscous liquid. [α] 20D = + 6.9(c = 0.01, MeOH). The molecular formula of compound 2 was established as C29H36O16 by HR-ESI-MS (m/z 663.1666 ([M + Na] +)). In the 1H NMR spectrum of 2, A α, β-unsaturated keto group δ 7.43 (d, J = 15.6 Hz, 1H) and δ 6.20 (d, J =15.6 Hz, 1H) were observed and hence, the double bond was concluded to be transconfiguration. There were two benzene ring signals observed in the 1H NMR spectrum. The presence of a 1,3,4-trisubstituted benzene ring was concluded from the following proton signals: 7.01 (s, 1H), 6.94 (d, J =7.6 Hz, 1H), 6.73 (d, J = 7.6 Hz, 1H). Also, the following proton signals: 6.58 (d, J =7.6 Hz, 1H), 6.48 (d, J = 7.6 Hz, 1H) showed a 1,2,3,4-tetrasubstituted benzene rings.
Structures
Position 1H NMR
Phenethyl alcohol 1 groups 2 3 4 5 6 α β Caffeoyl groups 1′ 2′ 3′ 4′ 5′ 6′ α′ β′ 7′ Caffeoyl groups 1″ 2″ 3″ 4″ 5″ 6″ α″ β″ 7″ Glc 1″′ 2″′ 3″′ 4″′ 5″′ 6″′a 6″′b Rha 1″″ 2″″ 3″″ 4″″ 5″″ 6″″
13 C NMR
131.3 115.9 146.3 145.1 6.74 (1H, d, 8.5 Hz) 116.0 6.50 (1H, d, 8.0 Hz) 119.7 2.66 (2H, m) 70.3 3.38 (2H, m) 35.1 125.8 7.06 (1H, s) 114.3 148.6 145.7 6.65 (1H, s) 115.8 6.95 (1H, d, 8.0 Hz) 121.7 6.23 (1H, d, 16.0 Hz) 145.7 7.50 (1H, d, 16.0 Hz) 114.9 165.8 129.5 7.06 (1H, s) 114.7 146.4 149.5 6.79 (1H, d, 8.0 Hz) 116.5 6.95 (1H, d, 8.0 Hz) 123.3 6.25 (1H, d, 16.0 Hz) 148.1 7.51 (1H, d, 16.0 Hz) 115.7 166.0 4.34 (1H, d, 7.0 Hz) 102.5 3.31 (1H, dd, 10.0, 8.0 Hz) 76.9 3.80 (1H, t, 10.0 Hz) 79.5 4.80 (1H, t, 9.5 Hz) 70.6 3.42 (1H, d, 9.5 Hz) 74.7 3.48 (1H, d, 15.0 Hz) 60.9 3.43 (1H, d, 15.0 Hz) 5.10 (1H, s) 101.4 3.92 (1H, s) 71.9 3.49 (1H, d, 10.0 Hz) 72.6 3.20 (1H, t, 10.0 Hz) 73.6 3.46 (1H, dd, 10.0, 7.0 Hz) 69.3 1.08 (3H, d, 7.0 Hz) 18.6 6.67 (1H, s)
HSQC
6.67 (115.9)
6.74 (116.0) 6.50 (119.7) 2.66 (70.3) 3.38 (35.1) 7.06 (114.3)
6.65 (115.8) 6.95 (121.7) 6.23 (145.7) 7.50 (114.9)
7.06 (114.7)
6.79 (116.5) 6.95 (123.3) 6.25 (148.1) 7.51 (115.7) 4.34 (102.5) 3.31 (76.9) 3.80 (79.5) 4.80 (70.6) 3.42 (74.7) 3.48 (60.9) 3.43 (60.9) 5.10 (101.4) 3.92 (71.9) 3.49 (72.6) 3.20 (73.6) 3.46 (69.3) 1.08 (18.6)
1 H, 13C NMR data and HSQC of 3 (at 500 and 125 MHz, in DMSO at 27°; δ in ppm).
The 13C NMR spectrum displayed 29 carbons. Twelve carbons corresponding to a Glucose moiety and a Rhamnose moiety were found at 102.1, 74.3, 79.3, 68.7, 74.4, 60.6 and 101.1, 70.3, 70.4, 71.6, 69.0, 18.2. When its 1H and 13C NMR data were compared with those analogous of Compound 4 [9], the difference between the two structures was a large signal change in the Phenethyl Alcohol C-2 in Compound 2, which revealed the presence of a hydroxyl located at C-2. As the impact of shielding effect of hydroxyl, C-2 was downfield from 115.9 to 128.4. On the basis of HMBC (Fig. 2), the correlations of H-2′ with C-α′, of H-α′, H-4″ with C-7′, revealed the presence of a double bond across C-α″/C-β′ and a carbonyl group located at C-7′. Similarly, correlations of H-6′ with C-4′, C-2′ and of H-5′ with C-3′, demonstrated that two hydroxyl groups were attached to C-3′ and C-4′, also a Caffeoyl group was attached to C-4″ of Glucose. While correlations of H-6 with C-2 and C-4, and of H-5 with C-3, demonstrated that three hydroxyl groups were attached to C-2, C-3 and C-4. While correlations of H-α with C-2, of H-β with C-1, and of H-1″ with C-β, demonstrated that a Phenethyl Alcohol group was attached to C-1″ of Glucose.
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Table 4 The minimal inhibitory concentration values of Compounds 1–7. Sample
Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7
Minimal inhibitory concentration (MICs) (μg/ml) Hela cell
Hep-6 cell
10 12 9 13 16 20 17
16 17 20 39 42 47 37
The minimal inhibitory concentration values (MICs) (μg/ml) of Compounds 1-7. Hela cell: clinical strain; Hep-6 cell: clinical strain.
While correlations of H-3″ with C-1″′, when coupled with information from the 1H and 13C NMR spectrum, demonstrated that the Rhamnose group was attached to C-3″ of
Glucose. This result was in accord with the ROESY correlations of 2 shown in Fig. 3. Compound 2 was determined to be 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose)-4-[(2E)-3(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside. Compound 3 was obtained as light viscous liquid, [α] 20D = + 9.7(c = 0.01, MeOH). It possessed the molecular formula C38H42O18, as derived from the HR-ESI-MS (m/z 809.2269 ([M + Na] +)). In the 1H NMR spectrum of 3, two α, β-unsaturated keto groups δ 7.49–δ 7.52 (d, J = 16.0 Hz, 2H) and δ 6.22–δ 6.25 (d, J = 16.0 Hz, 2H) were observed in Compound 3. The following proton signals: 6.67 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 7.06 (s, 2H), 6.65 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H) and 6.79 (d, J = 8.0 Hz, 1H) showed the presence of three 1,3,4-trisubstituted benzene rings. The 13C NMR spectrum displayed 38 carbons. Twelve carbons corresponding to a Glucose moiety and a Rhamnose moiety were found at 102.5, 76.9, 79.5, 70.6, 74.7, 60.9 and 101.4, 71.9, 72.6, 73.6, 69.3,
Fig. 2. Key HMBC (H → C) correlations of Compounds 1–3.
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Fig. 3. Key ROESY correlations of Compounds 1–3.
18.6. The 1H and 13C NMR data of 3 suggested the compound had the structure of phenylpropanoid glycoside. In the HMBC experiment (Fig. 2), the correlations of H-2′ with C-α′, of H-α′ and H-4″′with C-7′, of H-2″ with C-α″, of H-α″, and H-6″′a and H-6″′b with C-7″, revealed the presence of two double bonds across C-α′/C-β′, C-α″/C-β″ and two carbonyl groups located at C-7′ and C-7″. While correlations of H-6′ with C-2′ and C-4′, and of H-5′ with C-3′, demonstrated that two hydroxyl groups were attached to C-3′ and C-4′. While correlations of H-6″ with C2″ and C-4″, and of H-5″ with C-3″, demonstrated that two hydroxyl groups were attached to C-3″ and C-4″. Also two Caffeoyl groups were attached to C-4″′ and C-6″′ of Glucose.
Correlations of H-5 with C-3, and of H-6 with C-2 and C-4, demonstrated that two hydroxyl groups were attached to C-3 and C-4. Correlations of H-α with C-2, of H-β with C-1, and of H-1″′ with C-β, demonstrated that a Phenethyl Alcohol group was attached to C-1″′ of Glucose. Correlations of H-3″′ with C-7′, when coupled with information from the 1H and 13C NMR spectrum, demonstrated that the Rhamnose group was attached to C-3″′ of Glucose. This result was in accord with the ROESY correlations of 3 shown in (Fig. 3). Compound 3 was determined to be 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose − 4[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoate]-6-[(2E)-3(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside.
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The Compounds 4–13 were identified by comparison of their spectroscopic data with literature values. Cytotoxic activities of Compounds 1–7 against Hela cell and Hep-6 cell were tested. Especially, the three new compounds against Hela cell and Hep-6 cell revealed significant anti-tumor activity in vitro with MIC values ranging from 9 to 20 μg/ml.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.fitote.2011.04.005.
References Acknowledgment This work was supported by the National Natural Science Foundation of China (30770233) and the Foundation of Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research. We thank Prof. Shi-long Chen of Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material. We also thank Dr. Xue-lian Han (pharmacological research center of Southeast University, Nanjing, China) for the pharmacological research of the compounds.
[1] Editorial Committee of Chinese Flora. BeiJing: Science Press. 1980. [2] Northwest Plateau Institute of Biology. Xining: QingHai People's Press. 1991. [3] Marilyn JS, Frank RS. Phytochemistry 1990;29:1811. [4] Thomas B, Søren D, Soren RJ, Bent JN. Phytochemistry 1985;24:491. [5] Wang CZ, Jia ZJ. Phytochemistry 1997;45:159. [6] W. T. He. Development of Regeneration Systems of Four Chinese Herbs and Preliminary Determination PPG in Pedicularis Kansuensis Maxim Lanzhou University. 2006. [7] Li J, Zheng Y, Zheng RL, Liu ZM, Jia ZJ. Chin Pharm J 1995;130:131. [8] Li BZ, Wang BG, Jia ZJ. Phytochemistry 1998;49:2477. [9] Yang AM, Lu RH, Shi YP. Nat Prod Res Dev 2007;19:263.
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Phenylpropanoid glycosides and triterpenoid of Pedicularis kansuensis Maxim Bei-bei Zhang a, Keli Shi a, Zhi-xin Liao a, b,⁎, Yuan Dai a, Zhi-hong Zou a a b
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China
a r t i c l e
i n f o
Article history: Received 3 March 2011 Accepted in revised form 7 April 2011 Available online 4 May 2011
Keywords: Pedicularis kansuensis Maxim Triterpenoid Phenylpropanoid glycoside Anti-tumor activity
a b s t r a c t Phytochemical investigation of the ethanol extract of Pedicularis kansuensis Maxim. led to the isolation of one new triterpenoid and two new phenylpropanoid glycosides, along with ten known compounds. Their structures were established by extensive 1D and 2D NMR, as well as other spectrum analysis. Biological evaluation of the three new compounds against Hela cell and Hep-6 cell with MIC values ranging from 9 to 20 μg/ml. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The genus Pedicularis L. (Scrophulariceae) is comprised of 500 species of plants, 340 of which are found in China [1]. As important herb medicines, nearly sixteen species of Scrophulariceae were used in Tibetan medicine system, such as Pedicularis muscicola, Pedicularis oliveriana, Pedicularis kansuensis Maxim. and Pedicularis rhinanthoides, which were described to exert relieving heat and toxic activities [2]. Especially, the aerial parts of P. kansuensis Maxim. have been used as herbal medicines for treatment of edema and boils [2]. P. kansuensis Maxim. is distributed abundantly in Gansu, Qinghai, west of Sichan province and east of Tibet Autonomous Region of China. However, there were few studies on the constituents of P. kansuensis Maxim. Previous phytochemical studies on other Pedicularis L. plants led to identification of alkaloids [3], iridoid glycosides [4] and phenylpropanoid glycosides [5,6]. Among the identified components, phenylpropanoid glycosides were proved to have significant activity [7]. In order to find biologically active components from Chinese traditional folk medicines, the ethanol extracts of P. kansuensis Maxim. was investigated. We described herein ⁎ Corresponding author. Tel.: + 86 25 52090620; fax: + 86 25 52090618. E-mail address: [email protected] (Z. Liao). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.04.005
the isolation and structural elucidation of one new triterpenoid: 1,2,3,16,19,20-hexahydroxyolean-12-en-28-oic acid (1), two new phenylpropanoid glycosides: 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)-3-(3,4-dihydroxyphenyl)-2-prope-noate]-Glucopyranoside (2) and 1-(2,3, 4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)-3-(3,4dihydroxyphenyl)-2-propenoate]-6-[(2E)-3-(3,4-dihydroxyphenyl)-2-prope-noate]-Glucopyranoside (3) (Fig. 1), as well as ten known compounds Acteoside (4), Echinacoside (5), Leucoseepyoside A (6), Pedicularioside (7), Luteolin (8), Orientin (9), Apigenin (10), Acacetin (11), 1-Hydroxy-3,5,8trimethoxy-xanthone (12) and β-sitosterol-3-O-β-D-glucoside (13). Their structures were mainly determined by application of spectroscopic methods. Compounds 1–7 showed anti-tumor activities in vitro through biological assessing. Especially, the three new compounds showed strong activity against Hela cell and Hep-6 cell.
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 spectropolarimeter. IR spectra were recorded on an Avatar
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Fig. 1. Chemical structures of Compounds 1–3.
360-ESP spectrophotometer. 1H and 13C NMR spectra were recorded on a DRX-300 and DRX-500 spectrometer. HR-ESI-MS was carried out on a Bruker APEX 7.0 TESLA FT-MS apparatus. Column chromatography (CC) was performed using silica gel (200–300 mesh). MCI GEL CHP20p (75–150 μ) and Sephdex LH-20 were used for CC. TLC was carried out on precoated silica gel plates. Spots were visualized by UV light or by spraying with H2SO4–EtOH. 2.2. Plant material The dried aerial parts of P. kansuensis Maxim. were collected in July, 2005 in the Beishan National Forest Park of Qinghai Province, China. It was identified by Prof. Shi-long
Chen of Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 05-01-02) was deposited at the laboratory of Zhixin Liao, Southeast University, Nanjing, China.
2.3. Extraction and isolation The dried and powdered plant (3.0 kg) of P. kansuensis Maxim. was percolated three times with 95% ethanol at room temperature. The filtrates were combined and evaporated to dryness in vacuum. The residue (153 g) was suspended in H2O (1.5 L), extracted with petroleum ether (3× 500 ml), ethyl acetate (3× 500 ml), and n-BuOH (3× 500 ml), successively.
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The petroleum ether extract (25.7 g) was subjected to a silica gel column eluting with petroleum ether–ethyl acetate (40:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield 1 (21 mg). The ethyl acetate fraction (39.0 g) was fractionated by column chromatography over silica gel eluting with petroleum ether followed by increasing concentration of ethyl acetate to yield 4 fractions (Fr.1–Fr.4). Fr.1 was firstly subjected to petroleum ether–ethyl acetate (30:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield 10 (16.0 mg) and 11 (15.0 mg). Fr.2 was subjected to a silica gel column eluting with petroleum ether–ethyl acetate (15:1) to yield 8 (60.0 mg), 9 (23.0 mg) and 12 (15.0 mg). Fr.3 was further purified on silica gel (200–300 mesh) with petroleum ether–ethyl acetate (10:1–1:4) to give 13 (22.0 mg), 2 (14.0 mg) and 6 (19.0 mg). Also the n-BuOH extract (46.0 g) was fractionated by column chromatography over silica gel eluting with chloroform followed by increasing concentration of methanol to yield 2 fractions (Fr.1–Fr.2). Fr.1 was firstly subjected to chloroform–methanol (40:1 to 0:100) containing Sephadex LH-20 eluting with ethanol–water (7:3 to 9:1) to yield Compounds 3 (12 mg), 4 (21 mg) and 5 (28 mg). Fr.2 was subjected to a silica gel column eluting with chloroform–methanol (20:1) to yield 7 (21 mg). 1,2,3,16,19,20-hexahydroxyolean-12-en-28-oic acid (1): white powder. [α]D20 = + 3.0[α]D20 = + 3.0(c = 0.01, MeOH), IR (KBr): 3434, 2935, 2867, 1642, 1464, 1380, and 1210. 1H and 13C NMR (DMSO) see (Table 1). HR-ESI-MS: 536.0602 ([M–H] −, C30H47O8−; calc. 535.3271). 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose)-4-[(2E)3-(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside (2): light viscous liquid. [α]20D = + 6.9(c = 0.01, MeOH), IR (KBr): 3365, 2939, 1696, 1630, 1446, 1372, 1159, and 1024. 1H and 13C NMR (DMSO) see (Table 2). HR-ESI-MS: 663.1666 + ([M + Na] +, C29H36Na1O16 ; calc. 663.1901). 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose-4-[(2E)3-(3,4-dihydroxyphenyl)-2-propenoate]-6-[(2E)-3-(3,4dihydroxyphenyl)-2-propenoate]-Glucopyranoside (3): light viscous liquid. [α]20D = + 9.7(c = 0.01, MeOH), IR (KBr): 3391, 2935, 1701, 1635, 1448, 1377, 1159, and 1034. 1H and 13C NMR (DMSO) see (Table 3). HR-ESI-MS: 809.2269 + ([M + Na] +, C38H42 Na1O18 ; calc. 809.2269). 2.4. Anti-tumor activity experiments Anti-tumor activities of Compounds 1–7 against Hela cell and Hep-6 cell were tested. The microbial cells were suspended in Columbia agar containing 5% sheep blood to form a final density of 5 × 10 − 5– 10 − 6 cfu/ml and incubated at 36–37 °C for 3 days, in 5% O2, 10% CO2, and 85% N2 atmosphere with the respective compounds. Compounds 1–7 were dissolved in DMSO. The blank controls of microbial culture were incubated with limited DMSO under the same conditions. DMSO was not toxic at a limited amount under the experimental conditions (Table 4). 3. Results and discussion The petroleum ether soluble fraction, ethyl acetate soluble fraction and n-BuOH soluble fraction of the EtOH–H2O (95:5) extract of the whole parts of P. kansuensis Maxim. were
Table 1 1 H, 13C NMR and HSQC spectral data of 1. Position
1
1 2 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19 20 21a 21b 22a 22b 23 24 25 26 27 28 29 30
4.70 (1H, d, 5.0 Hz) 4.26 (1H, d, 5.0 Hz) 3.72 (1H, s)
H NMR
1.34 1.54 1.25 1.51 1.30
(1H, m) (1H, d, 4.0 Hz) (1H, d, 4.0 Hz) (1H, d, 4.5 Hz) (1H, d, 4.5 Hz)
1.41 (1H, m) 1.90 (1H, m) 1.86 (1H, m) 5.18 (1H, s)
1.51 (1H, d, 4.5 Hz) 1.25 (1H, d, 4.5 Hz) 3.02 (1H, t, 4.5 Hz) 2.39 (1H, s)
1.62 (1H, d, 4.0 Hz) 1.47 (1H, d, 4.0 Hz) 1.70 (1H, d, 4.0 Hz) 1.54 (1H, d, 4.0 Hz) 0.92 (3H, s) 0.70 (3H, s) 0.87 (3H, s) 0.72 (3H, s) 0.87 (3H, s) 11.80 (1H, s) 1.09 (3H, s) 0.85 (3H, s)
13
C NMR
71.6 78.9 80.7 38.3 53.1 18.0 32.6 41.3 46.6 38.1 25.9 126.7 138.5 41.0 28.2 78.3 46.8 54.8 76.8 79.1 26.3 37.2 28.0 15.9 15.0 16.2 25.1 178.8 26.9 16.5
HSQC 4.70 (71.6) 4.26 (78.9) 3.72 (80.7) 1.34 1.54 1.25 1.51 1.30
(53.1) (18.0) (18.0) (32.6) (32.6)
1.41 (46.6) 1.90 (25.9) 1.86 (25.9) 5.18 (126.7)
1.51 (28.2) 1.25 (28.2) 3.02 (78.3) 2.39 (54.8)
1.62 1.47 1.70 1.54 0.92 0.70 0.87 0.72 0.87 11.80 1.09 0.85
(26.3) (26.3) (37.2) (37.2) (28.0) (15.9) (15.0) (16.2) (25.1) (178.8) (26.9) (16.5)
1 H, 13C NMR data and HSQC of 1 (at 500 and 125 MHz, in DMSO at 27°; δ in ppm).
submitted to multiple chromatographic steps to afford Compounds 1–13. Compound 1, was obtained as white powder, [α]20D= +3.0 (c =0.01,MeOH). The negative HR-ESI-MS of Compound 1 showed a molecular ion peak at m/z 536.0602 [M–H]−. When considered in conjunction with its 13C NMR data, it indicated a molecular formula of C30H48O8. The assignment of 1H and 13C NMR spectroscopic data of 1 was based on HSQC, HMBC and ROESY spectra. In the 1H NMR spectrum of 1 showed seven methyl signals at δ 0.70, 0.72, 0.85, 0.87 × 2, 0.92, and 1.09, an olefinic proton at δ 5.18 (s, 1H), and a carboxylic acid proton at δ 11.80 (s, 1H), together with a singlet at δ 2.39 (1H, s), which is a characteristic signal for the H-18 of an ursane type with 19-Osubstitution [8]. Furthermore, the 13C NMR spectrum of 1 showed seven methyl signals at δ 15.0, 15.9, 16.2, 16.5, 25.1, 26.9 and 28.0, also a double bond across C-12/C-13 with a pair of olefinic carbon signals at δ 126.7 and 138.5 [8]. In addition, in the 1H NMR spectrum: four oxygen bearing methine protons at δ 3.02 (d, J = 4.5 Hz, 1H), 3.72 (s, 1H), 4.26 (d, J = 5.0 Hz, 1H) and 4.70 (d, J = 5.0 Hz, 1H), corresponding to four oxygen bearing methine carbons at δ 71.6, 78.3, 78.9 and 80.7 could be observed respectively. And two oxygen bearing quartery carbons at δ 76.8 and 79.1 could also be
B. Zhang et al. / Fitoterapia 82 (2011) 854–860 Table 2 1 H, 13C NMR and HSQC spectral data of 2. Structures
Position
Phenethyl alcohol 1 groups 2 3 4 5 6 α β Caffeoyl groups 1′ 2′ 3′ 4′ 5′ 6′ α′ β′ 7′ Glc 1″ 2″ 3″ 4″ 5″ 6″a 6″b Rha 1″′ 2″′ 3″′ 4″′ 5″′ 6″′
1
H NMR
6.58 6.48 2.66 3.38
(1H, d, 7.6 Hz) (1H, d, 7.6 Hz) (2H, m) (2H, m)
7.01 (1H, s)
6.73 (1H, d, 7.6 Hz) 6.94 (1H, d, 7.6 Hz) 6.20 (1H, d, 15.6 Hz) 7.43 (1H, d, 15.6 Hz) 4.32 3.30 3.80 4.68 3.43 3.53 3.45 4.98 3.90 3.49 3.18 3.46 1.03
857
Table 3 1 H, 13C NMR and HSQC spectral data of 3. 13 C NMR
129.2 128.4 144.8 143.4 115.7 119.6 70.1 34.8 125.5 113.5 148.4 145.5 115.4 121.5 145.6 116.2 165.8 102.1 74.3 79.3 68.7 74.4 60.6
HSQC
6.58 (115.7) 6.48 (119.6) 2.66 (70.1) 3.38 (34.8) 7.01 (113.5)
6.73 (115.4) 6.94 (121.5) 6.20 (145.6) 7.43 (116.2)
(1H, d, 7.8 Hz) 4.32 (102.1) (1H, m) 3.30 (74.3) (1H, m) 3.80 (79.3) (1H, m) 4.68 (68.7) (1H, m) 3.43 (74.4) (1H, m) 3.53 (60.6) (1H, m) 3.45 (60.6) (1H, brs) 101.1 4.98 (101.1) (1H, s) 70.3 3.90 (70.3) (1H, m) 70.4 3.49 (70.4) (1H, m) 71.6 3.18 (71.6) (1H, m) 69.0 3.46 (69.0) (3H, t, 6.9 Hz) 18.2 1.03 (18.2)
1 H and 13C NMR data and HSQC of 2 (at 300 and 75 MHz, in DMSO at 27°; δ in ppm).
observed in the 13C NMR spectrum. Thus, the structure of 1 was elucidated as a polyhydroxyolean-12-ene triterpenoid derivative. In the HMBC spectrum of 1 (Fig. 2), the correlations of H-3, H-5 with C-1, of H-2 with C-4, and of H-1 with C-3, revealed the presence of three hydroxyl groups located at C-1, C-2 and C-3. Correlations of H-18 with C-12, revealed the presence of a double bond across C-12/C-13. Correlations of H-18 with C-16, C-19 and C-20, demonstrated that three hydroxyl groups were attached to C-16, C-19 and C-20. Correlations of H-18 with C-28, revealed a carbonyl group located at C-17. The relative configuration of 1 was assigned on the basis of coupling constants and a ROESY (Fig. 3) experiment. Compound 1 was determined to be 1,2,3,16,19,20hexahydroxyolean-12-en-28-oic acid. Compound 2, was obtained as light viscous liquid. [α] 20D = + 6.9(c = 0.01, MeOH). The molecular formula of compound 2 was established as C29H36O16 by HR-ESI-MS (m/z 663.1666 ([M + Na] +)). In the 1H NMR spectrum of 2, A α, β-unsaturated keto group δ 7.43 (d, J = 15.6 Hz, 1H) and δ 6.20 (d, J =15.6 Hz, 1H) were observed and hence, the double bond was concluded to be transconfiguration. There were two benzene ring signals observed in the 1H NMR spectrum. The presence of a 1,3,4-trisubstituted benzene ring was concluded from the following proton signals: 7.01 (s, 1H), 6.94 (d, J =7.6 Hz, 1H), 6.73 (d, J = 7.6 Hz, 1H). Also, the following proton signals: 6.58 (d, J =7.6 Hz, 1H), 6.48 (d, J = 7.6 Hz, 1H) showed a 1,2,3,4-tetrasubstituted benzene rings.
Structures
Position 1H NMR
Phenethyl alcohol 1 groups 2 3 4 5 6 α β Caffeoyl groups 1′ 2′ 3′ 4′ 5′ 6′ α′ β′ 7′ Caffeoyl groups 1″ 2″ 3″ 4″ 5″ 6″ α″ β″ 7″ Glc 1″′ 2″′ 3″′ 4″′ 5″′ 6″′a 6″′b Rha 1″″ 2″″ 3″″ 4″″ 5″″ 6″″
13 C NMR
131.3 115.9 146.3 145.1 6.74 (1H, d, 8.5 Hz) 116.0 6.50 (1H, d, 8.0 Hz) 119.7 2.66 (2H, m) 70.3 3.38 (2H, m) 35.1 125.8 7.06 (1H, s) 114.3 148.6 145.7 6.65 (1H, s) 115.8 6.95 (1H, d, 8.0 Hz) 121.7 6.23 (1H, d, 16.0 Hz) 145.7 7.50 (1H, d, 16.0 Hz) 114.9 165.8 129.5 7.06 (1H, s) 114.7 146.4 149.5 6.79 (1H, d, 8.0 Hz) 116.5 6.95 (1H, d, 8.0 Hz) 123.3 6.25 (1H, d, 16.0 Hz) 148.1 7.51 (1H, d, 16.0 Hz) 115.7 166.0 4.34 (1H, d, 7.0 Hz) 102.5 3.31 (1H, dd, 10.0, 8.0 Hz) 76.9 3.80 (1H, t, 10.0 Hz) 79.5 4.80 (1H, t, 9.5 Hz) 70.6 3.42 (1H, d, 9.5 Hz) 74.7 3.48 (1H, d, 15.0 Hz) 60.9 3.43 (1H, d, 15.0 Hz) 5.10 (1H, s) 101.4 3.92 (1H, s) 71.9 3.49 (1H, d, 10.0 Hz) 72.6 3.20 (1H, t, 10.0 Hz) 73.6 3.46 (1H, dd, 10.0, 7.0 Hz) 69.3 1.08 (3H, d, 7.0 Hz) 18.6 6.67 (1H, s)
HSQC
6.67 (115.9)
6.74 (116.0) 6.50 (119.7) 2.66 (70.3) 3.38 (35.1) 7.06 (114.3)
6.65 (115.8) 6.95 (121.7) 6.23 (145.7) 7.50 (114.9)
7.06 (114.7)
6.79 (116.5) 6.95 (123.3) 6.25 (148.1) 7.51 (115.7) 4.34 (102.5) 3.31 (76.9) 3.80 (79.5) 4.80 (70.6) 3.42 (74.7) 3.48 (60.9) 3.43 (60.9) 5.10 (101.4) 3.92 (71.9) 3.49 (72.6) 3.20 (73.6) 3.46 (69.3) 1.08 (18.6)
1 H, 13C NMR data and HSQC of 3 (at 500 and 125 MHz, in DMSO at 27°; δ in ppm).
The 13C NMR spectrum displayed 29 carbons. Twelve carbons corresponding to a Glucose moiety and a Rhamnose moiety were found at 102.1, 74.3, 79.3, 68.7, 74.4, 60.6 and 101.1, 70.3, 70.4, 71.6, 69.0, 18.2. When its 1H and 13C NMR data were compared with those analogous of Compound 4 [9], the difference between the two structures was a large signal change in the Phenethyl Alcohol C-2 in Compound 2, which revealed the presence of a hydroxyl located at C-2. As the impact of shielding effect of hydroxyl, C-2 was downfield from 115.9 to 128.4. On the basis of HMBC (Fig. 2), the correlations of H-2′ with C-α′, of H-α′, H-4″ with C-7′, revealed the presence of a double bond across C-α″/C-β′ and a carbonyl group located at C-7′. Similarly, correlations of H-6′ with C-4′, C-2′ and of H-5′ with C-3′, demonstrated that two hydroxyl groups were attached to C-3′ and C-4′, also a Caffeoyl group was attached to C-4″ of Glucose. While correlations of H-6 with C-2 and C-4, and of H-5 with C-3, demonstrated that three hydroxyl groups were attached to C-2, C-3 and C-4. While correlations of H-α with C-2, of H-β with C-1, and of H-1″ with C-β, demonstrated that a Phenethyl Alcohol group was attached to C-1″ of Glucose.
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Table 4 The minimal inhibitory concentration values of Compounds 1–7. Sample
Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7
Minimal inhibitory concentration (MICs) (μg/ml) Hela cell
Hep-6 cell
10 12 9 13 16 20 17
16 17 20 39 42 47 37
The minimal inhibitory concentration values (MICs) (μg/ml) of Compounds 1-7. Hela cell: clinical strain; Hep-6 cell: clinical strain.
While correlations of H-3″ with C-1″′, when coupled with information from the 1H and 13C NMR spectrum, demonstrated that the Rhamnose group was attached to C-3″ of
Glucose. This result was in accord with the ROESY correlations of 2 shown in Fig. 3. Compound 2 was determined to be 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose)-4-[(2E)-3(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside. Compound 3 was obtained as light viscous liquid, [α] 20D = + 9.7(c = 0.01, MeOH). It possessed the molecular formula C38H42O18, as derived from the HR-ESI-MS (m/z 809.2269 ([M + Na] +)). In the 1H NMR spectrum of 3, two α, β-unsaturated keto groups δ 7.49–δ 7.52 (d, J = 16.0 Hz, 2H) and δ 6.22–δ 6.25 (d, J = 16.0 Hz, 2H) were observed in Compound 3. The following proton signals: 6.67 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 7.06 (s, 2H), 6.65 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H) and 6.79 (d, J = 8.0 Hz, 1H) showed the presence of three 1,3,4-trisubstituted benzene rings. The 13C NMR spectrum displayed 38 carbons. Twelve carbons corresponding to a Glucose moiety and a Rhamnose moiety were found at 102.5, 76.9, 79.5, 70.6, 74.7, 60.9 and 101.4, 71.9, 72.6, 73.6, 69.3,
Fig. 2. Key HMBC (H → C) correlations of Compounds 1–3.
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Fig. 3. Key ROESY correlations of Compounds 1–3.
18.6. The 1H and 13C NMR data of 3 suggested the compound had the structure of phenylpropanoid glycoside. In the HMBC experiment (Fig. 2), the correlations of H-2′ with C-α′, of H-α′ and H-4″′with C-7′, of H-2″ with C-α″, of H-α″, and H-6″′a and H-6″′b with C-7″, revealed the presence of two double bonds across C-α′/C-β′, C-α″/C-β″ and two carbonyl groups located at C-7′ and C-7″. While correlations of H-6′ with C-2′ and C-4′, and of H-5′ with C-3′, demonstrated that two hydroxyl groups were attached to C-3′ and C-4′. While correlations of H-6″ with C2″ and C-4″, and of H-5″ with C-3″, demonstrated that two hydroxyl groups were attached to C-3″ and C-4″. Also two Caffeoyl groups were attached to C-4″′ and C-6″′ of Glucose.
Correlations of H-5 with C-3, and of H-6 with C-2 and C-4, demonstrated that two hydroxyl groups were attached to C-3 and C-4. Correlations of H-α with C-2, of H-β with C-1, and of H-1″′ with C-β, demonstrated that a Phenethyl Alcohol group was attached to C-1″′ of Glucose. Correlations of H-3″′ with C-7′, when coupled with information from the 1H and 13C NMR spectrum, demonstrated that the Rhamnose group was attached to C-3″′ of Glucose. This result was in accord with the ROESY correlations of 3 shown in (Fig. 3). Compound 3 was determined to be 1-(2,3,4-trihydroxyphenyl) ethyl-3-O-rhamnose − 4[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoate]-6-[(2E)-3(3,4-dihydroxyphenyl)-2-propenoate]-Glucopyranoside.
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The Compounds 4–13 were identified by comparison of their spectroscopic data with literature values. Cytotoxic activities of Compounds 1–7 against Hela cell and Hep-6 cell were tested. Especially, the three new compounds against Hela cell and Hep-6 cell revealed significant anti-tumor activity in vitro with MIC values ranging from 9 to 20 μg/ml.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.fitote.2011.04.005.
References Acknowledgment This work was supported by the National Natural Science Foundation of China (30770233) and the Foundation of Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research. We thank Prof. Shi-long Chen of Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material. We also thank Dr. Xue-lian Han (pharmacological research center of Southeast University, Nanjing, China) for the pharmacological research of the compounds.
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