Four new compounds from Zygophyllum fabago L.

Phytochemistry Letters 15 (2016) 116–120

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Four new compounds from Zygophyllum fabago L. Jiangbo Hea,* , Xiaoman Lvb , Yanfen Niua , Jian Taoa , Bo Wanga , Jing Jiaa , Weiwei Chena a b

School of Medicine, Kunming University, Kunming, 650214, PR China Yunnan University of Traditional Chinese Medicine, Kunming 650500, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 September 2015 Received in revised form 12 December 2015 Accepted 21 December 2015 Available online xxx

Phytochemical investigation of Zygophyllum fabago L. afforded seven compounds, including a new valencane type sesquiterpenoid (1), a new phenylpropanoid derivative (2) and two new quinovic acid derivatives (3 and 4), and 3-O-[b-D-glucopyranosyl]-quinovic acid (5). Their structures were elucidated by the extensive use of 1D (1H NMR and 13C NMR) and 2D (COSY, HSQC, HMBC and ROESY) NMR experiments, as well as IR and HREIMS spectral data. These new compounds were evaluated for their cytotoxic activity against five cancer cell lines (HL-60, SMMC-721, A-549, MCF-7, and SW-480). All new compounds exhibited no cytotoxic activities (>40 mm). ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Zygophyllum fabago L Quinovic acid Structural elucidation Cytotoxic activity

1. Introduction Zygophyllum fabago L. (Z. fabago) is herbaceous plant mainly distributed in Mediterranean area which belongs to the Zygophyllaceae family. The genus Zygophyllum, with about 90 species, grows mainly between northern Africa and central Asia in mainly arid and semiarid areas (Cronquist, 1981). In China, Z. fabago. is distributed in the Gansu provinces and Xinjiang autonomous region (Wu, 1988). The previous mention of the Zygophyllaceae family as possible inducer of allergic diseases was by Small and Smell in 1946. The researchers confirmed that Z. fabago. is as a source of allergenic pollen (Belchi-Hernandez et al., 1997). The main chemical constituents described from Zygophyllum species are zygophyllin, quinovic acid, and glycosides, which have been shown to have anti-inflammatory and antipyretic activity (Smati et al., 2004). The extracts from Z. fabago. displayed inhibition against butyrylcholinesterase and lower activity against acetylcholinesterase, and both of the enzymes play a role in the pathology of Alzheimer’s Disease (Orhan et al., 2004). Some new sulfated triterpenoid saponins from the barks of Z. fabago have been isolated by researchers (Saleha et al., 2014; Viqar et al., 2007; Feng et al., 2008a,b). Based on the previous phytochemical work and the growing demand for active pharmaceutical components obtained from natural sources, we chose to evaluate the phytochemical

* Corresponding author at: 2# Puxin Road, Kunming Economic and Technological Development Zone, Kunming, Yunnan. Fax: +86 871 65098540. E-mail addresses: [email protected], [email protected] (J. He).

components of Z. fabago. and its biological activity. This paper reports the isolation and characterization of four new compounds and three known compounds (Fig. 1) from Z. fabago. Compounds 1–4 were evaluated for their inhibitory activities against human HL-60, SMMC-7721, A549, MCF-7 and SW480 cancer cell lines. 2. Results and discussion Compound 1 was obtained as a colorless oil. Its molecular formula was determined as C15H26O2 by HREIMS m/z 238.1929 (calc for C15H26O2, 238.1933 [M]+), with three degrees of unsaturation. The IR absorption band at 3424 cm
1 indicated the existence of hydroxyl group. Full assignments of all the individual protons and carbons were ascertained from a combined analysis of 1 H NMR, 13C NMR, COSY, DEPT, HSQC, HMBC and ROESY spectral data. The 1H NMR revealed the presence of four single methyls [dH 0.89 (3H, d, J = 6.4 Hz, H-14), 0.95 (3H, s, H-15), 1.17 (3H, s, H-12), 1.20 (3H, s, H-13)]. The 13C NMR and DEPT spectra showed 15 carbon resonances, including four methyls, four methylenes, four methines, and three quaternary carbons. The signals of dC121.4 and dC 141.4 should be a sp2 carbon proton, therefore compound 1 is a bicyclic sesquiterpenoid based on degrees of unsaturation and 1D NMR. Detailed analysis of 2D NMR data (Fig. 2) of compound 1, the HMBC correlations from H-14 to each of C-3 (dC 40.1), C-4 (dC 41.1), C-5 (dC 37.6) indicated the link between the C-14 and C-4. The HMBC cross peaks from H-15 to each of C-4 (dC 41.1), C-5 (dC 37.6), C-6 (dC 38.4) and C-10 (dC 141.4) indicated the situation of C-15 link with C-5. The downfield shift noticed for the carbon signal (dC 72.5) indicated its attachment to hydroxyl

http://dx.doi.org/10.1016/j.phytol.2015.12.004 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

J. He et al. / Phytochemistry Letters 15 (2016) 116–120

117

Fig. 1. Structures of compounds 1–5.

group. The HMBC correlations from H-12 (dH 1.17) and H-13 (dH 1.20) to C-11 (dC 72.5), verified the location of C-12 and C-13. The double bond was located between C-9 and C-10 because H-1, H-15 and H-8 showed the HMBC correlations with the olefinic quaternary carbon (dC 141.4, C-10). The HMBC correlations from H-7, H-8 to the olefinic methine (dC 121.4, C-9) further confirmed the location of the double bond. The 1H-1HCOSYcorrelations(Fig.2)ofH-1/H-2/H-3/H-4/H-14 and H-6/H-7/H-8/H-9 displayed the key spin systems. Therefore, the planar structure of 1 was unambiguously established. The relative configurationof1wasdetermined bytheanalysisofROESYspectrum (RecordedinDMSO-d6).Me-14and2-OHinthesamesideofringwere confirmed via the ROESY correlations of H-2/H-4. Meanwhile, ROESY correlation of H-4/CH3-15 suggested that H-2/H-4/CH3-15 were in the opposite of ring. In addition, correlation of CH3-15/H-7 revealed the relative configuration at C-7. Therefore, the structure of 1 was established and named fabagoin A. Compound 2, obtained as a colorless oil, gave the molecular formula of C21H28O9 by HREIMS m/z 424.1748 (calc for C21H26O9, 424.1733 [M]+), with eight degrees of unsaturation. The IR spectrum of 2 revealed the presence of hydroxyl group (3432 cm
1). The 1H, 13C NMR and DEPT data of 2 showed two

Fig. 2. Selected 2D NMR correlations of 1 and 2.

benzene ring and five methoxy groups, suggested that 2 should be phenylpropanoid derivatives. The 1H-1H COSY (Fig. 2) correlations of H-50 /H-60 and H-70 /H-80 /H-90 revealed two spin systems. In the HMBC spectrum (Fig. 2), the correlations from dH 3.31 to C-7 (dC 104.5), and from dH 3.82 to C-3 (dC 154.5), C-5 (dC 154.5) and C-30 (dC 148.8) indicated that methoxy groups were connected to C-3, C-30 , C-5 and C-7, respectively. The HMBC correlations from H-80 (dH 4.26) to C-4 (dC 136.9), C-70 (dC 74.2), and C-90 (dC 61.7) suggested that the C-80 link with C-4 by the oxygen-bearing carbons based on their downfield chemical shifts in the 13C NMR spectrum. The OH group located at C-40 (dC 147.1), according to the HMBC correlations from H-50 (dH 6.73) to C-40 . The C-70 (dC 74.2) link with C-10 (dC 133.9), also based on the HMBC correlations from H-70 (dH 4.90) to C-10, and from H-20 to C-70. The relative configurations of the H-70 and H-80 were erythro isomer, established by the coupling constant H-70 (1H, d, J = 5.6 Hz) (Takeshi et al., 1987). Based on the previous discussion, the planner structure of compound 2 was established, and named fabagoin B. Compound 3 was determined to have the molecular formula C30H46O6 (6 degrees of unsaturation) on the basis of HREIMS analysis. IR spectrum revealed the existence of hydroxyl groups due to the absorption bands at 3440 cm
1. The 1H and 13C NMR spectra of 3 (Table 1) were extremely similar with Metatrichosin A (Zhang and Tan, 2006), but the differences were that carbonyl of C-3 was reduced to hydroxyl group and C-30 was oxidized to hydroxymethyl. Detailed analysis of 2D NMR data (Fig. 3) of compound 3, the HMBC correlations from H-3 (dH 3.10, dd, J = 9.2, 4.0 Hz) to C-2, C-3, C-4, and from H-23 and H-24 to C-3, indicated that the OH group located at C-3, and 23-CH3 and 24-CH3 were connected to C-4. The HMBC correlations from H-29 to C-18, C-19, C-20, suggested that the 29-CH3 was located at C-19. The HMBC correlations from H-30 to C-19, C-20 and C-21, indicated that C-30 link with C-20, and C-30 was oxygen-bearing carbon based on their low field chemical shifts in the 13C NMR spectrum. The relative configuration of 3 was determined by the analysis of

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J. He et al. / Phytochemistry Letters 15 (2016) 116–120

Table 1 1 H NMR (400 MHz) and

13

C NMR data (100 MHz) of compound 3 and 4 in methanol-d4 (d in ppm, J in Hz).

3

4

Position

dH

Position

dH

dC

Position

dH

Position

dH

dC

1

1.70, m 1.02, m 1.59, m

40.0

21

1.70, m

25.7

1

39.9

21

22

37.4

2

27.1

22

37.6

3.10, dd (9.2, 4.0)

79.6 39.8 56.6 19.4

23 24 25 26

1.73, m 1.57, m 0.93, s 0.75, s 0.90, s 0.96, s

1.42, drd (8.8) 1.24, m 1.62, m

31.2

27.9

1.65, m 0.97, m 1.78, m

28.7 16.4 19.1 16.8

3 4 5 6

3.07, dd (9.2, 3.6)

90.6 40.1 56.9 19.3

23 24 25 26

0.80, s 0.98, s 0.95, s 0.87, s

17.0 28.5 16.9 18.2

38.1

27

178.9

7

38.0

27

40.6 48.0 38.1

28 29 30

181.5 17.7 66.2

8 9 10

2.19, d (4.4)

40.6 48.0 37.8

28 29 30

0.87, d (8.0) 0.89, d (8.0)

181.5 19.1 21.5

23.8

11

1.92, m

23.8

10

4.22, d (6.0)

107.4

12 13 14 15

5.58, dd (4.0, 1.6)

2.05, m

130.8 133.6 57.2 26.4

20 30 40 50 a 50 b

3.15, overlap 3.25, t (7.2) 3.43, m 3.79, dd (9.2, 4.4) 3.15, dd (8.8, 4.0)

75.4 78.0 71.2 66.7

16

1.35, m

25.5

16

2.02, m 1.70, m

17 18 19 20

2.27, d (9.2) 1.22, m 1.02, m

49.3 55.5 32.8 47.9

17 18 19 20

2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.72, brd (9.2) 1.53, m 1.36, m 1.65, m 1.21, m 2.22, dd (9.2, 4.0)

1.99, m 1.90, ddd (14.4, 9.2, 2.0) 5.61, dd (3.6, 2.0)

dC

0.91, d (5.2) 3.68, dd (8.8, 2.4) 3.34, dd (8.4, 2.0)

0.72, d (8.8) 1.32, d (6.4) 1.51, brd (10.8) 1.18, m

1.65, m

2.23, brd (7.6) 0.97, m 0.90, m

dC

130.4 133.8 57.2 26.4

179.0

25.7 49.5 55.5 38.3 40.4

Fig. 3. Selected 2D NMR correlations of 3 and 4.

ROESY spectrum, the OH group at C-3 of aglycone was disposed b and equatorial on the basis of coupling constant of H-3 (dd, J = 11.5, 4.5 Hz). The compound was therefore characterized as 3,30-hydroxy-urs-12-en-27-28 dioic acid, trivial name fabagoin C. Compound 4, obtained as a white amorphous powder, possessed the molecular formula as C15H26O3, which was deduced from the HREIMS m/z 618.3766 (calc for C35H54O9, 618.3768 [M]+), indicating seven degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3442 cm
1). The 1D NMR data of 4 (Table 1) were very similar to the known compound quinovic acid 3b-O-b-D-quinovopyranoside (Hassanean et al., 1993), the only difference that C-3 of compound 4 connected to xylose moiety (dC 107.4, dC 75.4, dC 78.0, dC 71.2, dC 66.7), didn’t link with rhamnose moiety (Saleha et al., 2014). The b-configuration of the xylose determined from the coupling constant (6.0 Hz) of the anomeric proton signal in the 1H NMR spectrum. The sugar obtained after acid hydrolysis of compound 4 was determined as b-D-xylose followed by comparing its TLC and specific rotation with an authentic sample. The signal due to C-3 was shifted downfield, suggested that the b-D-xylopyranosyl moiety was located at C-3, which was confirmed by the HMBC correlations (Fig. 3) of H-10 /C-3 (dC 90.6) Accordingly, the structure of the

new quinovic acid glycoside 4 was fully established to be 3-b-D-xylopyranosyl- quinovic acid and gave the name fabagoin D was given to 4. The known compound were identified as 3-O-[b-D-glucopyranosyl]-quinovic acid (5) (Matos et al., 1983), by comparison their spectroscopic data with literature data. All new compounds from Z. fabago L. were evaluated for their cytotoxicities against five cancer cell lines in HL-60, SMMC-7721, A-549, MCF-7, SW-480, and their cytotoxicity was measured in parallel with the determination of antitumor activity using Cisplatin as the positive control. Compounds 1–4 were noncytotoxic to all cell lines (IC50 > 40 mm). 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Jasco-P-1020 polarimeter. IR spectra were obtained by using Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with instruments of Bruker AV-400, DRX-500 or AV 600 MHz. HREIMS were measured on a waters Autospec Primier P776 instrument and API QSTAR pulsar l spectrometer. Optical rotations were recorded

J. He et al. / Phytochemistry Letters 15 (2016) 116–120

on a Horiba SEPA-300 polarimeter. Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Amersham Biosciences, Sweden), RP-18 silica gel (40–75 mm, Fuji Silysia Chemical Ltd.), MCI gel CHP 20 P (75–150 mm, Tokyo, Japan). Fractions were monitored by TLC and spots were visualized by heating silica gel plates immersed in vanillin-H2SO4 in EtOH, in combination with Agilent 1200 series HPLC system (Eclipse XDB-C18 column, 5 mm, 4.6  150 mm). 3.2. Plant material The whole plant of Z. fabago L. were collected at wulumuqi, Xinjiang autonomous region, China in 2010 and identified by Prof. Kai-Yun Guan. The voucher specimen (NO.LTTB2010) was deposited at herbarium of Kunming University. 3.3. Extraction and isolation The plant material of Z. fabago L. (8.0 kg) was extracted using refluxing MeOH (3  20 L  2 h) to give a crude extract, which was suspended in water followed by extraction with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (230 g) was separated by a silica gel CC (10  120 cm, 200–300 mesh, 2.0 kg) eluted with a gradient of CHCl3/MeOH to afford Fr. A–Fr. H. Fraction B (14.7 g) was separated into fractions B1–B4 by CC over silica gel with petroleum ether/acetone (50:1–4:1) as the eluent. Fraction B2 (3.3 g) was gel filtered over Sephadex LH-20 (CHCl3/MeOH, 1:1, v/v) to give sub-fractions B2-1 and B2-2, sub-fractions B2-1 was further purified by silica gel (petroleum ether–acetone, 20/1, v/v) to yield compound 1 (20.6 mg). Fraction H (8.9 g) was subjected to MCI gel CHP 20 P (75–150 mm, Tokyo, Japan) with MeOH/H2O (10:90–100:0, v/v) as the mobile phase to yield subfractions H1–H4. The subfraction H1 (190 mg) was further purified by Sephadex LH-20 (MeOH) to gave subfraction H1-1, followed by RP-18 silica gel (40–75 mm, Fuji Silysia Chemical Ltd.) with eluting MeOH/H2O (10:90–100:0, v/v) to afford 2 (8.0 mg). The subfraction H4 was separated by Sephadex LH-20 (MeOH) to yield 3 (19.0 mg) and a mixture. The mixture was subjected to RP-18 by using MeOH/H2O (10:90–100:0, v/v) as mobile phase to afford 4 (38.0 mg) and 5 (120.0 mg). 3.4. Fabagoin A (1) Coloress oil, [a]21.9D
15.3 (c 0.1 MeOH); IR (KBr) vmax 3424, 2968, 2923, 2855, 1639 cm
1; EIMS m/z 238 [M]; HREIMS m/z 238.1929 (calc for C15H26O2, 238.1933 [M]+). 1H NMR (400 MHz, CDCl3): d 5.38 (1H, brs, H-9), 3.56 (1H, m, H-2), 2.32 (1H, ddd, J = 12.4, 4.8, 2.2 Hz, H-1a), 2.11 (1H, overlap, H-1b), 2.04 (1H, m, H-8a), 1.86 (1H, brd, J = 12.4 Hz, H-6a), 1.76 (1H, overlap, H-8b), 1.73 (1H,m, H-3a), 1.64 (1H, m, H-7), 1.38 (1H, m, H-3b), 1.30 (1H, m, H-4), 1.20 (3H, s, H-13), 1.17 (3H, s, H-12), 0.95 (3H, s, H-15), 0.94 (1H, overlap, H-6b), 0.89 (3H, d, J = 6.4 Hz, H-14). 13C NMR (100 MHz, CDCl3): d 141.4 (C-10), 121.4 (C-9), 72.5 (C-11), 70.9 (C-2), 41.9 (C-1), 41.1 (C-4, C-7), 40.1 (C-3), 38.4 (C-6), 37.6 (C-5), 27.5 (C-13), 27.4 (C-8), 26.1 (C-12), 17.8 (C-15), 15.3 (C-14). 3.5. Fabagoin B (2) Coloress oil, [a]21.9D
6.4 (c 0.25 MeOH); IR (KBr) vmax 3432, 2937, 2840, 1596, 1464, 1124 cm
1; ESIMS m/z 447 [M + Na]+; HREIMS m/z 424.1748 (calc for C21H26O9, 424.1733 [M]+). 1H NMR (600 MHz, CD3OD): d 6.98 (1H, brs, H-20 ), 6.78 (1H, d, J = 7.8 Hz, H-60 ), 6.75 (2H, brs, H-2,6), 6.73 (1H, d, J = 7.8 Hz, H-50 ), 5.32 (1H, s, H-7), 4.90 (1H, d, J = 5.4 Hz, H-70 ), 4.26 (1H, m, H-8), 3.90 (1H, dd, J = 12.0, 6.0 Hz, H-90 a), 3.82 (9H, brs, 30 -OCH3, 3-OCH3, 5-OCH3),

119

3.58 (1H, dd, J = 12.0, 3.0 Hz, H-90 b), 3.31 (6H, brs, 2  -OCH3). C NMR (150 MHz, CD3OD): d 154.5 (C-3, 5), 148.8 (C-30 ), 147.1 (C-40 ), 136.9 (C-4), 135.8 (C-1), 133.9 (C-10 ), 120.8 (C-60 ), 115.8 (C-50 ), 111.4 (C-20 ), 105.2 (C-2, 6), 104.5 (C-7), 87.5 (C-80 ), 74.2 (C-70 ), 61.7 (C-90 ), 56.7 (3-OCH3, 5-OCH3), 56.4 (30 -OCH3), 53.4 (7-OCH3). 13

3.6. Fabagoin C (3) White amorphous powder, [a]26.9D +22.2 (c 0.26 MeOH); IR (KBr) vmax 3440, 2933, 2873, 2873, 1695, 1637 cm
1; 1H NMR data and 13C NMR data (see Table 1); ESIMS (neg.) m/z 501 [M-H]
; HREIMS m/z 502.3285 (calc for C30H46O6, 502.3294 [M]+). 3.7. Fabagoin D (4) White amorphous powder, [a]21.9D +27.6 (c 0.26 MeOH); IR (KBr) vmax 3442, 2965, 2926, 2874, 1691, 1639 cm
1; 1H NMR data and 13C NMR data (see Table 1); ESIMS (neg.) m/z 617 [M-H]
; HREIMS m/z 618.3766 (calc for C35H54O9, 618.3768 [M]+). 3.8. Cytotoxic assay The following human tumor cell lines were used: HL-60, SMMC7721, A-549, MCF-7 and SW-480. All the cells were cultured in RPMI1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37  C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) (Sigma). Briefly, 100 mL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 105 cells/mL in 100 mL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 mg) was added to each well, and the incubation continued for 4 h at 37  C. The cells were lysed with 100 mL of 20% SDS–50% DMF after removal of 100 mL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench’s method. Acknowledgments This work was financially supported by the “Kunming University Research Program (XJL14009)” and the National Natural Science Foundation of China (No. 31300302). References Belchi-Hernandez, J., Moreno-Grau, S., Bayo, J., Rosique, C., Bartolome, B., M, Moreno, J., 1997. Zygophyllum fabago L.: a new source of allergenic pollen. J. Allergy Clin. Immunol. 99, 493–496. Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York. Feng, Y.L., Wu, B., Li, H.R., Li, Y.Q., Xu, L.Z., Yang, S.L., Kitanaka, S., 2008a. Triterpenoidal saponins from the barks of Zygophyllum fabago L. Chem. Pharm. Bull. 56 (6), 858–860. Feng, Y.L., Li, H.R., Rao, Y., Luo, X.J., Xu, L.Z., Wang, Y.S., Yang, S.L., Kitanaka, S., 2008b. Two sulfated triterpenoidal saponins from the barks of Zygophyllum fabago L. Chem. Pharm. Bull. 57 (6), 612–614. Hassanean, H.H., Desoky, E.K., El-Hamouly, M.M.A., 1993. Quinovic acid glycosides from zygophyllum album. Phytochemistry 33 (3), 663–666. Matos, M.E.O., Sousa, M.P., Machado, M.I.L., Braz-Filho, R., 1983. Quinovic acid glycosides from guettarda angelica. Phytochemistry 25 (6), 1419–1422. Orhan, I., Sener, B., Choudhary, M.I., Khalid, A., 2004. Acetylcholinesterase butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. J. Ethnophar. 91, 57–60.

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