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Rhamnocitrin glycosides from Oxytropis chiliophylla
Phytochemistry Letters 19 (2017) 50–54
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Short communication
Rhamnocitrin glycosides from Oxytropis chiliophylla Jun Wang, Yang Liu, Norbo Kelsang, Kewu Zeng, Hong Liang, Qingying Zhang* , Pengfei Tu State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, PR China
A R T I C L E I N F O
Article history: Received 20 September 2016 Received in revised form 10 November 2016 Accepted 21 November 2016 Available online xxx Keywords: Oxytropis Oxytropis chiliophylla Tibetan medicines Rhamnocitrin glycosides Anti-inflammatory activity
A B S T R A C T
Repeated chromatography of the n-BuOH soluble fraction from ethanol extract of the aerial part of Oxytropis chiliophylla led to the isolation of six new rhamnocitrin glycosides, named oxychilioflavonosides AF (1–6), together with eight known rhamnocitrin glycosides (7–14) of which six were isolated from the genus Oxytropis for the first time. The structures of these compounds were elucidated by extensive spectroscopic techniques and chemical methods. All isolated compounds were evaluated for their anti-inflammatory activity in lipopolysaccharide (LPS)-induced BV-2 microglial cells, and compound 11 showed potent inhibition on NO production with IC50 value of 9.91 mM. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Oxytropis chiliophylla Royle (Leguminosae), along with Oxytropis falcata Bunge, is officially documented as the botanical origin of Tibetan medicine “Er-Da-Xia” that is known as “King of Herbs” and wildly used for the treatment of inflammation, pyreticsis and bleeding (Diamer, 1986; Committee of Chinses Pharmacopoeia, 2010). Inhabited at 2800–5200 m altitude, O. chiliophylla is mainly distributed in ravines, on slopes, in steppe meadows and shrubberies in Tibet Autonomous Region and Xinjiang Urgur Autonomous Region in China (The Editorial Committee of the Administration Bureau of Flora of China, 2005). Many chemical and pharmacological investigations focusing on O. falcata had been carried out previously, indicating that flavonoids (Zhang et al., 2014; Chen et al., 2010a), oleanane-type triterpenoids (Chen et al., 2009) and N-benzoylindole analogues (Chen et al., 2010b) were the principle constituents, among which flavonoids exhibited significant anti-inflammatory and analgesic activities (Yang et al., 2010; Chen et al., 2011). However, investigations on O. chiliophylla were rarely few and only one paper up to date reported the isolation of one triterpenoid, three flavonoids and four other compounds from the herb (Yao et al., 2007). Our comparative fingerprint analysis of O. chiliophylla and O. falcata using high performance liquid chromatography (HPLC) coupled with diode array detector
* Corresponding author. E-mail address: [email protected] (Q. Zhang).
(DAD) revealed the consistency of both herbs in main components of less polar portion and the slight differences in constituents of high polar portion (Kelsang et al., 2014). Flavonol 3-O-glycosides, especially 3-hydroxy-3-methylglutaryl (HMG) rhamnocitrin 3-Oglycosides, proved to be the characteristic and principle constituents of n-BuOH soluble fraction of ethanol extract of O. falcata (Wang et al., 2012). The present research focusing on the characteristic flavonoids of n-BuOH soluble fraction from ethanol extract of O. chiliophylla resulted in the discovery of six new rhamnocitrin glycosides, oxychilioflavonosides AF (1–6) (Fig. 1), together with eight known rhamnocitrin glycosides (7–14) of which six were isolated from the genus Oxytropis for the first time. All the flavonoid glycosides obtained were different from those of O. falcata. Furthermore, their anti-inflammatory activities against NO production using LPS-induced BV-2 microglial cells were also evaluated. 2. Results and discussions Compound 1 was isolated as an amorphous yellow powder, and its molecular formula was determined to be C32H38O19 by the quasimolecular ion peak [M+H]+ at m/z 727.2100 (calcd 727.2086) in HRESIMS. The UV spectrum showed maximum absorption bands at 266 and 340 nm, suggesting a flavonol skeleton. The 1H (Table 1) and 13C NMR (Table 2) data exhibited characteristic signals of 5,7,40 -trisubstituted flavonol glycoside, with one conjugated phenolic hydroxy at dH 12.49 (1H, s), one methoxyl group at dH 3.87 (3H, s), and three sugar units evidenced by signals
http://dx.doi.org/10.1016/j.phytol.2016.11.011 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54
51
Fig. 1. Chemical structures for compounds 1–6.
of three anomeric carbons at dC 101.3, 100.0 and 103.9 and their corresponding anomeric protons at dH 5.37 (d, J = 5.2 Hz, H-100 ), 4.98 (d, J = 7.2 Hz, H-1000 ) and 4.20 (d, J = 7.5 Hz, H-10000 ). The methoxyl group was assigned to C-7 of the aglycone by the HMBC correlations between dH 3.87 (OCH3) and dC 165.3 (C-7). Based on the above evidences, the aglycone of 1 was identified as rhamnocitrin, ie. 5, 40 -dihydroxy-7-methoxy flavonol, which was Table 1 1 H NMR data for compounds 1–6 No 3-OH 5-OH 6 8 20 /60 30 /50 7-OMe 3-O 100 200 300 400 500
1 12.49 s 6.38 d (1.8) 6.80 d (1.8) 8.19 d (8.9) 7.20 d (8.9) 3.87 s Ara 5.37 d (5.2) 3.75 c 3.52 c 3.663.69 b 3.63–3.66 b 3.23–3.26 b
600 40 -O 1000 2000 3000 4000 5000 6000
100 00 200 00 300 00 400 00 500 00 600 00
a b c
Glc 4.98 d (7.2) 3.26–3.30 b 3.26–3.30 b 3.19–3.23 b 3.57–3.63 b 3.97 d (10.2) 3.57–3.63 b Xyl 4.20 d (7.5) 3.01 t (8.2) 3.10 t (8.8) 3.26–3.30 b 2.94 t (10.8) 3.66–3.69 b
a
confirmed by acid hydrolysis. The three sugar units were identified as a-L-arabinose, b-D-glucose and b-D-xylose by the coupling patterns of anomeric protons (Halabalaki et al., 2011) and acid hydrolysis results. The arabinose and glucose moieties were linked to the aglycone at C-3 and C-40 , respectively, and the xylose unit was linked to C-6 of glucose, as indicated by the HMBC correlations of dH 5.37 (H-100 )/dC 134.4 (C-3), dH 4.98 (H-1000 )/dC 159.3 (C-40 ), and
in DMSO-d6. 2
3
12.53 s 6.39 d (2.0) 6.79 d (2.0) 8.17 d (8.9) 7.17 d (8.9) 3.87 s Gal 5.43 d (7.6) 3.53–3.55 b 3.29–3.39 b 3.63–3.69 b 3.29–3.39 b
12.55 s 6.39 d (1.5) 6.78 d (1.5) 8.14 d (8.8) 7.19 d (8.8) 3.87 s Glc 5.50 d (7.2) 3.21–3.25 b 3.21–3.25 b 3.00–3.11 b 3.00–3.11 b
3.39–3.45 b 3.29–3.39 b Glc 4.95 d (7.5) 3.29–3.39 b 3.29–3.39 b 3.20 c 3.57–3.59 b 3.97 d (9.8) 3.57–3.59 b Xyl 4.19 d (7.3) 2.98–3.01 b 3.08 t (8.6) 3.19–3.29 b 2.95 t (10.7) 3.63–3.69 b
3.58–3.61 b 3.36–3.38 b Glc 4.98 d (7.7) 3.25–3.30 b 3.25–3.30 b 3.21–3.25 b 3.58–3.61 b 3.98 d (10.0) 3.58–3.61 b Xyl 4.19 d (7.5) 2.98–3.00 b 3.08–3.11 b 3.25–3.30 b 2.97 t (10.8) 3.68 dd (11.1, 5.2)
4
5
12.58 s 6.39 d (2.0) 6.82 d (2.0) 8.20 d (8.9) 7.22 d (8.9) 3.87 s Ara 5.38 d (5.2) 3.75 c 3.53 c 3.64–3.66 b 3.58–3.64 b 3.23–3.25 b
Glc 4.99 d (7.0) 3.30–3.38 b 3.303.38 b 3.07 t (9.2) 3.64–3.66 b 4.00 d (11.0) 3.64–3.66 b Glc 4.24 d (7.7) 2.99–3.02 b 2.99–3.02 b 3.13–3.18 b 3.13–3.18 b 3.64–3.66 3.40–3.43
Measured at 500 MHz for 5 and 400 MHz for 1–4, 6, dH in ppm, J in Hz. Overlapped with other signals. Multiplicities can not be clearly defined; Ara = arabinose, Glc = glucose, Gal = galactose, Xyl = xylose.
b b
9.64 s 12.41 s 6.36 d (1.5) 6.79 d (1.5) 8.17 d (8.9) 7.23 d (8.9) 3.87 s
Glc 4.96 d (7.4) 3.30–3.32 b 3.30–3.32 b 3.17 c 3.58–3.60 b 3.96 d (9.8) 3.58–3.60 b Xyl 4.19 d (7.6) 2.97–3.00 b 3.07–3.10 b 3.30–3.32 b 2.94 t (11.0) 3.66 dd (11.0, 5.0)
6 12.54 s 6.37 d (1.8) 6.76 d (1.8) 8.21 d (8.9) 7.16 d (8.9) 3.86 s Ara 5.38 d (5.1) 3.70–3.74 b 3.51–3.53 b 3.66–3.70 b 3.57 dd (11.5, 5.4) 3.23 dd (11.5, 1.8)
Glc 5.03 d (7.2) 3.30–3.35 b 3.30–3.35 b 3.18–3.20 b 3.38–3.42 b 3.70–3.74 b 3.48 dd (11.5, 5.8)
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J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54
Table 2 13 C NMR data for compounds 1–6
a
in DMSO-d6.
No
1
2
3
4
5
6
2 3 4 5 6 7 8 9 10 10 20 /60 30 /50 40 7-OCH3 3-O 100 200 300 400 500 600 40 -O 1000 2000 3000 4000 5000 6000
156.0 134.4 177.8 160.9 98.0 165.3 92.5 156.4 105.1 123.5 130.8 116.1 159.3 56.2 Ara 101.3 70.8 71.6 66.2 64.4
156.2 134.0 177.8 160.9 98.1 165.3 92.5 156.5 105.1 123.7 130.9 116.0 159.3 56.2 Gal 101.5 71.2 73.1 68.0 75.9 60.2 Glc 100.2 73.2 76.4 69.6 76.0 68.2 Xyl 103.9 73.5 76.6 69.6 65.7
156.2 134.0 177.8 161.0 98.1 165.3 92.5 156.5 105.2 123.7 130.8 116.0 159.3 56.2 Glc 100.7 74.2 76.5 69.9 77.7 60.9 Glc 100.0 73.2 76.4 69.6 76.0 68.2 Xyl 103.9 73.5 76.6 69.6 65.7
156.1 134.3 177.8 160.9 98.1 165.3 92.5 156.4 105.1 123.4 130.9 116.1 159.3 56.2 Ara 101.3 70.8 71.6 66.2 64.3
146.6 136.5 176.2 160.3 97.5 165.0 92.2 156.2 104.1 124.3 129.3 116.2 158.5 56.1
156.0 134.5 177.9 161.0 98.1 165.4 92.5 156.5 105.2 123.5 130.9 116.0 159.5 56.3 Ara 101.4 70.9 71.7 66.2 64.4
Glc 99.9 73.2 76.5 70.1 76.1 68.3 Glc 103.3 73.7 76.9 69.7 76.7 61.0
Glc 99.9 73.1 76.5 69.6 76.1 68.1 Xyl 103.9 73.4 76.5 69.6 65.6
Glc 100.0 73.3 76.7 69.8 77.2 60.8
100 00 200 00 300 00 400 00 500 00 600 00
Glc 100.0 73.2 76.4 69.6 76.1 68.1 Xyl 103.9 73.5 76.5 69.6 65.7
a Measured at 125 MHz for 5 and 100 MHz for 1–4, 6, dC in ppm; Ara = arabinose, Glc = glucose, Gal = galactose, Xyl = xylose.
dH 4.20 (H-10000 )/dC 68.1 (C-6 of glucose). Thus, compound 1 was identified as rhamnocitrin-3-O-a-L-arabinopyranosyl-40 -O-b-Dxylopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside A. Compound 2 was obtained as an amorphous yellow powder. The molecular formula C33H40O20 was established by HRESIMS ion peak at m/z 757.2192 [M+H]+ (calcd 757.2191). The UV, 1H and 13C NMR data were similar to those of compound 1. The only difference between compounds 1 and 2 involved the substitution of a galactose unit at C-3 of the aglycone in 2 instead of the arabinose unit in 1, which was consistent with acid hydrolysis results that yielded D-galactose, D-glucose, D-xylose and rhamnocitrin. The HMBC correlation between the anomeric proton of galactose unit at dH 5.43 (H-100 ) and dC 134.0 (C-3) further confirmed the above conclusion. The b configuration of the galactose moiety was assigned from the large coupling constant (7.6 Hz) of the anomeric proton. On the basis of above evidences, the structure of 2 was characterized as rhamnocitrin-3-O-b-D-galactopyranosyl-40 -Ob-D-xylopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside B. Compound 3, isolated as an amorphous yellow powder, had the same molecular formula as 2, C33H40O20, on the basis of HRESIMS at m/z 757.2189 [M + H]+ (calcd 757.2191). The 1H and 13C NMR data of 3 were similar to those of 2 except for a glucose unit located at C3 of the aglycone in 3 replacing the galactose moiety in 2, which was supported by acid hydrolysis results that only yield D-glucose, D-xylose and rhamnocitrin. The b configuration of D-glucose and Dxylose was determined using the same method mentioned above. Therefore, the structure of compound 3 was identified as rhamnocitrin-3-O-b-D-glucopyranosyl-40 -O-b-D-xylopyranosyl-
(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside C. Compound 4, a yellow amorphous powder, gave a molecular formula of C33H40O20, the same as compounds 2 and 3, by HRESIMS at m/z 757.2188 [M+H]+ (calcd 757.2191). Acid hydrolysis and coupling patterns of anomeric protons confirmed the presence of b-D-glucose, a-L-arabinose and rhamnocitrin, and their linkages were determined by careful analysis of 2D NMR correlations. Unambiguously, compound 4 was assigned as rhamnocitrin-3-Oa-L-arabinopyranosyl-40 -O-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside D. Compound 5 was a yellow, amorphous powder. The HRESIMS showed a quasimolecular ion peak at m/z 595.1678 [M+H]+ (calcd 595.1663) indicating the molecular formula of C27H30O15. Its 1H and 13C NMR data differed from those of 1 by the absence of a set of arabinose signals, which was consistent with acid hydrolysis results that yielded only D-glucose, D-xylose and rhamnocitrin. Further analysis of the 2D NMR spectra confirmed the structure of compound 5 to be rhamnocitrin-40 -O-b-D-xylopyranosyl-(1 ! 6)b-D-glucopyranoside, and it was named oxychilioflavonoside E. Compound 6, a yellow amorphous powder, had a molecular formula of C27H30O15, the same as compound 5, by HRESIMS at m/z 595.1671 [M+H]+ (calcd 595.1663). The presence of b-D-glucose, a-L-arabinose and rhamnocitrin were confirmed by acid hydrolysis and their attachments were determined by 2D NMR correlations. Consequently, compound 6 was characterized as rhamnocitrin-3O-a-L-arabinopyranosyl-40 -O-b-D-glucopyranoside, and it was named oxychilioflavonoside F. The eight known compounds were identified as rhamnocitrin3-O-b-D-galactopyranosyl-40 -O-b-D-glucopyranoside (7) (Marco et al., 1986), rhamnocitrin-3-O-b-D-galactopyranoside (8) (Gong et al., 2010), rhamnocitrin-3-O-a-L-arabinopyranoside (9) (Chokchaisiri et al., 2012), rhamnocitrin-3-O-b-D-glucopyranoside (10) (Lu et al., 2009), rhamnocitrin-40 -O-b-D-glucopyranoside (11) (Lu et al., 2009), rhamnocitrin-3-O-rutinoside (12) (Wang et al., 2008), rhamnocitrin-3-O-b-D-glucopyranosyl-40 -O-b-D-glucopyranoside (13) (Lu et al., 2010), rhamnocitrin-3-O-a-L-rhamnopyranosyl(1 ! 6)-b-D-galactopyranoside (14) (Bodakhe et al., 2013), by comparison of the spectral data with those previously reported in the literatures. Compounds 9–14 were isolated from the genus Oxytropis for the first time, and compounds 7–8 were obtained from this herb for the first time. The present research definitely confirmed the differences between the constituents from high polar fraction of O. chiliophylla and those of O. falcata. From the n-BuOH soluble fraction of ethanol extract of O. falcata, a series of rhamnocitrin 3-O-glycosides and kaempferol 3-O-glycosides, a few of which were further glycosylated at C-40 by a glucose unit or at C-7 by a rhamnose unit, had been obtained. The sugar chains located at C-3 of the aglycone consisted of one to four sugar units of galactose, rhamnose and xylose and the obvious characteristic was that the galactose moiety was always linked directly to the aglycone. In addition, a HMG group was occasionally conjugated to C-6, C-4 or C-3 of galactose, and the HMG conjugated rhamnocitrin 3-O-glycosides might be considered as the characteristic chemical markers of O. falcata. However, the constituents from n-BuOH soluble fraction of O. chiliophylla obtained herein were totally different from those of O. falcata. All the flavonoid glycosides of O. chiliophylla were derived from the same aglycone of rhamnocitrin, while the sugars linked directly to C-3 of rhamnocitrin were varied, including galactose, glucose and arabinose. Moreover, flavonol-40 -O-diglycosides, which were not so popular in the plant kingdom, were relatively abundant in O. chiliophylla, and the disaccharide chains, i.e. xylosyl-(1 ! 6)-glucose or gentiobiose, were different from those previously reported (Leitao et al., 2000; Zahid et al., 2002; Kumar
J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54 Table 3 Anti-inflammatory data for compounds 1–14. Compounds
IC50 (mM)
Compounds
IC50 (mM)
1 2 3 4 5 6 7 Quercetin
>100 >100 >100 >100 >100 >100 >100 5.31 0.03
8 9 10 11 12 13 14
>100 31.70 2.26 >100 9.91 0.07 >100 >100 >100
a
a
Quercetin was used as positive control for anti-inflammatory activity.
et al., 2010). What’s more, no HMG conjugated rhamnocitrin glycosides were obtained from O. chiliophylla. All the isolated compounds were further evaluated for their anti-inflammatory activity through LPS-induced NO production in BV-2 microglial cells. Colorimetric MTT assay was firstly used to test cell viability, and it was confirmed that all tested compounds had no or little cytotoxicity at the concentration tested (data not shown). Rhamnocitrin-40 -O-b-D-glucopyranoside (11) exhibited potent inhibitory activity with IC50 value of 9.91 mM, rhamnocitrin-3-O-a-L-arabinopyranoside (9) had weak inhibitory activity with IC50 value of 31.70 mM, while the others showed no remarkable effect with IC50 values greater than 100 mM (Table 3). The reasons why rhamnocitrin monoglycoside exhibited more potential activity than diglycosides and triglycosides might be attributed to the lipophilic nature that might result in the increasing of cellular penetration. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured with Rudolph Research Analytical Autopol IV automatic polarimeter. UV spectra were obtained using a Shimadzu UV-260 spectrometer. NMR spectra were recorded on a Bruker AVANCE III-400/600 or a Varian INOVA500 instrument with TMS as internal standard. HRESIMS was carried out on an Agilent QTOF 6538 or a Abseciex QSTAR spectrometer. HPLC analysis was performed on an Agilent 1260 LC system with a Phenomenex column (250 4.6 mm, 5 mm). HPLC separations were performed on an Alltech semi-preparative instrument equipped with a YMC-Pack ODS-A column (250 10 mm, 5 mm). Column chromatography (CC) was performed on Diaion HP20 (200–300 mesh, Mitsubishi Chemical Co., Japan), silica gel (200–300 mesh, Qingdao Marine Chemical Inc.), ODS-A (50 mm, YMC Co. Ltd., Japan) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). Analytical TLC was carried out on silica GF254 (10–40 mm, Qingdao Marine Chemical, Inc.), and spots were observed by UV light and 10% H2SO4-EtOH reagent. HPLC grade solvents used for HPLC analysis and separations were purchased from Fisher Scientific International (Fair Lawn, New Jersey, USA), and deionized water was purified by Milli-Q Synthesis A10 (Bedford, MA). Other solvents used for extraction and isolation were of analytical grade and purchased from Beijing Tongguang Chemicals (China). All chemicals were purchased from J & K Co. Ltd. (China). 3.2. Plant material The whole plant of O. chiliophylla was collected from Langkazi County, Tibet Autonomous Region, China, in September 2012, and was authenticated by Associate Professor Ying-Tao Zhang, Department of Natural Medicines, School of Pharmaceutical Science, Peking University Health Science Center. A voucher specimen (No
53
20120901) was deposited in the Herbarium of Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center. 3.3. Extraction and isolation The aerial part of O. chiliophylla (10.0 kg) was pulverized and in turn extracted with 95% EtOH and 50% EtOH for three times. After removal of solvent under reduced pressure, the residue was dissolved in water and then partitioned successively with petroleum ether, EtOAc and n-BuOH, respectively. The n-BuOH extract (250 g) was separated by chromatography on Dianion HP20 macroporous absorbent resin using an EtOH/H2O gradient (0, 20, 40, 60, 80, 95% EtOH) to obtain six fractions (DK1 DK6). DK4 (eluted with 60% EtOH, 80 g) was subjected to silica gel CC eluted with CH2Cl2/CH3OH gradient system (10:1, 5:1, 3:1, 1:1, 1:5) to give six subfractions (DK4–1 DK4-6). DK4-3 (13.2 g) was purified by chromatography on ODS CC with a gradient system of MeOH/H2O (30, 50, 70, 100% MeOH) to yield four subfractions (DK4-3A DK4-3-D). Further purification of DK4-3-A (3.0 g) by CC on Sephadex LH-20 (MeOH/H2O, 1:1) and semi-preparative HPLC (MeCN H2O, 23:77) afforded compounds 1 (100 mg), 6 (500 mg), 7 (10 mg) and 8 (12 mg). Compound 9 (200 mg) was obtained by further separation of fraction DK4-3-B (1.0 g) on repeated Sephadex LH-20 CC (MeOH/H2O, 4:1). Fraction DK4-4 (20.0 g) was purified by CC over Sephadex LH-20 eluted with MeOH/H2O (30, 50, 70, 100% MeOH) to yield four subfractions (DK4-4A DK4-4-D). Further separation of DK4-4-B by repeated Sephadex LH-20 CC (MeOH/H2O, 1:1) and isocratic semipreparative HPLC (MeCN H2O, 20:80) afforded compounds 2 (18 mg), 3 (30 mg) and 4 (7 mg). DK3 (eluted with 60% EtOH, 10.0 g) was separated by chromatography on ODS CC with a gradient system of MeOH/H2O (30, 50, 70, 100% MeOH) to give three subfractions (DK3–1 DK33), and compounds 5 (3 mg), 10 (10 mg), 11 (20 mg), 12 (30 mg), 13 (8 mg), 14 (7 mg) were afforded through further purification of fraction DK3-3 by repeated Sephadex LH-20 CC (MeOH/H2O, 1:1) and then isocratic semi-preparative HPLC (MeCN/H2O, 30:70). 25 Oxychilioflavonoside A (1): amorphous, yellow powder; ½a D 55.6 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 727.2100 [M+H]+ (calcd 727.2086); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside B (2): amorphous, yellow powder; ½a D 75.8 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2192 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside C (3): amorphous, yellow powder; ½a D 47.7 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2189 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside D (4): amorphous, yellow powder; ½a D 79.1 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2188 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside E (5): amorphous, yellow powder; ½a D 71.3 (c 0.1, MeOH); UV (MeOH) lmax 266, 360 nm; HRESIMS (positive mode) m/z 595.1678 [M+H]+ (calcd 595.1663); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside F (6): amorphous, yellow powder; ½a D 55.6 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS
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(positive mode) m/z 595.1671 [M+H]+ (calcd 595.1663); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 3.4. Determination of absolute configurations of sugar moieties Compounds 1–6 (each 2.0 mg) were hydrolyzed with 1 M HCl for 5 h at 85 C. The reaction product was dissolved in H2O after evaporation, and then extracted with CH2Cl2 for three times. After being concentrated to dryness, the aqueous residue was added into 0.5 mL anhydrous pyridine containing 2.0 mg L-cysteine methyl ester hydrochloride, and heated at 60 C for 1 h. Then, o-tolyl isothiocyanate (5 mL) was added and heated at 60 C for another 1 h (Tanaka et al., 2007). Subsequently, the reaction mixture was directly analyzed by HPLC under the following conditions: an Agilent 1260 chromatograph equipped with a Phenomenex column (250 4.6 mm, 5 mm); column temperature: 35 C; mobile phase: isocratic elution of 25% MeCN H2O (V:V) containing 0.08% formic acid; flow rate: 0.8 mL/min; UV detection wavelength: 250 nm. The tR values (min) of standard monosaccharide derivatives prepared in the same way were 18.9 (D-Gal), 20.5 (LGlc), 21.5 (D-Glc), 24.2 (L-Ara), 25.3 (D-Xyl) min, respectively. By comparison of the retention times, D-Glc, L-Ara and D-Xyl were identified from compound 1, D-Gal, D-Glc and D-Xyl from compound 2, D-Glc and D-Xyl from compounds 3 and 5, and DGlc and L-Ara from compounds 4 and 6. 3.5. Cell culture and viability detection BV-2 microglial cells were from Peking Union Medical College, Cell Bank (Beijing, China). Previous literature (Zeng et al., 2010) had described cell maintenance, experimental procedures, and viability assay. Briefly, BV-2 cells were seeded into 48-well culture plates (5.0 104 cells per well), then challenged with different compounds and LPS (1.0 mg/mL, Escherichia coli 0111:B4, Sigma, MO, USA). MTT method was used to detect the cell viability. BV-2 cells were incubated with 500 mL MTT solution (0.5 mg/mL in culture medium) at 37 C for 4 h, then the supernatant was removed and the residue was dissolved in 500 mL DMSO. The absorbance (540 nm) was detected by using a microplate reader (Tecan Trading AG, Switerland). Data are presented as mean S.D. (n = 3) for three independent experiments. 3.6. Anti-inflammatory activity assay The anti-inflammatory effects were investigated by detecting the production of nitric oxide (NO), which was detected by Griess Reagent (Green et al., 1982). Briefly, BV-2 cells (5.0 104 cells per well) were treated with LPS (1 mg/mL) and different concentrations of compounds for 24 h. Then, cell culture supernatants (300 mL) were collected and reacted with 100 mL of Griess reagent (0.1% naphthylethylene diamine dihydrochloride/1% sulfanilamide/2% phosphoric acid). After incubation for 10 min at room temperature, the optical density was detected at 540 nm using a microplate reader (Tecan Trading AG, Switzerland). The experiments were executed in parallel three times. The IC50 value, means the sample concentration resulting in a 50% inhibition of NO production, was calculated from the concentration–response curves which were generated by Graph Pad Prism 5 software (Graph Pad Software, Inc., San Diego, California). Quercetin was used as a positive control. Conflict of interest statement The authors have declared no conflict of interest.
Acknowlegement This work was financially supported by the National Natural Science Foundation of China (No. 21372015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2016.11.011. References Bodakhe, K.S., Namdeo, K.P., Bodakhe, S.H., Pandey, D.P., 2013. A new flavonol glycoside from Kalancho epinnata leaves. Asian J. Chem. 25, 9763–9765. Chen, W.H., Qi, H.Y., Shi, Y.P., 2009. 24-hydroxyoleanane-type triterpenes from the aerial parts and roots of Oxytropis falcata. J. Nat. Prod. 72, 1410–1413. Chen, W.H., Wang, R., Shi, Y.P., 2010a. Flavonoids in the poisonous plant Oxytropis falcata. J. Nat. Prod. 73, 1398–1403. Chen, W.H., Wu, Q.X., Wang, R., Shi, Y.P., 2010b. Oxytrofalcatins A-F, N-benzoylindole analogues from the roots of Oxytropis falcata (Leguminosae). Phytochemistry 71, 1002–1006. Chen, Z.P., Qu, M.M., Chen, H.X., Liu, D., Xiao, Y.Y., Chen, J., Lu, T.L., Cai, B.C., 2011. The studies of anti-inflammatory and analgesic activities and pharmacokinetics of Oxytropis falcata Bunge extraction after transdermal administration in rats. Fitoterapia 82, 426–433. Chokchaisiri, R., Innok, P., Suksamrarn, A., 2012. Flavonoid glycosides from the aerial part of Curcuma Comosa. Phytochem. Lett. 5, 361–366. Committee of Chinses Pharmacopoeia, 2010. Chinese Pharmacopoeia. Chinese Medical Science and Technology Press, Beijing. Diamer, D.Z.P.C., 1986. Jingzhu Materia Medica. Shanghai Science and Technology Press, Shanghai. Gong, H.F., Yang, A.M., Liu, J.X., Di, D.L., 2010. Studies on chemical constituents of Oxytropis kansuensis. Chin. Tradit. Herbs Drugs 41, 187–190. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S. R., 1982. Analysis of nitrate, nitrite,and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Halabalaki, M., Urbain, A., Paschali, A., Mitakou, S., Tillequin, F., Skaltsounis, A.L., 2011. Quercetin and kaempferol 3-O-[a-L-Rhamnopyranosyl-(1 ! 2)-a-Larabinopyranoside]-7-O-a-L-rhamnopyranosides from anthyllis hermanniae: structure determination and conformational studies. J. Nat. Prod. 74, 1939– 1945. Kelsang, N., Que, S., Zhang, X.J., Samnor Liang, H., Zhang, Q.Y., 2014. Highperformance liquid chromatographic fingerprint analysis of Oxytropis falcata Bunge and Oxytropis chiliophylla Royle. J. Chin. Pharm. Sci. 23, 783–789. Kumar, M., Ahmad, A., Rawat, P., Khan, M.F., Rasheed, N., Gupta, P., Sathiamoorthy, B., Bhatia, G., Palit, G., Maurya, R., 2010. Antioxidant flavonoids from Evolvulus alsinoides. Fitoterpia 81, 234–242. Leitao, G.G., Soares, S.S.V., Brito, T.B.M., Monache, F.D., 2000. Kaempferol glycosides from Siparuna apiosyce. Phytochemistry 55, 679–682. Lu, C.L., Zhou, G.X., Wang, H.S., Li, Y.L., Yao, X.S., 2009. Studies on the chemical constituents of Nerviliae fordii. J. Chin. Med. Mater. 32, 373–375. Lu, C.L., Zhou, G.X., Wang, H.S., Ye, W.C., Yao, X.S., 2010. Water-soluble constituents from Nerviliae fordii. Lishizhen Med. Mater. Med. Res. 21, 3087–3088. Marco, J.A., Barbera, O., Sanz, J.F., Parareda, J.S., 1986. A new flavonol diglycoside from anthyllis onobrychioides. J. Nat. Prod. 49, 151–152. Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of aldose enantiomers by reversed-phased HPLC. Chem. Pharm. Bull. 55, 899–901. The Editorial Committee of the Administration Bureau of Flora of China, 2005. Flora of China, 42 vols. Beijing Science and Technology Press, Beijing. Wang, H., Leach, D.N., Forster, P.I., Waterman, P.G., 2008. Secondary metabolites from Grevillea robusta. Biochem. Syst. Ecol. 36, 452–453. Wang, S.S., Zhang, X.J., Que, S., Tu, G.Z., Wan, D., Cheng, W., Liang, H., Ye, J., Zhang, Q. Y., 2012. 3-Hydroxy-3-Methylglutaryl flavonol glycosides from Oxytropis falcata. J. Nat. Prod. 75, 1359–1364. Yang, G.M., Wang, D., Tang, W., Chen, X., Fan, L.Q., Zhang, F.F., Yang, H., Cai, B.C., 2010. Anti-inflammatory and antioxidant activities of Oxytropis falcata fractions and its possible anti-inflammatory mechanism. Chin. J. Nat. Med. 8, 285–292. Yao, S.Y., Ma, Y.B., Tang, Y., Chen, J.J., Zhang, X.M., 2007. Chemical constituents of Oxytropis chiliophylla. China. J. Chin. Mater. Med. 32, 1660–1662. Zahid, M., Ishrud, O., Pan, Y.J., Asim, M., Riaz, M., Ahmad, V.U., 2002. Flavonoid glycosides from Salvia moorcroftiana wall. Carbohyd. Res. 337, 403–407. Zeng, K.W., Fu, H., Liu, G.X., Wang, X.M., 2010. Icariin attenuates lipopolysaccharideinduced microglial activation and resultant death of neurons by inhibiting TAK1/IKK/NF-kB and JNK/p38 MAPK pathways. Int. Immunopharmacol. 10, 668–678. Zhang, X.J., Li, L.Y., Kelsang, N., Wang, S.S., Que, S., Cheng, W., Liang, H., Zhao, Y.Y., Zhang, Q.Y., 2014. Flavonoids from Tibetan medicine Oxytropis falcata Bunge. J. Chin. Pharm. Sci. 23, 99–105.
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Short communication
Rhamnocitrin glycosides from Oxytropis chiliophylla Jun Wang, Yang Liu, Norbo Kelsang, Kewu Zeng, Hong Liang, Qingying Zhang* , Pengfei Tu State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, PR China
A R T I C L E I N F O
Article history: Received 20 September 2016 Received in revised form 10 November 2016 Accepted 21 November 2016 Available online xxx Keywords: Oxytropis Oxytropis chiliophylla Tibetan medicines Rhamnocitrin glycosides Anti-inflammatory activity
A B S T R A C T
Repeated chromatography of the n-BuOH soluble fraction from ethanol extract of the aerial part of Oxytropis chiliophylla led to the isolation of six new rhamnocitrin glycosides, named oxychilioflavonosides AF (1–6), together with eight known rhamnocitrin glycosides (7–14) of which six were isolated from the genus Oxytropis for the first time. The structures of these compounds were elucidated by extensive spectroscopic techniques and chemical methods. All isolated compounds were evaluated for their anti-inflammatory activity in lipopolysaccharide (LPS)-induced BV-2 microglial cells, and compound 11 showed potent inhibition on NO production with IC50 value of 9.91 mM. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Oxytropis chiliophylla Royle (Leguminosae), along with Oxytropis falcata Bunge, is officially documented as the botanical origin of Tibetan medicine “Er-Da-Xia” that is known as “King of Herbs” and wildly used for the treatment of inflammation, pyreticsis and bleeding (Diamer, 1986; Committee of Chinses Pharmacopoeia, 2010). Inhabited at 2800–5200 m altitude, O. chiliophylla is mainly distributed in ravines, on slopes, in steppe meadows and shrubberies in Tibet Autonomous Region and Xinjiang Urgur Autonomous Region in China (The Editorial Committee of the Administration Bureau of Flora of China, 2005). Many chemical and pharmacological investigations focusing on O. falcata had been carried out previously, indicating that flavonoids (Zhang et al., 2014; Chen et al., 2010a), oleanane-type triterpenoids (Chen et al., 2009) and N-benzoylindole analogues (Chen et al., 2010b) were the principle constituents, among which flavonoids exhibited significant anti-inflammatory and analgesic activities (Yang et al., 2010; Chen et al., 2011). However, investigations on O. chiliophylla were rarely few and only one paper up to date reported the isolation of one triterpenoid, three flavonoids and four other compounds from the herb (Yao et al., 2007). Our comparative fingerprint analysis of O. chiliophylla and O. falcata using high performance liquid chromatography (HPLC) coupled with diode array detector
* Corresponding author. E-mail address: [email protected] (Q. Zhang).
(DAD) revealed the consistency of both herbs in main components of less polar portion and the slight differences in constituents of high polar portion (Kelsang et al., 2014). Flavonol 3-O-glycosides, especially 3-hydroxy-3-methylglutaryl (HMG) rhamnocitrin 3-Oglycosides, proved to be the characteristic and principle constituents of n-BuOH soluble fraction of ethanol extract of O. falcata (Wang et al., 2012). The present research focusing on the characteristic flavonoids of n-BuOH soluble fraction from ethanol extract of O. chiliophylla resulted in the discovery of six new rhamnocitrin glycosides, oxychilioflavonosides AF (1–6) (Fig. 1), together with eight known rhamnocitrin glycosides (7–14) of which six were isolated from the genus Oxytropis for the first time. All the flavonoid glycosides obtained were different from those of O. falcata. Furthermore, their anti-inflammatory activities against NO production using LPS-induced BV-2 microglial cells were also evaluated. 2. Results and discussions Compound 1 was isolated as an amorphous yellow powder, and its molecular formula was determined to be C32H38O19 by the quasimolecular ion peak [M+H]+ at m/z 727.2100 (calcd 727.2086) in HRESIMS. The UV spectrum showed maximum absorption bands at 266 and 340 nm, suggesting a flavonol skeleton. The 1H (Table 1) and 13C NMR (Table 2) data exhibited characteristic signals of 5,7,40 -trisubstituted flavonol glycoside, with one conjugated phenolic hydroxy at dH 12.49 (1H, s), one methoxyl group at dH 3.87 (3H, s), and three sugar units evidenced by signals
http://dx.doi.org/10.1016/j.phytol.2016.11.011 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54
51
Fig. 1. Chemical structures for compounds 1–6.
of three anomeric carbons at dC 101.3, 100.0 and 103.9 and their corresponding anomeric protons at dH 5.37 (d, J = 5.2 Hz, H-100 ), 4.98 (d, J = 7.2 Hz, H-1000 ) and 4.20 (d, J = 7.5 Hz, H-10000 ). The methoxyl group was assigned to C-7 of the aglycone by the HMBC correlations between dH 3.87 (OCH3) and dC 165.3 (C-7). Based on the above evidences, the aglycone of 1 was identified as rhamnocitrin, ie. 5, 40 -dihydroxy-7-methoxy flavonol, which was Table 1 1 H NMR data for compounds 1–6 No 3-OH 5-OH 6 8 20 /60 30 /50 7-OMe 3-O 100 200 300 400 500
1 12.49 s 6.38 d (1.8) 6.80 d (1.8) 8.19 d (8.9) 7.20 d (8.9) 3.87 s Ara 5.37 d (5.2) 3.75 c 3.52 c 3.663.69 b 3.63–3.66 b 3.23–3.26 b
600 40 -O 1000 2000 3000 4000 5000 6000
100 00 200 00 300 00 400 00 500 00 600 00
a b c
Glc 4.98 d (7.2) 3.26–3.30 b 3.26–3.30 b 3.19–3.23 b 3.57–3.63 b 3.97 d (10.2) 3.57–3.63 b Xyl 4.20 d (7.5) 3.01 t (8.2) 3.10 t (8.8) 3.26–3.30 b 2.94 t (10.8) 3.66–3.69 b
a
confirmed by acid hydrolysis. The three sugar units were identified as a-L-arabinose, b-D-glucose and b-D-xylose by the coupling patterns of anomeric protons (Halabalaki et al., 2011) and acid hydrolysis results. The arabinose and glucose moieties were linked to the aglycone at C-3 and C-40 , respectively, and the xylose unit was linked to C-6 of glucose, as indicated by the HMBC correlations of dH 5.37 (H-100 )/dC 134.4 (C-3), dH 4.98 (H-1000 )/dC 159.3 (C-40 ), and
in DMSO-d6. 2
3
12.53 s 6.39 d (2.0) 6.79 d (2.0) 8.17 d (8.9) 7.17 d (8.9) 3.87 s Gal 5.43 d (7.6) 3.53–3.55 b 3.29–3.39 b 3.63–3.69 b 3.29–3.39 b
12.55 s 6.39 d (1.5) 6.78 d (1.5) 8.14 d (8.8) 7.19 d (8.8) 3.87 s Glc 5.50 d (7.2) 3.21–3.25 b 3.21–3.25 b 3.00–3.11 b 3.00–3.11 b
3.39–3.45 b 3.29–3.39 b Glc 4.95 d (7.5) 3.29–3.39 b 3.29–3.39 b 3.20 c 3.57–3.59 b 3.97 d (9.8) 3.57–3.59 b Xyl 4.19 d (7.3) 2.98–3.01 b 3.08 t (8.6) 3.19–3.29 b 2.95 t (10.7) 3.63–3.69 b
3.58–3.61 b 3.36–3.38 b Glc 4.98 d (7.7) 3.25–3.30 b 3.25–3.30 b 3.21–3.25 b 3.58–3.61 b 3.98 d (10.0) 3.58–3.61 b Xyl 4.19 d (7.5) 2.98–3.00 b 3.08–3.11 b 3.25–3.30 b 2.97 t (10.8) 3.68 dd (11.1, 5.2)
4
5
12.58 s 6.39 d (2.0) 6.82 d (2.0) 8.20 d (8.9) 7.22 d (8.9) 3.87 s Ara 5.38 d (5.2) 3.75 c 3.53 c 3.64–3.66 b 3.58–3.64 b 3.23–3.25 b
Glc 4.99 d (7.0) 3.30–3.38 b 3.303.38 b 3.07 t (9.2) 3.64–3.66 b 4.00 d (11.0) 3.64–3.66 b Glc 4.24 d (7.7) 2.99–3.02 b 2.99–3.02 b 3.13–3.18 b 3.13–3.18 b 3.64–3.66 3.40–3.43
Measured at 500 MHz for 5 and 400 MHz for 1–4, 6, dH in ppm, J in Hz. Overlapped with other signals. Multiplicities can not be clearly defined; Ara = arabinose, Glc = glucose, Gal = galactose, Xyl = xylose.
b b
9.64 s 12.41 s 6.36 d (1.5) 6.79 d (1.5) 8.17 d (8.9) 7.23 d (8.9) 3.87 s
Glc 4.96 d (7.4) 3.30–3.32 b 3.30–3.32 b 3.17 c 3.58–3.60 b 3.96 d (9.8) 3.58–3.60 b Xyl 4.19 d (7.6) 2.97–3.00 b 3.07–3.10 b 3.30–3.32 b 2.94 t (11.0) 3.66 dd (11.0, 5.0)
6 12.54 s 6.37 d (1.8) 6.76 d (1.8) 8.21 d (8.9) 7.16 d (8.9) 3.86 s Ara 5.38 d (5.1) 3.70–3.74 b 3.51–3.53 b 3.66–3.70 b 3.57 dd (11.5, 5.4) 3.23 dd (11.5, 1.8)
Glc 5.03 d (7.2) 3.30–3.35 b 3.30–3.35 b 3.18–3.20 b 3.38–3.42 b 3.70–3.74 b 3.48 dd (11.5, 5.8)
52
J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54
Table 2 13 C NMR data for compounds 1–6
a
in DMSO-d6.
No
1
2
3
4
5
6
2 3 4 5 6 7 8 9 10 10 20 /60 30 /50 40 7-OCH3 3-O 100 200 300 400 500 600 40 -O 1000 2000 3000 4000 5000 6000
156.0 134.4 177.8 160.9 98.0 165.3 92.5 156.4 105.1 123.5 130.8 116.1 159.3 56.2 Ara 101.3 70.8 71.6 66.2 64.4
156.2 134.0 177.8 160.9 98.1 165.3 92.5 156.5 105.1 123.7 130.9 116.0 159.3 56.2 Gal 101.5 71.2 73.1 68.0 75.9 60.2 Glc 100.2 73.2 76.4 69.6 76.0 68.2 Xyl 103.9 73.5 76.6 69.6 65.7
156.2 134.0 177.8 161.0 98.1 165.3 92.5 156.5 105.2 123.7 130.8 116.0 159.3 56.2 Glc 100.7 74.2 76.5 69.9 77.7 60.9 Glc 100.0 73.2 76.4 69.6 76.0 68.2 Xyl 103.9 73.5 76.6 69.6 65.7
156.1 134.3 177.8 160.9 98.1 165.3 92.5 156.4 105.1 123.4 130.9 116.1 159.3 56.2 Ara 101.3 70.8 71.6 66.2 64.3
146.6 136.5 176.2 160.3 97.5 165.0 92.2 156.2 104.1 124.3 129.3 116.2 158.5 56.1
156.0 134.5 177.9 161.0 98.1 165.4 92.5 156.5 105.2 123.5 130.9 116.0 159.5 56.3 Ara 101.4 70.9 71.7 66.2 64.4
Glc 99.9 73.2 76.5 70.1 76.1 68.3 Glc 103.3 73.7 76.9 69.7 76.7 61.0
Glc 99.9 73.1 76.5 69.6 76.1 68.1 Xyl 103.9 73.4 76.5 69.6 65.6
Glc 100.0 73.3 76.7 69.8 77.2 60.8
100 00 200 00 300 00 400 00 500 00 600 00
Glc 100.0 73.2 76.4 69.6 76.1 68.1 Xyl 103.9 73.5 76.5 69.6 65.7
a Measured at 125 MHz for 5 and 100 MHz for 1–4, 6, dC in ppm; Ara = arabinose, Glc = glucose, Gal = galactose, Xyl = xylose.
dH 4.20 (H-10000 )/dC 68.1 (C-6 of glucose). Thus, compound 1 was identified as rhamnocitrin-3-O-a-L-arabinopyranosyl-40 -O-b-Dxylopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside A. Compound 2 was obtained as an amorphous yellow powder. The molecular formula C33H40O20 was established by HRESIMS ion peak at m/z 757.2192 [M+H]+ (calcd 757.2191). The UV, 1H and 13C NMR data were similar to those of compound 1. The only difference between compounds 1 and 2 involved the substitution of a galactose unit at C-3 of the aglycone in 2 instead of the arabinose unit in 1, which was consistent with acid hydrolysis results that yielded D-galactose, D-glucose, D-xylose and rhamnocitrin. The HMBC correlation between the anomeric proton of galactose unit at dH 5.43 (H-100 ) and dC 134.0 (C-3) further confirmed the above conclusion. The b configuration of the galactose moiety was assigned from the large coupling constant (7.6 Hz) of the anomeric proton. On the basis of above evidences, the structure of 2 was characterized as rhamnocitrin-3-O-b-D-galactopyranosyl-40 -Ob-D-xylopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside B. Compound 3, isolated as an amorphous yellow powder, had the same molecular formula as 2, C33H40O20, on the basis of HRESIMS at m/z 757.2189 [M + H]+ (calcd 757.2191). The 1H and 13C NMR data of 3 were similar to those of 2 except for a glucose unit located at C3 of the aglycone in 3 replacing the galactose moiety in 2, which was supported by acid hydrolysis results that only yield D-glucose, D-xylose and rhamnocitrin. The b configuration of D-glucose and Dxylose was determined using the same method mentioned above. Therefore, the structure of compound 3 was identified as rhamnocitrin-3-O-b-D-glucopyranosyl-40 -O-b-D-xylopyranosyl-
(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside C. Compound 4, a yellow amorphous powder, gave a molecular formula of C33H40O20, the same as compounds 2 and 3, by HRESIMS at m/z 757.2188 [M+H]+ (calcd 757.2191). Acid hydrolysis and coupling patterns of anomeric protons confirmed the presence of b-D-glucose, a-L-arabinose and rhamnocitrin, and their linkages were determined by careful analysis of 2D NMR correlations. Unambiguously, compound 4 was assigned as rhamnocitrin-3-Oa-L-arabinopyranosyl-40 -O-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside, and it was named oxychilioflavonoside D. Compound 5 was a yellow, amorphous powder. The HRESIMS showed a quasimolecular ion peak at m/z 595.1678 [M+H]+ (calcd 595.1663) indicating the molecular formula of C27H30O15. Its 1H and 13C NMR data differed from those of 1 by the absence of a set of arabinose signals, which was consistent with acid hydrolysis results that yielded only D-glucose, D-xylose and rhamnocitrin. Further analysis of the 2D NMR spectra confirmed the structure of compound 5 to be rhamnocitrin-40 -O-b-D-xylopyranosyl-(1 ! 6)b-D-glucopyranoside, and it was named oxychilioflavonoside E. Compound 6, a yellow amorphous powder, had a molecular formula of C27H30O15, the same as compound 5, by HRESIMS at m/z 595.1671 [M+H]+ (calcd 595.1663). The presence of b-D-glucose, a-L-arabinose and rhamnocitrin were confirmed by acid hydrolysis and their attachments were determined by 2D NMR correlations. Consequently, compound 6 was characterized as rhamnocitrin-3O-a-L-arabinopyranosyl-40 -O-b-D-glucopyranoside, and it was named oxychilioflavonoside F. The eight known compounds were identified as rhamnocitrin3-O-b-D-galactopyranosyl-40 -O-b-D-glucopyranoside (7) (Marco et al., 1986), rhamnocitrin-3-O-b-D-galactopyranoside (8) (Gong et al., 2010), rhamnocitrin-3-O-a-L-arabinopyranoside (9) (Chokchaisiri et al., 2012), rhamnocitrin-3-O-b-D-glucopyranoside (10) (Lu et al., 2009), rhamnocitrin-40 -O-b-D-glucopyranoside (11) (Lu et al., 2009), rhamnocitrin-3-O-rutinoside (12) (Wang et al., 2008), rhamnocitrin-3-O-b-D-glucopyranosyl-40 -O-b-D-glucopyranoside (13) (Lu et al., 2010), rhamnocitrin-3-O-a-L-rhamnopyranosyl(1 ! 6)-b-D-galactopyranoside (14) (Bodakhe et al., 2013), by comparison of the spectral data with those previously reported in the literatures. Compounds 9–14 were isolated from the genus Oxytropis for the first time, and compounds 7–8 were obtained from this herb for the first time. The present research definitely confirmed the differences between the constituents from high polar fraction of O. chiliophylla and those of O. falcata. From the n-BuOH soluble fraction of ethanol extract of O. falcata, a series of rhamnocitrin 3-O-glycosides and kaempferol 3-O-glycosides, a few of which were further glycosylated at C-40 by a glucose unit or at C-7 by a rhamnose unit, had been obtained. The sugar chains located at C-3 of the aglycone consisted of one to four sugar units of galactose, rhamnose and xylose and the obvious characteristic was that the galactose moiety was always linked directly to the aglycone. In addition, a HMG group was occasionally conjugated to C-6, C-4 or C-3 of galactose, and the HMG conjugated rhamnocitrin 3-O-glycosides might be considered as the characteristic chemical markers of O. falcata. However, the constituents from n-BuOH soluble fraction of O. chiliophylla obtained herein were totally different from those of O. falcata. All the flavonoid glycosides of O. chiliophylla were derived from the same aglycone of rhamnocitrin, while the sugars linked directly to C-3 of rhamnocitrin were varied, including galactose, glucose and arabinose. Moreover, flavonol-40 -O-diglycosides, which were not so popular in the plant kingdom, were relatively abundant in O. chiliophylla, and the disaccharide chains, i.e. xylosyl-(1 ! 6)-glucose or gentiobiose, were different from those previously reported (Leitao et al., 2000; Zahid et al., 2002; Kumar
J. Wang et al. / Phytochemistry Letters 19 (2017) 50–54 Table 3 Anti-inflammatory data for compounds 1–14. Compounds
IC50 (mM)
Compounds
IC50 (mM)
1 2 3 4 5 6 7 Quercetin
>100 >100 >100 >100 >100 >100 >100 5.31 0.03
8 9 10 11 12 13 14
>100 31.70 2.26 >100 9.91 0.07 >100 >100 >100
a
a
Quercetin was used as positive control for anti-inflammatory activity.
et al., 2010). What’s more, no HMG conjugated rhamnocitrin glycosides were obtained from O. chiliophylla. All the isolated compounds were further evaluated for their anti-inflammatory activity through LPS-induced NO production in BV-2 microglial cells. Colorimetric MTT assay was firstly used to test cell viability, and it was confirmed that all tested compounds had no or little cytotoxicity at the concentration tested (data not shown). Rhamnocitrin-40 -O-b-D-glucopyranoside (11) exhibited potent inhibitory activity with IC50 value of 9.91 mM, rhamnocitrin-3-O-a-L-arabinopyranoside (9) had weak inhibitory activity with IC50 value of 31.70 mM, while the others showed no remarkable effect with IC50 values greater than 100 mM (Table 3). The reasons why rhamnocitrin monoglycoside exhibited more potential activity than diglycosides and triglycosides might be attributed to the lipophilic nature that might result in the increasing of cellular penetration. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured with Rudolph Research Analytical Autopol IV automatic polarimeter. UV spectra were obtained using a Shimadzu UV-260 spectrometer. NMR spectra were recorded on a Bruker AVANCE III-400/600 or a Varian INOVA500 instrument with TMS as internal standard. HRESIMS was carried out on an Agilent QTOF 6538 or a Abseciex QSTAR spectrometer. HPLC analysis was performed on an Agilent 1260 LC system with a Phenomenex column (250 4.6 mm, 5 mm). HPLC separations were performed on an Alltech semi-preparative instrument equipped with a YMC-Pack ODS-A column (250 10 mm, 5 mm). Column chromatography (CC) was performed on Diaion HP20 (200–300 mesh, Mitsubishi Chemical Co., Japan), silica gel (200–300 mesh, Qingdao Marine Chemical Inc.), ODS-A (50 mm, YMC Co. Ltd., Japan) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). Analytical TLC was carried out on silica GF254 (10–40 mm, Qingdao Marine Chemical, Inc.), and spots were observed by UV light and 10% H2SO4-EtOH reagent. HPLC grade solvents used for HPLC analysis and separations were purchased from Fisher Scientific International (Fair Lawn, New Jersey, USA), and deionized water was purified by Milli-Q Synthesis A10 (Bedford, MA). Other solvents used for extraction and isolation were of analytical grade and purchased from Beijing Tongguang Chemicals (China). All chemicals were purchased from J & K Co. Ltd. (China). 3.2. Plant material The whole plant of O. chiliophylla was collected from Langkazi County, Tibet Autonomous Region, China, in September 2012, and was authenticated by Associate Professor Ying-Tao Zhang, Department of Natural Medicines, School of Pharmaceutical Science, Peking University Health Science Center. A voucher specimen (No
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20120901) was deposited in the Herbarium of Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center. 3.3. Extraction and isolation The aerial part of O. chiliophylla (10.0 kg) was pulverized and in turn extracted with 95% EtOH and 50% EtOH for three times. After removal of solvent under reduced pressure, the residue was dissolved in water and then partitioned successively with petroleum ether, EtOAc and n-BuOH, respectively. The n-BuOH extract (250 g) was separated by chromatography on Dianion HP20 macroporous absorbent resin using an EtOH/H2O gradient (0, 20, 40, 60, 80, 95% EtOH) to obtain six fractions (DK1 DK6). DK4 (eluted with 60% EtOH, 80 g) was subjected to silica gel CC eluted with CH2Cl2/CH3OH gradient system (10:1, 5:1, 3:1, 1:1, 1:5) to give six subfractions (DK4–1 DK4-6). DK4-3 (13.2 g) was purified by chromatography on ODS CC with a gradient system of MeOH/H2O (30, 50, 70, 100% MeOH) to yield four subfractions (DK4-3A DK4-3-D). Further purification of DK4-3-A (3.0 g) by CC on Sephadex LH-20 (MeOH/H2O, 1:1) and semi-preparative HPLC (MeCN H2O, 23:77) afforded compounds 1 (100 mg), 6 (500 mg), 7 (10 mg) and 8 (12 mg). Compound 9 (200 mg) was obtained by further separation of fraction DK4-3-B (1.0 g) on repeated Sephadex LH-20 CC (MeOH/H2O, 4:1). Fraction DK4-4 (20.0 g) was purified by CC over Sephadex LH-20 eluted with MeOH/H2O (30, 50, 70, 100% MeOH) to yield four subfractions (DK4-4A DK4-4-D). Further separation of DK4-4-B by repeated Sephadex LH-20 CC (MeOH/H2O, 1:1) and isocratic semipreparative HPLC (MeCN H2O, 20:80) afforded compounds 2 (18 mg), 3 (30 mg) and 4 (7 mg). DK3 (eluted with 60% EtOH, 10.0 g) was separated by chromatography on ODS CC with a gradient system of MeOH/H2O (30, 50, 70, 100% MeOH) to give three subfractions (DK3–1 DK33), and compounds 5 (3 mg), 10 (10 mg), 11 (20 mg), 12 (30 mg), 13 (8 mg), 14 (7 mg) were afforded through further purification of fraction DK3-3 by repeated Sephadex LH-20 CC (MeOH/H2O, 1:1) and then isocratic semi-preparative HPLC (MeCN/H2O, 30:70). 25 Oxychilioflavonoside A (1): amorphous, yellow powder; ½a D 55.6 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 727.2100 [M+H]+ (calcd 727.2086); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside B (2): amorphous, yellow powder; ½a D 75.8 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2192 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside C (3): amorphous, yellow powder; ½a D 47.7 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2189 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside D (4): amorphous, yellow powder; ½a D 79.1 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS (positive mode) m/z 757.2188 [M+H]+ (calcd 757.2191); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside E (5): amorphous, yellow powder; ½a D 71.3 (c 0.1, MeOH); UV (MeOH) lmax 266, 360 nm; HRESIMS (positive mode) m/z 595.1678 [M+H]+ (calcd 595.1663); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 25 Oxychilioflavonoside F (6): amorphous, yellow powder; ½a D 55.6 (c 0.1, MeOH); UV (MeOH) lmax 266, 340 nm; HRESIMS
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(positive mode) m/z 595.1671 [M+H]+ (calcd 595.1663); 1H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2. 3.4. Determination of absolute configurations of sugar moieties Compounds 1–6 (each 2.0 mg) were hydrolyzed with 1 M HCl for 5 h at 85 C. The reaction product was dissolved in H2O after evaporation, and then extracted with CH2Cl2 for three times. After being concentrated to dryness, the aqueous residue was added into 0.5 mL anhydrous pyridine containing 2.0 mg L-cysteine methyl ester hydrochloride, and heated at 60 C for 1 h. Then, o-tolyl isothiocyanate (5 mL) was added and heated at 60 C for another 1 h (Tanaka et al., 2007). Subsequently, the reaction mixture was directly analyzed by HPLC under the following conditions: an Agilent 1260 chromatograph equipped with a Phenomenex column (250 4.6 mm, 5 mm); column temperature: 35 C; mobile phase: isocratic elution of 25% MeCN H2O (V:V) containing 0.08% formic acid; flow rate: 0.8 mL/min; UV detection wavelength: 250 nm. The tR values (min) of standard monosaccharide derivatives prepared in the same way were 18.9 (D-Gal), 20.5 (LGlc), 21.5 (D-Glc), 24.2 (L-Ara), 25.3 (D-Xyl) min, respectively. By comparison of the retention times, D-Glc, L-Ara and D-Xyl were identified from compound 1, D-Gal, D-Glc and D-Xyl from compound 2, D-Glc and D-Xyl from compounds 3 and 5, and DGlc and L-Ara from compounds 4 and 6. 3.5. Cell culture and viability detection BV-2 microglial cells were from Peking Union Medical College, Cell Bank (Beijing, China). Previous literature (Zeng et al., 2010) had described cell maintenance, experimental procedures, and viability assay. Briefly, BV-2 cells were seeded into 48-well culture plates (5.0 104 cells per well), then challenged with different compounds and LPS (1.0 mg/mL, Escherichia coli 0111:B4, Sigma, MO, USA). MTT method was used to detect the cell viability. BV-2 cells were incubated with 500 mL MTT solution (0.5 mg/mL in culture medium) at 37 C for 4 h, then the supernatant was removed and the residue was dissolved in 500 mL DMSO. The absorbance (540 nm) was detected by using a microplate reader (Tecan Trading AG, Switerland). Data are presented as mean S.D. (n = 3) for three independent experiments. 3.6. Anti-inflammatory activity assay The anti-inflammatory effects were investigated by detecting the production of nitric oxide (NO), which was detected by Griess Reagent (Green et al., 1982). Briefly, BV-2 cells (5.0 104 cells per well) were treated with LPS (1 mg/mL) and different concentrations of compounds for 24 h. Then, cell culture supernatants (300 mL) were collected and reacted with 100 mL of Griess reagent (0.1% naphthylethylene diamine dihydrochloride/1% sulfanilamide/2% phosphoric acid). After incubation for 10 min at room temperature, the optical density was detected at 540 nm using a microplate reader (Tecan Trading AG, Switzerland). The experiments were executed in parallel three times. The IC50 value, means the sample concentration resulting in a 50% inhibition of NO production, was calculated from the concentration–response curves which were generated by Graph Pad Prism 5 software (Graph Pad Software, Inc., San Diego, California). Quercetin was used as a positive control. Conflict of interest statement The authors have declared no conflict of interest.
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