Chemical Constituents from Gnaphalium Affine and Their Xanthine Oxidase Inhibitory Activity

Chinese Journal of

Natural Chinese Journal of Natural Medicines 2018, 16(5): 03470353 Medicines

Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity

ZHANG Wei1, 2, WU Chun-Zhen1, 3, FAN Si-Yang 1, 2*

1 Department of Traditional Chinese Medicine, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China; 2 Innovation Center of Traditional Chinese Medicine, China State Institute of Pharmaceutical Industry, Shanghai 201203, China; 3 Sinopharm Health Industry Research Co., Ltd., Shanghai 201203, China Available online 20 May, 2018

[ABSTRACT] Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (−) ethyl 1, 4-di-O-caffeoylquinate (1) and (−) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3−37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening −1 −1 assay. Among the tested compounds, 1 (IC50 11.94 μmol·L ) and 2 (IC50 15.04 μmol·L ) showed a good inhibitory activity. The current results supported the medical use of the plant.

[KEY WORDS] Gnaphalium affine; Compositae; Caffeoylquinate; Flavonoid; Xanthine oxidase inhibition [CLC Number] R284 [Document code] A [Article ID] 2095-6975(2018)05-0347-07

 vestigation of the aerial part of G. affine that showed a re- Introduction markable secondary metabolite pattern. Current study led to a Gnaphalium affine D. Don (Compositae), commonly finding of 37 compounds (Fig. 1) – flavonoids, caffe- known as cudweed or Ching Ming vegetable, is an annual oylquinates, phenolic acids and adenine derivatives, including species of the genus Gnaphalium. As the name described, the two new caffeoylquinates, (−) Ethyl 1, 4-di-O-caffeoylquinate species is extensively harvested around the Ching Ming Fes- (1), and (−) Methyl 1, 4-di-O-caffeoylquinate (2). Further- tival (Tomb Sweeping Day) and used to flavor the qingtuan more, xanthine oxidase inhibitory activity of these isolated (green dumplings) consumed by Chinese families. In tradi- compounds was also determined in the present study. tional Chinese medicine, G. affine has been used for the Results and Discussion treatment of cough, asthma, rheumatic arthritis, and gout for a [1] long time . Recent pharmacological studies has proven that Compound 1 was obtained as yellow powder and had the [2] this plant possesses anti-histamine, anti-bacterial , anti-fungal, molecular formula C27H28O12 (14 unsaturations) as deter- [3] [4] [5] antioxidant , anti-inflammatory , anti-complement , and mined by the HR-ESI-MS [M−H]- ion at m/z 543.149 9. In the [6] xanthine oxidase inhibitory activities. More than 77 chemical 1H NMR spectra, the two sets of aromatic signals were dif- constituents have been reported from this plant, including ferentiated as two 1, 2, 4-trisubstituted aromatic rings whose flavonoids, triterpenes, phytosterols, anthraquinones, caffe- protons resonated at H 7.04 (1H, d, J = 1.9 Hz ), 6.92 (1H, dd, [1] oylquinic acid derivatives, and other compounds . J = 8.2, 1.9 Hz) and 6.76 (1H, d, J = 8.2 Hz), and protons at In the present study, we conducted a phytochemical in- H 7.03 (1H, d, J = 1.9 Hz), 6.89 (1H, dd, J = 8.2, 1.9 Hz) and

6.74 (1H, d, J = 8.2 Hz). The signals at H 7.56 (1H, d, J =  [Received on] 30-July-2017 15.9 Hz), 6.27 (1H, d, J = 15.9 Hz), 7.55 (1H, d, J = 15.9 Hz) [Research funding] The work was supported by the Natural Science and 6.26 (1H, d, J = 15.9 Hz) were assigned to two trans Foundation of Shanghai (No. 15ZR1440100) and the National Natu- double bonds. These evidences suggested the presence of two ral Science Foundation of China (No. 81603279). [*Corresponding author] E-mail : [email protected] caffeoyl groups. The chemical shifts at H 5.63 (1H, ddd, J = These authors have no conflict of interest to declare. 5.3, 3.8, 3.3 Hz), 5.03 (1H, dd, J = 8.3, 3.3 Hz), 4.33 (1H, ddd, Published by Elsevier B.V. All rights reserved J = 8.5, 8.3, 4.4 Hz), and 2.11−2.36 (4H, m) indicated a

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Fig. 1 Structures of compounds 1-37 obtained from G. affine quinic acid moiety. Besides, the protons at 4.21 (2H, dq) and two diaxial and an axial-equatorial coupling with H-4, H-2ax, 1 1 1.31 (3H, t, J = 7.2 Hz) and their correlations in H- H COSY and H-2eq. Three J values (Jmedium = 5.3, Jsmall 1 = 3.8, Jsmall 2 = experiment indicated an ethoxyl group (Fig. 2). The HMBC 3.3 Hz) of H-5 indicated a diequatorial and two ax- cross peaks at H/C 5.03 (H-4)/168.53 (C-9'), and 4.21 ial-equatorial couplings with H-6eq, H-6ax, and H-4. Fur- (H-8)/175.69 (C-7) suggested the presence of a caffeoyl moi- thermore, its relative configuration was also confirmed by ety at C4-O and the ethoxyl group at C-7. Another caffeoyl NOESY spectrum, indicating that H-2ax (H 2.12) was corre- moiety was confirmed to link with C1-O by comparing the lated to H-8 (H 4.21) and H-4 (H 5.03), H-6ax (H 2.36) cor- chemical shifts of C-1 and H-3, 4, 5 with those of the known related to H-4 (H 5.03) and H-2ax (H 2.12). No cross peaks [7] 1, 4-Di-O-caffeoylquinic acid . The relative configuration of were observed between H-5 (H 5.63) and H-3 (H 4.33), H-3

1 was elucidated by first-order multiplet analysis of H-3, H-4, (H 5.03), and H-6 (H 2.36, 2.11). The results of the two and H-5 in 1HNMR spectroscopy (Fig. 3). Two J values of NMR methods suggested the β-configurations of C-4-H,

H-4 (Jlarge = 8.3, Jsmall = 3.3 Hz) indicated a diaxial and an C-5-H, C-3-OH and C-1-COOEt group. The absolute con- axial-equatorial coupling with H-3 and H-5. Three J values figuration of 1 was also determined by the negative specific 20 (Jlarge 1 ≈ Jlarge 2 ≈ 8.3, Jsmall = 4.4 Hz) of H-3 indicated rotation ([α]D –82.9°), which was in agreement with the re-

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20 time, and the full NMR, IR assignments and optical charac- ported data of (−) 1, 4-Di-O-caffeoylquinic acid ([α]D – 62° − [8] teristics were provided for the first time. 82°) . On the basis of the above evidences, the basic skele- 35 known compounds (Fig. 1) were isolated and identi- ton of 1 was proposed to be (−) Quinic acid ((1α, 3R, 4α, fied in the extract: 1, 3, 5-Tri-O-caffeoylquinic acid (3) [10], 5R)-1, 3, 4, 5-Tetrahydroxycyclohexanecarboxylic acid). Thus, Methyl 3, 5-di-O-caffeoylquinate (4) [11], 3, 4, 5-Tri-O-caffeo- the structure of 1 was established as (−) ethyl 1, 4-di-O-ca- ylquinic acid (5) [12], 1, 3, 4-Di-O-caffeoylquinic acid (6) [13], ffeoylquinate. To the best of our knowledge, compound 1 was Methyl 3, 4-di-O-caffeoylquinate (7) [14], Chlorogenic acid (8), a new ethyl ester of caffeoylquinic acid. 5-Hydroxy-3, 6, 7, 8-tetramethoxyflavone (9) [15], Gnaphaliin Compound 2 displayed a molecular formula of C26H26O12 [16] − A (10) , 5, 3'-Dihydroxy-3, 6, 7, 8, 4'-pentamethoxyflavone (14 unsaturations) by HR-ESIMS ([M − H] at m/z 529.134 7). (11) [17], Calycopterin (12) [18], 5, 4'-Dihydroxy-3, 7, Comparison of NMR spectra of 2 with those of 1 revealed an 8-trimethoxyflavone (13) [19-20], Galangin (14) [21], 3, 5, absence of ethoxyl group and the presence of methoxyl group 7-Trihydroxy-8-methoxyflavone (15) [22], 5, 7-Dihydroxy-3, 6, at C-7 of 2. The data of 2D−NMR (1H-1H COSY, HMQC and 8-trimethoxyflavone (16) [23], 5, 7-Dihydroxy-3, 8, 4'-trime- HMBC) experiments (Fig. 2) confirmed the structure of 2 to thoxyflavone (17) [24], 5-Hydroxy-3, 6, 7, 8, 3', 4'-hexame- be methyl 1, 4-di-O-caffeoylquinate. On the basis of NOESY thoxyflavone (18) [25], Apigenin (19), Luteolin (20) [26], Lute- experiment (Fig. 2) and first-order multiplet analysis (Fig. 4), olin-4'-O-β-D-glucoside (21) [27], Kaempferol (22), Quercetin α-configurations of caffeoyl moieties at C-1 and C-4, (23) [28], 4, 4', 6'-Trihydroxy-2'-methoxy-chalcone (24) [29], α-configuration of hydroxy group at C-5, and β-configuration Dibutyl phthalate (25) [30], 2-Ethylhexyl phthalate (26) [31], of hydroxy group at C-3 were confirmed. The negative spe- Ethyl 3, 4-dihydroxybenzoate (27) [32], Methylparaben (28) [33, 34], cific rotation of 2 ([α]20 – 70.9 °) was also in agreement with D Ethyl caffeate (29) [35], trans-Caffeic acid (30) [28], Methyl caf- the negative specific rotation of (-) 1, 4-Di-O-caffeoylquinic feate (31) [36], 1-O-caffeoyl-β-D-glucopyranose (32) [37], Gna- 20 [8] [5], acid ([α]–D 62 ° − 82 °) . Thus, the structure of 2 was iden- phaffine A (33) Adenosine (34), 5'-Deoxy-adenosine (35), [38] tified as (-) methyl 1, 4-di-O-caffeoylquinate. This compound Adenine (36), and Octadecanoic acid (37) . 14 compounds (4, has been analyzed from mixture by HPLC-MS/MS with the 6, 7, 11, 13, 14, 15, 25, 26, 28, 31, 32, 35, and 36) were firstly only MS data [9], but in our study, was isolated for the first reported from the Gnaphalium species.

Fig. 2 Key 1H-1H COSY, HMBCand NOEcorrelations of 1 and 2

Xanthine oxidase catalyzes the oxidation of hypoxan- Experimental thine and xanthine to uric acid. The enzyme plays a crucial role in hyperuricemia and gout. Due to the therapeutic appli- General experimental procedures cation for gout, all the compounds were tested against xan- Optical rotations were measured on an Anton Paar MCP thine oxidase. As shown in Table 2, compounds 1 − 7, 14, 19, 500 (Anton Paar, Graz, Austria) in MeOH. The circular di- 20, 22, 23, and 24 displayed a good activity. The introduction chroism spectra were obtained by a JASCO J-815 CD spec- of two or more caffeoyl groups in the quinic acid skeleton trometer (JASCO, Hachioji, Tokyo, Japan) in MeOH. IR gave an increase in potency. While methoxylation of fla- spectra were acquired using a Nicolet 6700 FTIR spec- vones had a negative influence. From the assay data, the trometer (Thermo Scientific, Madison, WI, USA). ESIMS components including polyhydroxyflavones, chalcone, and were performed in positive and negative ion mode on a caffeoylquinates might be responsible for the anti-gout ac- Q-TOF Micro YAO19 mass spectrometer (Waters, Milford, tivity. These results supported the traditional medicinal use MA, USA). High-resolution mass spectra (HR-ESI-MS) of G. affine. were obtained using a Xevo G2 QTOF MS, coupled with an

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Acquity I class Ultra Performance Liquid Chromatography CDCl3. Xanthine oxidase (ammonium sulfate suspension) system (Waters, Milford, MA, USA) . NMR experiments and uric acid (≥ 99%, HPLC) were purchased from were recorded on Bruker Avance DRX-400 (Bruker, Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Al- Waltham, MA, USA) and Varian INOVA-500 (Varian, Palo lopurinol (98%, HPLC) and xanthine (98%, HPLC) were

Alto, CA, USA) spectrometers in acetone-d6, CD3OD or obtained from J&K scientific co., Ltd. (Beijing, China).

Fig. 3 Results of first-order multiplet analysis of H-4, H-5, and H-3 of compound 1

Fig. 4 Results of first-order multiplet analysis of H-4, H-5, and H-3 of compound 2

Plant materials 20140413). The aerial parts of G. a ff i ne (Compositae family) were Extraction and isolation collected in Jinhua, Zhejiang Province, China (29°20'30'' N, The dried plant materials (6.1 kg) were cut and extracted 119°54'25'' E) in April 2014. Voucher specimens were iden- four times with 60% (V/V) ethanol under reflux. Combined tified by Prof. Tong Wu of Shanghai Institute of Pharmaceu- solution was concentrated to crudeness in-vacuo and dried to tical Industry and preserved at the Traditional Chinese obtain crude extract. The crude extract was suspended in wa- Medicine Department of the institute (Herbarium No. ter (6 L) and sequentially partitioned with 6 L of petroleum

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1 13 Table 1 H- (500 MHz) and C NMR (125 MHz) data of compounds 1 and 2 (in CD3OD, J in Hz) NO. 1H 1 13C HMBC H→C 1H 2 13C HMBC H→C 1 75.70 75.58 − 2 2.12, 2.15 m 41.36 C-1, 3, 5, 6, 7 2.13, 2.17 m 41.31 C-1, 3, 4, 5, 7 3 4.33 ddd (8.5, 8.3, 4.4) 66.03 C-1, 4 4.32 ddd (8.5, 8.4, 4.1) 66.03 − 4 5.03 dd (8.3, 3.3) 75.12 C-3, 9' 5.03 dd (8.4, 3.2) 75.16 C-9' 5 5.63 ddd (5.3, 3.8, 3.3) 69.86 C-1, 4 5.62 ddd (5.2, 4.0, 3.2) 69.82 − 6 2.11, 2.36 m 36.78 C-1, 2, 3, 4, 5, 7 2.10, 2.36 m 36.8 C−1, 2, 3, 4, 5, 7 7 175.69 − 176.12 − 8 4.21 dq 62.59 C-9 3.76 s 52.96 C−7 9 1.31 t (7.2) 14.39 C-8 − − − 1' 127.73 − 127.72 − 2' 7.04 d (1.9) 115.15 C-1', 3', 4' 6', 7' 7.03 d (1.9) 115.13 C-3', 5', 6', 7' 3' 146.79 − 146.81 − 4' 149.65 − 149.66 − 5' 6.76 d (8.2) 116.47 C-1', 3', 4', 6' 6.76 d (8.2) 116.46 C-1', 3', 4', 6' 6' 6.92 dd (8.2, 1.9) 123.24 C-2', 4', 5', 7', 8' 6.92 dd (8.2, 1.9) 123.25 C-2', 4', 5', 7', 8' 7' 7.56 d (15.9) 147.38 C-1', 2', 6', 8', 9’ 7.56 d (15.9) 147.39 C-1', 2', 6', 8', 9' 8' 6.27 d (15.9) 114.96 C-1', 7', 9' 6.27 d (15.9) 114.95 C-2', 9' 9' 168.53 − 168.52 − 1'' 127.65 − 127.64 − 2'' 7.03 d (1.9) 115.05 C-1'', 3'', 4'', 6'', 7'' 7.02 d (1.9) 115.04 C-3'', 4'', 6'', 7'' 3'' 146.78 − 146.80 − 4'' 149.61 − 149.63 − 5'' 6.74 d (8.2) 116.45 C-1'', 3'', 4'', 6'' 6.74 d (8.2) 116.44 C-3'', 4'', 6'' 6'' 6.89 dd (8.2, 1.9) 123.13 C-2'', 4'', 5'', 8'' 6.89 dd (8.2, 1.9) 123.14 C-2'', 4'', 5'', 7'', 8'' 7'' 7.55 d (15.9) 147.37 C-2'', 6'', 8'', 9'' 7.55 d (15.9) 147.38 C-1'', 2'', 6'', 8'', 9'' 8'' 6.26 d (15.9) 114.84 C-1'', 7'', 9'' 6.26 d (15.9) 114.82 C-1'', 9'' 9'' 168.45 − 168.44 −

Table 2 Effects of compounds on xanthine oxidase inhibition ether, EtOAc and n-BuOH, respectively. The petroleum ether, activities EtOAc (163.4 g), n-BuOH (297.78 g) and H2O portions were −1 −1 Compound IC50 (μmol·L ) Compound IC50 (μmol·L ) obtained by evaporation under reduced pressure. allopurinol 1.62 ± 0.16 19 7.82 ± 0.22 The EtOAc portion (157.68 g) was chromatographed on a 1 11.94 ± 1.70 20 2.42 ± 0.13 silica gel column (6 cm  72 cm) eluted by petroleum-EtOAc 2 15.04 ± 1.82 21 > 50 (74.29 ± 0.26) (V/V, 100 : 0, 100 : 1, 80 : 1, 40 : 1, 10 : 1, 4 : 1, 2 : 1 and 0 : 3 17.56 ± 4.21 22 8.33 ± 2.68 100, each 30 L) to afford 8 fractions (Et-1 to Et-8). Et-5 was 4 6.36 ± 1.71 23 4.09 ± 0.51 then partitioned by silica gel chromatography (petroleum- 5 20.45 ± 1.00 24 3.12 ± 1.42 EtOAc 10 : 1) to obtain 3 subfractions (Et-5-1 to Et-5-3).

6 3.11 ± 0.22 25 > 50 Et-5-1 was chromatographed over a C18 reversible phase sil- 7 20.42 ± 0.87 26 > 50 ica gel column (2 cm 40 cm) using aqueous methanol (step- 8 > 50 27 > 50 wise, 10%, 30%, 50%, 70%, and 90%, 1 L each) to obtain 9 > 50 28 − Et-5-1-1 to Et-5-1-5. Et 5-2 was fractioned by Sephadex 10 > 50 29 − LH-20 (1.5 cm  90 cm) using methanol to acquire Et-5-2-1

11 − 30 > 50 to Et-5-2-10. Et 5-3 was separated by C18 silica gel column 12 − 31 > 50 (2.5 cm  36 cm) using aqueous methanol (stepwise, 20, 40%, 13 − 32 > 50 60%, 80%, and 100%, 2 L each) to afford Et-5-3-1 to Et-5-3-5. 14 27.87 ± 2.68 33 > 50 Compounds 9 (9 mg), 25 (1.2 mg), 26 (29.4 mg) and 37 ( 15 > 50 34 > 50 100 mg) from Et-5-1, 10 (84.3 mg) from Et-5-2, 11 (12.3 mg), 16 > 50 35 > 50 12 (27.1 mg), 13 (9.5 mg), 14 (2.7 mg), 15 (3.3 mg), 16 (30.9 mg), 17 > 50 36 − 17 (19.9 mg), 18 (26.1 mg), 27 (27.5 mg), 28 (3.3 mg), and 18 − 37 > 50 29 (7.9 mg) were isolated from Et-5-3 and purified by prepa-

The symbol “−” represented the compounds with no activities rative TLC and HPLC. Et-6 was subjected to C18 reversible

– 351 – ZHANG Wei, et al. / Chin J Nat Med, 2018, 16(5): 347353 phase silica gel column (4 cm × 36 cm) using aqueous methanol NMR data, see Table 1; HR-ESI-MS m/z 529.134 7 [M − − (stepwise, 20%, 40%, 60%, 80%, and 100 %, 1 L each) to H] (Calcd. for C26H25O12, 529.134 6). obtain 5 fractions (Et-6-1 to Et-6-5). Et-6-2 was separated by In vitro inhibition assay for xanthine oxidase activity Sephadex LH-20 (3 cm × 90 cm) with methanol to obtain Xanthine oxidase inhibitory activity was assayed in subfractions, which were further purified by preparative HPLC 96-well plates, according to the procedure described by Nguyen or TLC to obtain compounds 21 (7 mg), 30 (12 mg), 31 (23 mg), et al. [39], with a slight modification. Briefly, 30 μL of enzyme 7 (20 mg), 6 (8 mg), and 4 (21 mg). Et-6-3 was subjected to solution (0.01 U·mL−1 in 70 mmol·L−1 phosphate buffer) was silica gel column (3 × 20 cm) eluted by chloroform-acetone mixed with 35 μL of 70 mmol·L−1 phosphate buffer (pH 7.5) (V/V, 10 : 1, 4 : 1 and 2 : 1, 500 mL each) to afford 3 fractions and 50 μL of test solution. After incubation at 25 °C for (Et-6-3-1 to Et-6-3-3). Compounds 19 (30 mg) and 20 (16 mg) 15 min, 60 μL of xanthine solution (300 μmol·L−1 in from Et-6-3-1, and Compounds 22 (12 mg) and 23 (50 mg) 70 mmol·L−1 phosphate buffer) was added. After re-incuba- from Et-6-3-2 were isolated by preparative TLC. tion at 25 °C for 30 min, the mixture was added with 25 μL of The n-BuOH portion (291.76 g) was loaded onto LS-305 1 mol·L−1 HCl. The absorbance at 295 nm was measured us- resin column ((polystyrene skeleton, 4 cm  72 cm; Lanshen ing a "Biotek Synergy 2" microplate reader. A blank control corp., China) and further fractionated by aqueous ethanol elu- was set in the same way, but the enzyme solution was added tion (V/V, 20%, 40%, 60%, 80%, and 95%, 21 L each) to to the mixture after adding 1 mol·L−1 HCl. For all the tests, yield 5 fractions (Bu-1 to Bu-5). First, Bu-1 was subjected to the inhibition assay was performed in triplicate.

C18 reversible phase silica gel column (4 cm × 36 cm) using Xanthine oxidase inhibitory activity (%) = (1 − B/A) × aqueous methanol (stepwise, 20%, 40%, 60%, 80%, and 100, where A and B were the optical densities of the enzyme 100%, 1 L each) to obtain 5 fractions (Bu-1-1 to Bu-1-5). without and with test material. Bu-1-2 was separated by Sephadex LH-20 (3 cm × 90 cm) The compounds and positive control drug (allopurinol) with 50% (V/V) aqueous methanol to obtain subfractions, were dissolved in DMSO to obtain stock solutions, respec- which were further purified by preparative TLC to obtain tively. The stock solutions were diluted with phosphate buffer compounds 8 (15 mg), 32 (27 mg), 34 (56 mg), 35 (25 mg), (70 mmol·L−1, pH7.5) to obtain test solutions (DMSO < and 36 (40 mg). Second, Bu-2 (97.79 g) was subjected to a 0.25%, V/V) at different concentrations (50, 25, 12.5, 6.25, silica gel column (4 cm  50 cm) eluted by EtOAc−MeOH 3.125, and 1.5 625 μmol·L−1 ). (V/V, 100 : 1, 50 : 1, 20 : 1, 8 : 1, 3 : 1 and 0 : 100, 20 L each) to obtain 6 fractions (Bu-2-1 to Bu-2-6). Bu-2-1 was sepa- References rated by a C18 reversible phase silica gel column (2.5 cm  36 [1] Zhang W, Fan SY, Wu CZ. Review on chemical constituents cm) using aqueous methanol (stepwise, 20%, 40%, 60%, 80%, and pharmacological activities of Gnaphalium affine D. Don [J]. and 100%, 3 L each) to acquire Bu-2-1-1 to Bu-2-1-5. Com- Chin J Pharm, 2016, 47(8): 1057-1064. pounds 24 (14.1 mg) from Bu-2-1-3, 3 (74.2 mg) and 7 [2] Zeng WC, Zhu RX, Jia LR, et al. Chemical composition, an- (11.6 mg) from Bu-2-1-1, 1 (11.6 mg), 2 (3.9 mg), 4 (3.6 mg), timicrobial and antioxidant activities of essential oil from 5 (22.5 mg) and 6 (15.8 mg) from Bu-2-1-2, were isolated and Gnaphalium affine [J]. Food Chem Toxicol, 2011, 49(6): 1322- purified by preparative TLC and HPLC. Finally, Bu-3 was 1328. separated on a polyamide (100 − 200 mesh) column (6 cm × [3] Zeng WC, Zhang WC, Zhang WH, et al. The antioxidant activity and active component of Gnaphalium affine extract [J]. 40 cm) using aqueous ethanol (stepwise, 10%, 30%, 50%, and Food Chem Toxicol, 2013, 58(7): 311-317. 100%, 2 L each) to obtain 4 fractions (Bu-3-1 to Bu-3-4). [4] Huang XJ, Li YJ, Li J, et al. Total flavonoids from Gnaphalium Bu-3-3 was then chromatographed on Sephadex LH-20 affine D. Don exert analgesic effect via inhibiting the produc- (3 cm × 90 cm) with methanol to afford subfractions, which tion of pro-inflammatory mediators in mouse model of pain [J]. were further purified by preparative TLC to obtain compound Food Sci, 2014, 35(21): 240-243. 33 (28 mg). [5] Li JL, Huang DD, Chen WS, et al. Two new phenolic gly- (−) Ethyl 1, 4-di-O-caffeoylquinate (1) cosides from Gnaphalium affine D. Don and their anti-comple- 20 mentary activity [J]. Molecules, 2013, 18(7): 7751-7760. Yellowish powder; [α]D −82.9 ° (c 0.11, MeOH); CD [6] Lin WQ, Xie JX, Wu XM, et al. Inhibition of xanthine oxidase (MeOH, c 0.0019 M): ∆ε (nm): − 21.91 (344), + 9.19 (282); activity by Gnaphalium affine extract [J]. Chin Med Sci J, 2014, IR (KBr) νmax 3 426.5, 1 696.6, 1 601.4, 1 522.3, 1 445.0, 29(4): 225-230. -1 1 13 1 368.2, 1 277.9, 1 261.9, 1 117.3 cm . H and C NMR data, [7] Zhang DZ. Isolation and identification of two new ent-kau- see Table 1; HR-ESI-MS m/z 543.1499 [M − H]−(Calcd. for ranoid diterpenes from Artemisia sacrorum Ledeb [J]. Nat Prod

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Cite this article as: ZHANG Wei, WU Chun-Zhen, FAN Si-Yang. Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity [J]. Chin J Nat Med, 2018, 16(5): 347-353.

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