Chinese Journal of Natural Chinese Journal of Natural Medicines 2018, 16(5): 03470353 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 isola- tion 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 analy- sis. 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 cur- rent 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 – 347 – ZHANG Wei, et al. / Chin J Nat Med, 2018, 16(5): 347353 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- – 348 – ZHANG Wei, et al. / Chin J Nat Med, 2018, 16(5): 347353 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.