A new phenolic compound with antioxidant activity from the branches and leaves of Pyrus pashia

The branches and leaves of Pyrus pashia are used to cure abdominal pain and diarrhoea in Chinese folk medicine. A new phenilic compound, 4-O-β-d-glucopyranosylbenzyl-benzoate ester (1), along with 21 known ones (2–22) were isolated from the branches and leaves of this plant. Compounds 2 and 3 displayed remarkable antioxidant activities against 1,1-diphenyl-2-picrylhydrazyl radical (IC50 = 13.26 ± 0.04 μM, 13.28 ± 0.11 μM, respectively), which were at the same grade as positive control rutin. The caffeoyl group in compounds 2 and 3 was supposed to play an important role in the antioxidant activities.


Introduction
Pyrus pashia Buch.-Ham. ex D. Don is a kind of arbor distributed in Yunnan, Sichuan and Guizhou Provinces of China growing at an elevation of 650 -3000 m (Yu 1986). The flower of P. pashia is a favourite health food for lowering blood lipid in the Yunnan Province of China. Its branches and leaves are used to cure abdominal pain and diarrhoea in Chinese folk medicine. Thirteen compounds were isolated from P. pashia flowers (Liu et al. 2013) and 28 compounds were isolated from branches and leaves of this plant (Bhakuni, Gupta et al. 1971;Zhao et al. 2013;Cai et al. 2014). Our previous work indicated that the n-butanol extract of the branches and leaves of P. pashia possesses potent antioxidant activities. Some studies have indicated that antioxidants can reduce the pain. For example, natural antioxidants such as vitamin E and C at low doses are efficient in relieving chronic pelvic pain in women with endometriosis (Santanam et al. 2013), and Combretum zeyheri and Burkea africana leaves with antioxidant activities could alleviate pain and inflammation (Dzoyem & Eloff, 2015). To find more antioxidant constituents from the branches and leaves of P. pashia, a detailed phytochemical investigation on this plant was carried out, which led to the isolation of a new compound, 4-O-b-D-glucopyranosylbenzyl-benzoate ester (1), and 21 known ones (2 -22) ( Figure 1). The antioxidant activities of the isolated compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The results showed that compounds 2 and 3 exhibited strong antioxidant activities and compounds 4, 5, 6, 9 and 16 displayed moderate antioxidant activities (Tai et al. 2011;Zhou et al. 2014) against DPPH radical (Table 1). This paper describes the isolation, structural elucidation of the new compound, as well as the DPPH radical scavenging activities of the isolated compounds from this plant.
In the structure-activity relationships, compounds 2 and 3, both bearing two caffeic acid units, were more active than compounds 4 and 16 which both possess only one caffeic acid unit, indicating that caffeic acid moieties was likely responsible for the antioxidant activity in DPPH radical scavenging assay. The antioxidant activities of caffeic acid moieties in compounds 2, 3 and 4 were mainly due to catechol moiety in their structures (Cai et al. 2006;Kim et al. 2006;Kim et al. 2011). Compound 3, which was a methyl ester of 2, had the same strong antioxidant activity as 2, indicating that the carboxyl in 2 and 3 was not essential for the antioxidant activities. Notably, antioxidant activity of compound 6 (4-hydroxy-cis-cinnamomic acid 4-b-D-glucopyranosyloxybenzyl ester) was three times higher than 5 (4-hydroxy-trans-cinnamomic acid 4-b-Dglucopyranosyloxybenzyl ester) in the assay, implying that cis-p-hydroxy cinnamic acid was more important than trans-p-hydroxy cinnamomi acid in DPPH radical scavenging activities.

General procedure
Melting points were determined on an XRC-1 Melting Point Apparatus (Sichuan University Science Instrument Co., Chengdu, China) and uncorrected. Methanol (HPLC grade) was purchased from Fisher Chemicals (NJ, Fair Lawn, USA). A Shimanzu UV-VISPS 2550 spectrometer (Shimadzu, Kyota, Japan) was used for UV-VIS spectrum. NMR spectra were recorded with a Bruker DRX-600 or DRX-400 NMR spectrometer (Bruker Corporation, Larlsruhe, Germany) using Me 4 Si as an internal standard. GC was determined at Agilent 7890A (Agilent, Santa Clara, USA). MS was measured at an Agilent 1100 LC/MSD TOF spectrometer (Agilent, Santa Clara, USA). The water was purified with a purity water system (Chengdu YouPu Electronic Products Technology Co., Ltd., Chengdu, China and the system model name is UPTL-1-100L). Silica gel (200 -300 mesh), RP-18 and Sephadex LH-20 for column chromatography were supplied by the Merck Corporation (Darmstadt, Germany). All other reagents were of analytical grade. GF 254 plates (Qingdao Marine Chemical Corporation, Qingdao, China) were used for TLC, and Spots were visualised under UV light or by spraying with 3% FeCl 3 in ethanol or 10% H 2 SO 4 in ethanol followed by heating. The 2,2-Diphenyl-1picrylhydrazyl radical (Beijing J & K Scientific Ltd, Beijing, China) radical was used in radical scavenging assay. L-cysteine methyl ester hydrochloride (Tcishanghai Tech. Ltd, Shanghai, China), N-trimethylsilylimidazole (Beijing J & K Scientific Ltd) and silver carbonate (Beijing J & K Scientific Ltd) were used in acid hydrolysis assay of 1. Microplate reader (Bio-Tek Company, Vermont, USA) was employed to read OD date.

Plant material
The branches and leaves of P. pashia were collected in Shuangshao County, Kunming, Yunnan Province, China, in March 2008, and identified by Prof. Shu-gang Lu (School of Life Science, Yunnan University, Kunming, China). A voucher specimen (2010-tl-1) was deposited in the Key Laboratory of Medicinal Chemistry for Nature Resource of Yunnan University.

Acid hydrolysis of compound 1and determination of sugar
The experiment was implemented using the method previously described . A solution of compound (2.1 mg) dissolved in 1 mM HCl (dioxane/H 2 O, 1:1, 2.0 mL) was heated to 90 8C for 2 h. After cooling, the reaction mixture was neutralised with silver carbonate (5 mg), and the solvent was evaporated to dryness under reduce pressure and then extracted with CHCl 3 (2.0 mL) and H 2 O (2.0 mL). The aqueous layer was concentrated to dryness, the residue was dissolved in dry pyridine (0.2 mL), and then L-cysteine methyl ester hydrochloride in dry pyridine (0.03 M, 0.1 mL) was added. The mixture was reacted at 60 8C for 2 h, and 0.1 mL of (trimethylsilyl)-imidazole dissolved in dry pyridine was added. After being held at 60 8C for another 2 h, the reactant was partitioned with n-hexane and H 2 O (0.1 mL, each). The n-hexane fraction was subjected to gas chromatographic analysis. Agilent 7890A gas chromatograph equipped with an H 2 flame ionisation detector. Column: TC-1 capillary column (30 m £ 0.25 mm). Column temperature: 280 8C/300 8C, programmed increase: 18C/min, carrier gas: N 2 (1 mL/min). Injector and detector temperature: 260 8C; injection volume: 1 mL; split ratio: 1/10. The sugar residue in compounds 1 was identified by comparison with the standard references: D-glucose (t R 5.78 min) 3.6. Antioxidant activities of some compounds in DPPH radical scavenging assay The antioxidant activities of some compounds were measured in the DPPH radical scavenging assay with a slight modification of the reported method (Muhammad et al. 2015). Briefly, 150 mL of 0.2 mM DPPH prepared in MeOH were added to 96-well dishes with compounds at different concentrations. The samples were incubated for 30 min in the dark at room temperatures, and the decrease of absorbance at 515 nm was measured against MeOH using a Microplate reader (M200 PRO, Tecan Group Ltd., Switzerland). The DPPH solution was freshly prepared and used within 12 h. All determinations were performed in more than triplicate. The radical scavenging activity of the tested samples was calculated as 'inhibition percentage' according to the equation: where A s is an absorbance of test samples at 30 min; A b is an absorbance of control DPPH solution at 30 min and A ref is an absorbance of methanol at 30 min. IC 50 values denote the concentration of samples required to scavenge 50% of the DPPH free radicals and were calculated from the graph plotted as inhibition percentage against the concentration (Lee et al. 2002;Kim et al. 2006).

Conclusion
A new phenilic compound, 4-O-b-D-glucopyranosylbenzyl-benzoate ester (1), along with twenty-one known ones (2-22) were isolated from the branches and leaves of P. pashia. Some compounds exhibited potent antioxidant activities in the DPPH radical scavenging assay. Especially, 3,5-dicaffeoylquinic acid (2) and methyl 3,5-dicaffeoylquinate (3) displayed remarkable antioxidant activities which was at the same level as rutin in our assay. The result indicated that these compounds were responsible for the antioxidant activity of n-butanol extract of the branches and leaves of P. pashia. The efficiency of the branches and leaves of P. pashia in treating abdominal pain might be related with these phenolic constituents.

Supplementary material
Experimental details relating to this paper are available online.

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1141 Natural Product Research