Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus Aurantium L

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Article Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits

Kun Liu, Wei Wang *, Bing-Hua Guo, Hua Gao, Yang Liu, Xiao-Hong Liu, Hui-Li Yao and Kun Cheng School of Pharmacy, Qingdao University, Qingdao 266021, Shandong, China; [email protected] (K.L.); [email protected] (B.-H.G.); [email protected] (H.G.); [email protected] (Y.L.); [email protected] (X.-H.L.); [email protected] (H.-L.Y.); [email protected] (K.C.) * Correspondence: [email protected]; Tel./Fax: +86-532-869-91172

Academic Editor: Isabel C. F. R. Ferreira Received: 24 January 2016 ; Accepted: 29 February 2016 ; Published: 2 March 2016 Abstract: Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 µM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia.

Keywords: Citrus; xanthine oxidase; aurantii fructus immaturus; hesperetin

1. Introduction Epidemiological data show a rise in the prevalence of gout that is potentially attributable to shifts in diet and lifestyle and increased longevity [1]. Gout is a disorder of purine metabolism and results from urate crystal deposition in and around the joints caused by longstanding hyperuricemia. Xanthine oxidase (XO), an enzyme present in significant concentrations in the gastrointestinal and liver, is responsible for the metabolism of hypoxanthine and xanthine to uric acid in the purine catabolic pathway. Accordingly, the use of the XO inhibitor that blocks the synthesis of uric acid in the body should be one of the therapeutic approaches for the treatment of hyperuricemia and chronic gout [2]. Some synthetic XO inhibitors, such as allopurinol, febuxostat, and Y-700, have shown good efficacies against hyperuricemia and chronic gout [3–5]. However, they also cause side effects, such as allergic and hypersensitivity reactions, renal failure, skin rash, gastrointestinal distress, and enhancement of 6-mercaptopurine toxicity [6]. As a result of increasing consumer preference toward more natural and healthier products, scientific research has begun to focus on the screen of natural compounds as possible XO inhibitors [7–10]. Citrus is a genus of small trees or large shrubs that comprises more than 20 species widely distributed throughout the worldwide tropics and subtropics, the original range of this genus can be traced to Southeast Asia and India. The main uses of Citrus in food industries include fresh juice or Citrus-based drinks. Additionally, Citrus fruits are well-known sources of flavonoids, terpenes, coumarins, and alkaloids with potential health-promoting properties related to their anticancer, antioxidant, anti-inflammatory, antimicrobial, hypolipidemic, hepatoprotective, neuroprotective, antiedematogenic, and cardiovascular activities [11,12]. As a continuation of our screening program

Molecules 2016, 21, 302; doi:10.3390/molecules21030302 www.mdpi.com/journal/molecules Molecules 2016, 21, 302 2 of 10 relating to plants with XO inhibitory activity [13,14], the present work describes, for the first time, a systematic in vitro XO inhibitory assay of 24 organic extracts of four species belonging to Citrus genus. The results revealed that the ethyl acetate extract of C. aurantium dried immature fruits has potent XO inhibitory activity. However, it is still unclear which compounds are the active ingredients in the extract, and the contributions of the main identified compounds to the XO inhibitory activity of the extract are unknown. The present study seeks to investigate the potent XO inhibitors from the ethyl acetate extract of C. aurantium dried immature fruits. The study benefits would explain and support application of the ethyl acetate extract of C. aurantium dried immature fruits as a complementary medicine for the prevention and treatment of hyperuricemia.

2. Results and Discussion

2.1. XO Inhibitory Activity of the Extracts XO belongs to molybdenum hydroxylase superfamily and consists of two identical subunits of 145 kDa, which participates in purine degradation. During these reactions, it uses molecular oxygen as the electron acceptor, thereby resulting in production of superoxide anion radical and hydrogen peroxide [15]. The antioxidant property of extract from Citrus genus are well documented, but considerably less work has been perform regarding the inhibition of XO [16–18]. As extending studies, the XO inhibitory activities of 24 organic extracts of four species belonging to Citrus genus were evaluated. The results of the assays are listed in Table1. The extracts were prepared with petroleum ether (A), ethyl acetate (B), butanol (C), and ethanol–water (75:25, D) from selected plant organs. Although there is no generally accepted threshold for efﬁcacy in our experiment, XO inhibitory effects of <10% were considered irrelevant and are, therefore, not presented in Table1. A total of nine extracts demonstrated substantial XO inhibitory activity (>30%) at 200 µg/mL, while C. aurantium dried immature fruit extracts exhibited much higher inhibitions than those of other selected plant same solvent extracts (p < 0.05). In the case of C. aurantium dried immature fruits, ethyl acetate extract showed the highest inhibition of XO activity than did petroleum ether, butanol, and ethanol–water (75:25) extracts (p < 0.05). Percent inhibition was calculated to be 89.24% ˘ 0.69% for allopurinol, a clinical XO inhibitory drug, at 1 µg/mL.

Table 1. Yield and XO inhibitory effects of the prepared extracts.

XO Inhibition (Mean ˘ S.D.%) Species Parts Solvent Yield (w/w %) 200 µg/mL A 0.08 31.64 ˘ 0.25 B 2.55 60.70 ˘ 0.74 Citrus aurantium L. Immature fruits C 11.45 46.25 ˘ 0.27 D 21.01 37.26 ˘ 0.35 A 0.28 27.63 ˘ 0.75 B 4.52 19.08 ˘ 0.07 Citrus medica L. Mature fruit pericarp C 13.87 - D 16.69 - A 0.33 21.38 ˘ 0.99 Citrus medica L. B 2.05 23.36 ˘ 0.53 Mature fruit var. sarcodactylis Swingle C 5.33 17.60 ˘ 0.05 D 20.08 12.85 ˘ 0.48 A 0.27 39.76 ˘ 0.31 B 2.47 50.15 ˘ 0.44 Citrus reticulata Blanco Immature fruits pericarp C 8.00 38.73 ˘ 0.24 D 12.95 29.01 ˘ 1.11 A 0.22 41.46 ˘ 0.25 B 1.78 33.11 ˘ 0.70 Citrus reticulata Blanco Mature fruit pericarp C 17.68 12.32 ˘ 1.29 D 23.89 - A 0.45 26.44 ˘ 0.56 B 3.80 23.41 ˘ 0.88 Citrus reticulata Blanco Mature fruit exocarp C 15.97 10.90 ˘ 0.44 D 18.34 - Molecules 2016, 21, 302 3 of 10

2.2. Identification of the Constituents of the Most Active Extract The ethyl acetate extract of C. aurantium dried immature fruits was fractionated by RP-18 reversed-phase silica gel column chromatography into six fractions. The active fractions were further purified by preparative HPLC to isolated seven compounds. Spectroscopic analysis and comparison with literature values were used to identify the structure of isolated compounds as naringin (1)[19,20], hesperidin (2), neohesperidin (3)[19,21], naringenin (4)[19,22], hesperetin (5)[19,23], nobiletin (6), and tangeretin (7)[24]. The confirming spectroscopic data for naringin are as follows: ESI-MS at m/z 581 + 1 1 1 [M + H] ; H-NMR (500 MHz, DMSO-d6) 7.32 (2H, d, J = 8.6 Hz, H-2 , 6 ), 6.80 (2H, d, J = 8.4 Hz, H-31,51), 6.12 (1H, d, J = 2.2 Hz, H-6), 6.11 (1H, d, J = 2.2 Hz, H-8), 5.52 (1H, dd, J = 13.2, 2.8 Hz, H-2), 11 111 5.14 (1H, d, J = 7.4 Hz, H-1 ), 5.10 (1H, d, J = 2.2 Hz, H-1 ), 2.72 (1H, dd, J = 17.1, 2.8 Hz, H-3ax), 111 13 1.15 (3H, d, J = 6.2 Hz, H-6 ); C-NMR (125 MHz, DMSO-d6) 197.3 (C-4), 164.8 (C-7), 162.9 (C-5), 162.8 (C-9), 157.8 (C-41), 146.8 (C-5), 130.5 (C-6), 128.6 (C-11), 128.5 (C-21, 61), 115.2 (C-31, 51), 103.3 (C-10), 100.4 (C-1111), 97.4 (C-111), 96.3 (C-6), 95.1 (C-8), 78.8 (C-2), 77.1 (C-211), 76.9 (C-311), 76.2 (C-511), 71.8 (C-4111), 70.4 (C-3111), 70.3 (C-2111), 69.6 (C-411), 68.2 (C-5111), 60.4 (C-611), 42.1 (C-3), 18.0 (C-6111). The confirming spectroscopic data for hesperidin are as follows: ESI-MS at m/z 611 [M + H]+; 1H-NMR 1 1 1 (500 MHz, DMSO-d6) 6.89–6.95 (3H, overlap, H-2 , 5 , 6 ), 6.15 (1H, d, J = 2.4 Hz, H-6), 6.12 (1H, d, J = 2.4 Hz, H-8), 5.50 (1H, dd, J = 12.2, 3.2 Hz, H-2), 4.99 (1H, d, J = 7.4 Hz, H-111), 4.52 (1H, br s, 111 111 H-1 ), 3.77 (3H, s, OCH3), 2.76 (1H, dd, J = 17.1, 3.2 Hz, H-3ax), 1.08 (3H, d, J = 6.2 Hz, H-6 ); 13 1 1 C-NMR (125 MHz, DMSO-d6) 196.9 (C-4), 165.1 (C-7), 163.0 (C-5), 162.5 (C-9), 147.9 (C-4 ), 146.4 (C-3 ), 131.0 (C-11), 117.9 (C-61), 114.1 (C-51), 112.1 (C-21), 103.3 (C-10), 100.6 (C-1111), 99.4 (C-111), 96.3 (C-6), 95.6 (C-8), 78.4, 78.3 (C-2), 76.2 (C-311), 75.5 (C-511), 73.0 (C-211), 72.0 (C-4111), 70.7 (C-3111), 70.2 (C-2111), 11 111 11 111 69.6 (C-4 ), 68.3 (C-5 ), 66.0 (C-6 ), 56.0 (OCH3), 42.0 (C-3), 17.8 (C-6 ). The confirming spectroscopic + 1 data for neohesperidin are as follows: ESI-MS at m/z 611 [M + H] ; H-NMR (500 MHz, DMSO-d6, δ) 6.94 (1H, d, J = 8.3 Hz, H-51), 6.93 (1H, d, J = 2.0 Hz, H-21), 6.88 (1H, dd, J = 8.3, 2.0 Hz, H-61), 6.12 (1H, d, J = 2.2 Hz, H-6), 6.09 (1H, d, J = 2.2 Hz, H-8), 5.51 (1H, dd, J = 12.2, 3.0 Hz, H-2), 5.12 11 111 (1H, d, J = 7.6 Hz, H-1 ), 5.11 (1H, d, J = 2.3 Hz, H-1 ), 3.78 (3H, s, OCH3), 2.77 (1H, dd, J = 17.2, 111 13 3.0 Hz, H-3ax), 1.16 (3H, d, J = 6.2 Hz, H-6 ); C-NMR (125 MHz, DMSO-d6, δ) 196.9 (C-4), 164.8 (C-7), 162.9 (C-5), 162.5 (C-9), 148.0 (C-41), 146.5 (C-31), 130.9 (C-11), 117.7 (C-61), 114.1 (C-51), 112.0 (C-21), 103.3 (C-10), 100.3 (C-1111), 97.4 (C-111), 96.2 (C-6), 95.1 (C-8), 78.4 (C-2), 77.1 (C-211), 76.9 (C-311), 11 111 111 111 11 111 11 76.0 (C-5 ), 71.8 (C-4 ), 70.4 (C-3 ), 70.3 (C-2 ), 69.6 (C-4 ), 68.2 (C-5 ), 60.4 (C-6 ), 55.7 (OCH3), 42.1 (C-3), 18.0 (C-6111). The confirming spectroscopic data for naringenin are as follows: ESI-MS at ´ 1 1 1 m/z 271 [M ´ H] ; H-NMR (500 MHz, DMSO-d6, δ) 7.31 (2H, d, J = 8.5 Hz, H-2 , 6 ), 6.80 (2H, dd, J = 8.5 Hz, H-31, 61), 5.88 (2H, s, H-6, 8), 5.44 (1H, dd, J = 12.8, 2.9 Hz, H-2), 3.26 (1H, dd, J = 17.1, 13 12.8 Hz, H-3eq), 2.68 (1H, dd, J = 17.1, 2.9 Hz, H-3ax); C-NMR (125 MHz, DMSO-d6, δ) 196.3 (C-4), 166.6 (C-7), 163.4 (C-5), 162.9 (C-9), 157.7 (C-41), 128.8 (C-11), 128.2 (C-21, 61), 115.1 (C-31, 51), 101.7 (C-10), 95.8 (C-6), 94.9 (C-8), 78.4 (C-2), 41.9 (C-3). The confirming spectroscopic data for hesperetin ´ 1 are as follows: ESI-MS at m/z 301 [M ´ H] ; H-NMR (500 MHz, DMSO-d6, δ) 6.93 (1H, d, J = 8.2 Hz, H-51), 6.92 (1H, br s, H-21), 6.87 (1H, dd, J = 8.2, 2.0 Hz, H-61), 5.90 (1H, d, J = 2.0 Hz, H-8), 5.88 (1H, d, J = 2.0 Hz, H-6), 5.43 (1H, dd, J = 12.3, 3.0 Hz, H-2), 3.78 (3H, s, OCH3), 3.19 (1H, dd, J = 17.1, 12.3 Hz, 13 H-3eq), 2.71 (1H, dd, J = 17.1, 3.0 Hz, H-3ax); C-NMR (125 MHz, DMSO-d6, δ) 196.1 (C-4), 166.6 (C-7), 163.4 (C-5), 162.8 (C-9), 147.8 (C-41), 146.4 (C-31), 131.1 (C-11), 117.6 (C-61), 114.0 (C-51), 112.0 (C-21), 101.8 (C-10), 95.8 (C-6), 95.0 (C-8), 78.2 (C-2), 55.7 (OCH3), 42.0 (C-3). The confirming spectroscopic + 1 data for nobiletin are as follows: ESI-MS at m/z 403 [M + H] ; H-NMR (500 MHz, DMSO-d6, δ) 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-61), 7.55 (1H, d, J = 2.0 Hz, H-21), 7.16 (1H, d, J = 8.4 Hz, H-51), 6.86 (1H, 1 1 s, H-3), 4.03 (3H, s, 7-OCH3), 3.98 (3H, s, 5-OCH3), 3.88 (3H, s, 3 -OCH3), 3.85 (3H, s, 4 -OCH3), 3.84 13 (3H, s, 6-OCH3), 3.79 (3H, s, 8-OCH3); C-NMR (125 MHz, DMSO-d6, δ) 175.8 (C-4), 160.2 (C-2), 151.7 (C-41), 150.9 (C-7), 149.0 (C-31), 147.5 (C-8), 147.1 (C-9), 143.5 (C-5), 137.6 (C-6), 123.1 (C-11), 119.3 (C-61), 1 1 1 114.6 (C-10), 11.8 (C-5 ), 108.9 (C-2 ), 106.3 (C-3), 61.8 (5-OCH3), 61.7 (7-OCH3), 61.4 (3 -OCH3), 61.3 1 (4 -OCH3), 55.7 (8-OCH3), 55.6 (6-OCH3). The confirming spectroscopic data for tangeretin are as + 1 1 follows: ESI-MS at m/z 373 [M + H] ; H-NMR (500 MHz, DMSO-d6, δ) 8.00 (2H, d, J = 8.9 Hz, H-2 , Molecules 2016,, 21,, 302302 44 ofof 1010

160.3 (C-2), 150.9 (C-4′), 147.5 (C-5), 147.1 (C-9), 143.5 (C-6), 137.7 (C-8), 127.7 (C-2′, 6′), 123.0 (C-1′), 1 1 1 6114.6), 7.14 (C-10), (2H, 114.2 dd, J (C-3= 8.9′, 5 Hz,′), 106.0 H-3 (C-3),, 6 ), 6.7661.8 (1H,(5, 7-OCH s, H-3),3), 61.4 4.02 (4 (3H,′-OCH s, 7-OC3), 61.3H (8-OCH3), 3.97 (3H,3), 55.5 s, (6-OCH 5-OCH33).), 1 13 3.86The structures (3H,Molecules s, 4 -OC 2016 of , Hthe213, 302), seven 3.84 (3H, compounds s, 6-OCH are3), shown 3.78 (3H, in s,Figure 8-OC 1.H 3From); C-NMR the HPLC (125 analysis MHz, DMSO-4 of of 10the ethyld6, δ) 1 175.7acetate (C-4), extract 162.0 of (C-7), C. aurantium 160.3 (C-2), dried 150.9 immature (C-4 ), 147.5 fruits (C-5), (Figure 147.1 2), (C-9), naringin 143.5 and (C-6), neohesperidin, 137.7 (C-8), 127.7 are 1 1160.3 (C-2), 150.91 (C-4′), 147.5 (C-5), 147.1 (C-9),1 1 143.5 (C-6), 137.7 (C-8), 127.7 (C-2′, 6′), 123.01 (C-1′), (C-2responsible, 6 ), 123.0 for (C-1predominant), 114.6 (C-10), peaks. 114.2 (C-3 , 5 ), 106.0 (C-3), 61.8 (5, 7-OCH3), 61.4 (4 -OCH3), 61.3 114.6 (C-10), 114.2 (C-3′, 5′), 106.0 (C-3), 61.8 (5, 7-OCH3), 61.4 (4′-OCH3), 61.3 (8-OCH3), 55.5 (6-OCH3). (8-OCH3), 55.5 (6-OCH3). The structures of the seven compounds are shown in Figure1. From the The structures of the seven compounds are shown in Figure 1. From the HPLC analysis of the ethyl C aurantium OR2 HPLC analysisacetate extract of the of ethyl C. aurantium acetate dried extract immature of . fruits (Figuredried 2), immaturenaringin and fruits neohesperidin, (Figure2), are naringin and neohesperidin,responsible for are predominant responsible peaks. for predominant peaks. R3O O R1 OR2

R3O O OH O R1

1 R1=H R2=H R3=glu-(2-1)-rha 2 R1=OH R2OH=CHO3 R3=glu-(6-1)-rha 3 R1 1 R=OH=H R R2=CH=H 3 RR=glu-(2-1)-rha3=glu-(2-1)-rha 4 R =H1 R 2=H R3 =H 2 1 R1=OH R22=CH 3 R 3=glu-(6-1)-rha3 5 R3 1 R=OH1=OH R R22=CH=CH3 RR33=glu-(2-1)-rha=H 4 R1=H R2=H R3=H 5 R1=OH R2=CH3 R3=H OCH3 H3CO OCH3 H3CO H3CO O R1 H3CO O R1 H3CO H3CO H3CO O H3CO O

66 R11=OCH=OCH3 3 77 R =H=H 11 Figure 1.Figure StructuresStructures 1. Structures of of the of the compoundsthe compounds compounds identifi identifi identiﬁededed from from the the ethyl theethyl acetate ethyl acetate extract acetate extract of extractC. aurantiumof C. ofaurantiumC dried. aurantium dried immature fruits: naringin (1); hesperidin (2); neohesperidin (3); naringenin (4); hesperetin (5); nobiletin driedimmature immature fruits: fruits:naringin naringin (1); hesperidin (1); hesperidin (2); neohesperidin (2); neohesperidin (3); naringenin (3); naringenin(4); hesperetin (4); (5 hesperetin); nobiletin (6); and tangeretin (7). ((56); and nobiletin tangeretin (6); and (7). tangeretin (7). mAU 2000 mAU 1 3 2000 1500 1 3 1500 5 1000 4 2 5 1000 500 4 6 7 2 0 500 6 7 0 10 20 30 40 50 60 min

0 Figure 2. HPLC profile of the ethyl acetate extract of C. aurantium dried immature fruits and 0 10 20 30 40 50 60 min compound-identified peaks: naringin (1); hesperidin (2); neohesperidin (3); naringenin (4); hesperetin (5); nobiletin (6); and tangeretin (7). Figure 2.2. HPLCHPLC profile profile of of the the ethyl acetate extract of C. aurantium dried immature fruits and compound-identified2.3. Contribution of the peaks: peaks: Identified naringin naringin Compounds (1 );(1 hesperidin); hesperidin to XO Inhibitory (2 );(2 neohesperidin); neohesperidin Activity (3); (3 naringenin); naringenin (4); ( hesperetin4); hesperetin (5); nobiletin(5); nobiletin (6); ( and6); and tangeretin tangeretin (7 ).(7). Seven identified compounds were evaluated for their ability to inhibit the production of uric acid from xanthine by xanthine oxidase. An overview about the effects of these substances on XO 2.3. Contributionactivity is given of the in IdentifiedIdentified Table 2. Neohesperidin, CompoundsCompounds tohesperidin,to XOXO InhibitoryInhibitory naringin, ActivityActivity and tangeretin showed weak inhibitory Sevenactivities, identifiedidentified while hesperetin, compoundscompounds nobiletin, were were evaluatedand evaluated naringenin for for displayed their their ability ability either to potent inhibitto inhibit or themoderate productionthe production activities of at uric of aciduric 200 μM with inhibition rates of 81.3%, 59.4%, and 49.8%, respectively. As shown in Figure 3, micromolar fromacid from xanthine xanthine by xanthine by xanthine oxidase. oxidase. An overview An over aboutview theabout effects the ofeffects these of substances these substances on XO activityon XO concentrations of hesperetin elicited dose-dependent inhibition of xanthine oxidase with an IC50 isactivity given valueis in given Table of 16.48 in2 .Table Neohesperidin,μM, 2.comparable Neohesperidin, to hesperidin,that 2.07 hesperidin, μM of naringin, the naringin, positive and control and tangeretin tangeretin allopurinol, showed showed a drug weak clinicallyweak inhibitory inhibitory activities,prescribed while hesperetin, hesperetin,in clinic for goutnobiletin, nobiletin, treatment. and and Narinaringenin naringeninngenin, having displayed displayed a hydroxyl either either substituent potent potent or oratmoderate C-4 moderate′ in ring activities C, activities at at200 200 μMµ displayedwithM with inhibition lower inhibition activity rates ratesof than 81.3%, ofhesperetin, 81.3%, 59.4%, 59.4%,withand 49.8%,a methoxyl and respectively. 49.8%, group respectively. at C-4As′ shownand a hydroxyl Asin Figure shown group 3, inmicromolar at Figure 3, micromolarconcentrations concentrations of hesperetin of hesperetinelicited dose-depen elicited dose-dependentdent inhibition inhibitionof xanthine of xanthineoxidase oxidasewith an withIC50 anvalue IC50 ofvalue 16.48 ofμM, 16.48 comparableµM, comparable to that 2.07 to thatμM 2.07of theµ Mpositive of the control positive allopurinol, control allopurinol, a drug clinically a drug clinicallyprescribed prescribed in clinic for in gout clinic treatment. for gout treatment. Naringenin, Naringenin, having a hydroxyl having a substituent hydroxyl substituent at C-4′ in ring at C-4 C,1 indisplayed ring C, displayedlower activity lower than activity hesperetin, than hesperetin, with a methoxyl with a methoxylgroup at C-4 group′ and at a C-4 hydroxyl1 and a hydroxylgroup at

Molecules 2016,, 21,, 302302 55 ofof 1010

C-3′ in ring C. These results are in agreement with the previous report on their XO inhibitory activities 1 group[25]. Previous at C-3 in studies ring C. have These demonstrated results are in agreementthat the substitution with the previous of the hydrox reportyl on group their XOat C-7 inhibitory of the activitiesbasic flavonoid [25]. Previous structure studies by a glycoside have demonstrated seems to thatdecrease the substitution the XO inhibitory of the hydroxyl effect [8]. group Therefore, at C-7 ofthe the presence basic ﬂavonoid of the glycosides structure byare a glycosideresponsible seems for the to decrease lower inhibitory the XO inhibitory activities effect of neohesperidin, [8]. Therefore, thehesperidin, presence and of thenaringin. glycosides are responsible for the lower inhibitory activities of neohesperidin, hesperidin, and naringin. Table 2. Xanthine oxidase inhibitory activities of the isolated compounds. Table 2. Xanthine oxidase inhibitory activities of the isolated compounds. XO Inhibition IC50 Ki Compounds Inhibition Mode (MeanXO ± S.D.%, Inhibition 200 μM) (μM) (μM) Compounds IC (µM) Inhibition Mode K (µM) 1 Naringin (Mean 15.29˘ S.D.%, ± 0.64 200 µM) 50 i 2 1 Hesperidin Naringin 15.29 7.28 ˘± 0.210.64 3 2 Neohesperidin Hesperidin 7.28 5.36˘ ± 0.210.01 4 3 Naringenin Neohesperidin 49.82 5.36 ˘ ±0.01 1.17 4 Naringenin 49.82 ˘ 1.17 5 Hesperetin 81.31 ± 0.36 16.48 mixed 1.40 5 Hesperetin 81.31 ˘ 0.36 16.48 mixed 1.40 6 6 Nobiletin Nobiletin 59.44 59.44˘ ± 0.83 107.53 107.53 noncompetitive noncompetitive 77.24 77.24 7 7 Tangeretin Tangeretin 28.82 28.82˘ ± 0.02 AllopurinolAllopurinol 2.07 2.07 competitive competitive 1.92 1.92

100 100

80 80

60 60

40 40

20 20 Inhibition of XO Activity(%) XO of Inhibition Inhibition of XO Activity(%) of XO Inhibition 0 0 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 Log10( hespere tin) μM Log10( allopurinol) μM Figure 3. Concentration-dependent inhibition of XO activity by hesperetin and allopurinol. Data Figure 3. Concentration-dependent inhibition of XO activity by hesperetin and allopurinol. Data represent mean ˘ standard deviation (S.D.) of triplicate experiments. represent mean ± standard deviation (S.D.) of triplicate experiments.

To understandunderstand the enzymeenzyme inhibitioninhibition mode of hesperetin,hesperetin, the Lineweaver-Burk plots were established, as shown in Figure 44.. SinceSince thethe Lineweaver-BurkLineweaver-Burk plotsplots ofof hesperetinhesperetin crosscross toto thethe leftleft ofof the 1/V axis axis but but above above the the 1/[S] 1/[S] axis, axis, it it belongs belongs to to the the mixed mixed type type of of inhibition. inhibition. In In mixed mixed inhibition, inhibition, the inhibitor can bind to thethe freefree enzymeenzyme asas wellwell asas toto thethe enzyme-substrateenzyme-substrate complex.complex. The inhibitor constant Ki, the dissociation constant of the enzymeenzyme of the-inhibitorthe-inhibitor complex,complex, can be calculatedcalculated from the slopeslope ofof thethe inhibitedinhibited curve,curve, andand thethe inhibitorinhibitor constantconstant KKII, the dissociationdissociation constant of the enzyme-substrate-inhibitor complex,complex, can can be be calculated calculated from from the the y-intercept y-intercept of theof the inhibited inhibited curve. curve. The KThei and Ki KandI of K hesperetinI of hesperetin were were determined determined to be 1.40to beµ M1.40 and μM 53.85 andµ M,53.85 respectively. μM, respectively. Parallel Parallel studies werestudies carried were withcarried allopurinol, with allopurinol, the data the indicate data indica that thete that mode the ofmode inhibition of inhibition by allopurinol by allopurinol is of the is competitiveof the competitive type with type a with Ki of a 1.92 Ki ofµ M.1.92 μM. For thethe ﬁrstfirst time, time, nobiletin nobiletin and and tangeretin, tangeretin, the mainthe main polymethoxyﬂavones polymethoxyflavones in the in ethyl the acetate ethyl extractacetate extract of C. aurantium of C. aurantiumimmature immature fruits, fruits, were were tested tested for their for th abilityeir ability to inhibit to inhibit the the production production of uricof uric acid acid from from xanthine xanthine by by xanthine xanthine oxidase oxidase exhibiting exhibiting only only moderate moderate oror weakweak effects.effects. Moreover, Moreover, pharmacokinetic investigations have demonstrated that neohesperidin and hesperidin are transformedtransformed into hesperetin byby humanhuman intestinalintestinal bacteriabacteria [[26,27].26,27]. These data strongly indicate that thethe actualactual inin vivovivo effect of ethylethyl acetateacetate extract from CC.. aurantiumaurantium dried immatureimmature fruits isis considerablyconsiderably strongerstronger than indicated by the present in vitrovitro experiment. Further in vivo research is being carried out to identify a potential inhibition for for complementary complementary use use in in the the prevention prevention and and treatment treatment of of hyperuricemia. hyperuricemia.

Molecules 2016, 21, 302 6 of 10 Molecules 2016, 21, 302 6 of 10

A 600 20 μM 15 μM 10 μM 400 5 μM

1/n moluric acid 0 μM v, 200 1/ -0.10 -0.05 0.00 0.05 0.10 1/[S], 1/μM xanthine 5000 200 B C

4000 180

3000 160

slope 2000 140 intercept

1000 120

0 100 0 5 10 15 20 0 5 10 15 20 concentration of hesperetin / μM concentration of hesperetin / μM

FigureFigure 4. InhibitionInhibition kinetics kinetics of of xanthine xanthine oxidase oxidase by by hesperetin. hesperetin. ( (AA)) Lineweaver-Burk Lineweaver-Burk plots plots in in the the absenceabsence or or at at the the different different concentrations concentrations of of hesper hesperetin.etin. Each Each point point is is the the av averageerage value value from from triplicate triplicate experiments. Secondary plots to calculate the inhibition constants are shown in (B) (Ki) and (C) (KI). experiments. Secondary plots to calculate the inhibition constants are shown in (B) (Ki) and (C) (KI).

3. Materials and Methods 3. Materials and Methods

3.1.3.1. Chemicals Chemicals AnalyticalAnalytical grade grade petroleu petroleumm ether, ethyl acetate, n-butanol, methanol, ethanol, ethanol, dimethyl dimethyl sulphoxide,sulphoxide, and and hydrochloric hydrochloric acid acid were were obtained obtained from from Beijing Beijing Chemical Chemical Industry Industry Group Group Co., Co., Ltd, Ltd, (Beijing,(Beijing, China). China). Methanol Methanol and and acetonitrile acetonitrile employed employed for for high-performance high-performance liquid liquid chromatography chromatography (HPLC)(HPLC) were were HPLC HPLC super super gradient gradient quality quality (Tedia (Tedia Company Company Inc., Inc., Fairfield, Fairﬁeld, OH, OH, USA). USA). Allopurinol, Allopurinol, XO,XO, and and xanthine xanthine were were purchased from Sigm Sigma-Aldricha-Aldrich Chemicals (St. Louis, MO, USA).

3.2.3.2. General General Experimental Experimental Procedures Procedures AA Bruker AV-500 FT-NMR FT-NMR spectrometer spectrometer was was used, used, operating operating at 500.1at 500.1 MHz MHz for 1 forH and 1H atand 125.8 at 125.8 MHz 13 13 MHzfor C,for chemicalC, chemical shifts shifts are expressed are expressed in δ referringin δ referring to the to residual the residual solvent solvent signals signalsδH 2.50 δH 2.50 and δandC 39.5 δC 39.5for DMSO-for DMSO-d6, couplingd6, coupling constants, constants,J, areJ, are in in hertz. hertz. ESI-MS ESI-MS was was acquired acquired withwith aa BruckBruck micro-TOFQ massmass spectrometer spectrometer (Bruck, (Bruck, Bremen, Bremen, Germany). Germany). Colu Columnmn chromatography chromatography was was performed performed over over a a RP-18 RP-18 reversed-phasereversed-phase silica silica gel gel (S-50 (S-50 μµm;m; YMC, YMC, Kyoto, Kyoto, Japan). Japan). Thin-layer Thin-layer chromatography chromatography (TLC) (TLC) analysis analysis waswas carried carried out out on on a a pre-coated pre-coated TLC TLC plate plate with with silica silica gel gel RP-18 RP-18 60 60 F254 F254 (0.25 (0.25 mm, mm, Merck, Merck, Darmstadt, Darmstadt, Germany).Germany). Detection Detection was was achieved achieved by by spraying spraying the the sample sample with with 10% H 2SOSO44 inin MeOH MeOH followed followed by by heating.heating. PreparativePreparative HPLC HPLC was was performed performed using using a Shimadzu a Shimadzu LC-6AD LC-6AD pump connectedpump connected to a Shimadzu to a ShimadzuSPD-20A UV-VISSPD-20A detector UV-VIS (at detector 254 nm) (at with 254 a nm) Shim with Pak a ODS Shim column Pak ODS (250 column mm ˆ (25021.2 mm,mm × i.d., 21.2 10 mm,µm, i.d.,Shimadzu, 10 μm, Kyoto,Shimadzu, Japan) Kyoto, and a Japan) Megress and ODS a Megress column (250ODS mm columnˆ 20 mm,(250 mm i.d., 10× 20µm, mm, Jiangsu i.d., 10 hanbon μm, JiangsuScience hanbon and Technology Science an Co.,d Technology Ltd., Huai1an, Co., China). Ltd., Huai Analytical′an, China). HPLC Analytical was performed HPLC with was two performed LC-20AT withsolvent two delivery LC-20AT pumps, solvent an delivery online mixer, pumps, a SIL-20A an online autosampler, mixer, a SIL-20A a CTO-20A autosampler, column temperaturea CTO-20A columncontroller, temperature and a SPD-M20A controller, photodiode-array and a SPD-M20A detector photodiode-array (Shimadzu). detector HPLC-grade (Shimadzu). water HPLC-grade was puriﬁed waterusing was a Milli-Q purified system using (Millipore, a Milli-Q system Boston, (Millipore, MA, USA). Boston, All solvents MA, USA). used All for solvents the chromatographic used for the chromatographicseparations were separations distilled before were use. distilled before use.

Molecules 2016, 21, 302 7 of 10

3.3. Plant Materials Dry immature fruits of Citrus aurantium L., mature fruit pericarp of Citrus medica L., mature fruit of Citrus medica L. var. sarcodactylis Swingle, and immature fruit pericarp, mature fruit pericarp, and mature fruit exocarp of Citrus reticulata Blanco, sold commercially as medicinal herbs, were purchased from Zhewan Pharmaceutical Company, Bozhou, Anhui province of China, and identiﬁed by Prof. Baomin Feng, Dalian University, China. Voucher specimens for each herb (2014AFI-01, 2014CF-01, 2014CSF-01, 2014CRPV-01, 2014CRP-01, and 2014CER-01, respectively) were deposited at the School of Pharmacy, Qingdao University, China.

3.4. Extraction and HPLC Analysis of the Extracts Four sets of each medicinal herb (2 kg) were reflux extracted separately twice with petroleum ether, ethyl acetate, butanol, and ethanol–water (75:25, v/v). The resultant extracts were then filtered, concentrated under reduced pressure, and evaporated to dryness and the percentage yield (w/w) of the extracts are given in Table1. HPLC analysis of the extracts was performed as follows. 64 mg of each extract was dissolved in a mixture of methanol and water (1:1, v/v, 2 mL) and filtered with a membrane filter (Jinteng Nylon66, 0.45 µm, Tianjin Jinteng Experimental Equipment Co., Ltd., Tianjin, China), and 5 µL of the solution was subjected to HPLC under the following conditions. The chromatographic separation was carried out on a XDB-C18 (250 mm ˆ 4.6 mm, 5 µm, Agilent Technologies, Inc., Santa Clara, CA, USA). The mobile phase consisted of acetonitrile (A) and water containing 2% acetic acid (B). A gradient program was used: 0–30 min, linear change from A–B (10:90, v/v) to A–B (20:80, v/v); 30–50 min, linear change from A–B (20:80, v/v) to A–B (40:60, v/v); 50–60 min, linear change from A–B (40:60, v/v) to A–B (60:40, v/v); and 60–70 min, isocratic elution with A–B (60:40, v/v). The flow rate was 1.0 mL/min, and the column temperature was maintained at 25 ˝C. The product was detected by monitoring UV absorption (SPD-M20A, Shimadzu) at 280 nm.

3.5. XO Inhibitory Activity Assay All the extracts, fractions and isolated compounds were assayed for their XO inhibitory activity. The assay was performed according to the method modified by our group [13], based on the procedure reported by Masuda et al. [28]. The assay mixture consisting of 200 µL of test solution of various concentration, 140 µL of 70 mM phosphate buffer (pH = 7.5), and 120 µL enzyme solution (0.02 units/mL in the same buffer) was prepared immediately before use. After pre-incubation at 25 ˝C for 15 min, the reaction was initiated by the addition of 240 µL of substrate solution (300 µM xanthine in the same buffer). The assay mixture was incubated at 25 ˝C for 30 min. The reaction was stopped by adding 100 µL of 1 M HCl, and uric acid level was determined by an HPLC method using a Diamonsil ODS-C18 column (250 ˆ 4.6 mm i.d., 5 µm, Dikma Technologies, Beijing, China) on an Agilent 1260 system equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A thermostatted column compartment, and a G1314F variable wavelength detector coupled with an analytical workstation (Agilent Technologies, Inc.). After filtration, 20 µL of the sample was injected into the column and eluted with 100 mM sodium dihydrogen phosphate in water at a flow rate of 1 mL/min. The eluate was monitored for absorbance at 290 nm. All assays were conducted in triplicate, thus inhibition percentages are the mean of triplicate observations. Inhibition percent was calculated by the following equation: inhibition (%) = [(peak area of uric acid in control experiment) – (peak area of uric acid in sample experiment)]/(peak area of uric acid in control experiment) ˆ 100. The IC50 values of the active compounds were calculated from regression lines of a plot of the percentage of inhibition on XO activity versus the concentrations of the samples. Different concentration of the tested samples were dissolved in dimethyl sulphoxide (DMSO) and, subsequently, diluted with 70 mM phosphate buffer (pH = 7.5) to a final concentration containing less than 1% DMSO (v/v), which did not affect the enzyme assay. Allopurinol, a known inhibitor of XO, was used as the positive control. Molecules 2016, 21, 302 8 of 10

3.6. XO Inhibitory Modes of Action Assay Enzyme kinetics were determined in the absence and presence of the tested samples with varying concentrations of xanthine (37.5, 50, 75, and 100 µM) as the substrate, where tested samples were at various concentration (2–20 µM) and xanthine at a certain concentration, using the XO assay methodology. The plots were drawn by the reciprocal velocity (ν-1) versus the concentration of the substrate (1/[xanthine]).

3.7. Isolation and Identification of Compounds The ethyl acetate extract of C. aurantium dried immature fruits (20 g), which showed the highest XO inhibitory activity, was subjected to RP-18 reversed-phase silica gel column eluted with a gradient increasing MeOH (20%–100%) in water to give six subfractions (Fr. 1, 4.3 g; Fr. 2, 3.7 g; Fr. 3, 1.5 g; Fr. 4, 2.6 g; Fr. 5, 3.9 g; Fr. 6, 3.2 g) on the basis of TLC analyses. Fr.2 was purified by preparative HPLC using MeOH–H2O (40:60) at a flow rate of 2.0 mL/min (Megres C18 column, 250 mm ˆ 20 mm, i.d., 10 µm) to yield compound 1 (428.5 mg, tR = 71 min), 2 (51.4 mg, tR = 87 min), and 3 (97.5 mg, tR = 102 min). Compound 4 (65.3 mg, tR = 85 min) and 5 (71.8 mg, tR = 112 min) were obtained from Fr. 4 by preparative HPLC (Shim Pak ODS column, 250 mm ˆ 21.2 mm, i.d., 10 µm; flow rate, 3.0 mL/min) employing MeOH–H2O (60:40) as the mobile phase. Fr. 5 was isolated by preparative HPLC using MeOH–H2O (80:20) at a flow rate of 2.8 mL/min (Megres C18 column, 250 mm ˆ 20 mm, i.d., 10 µm) to yield compound 6 (40.2 mg, tR = 62 min) and 7 (33.3 mg, tR = 78 min). Their structures were elucidated on the basis of spectroscopic methods, including MS, 1-D, and 2-D NMR spectral techniques.

3.8. Statistical Analysis Data from the XO inhibitory assay were expressed as the mean ˘ standard deviation (S.D.) and were analyzed by one-way analysis of variance using the SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). A value of p < 0.05 was considered statistically signiﬁcant. IC50 values of the samples were calculated with origin version 8.0 software (OriginLab Corporation, Northampton, UK). The inhibitory type and Ki value were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA).

4. Conclusions In summary, 24 organic extracts of four species belonging to Citrus genus recording in the Chinese Pharmacopeia were prepared and their XO inhibitory activities evaluated in order to develop therapeutic or preventive agents for hyperuricemia. The results demonstrated for the first time that the ethyl acetate extract of C. aurantium dried immature fruits possessed XO inhibitory activity. Bioguided fractionation by chromatography of the extract to obtain five flavanones and two polymethoxyflavones showed that hesperetin was a putative candidate for the XO inhibitory activity.

Acknowledgments: This project was financially supported by the Fostering Plan for Outstanding Young Teachers in College of Meicine of Qingdao University. Author Contributions: The contributions of the respective authors are as follows: K.L., H.-L.Y., and K.C. performed the extraction, isolation and elucidation of the constituents. B.-H.G. and X.-H.L. engaged in the bioassay of the compounds in vitro. Y.L. and H.G. contribute to checking and confirming all of the procedures of the isolation and identification. This study was performed based on the design of W.W., the corresponding author. All the authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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