Flavonoids: Separation and Quantitation

The Scientific World Journal

Guest Editors: Wanchai De-Eknamkul, Kaoru Umehara, and Jan Frederik Stevens Flavonoids: Separation and Quantitation The Scientific World Journal Flavonoids: Separation and Quantitation

This is a special issue published in “The Scientific World Journal.”All articles are open access articles distributed under the Creative Com- mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

Flavonoids: Separation and Quantitation, Wanchai De-Eknamkul, Kaoru Umehara, and Jan Frederik Stevens Volume2015,ArticleID874148,2pages Antimycobacterial and Nitric Oxide Production Inhibitory Activities of Ocotea notata from Brazilian Restinga, Isabela Francisca Borges Costa, Sanderson Dias Calixto, Marlon Heggdorne de Araujo, Tatiana Ungaretti Paleo Konno, Luzineide Wanderley Tinoco, Denise Oliveira Guimaraes,˜ Elena B. Lasunskaia, Ivana Ramos Correa Leal, and Michelle Frazao˜ Muzitano Volume 2015, Article ID 947248, 9 pages Microwave-Assisted Simultaneous Extraction of Luteolin and Apigenin from Tree Peony Pod and Evaluation of Its Antioxidant Activity, Hongzheng Wang, Lei Yang, Yuangang Zu, and Xiuhua Zhao Volume 2014, Article ID 506971, 12 pages Analysis of Flavone C-Glycosides in the Leaves of Clinacanthus nutans (Burm. f.) Lindau by HPTLC and HPLC-UV/DAD, June Lee Chelyn, Maizatul Hasyima Omar, Nor Syaidatul Akmal Mohd Yousof, Ramesh Ranggasamy, Mohd Isa Wasiman, and Zakiah Ismail Volume 2014, Article ID 724267, 6 pages Phenolic Composition and Antioxidant Activity of Malus domestica Leaves, Mindaugas Liaudanskas, Pranas Viˇskelis, Raimondas Raudonis, Darius Kviklys, Norbertas Uselis, and Valdimaras Janulis Volume2014,ArticleID306217,10pages Optimization of Ionic Liquid Based Simultaneous Ultrasonic- and Microwave-Assisted Extraction of Rutin and Quercetin from Leaves of Velvetleaf (Abutilon theophrasti)byResponseSurface Methodology, Chunjian Zhao, Zhicheng Lu, Chunying Li, Xin He, Zhao Li, Kunming Shi, Lei Yang, Yujie Fu, and Yuangang Zu Volume 2014, Article ID 283024, 11 pages Phytochemical Profiles and Antioxidant and Antimicrobial Activities of the Leaves of Zanthoxylum bungeanum, Yujuan Zhang, Ziwen Luo, Dongmei Wang, Fengyuan He, and Dengwu Li Volume 2014, Article ID 181072, 13 pages Optimization of Reflux Conditions for Total Flavonoid and Total Phenolic Extraction and Enhanced Antioxidant Capacity in Pandan (Pandanus amaryllifolius Roxb.) Using Response Surface Methodology, Ali Ghasemzadeh and Hawa Z. E. Jaafar Volume 2014, Article ID 523120, 10 pages HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora, Fang-Ju Lin, Feng-Lin Yen, Pei-Chun Chen, Moo-Chin Wang, Chun-Nan Lin, Chiang-Wen Lee, and Horng-Huey Ko Volume 2014, Article ID 528653, 8 pages Flavonoids in Juglans regia L. Leaves and Evaluation of In Vitro Antioxidant Activity via Intracellular and Chemical Methods,Ming-HuiZhao,Zi-TaoJiang,TaoLiu,andRongLi Volume 2014, Article ID 303878, 6 pages Chemical Characterization of Fruit Wine Made from Oblacinskaˇ Sour Cherry, Milica Pantelic,´ Dragana Dabic,´ Saˇsa Matijaˇsevic,´ Sonja Davidovic,´ Biljana Dojcinoviˇ c,´ Duˇsanka Milojkovic-Opsenica,´ Zivoslav˜ Teˇsic,´ and Maja Natic´ Volume 2014, Article ID 454797, 9 pages Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 874148, 2 pages http://dx.doi.org/10.1155/2015/874148

Editorial Flavonoids: Separation and Quantitation

Wanchai De-Eknamkul,1 Kaoru Umehara,2 and Jan Frederik Stevens3

1 Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan 3Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA

Correspondence should be addressed to Wanchai De-Eknamkul; [email protected]

Received 13 January 2015; Accepted 13 January 2015

Copyright © 2015 Wanchai De-Eknamkul et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Flavonoids, products of secondary metabolism, are widely In this special issue, the contributions consist of ten distributed in the plant kingdom. They belong to the large papers covering several aspects of flavonoids analysis, rang- group of plant polyphenols and represent one of the most ing from methods and techniques of extraction, separation, abundant classes of phytochemicals that are ubiquitously and quantitation to phytochemical profiling and identifica- present in fruits, vegetables, medicinal plants, and their tion of bioactive flavonoids. products. Thousands of flavonoids, existing both in free form For the optimization of extraction, a chemometric and as glycosides, have been characterized and reported in approach based on response surface methodology was used the literature. Many of them are known to play ecological in two studies. One is for the optimization of ionic liquid- roles in the interactions of plants with their environment. based simultaneous ultrasonic and microwave-assisted Some flavonoids are known to act as signaling molecules and extraction for isolating rutin and quercetin from leaves of others as defense agents to fend off herbivores. Abutilon theophrasti (C. Zhao et al.) and the other for the In addition, flavonoids are renowned for their numerous optimization of reflux conditions for total flavonoid and health benefits. Scientific evidence supports epidemiological total phenolic extraction and enhanced antioxidant capacity observations that regular intake of dietary flavonoids reduces in Pandanus amaryllifolius (A.GhasemzadehandH.Z.E. the risk of oxidative-stress mediated pathogenesis of human Jaafar). A similar microwave-assisted method was used for diseases as well as of age-related ailments. In many cases, the simultaneous extraction of luteolin and apigenin from however, it is necessary to release the flavonoids from plant Paeonia ostii pods (H. Wang et al.). materials by extraction before they can be evaluated for For the separation and quantitation of flavonoids, in addi- biological and pharmacological activity. For further develop- tion to the generally used HPLC-UV,the technique of HPLC- ment of health products or herbal drugs, plant extracts are MS/MS is exemplified in two papers: “Flavonoids in Juglans usually characterized by chromatographic means for quality regia L. Leaves and Evaluation of In Vitro Antioxidant Activity control purposes or subjected to separation to obtain the via Intracellular and Chemical Methods” (M.-H. Zhao et al.) bioactive flavonoid constituents. and “Chemical Characterization of Fruit Wine Made from The increasing interest in bioactive flavonoids from an Oblacinska Sour Cherry” (M. Pantelicetal.).Thelatteralso´ ecological or health perspective has resulted in the isolation used the powerful UHPLC system for separation of the con- and structure elucidation of many novel and minor natural stituents. Furthermore, the technique of HPTLC was shown flavonoids with interesting biological activities. At the same to obtain good separation of the group flavone C-glycosides time, several techniques of chromatography have been fur- as reported in the paper entitled “Analysis of Flavone C- ther developed for efficient separation, identification, and Glycosides in the Leaves of Clinacanthus nutans (Burm. f.) quantitation of the flavonoids. Lindau by HPTLC and HPLC-UV/DAD” by J. L. Chelyn et al. 2 The Scientific World Journal

Interestingly, the paper “Phytochemical Profiles and Antioxidant and Antimicrobial Activities of the Leaves of Zanthoxylum bungeanum” by Y. Zhang et al. showed very nicely the simultaneous separation of 12 flavonoids and related compounds in a single HPLC run to obtain phytochemical profiles of the plant material. Equally impressive is the paper “Phenolic Composition and Antioxidant Activity of Malus domestica Leaves” (M. Liaudanskas et al.) which showed complete separation of all 13 flavonoids and phenolic present in apple leaf extracts. Similarly, nine closely related flavones differing from each other by variation in ring substi- tution were also successfully separated as demonstrated in the report “HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora”byF.-J.Linetal. Finally, there is one paper reporting on the isolation and structure elucidation of the flavonoids afzelin and isoquercitrin from Ocotea notata (I.F.B.Costaetal.).Both flavonoids strongly exhibited antimycobacterial activity and inhibition of nitric oxide production by macrophages. We hope that all the interesting results published in this special issue will stimulate the continuing efforts to develop superior methods of flavonoid separation and quantitative analysis.

Acknowledgments Wethank the authors of the submitted papers for their contribution. The preparation of this special issue would not have been possible without the generous support and dedication of experts who evaluated the submitted papers. Wanchai De-Eknamkul Kaoru Umehara Jan Frederik Stevens Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 947248, 9 pages http://dx.doi.org/10.1155/2015/947248

Research Article Antimycobacterial and Nitric Oxide Production Inhibitory Activities of Ocotea notata from Brazilian Restinga

Isabela Francisca Borges Costa,1 Sanderson Dias Calixto,2 Marlon Heggdorne de Araujo,1,2 Tatiana Ungaretti Paleo Konno,3 Luzineide Wanderley Tinoco,4 Denise Oliveira Guimarães,1 Elena B. Lasunskaia,2 Ivana Ramos Correa Leal,1,5 and Michelle Frazão Muzitano1

1 Laboratorio´ de Produtos Naturais (LaProN), Curso de Farmacia,´ Universidade Federal do Rio de Janeiro, Campus Macae,´ Polo Novo Cavaleiro-IMMT, R. Alu´ısiodaSilvaGomes50,27930-560Macae,´ RJ, Brazil 2 Laboratorio´ de Biologia do Reconhecer, Centro de Biocienciasˆ e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602 Campos dos Goytacazes, RJ, Brazil 3 Nucleo´ de Estudos em Ecologia e Desenvolvimento Socio-Ambiental´ de Macae,´ Universidade Federal do Rio de Janeiro, 27910-970 Macae,´ RJ, Brazil 4 InstitutodePesquisasdeProdutosNaturais,UniversidadeFederaldoRiodeJaneiro,21941-902RiodeJaneiro,RJ,Brazil 5 Laboratorio´ de Produtos Naturais e Ensaios Biologicos´ (LaProNEB), Departamento De Produtos Naturas e Alimentos, Faculdade de Farmacia,UniversidadeFederaldoRiodeJaneiro,21941-902RiodeJaneiro,RJ,Brazil´

Correspondence should be addressed to Michelle Frazao˜ Muzitano; [email protected]

Received 4 July 2014; Revised 13 September 2014; Accepted 1 October 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2015 Isabela Francisca Borges Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The genus Ocotea (Lauraceae) is distributed mainly in tropical and subtropical regions. Some species of this genus as O. puberula and O. quixos have been described in the literature, showing antibacterial activity. And Ocotea macrophylla showed anti-inflammatory activity with inhibition of COX-1, COX-2, and LOX-5. The purpose of this study was the phytochemical investigation of the plant species Ocotea notata from Restinga Jurubatiba National Park, Macae,´ RJ, Brazil, and the search for antimycobacterial fractions and compounds. The crude extract was evaluated for antimycobacterial activity and presented 95.75 ± 2.53% of growth inhibition at 100 𝜇g/mL. Then, it was subjected to a liquid-liquid partition and subsequently was chemically investigated by HPLC, revealing the major presence of flavonoids. In this process the partition fractions hexane, ethyl acetate, and butanol are shown to be promising in the antimycobacterial assay. In addition, ethyl acetate fraction was chromatographed and afforded two flavonoids identified byMS and NMR as afzelin and isoquercitrin. The isolated flavonoids afzelin and isoquercitrin were evaluated for their antimycobacterial activity and for their ability to inhibit NO production by macrophages stimulated by LPS; both flavonoids isoquercitrin (Acet22) and afzelin (Acet32) were able to inhibit the production of NO by macrophages. The calculated50 IC of Acet22 and Acet32 was 1.03 and 0.85 𝜇g/mL, respectively.

1. Introduction The plant communities at the periphery of the Atlantic rainforest complex, such as restingas, differ from the core Brazilian Atlantic forest areas were considered the fourth in formation in that they exhibit more extreme environmental relevance among a total of 25 hotspots worldwide. Hotspots conditions found in these systems; drought, salinity, high are areas that hold an exceptional concentration of endemic temperatures, and low soil nutrient contents are the main plants and vertebrates experiencing exceptional loss of habitat limiting factors in the open scrub habitat of the restinga veg- [1]. etation [2]. 2 The Scientific World Journal

Ocotea notata (Nees & Mart.) Mez, Lauraceae, is a observe a single spot in these subfractions, suggesting the medium sized tree, popularly known as white cinnamon. purity of the sample. The genus is distributed throughout tropical and subtropical regions, especially along the Brazilian coast. Ocotea species 2.4. Analysis by High-Performance Liquid Chromatography have been studied for their diversity in secondary metabo- (HPLC). Extracts, fractions, and isolated compounds were lites such as alkaloids, neolignans, lignans, terpenes, and submitted to high-performance liquid chromatography using flavonoids3 [ –8] and stand out for their biological activi- a Shimadzu Prominence LC-20. The detection was performed ties such as anti-inflammatory9 [ , 10], antioxidant [11, 12], at fixed wavelengths of 254 nm and 332 nm. The column used antiprotozoan [13], antiallergic [14], central nervous system was a Nucleosil 100-5 RP-18 (5 mM, 4.0 × 250 mm) main- ∘ depressant [15], antimicrobial [11, 16], and anti-herpetic [8]. tained at 30 C. The eluents were (A): H2OadjustedtopH3.0 The present study aims at the investigation of the by H3PO4 and (B): CH3CN. The following solvent gradients chemical profile of Ocotea notata ethanol extract collected (v/v) were applied: from H2O (pH 3.0)-CH3CN (10 : 0) to in Jurubatiba Restinga (Macae,´ RJ, Brazil) through HPLC H2O (pH 3.0)-CH3CN (8.5 : 1.5) within 10 min; from H2O analyses and phytochemical study. In addition, due to the (pH 3.0)-CH3CN (7.5: 2.5) to H2O (pH 3.0) within 30 min- previously described antimicrobial activity of O. notata,the CH3CN (5 : 5) within 45 min; from H2O (pH 3.0)-CH3CN antimycobacterial activity of the samples obtained in this (0 : 10) to H2O (pH 3.0) within 60 min; from 60 minutes as −1 chemical study and their ability to inhibit NO production by total time of analysis. Flow elution was 1 mL/min and 10 𝜇L macrophages stimulated by LPS were also investigated. samples were injected. HPLC analyses were performed after dilution of 10 mg of the extract in 1 mL of purified water. 2. Materials and Methods 1 13 2.1. General Experimental Procedures. Hand C-NMR 2.5. Quantification of Flavonoids. The flavonoid quantifica- spectra were recorded on a Bruker DRX-400 NMR spectrom- tion was carried out using calibration graph with ten data 1 13 eter ( H: 400 MHz; C: 100 MHz). Chromatography was points. Calibration graph for HPLC was recorded with rutin (quercetin 3-O-rutinoside) amounts ranging from 0.20 to performed on reversed-phase silica Kieselgel 60 silanisiert 𝜇 (0,063–0,200 mm). Eluates were monitored by thin-layer 10.0 g. The linearity range of the detector response was verified using a series of twofold diluted solutions of rutin. chromatography (TLC) on silica 60 F254 (Merck) using The relationship between peak areas (detector responses) butanol/acetic acid/water (BAW 8 : 1 : 1), visualized under UV 𝜇 𝑟2 = light and revealed with NP-PEG. and amount of rutin was linear over 1000–20 g/mL ( 0.9999). To evaluate the repeatability of the injection integra- 2.2. Botanical Material. The plant species studied was col- tion, the rutin standard solution and the extract were injected lected in Restinga de Jurubatiba National Park, Quis- three times and the relative standard deviation values were ∘ sama˜ (southeastern Rio de Janeiro state, Brazil): 22.19828 S; calculated. ∘ 41.46338 W. Voucher specimens were deposited at the Herbarium of the Rio de Janeiro Federal University, Brazil 2.6. Antimycobacterial Activity of the Extract, Fractions and (RFA38751), after identification by the botanist Tatiana U. P. Isolated Compounds. All samples were evaluated for their Kono. This research was complied with all relevant federal antimycobacterial activity, at concentrations of 0.8, 4, 20, and guidelines and institutional policies related to the botanical 100 𝜇g/mL, using a tetrazole salt assay in 96-well microplate material for research purposes. [17]. Initially, 300 𝜇L of a suspension of Mycobacterium 7 bovis BCG strain Moreau (3 × 10 CFU/mL) was incubated 2.3. Ethanol Extraction and Partitions. Fresh leaves with 7.2 mL of Middlebrook 7H9 medium supplemented (1,300.00 g) were triturated and extracted exhaustedly with with 0.05% Tween 80 and albumin-dextrose-catalase (ADC). ethanol ACS at room temperature (crude extract). An aliquot When at logarithmic growth phase, 50 𝜇Lwasplatedina96- 6 (60 g) of the dried extract (78.07 g) was resuspended with well microplate at 1 × 10 CFU/well, and 50 𝜇L of each sample methanol and partitioned with hexane to obtain the hexane was added in four concentrations. The sealed microplate ∘ fraction (26.68 g). Methanol residual phase was dried and was incubated at 37 Cand5%CO2 for 7 days. After this resuspended with pure water and partitioned sequentially period, 10 𝜇L of tetrazolium salt (3-[4,5-dimethylthiazol- with ethyl acetate and butanol, affording ethyl acetate 2-yl]-2,5-diphenyltetrazole, 5 mg/mL in sterile PBS) was fraction (3.40 g) and butanol fraction (10.50 g), respectively. added, and after 3 hours 100 𝜇L of lyses buffer (20% w/v The residual aqueous phase was named the aqueous fraction sodium dodecyl sulfate, SDS/50% dimethylformamide, DMF (13.99 g). An aliquot of ethyl acetate fraction (1.50 g) was in distilled water, pH 4.7) was added. The microplate was chromatographed on a silica Kieselgel 60 silanisiert (0,063– incubated overnight, and the reading was made using a 0,200 mm) (H2O/MeOH gradient), yielding 295 fractions. spectrophotometer at 570 nm. As a positive control, a culture Similar fractions between 92–96 (Acet22) and 166–168 medium with bacteria and the antibiotic rifampin (Sigma- (Acet32) were grouped observing the chromatographic Aldrich, 95% purity), at concentrations of 0.0011, 0.0033, 0.01, similarities, after TLC revealed with NP-PEG solution and 0.03 𝜇g/mL, were used. As a negative control, a culture with 1% ethanolic diphenylboryloxy-ethylamine acid (NP) medium with bacteria untreated with the samples was used. followed by 5% polyethylene glycol-4000 (PEG) with 10 mL The test was performed in triplicate and the average value and and 8 mL, respectively. By TLC analysis, it was possible to standard deviation were calculated. The Scientific World Journal 3

150 254 nm, 4 nm (1.00) 41.206 140 130 44.253 120 110 100 39.126 90 80

70 39.877 35.576 60

(mAU) 50 40 22.493 59.186 37.697 46.064 30 20 4.017 10 0 −10 −20 0 10 20 30 40 50 60 70 (min)

175 250 150 35.57/1.00 39.13/1.00 39.95/1.00 150 125 200 240 125 100 150 100 75 254 240 75 100 255 (mAU) (mAU) 354 (mAU) 354 50 282 50 294 299 354 50 25 25 420 420 0 0 0

200 300 400 200 300 400 200 300 400 (nm) (nm) (nm)

41.22/1.00 350 44.25/1.00 300 300 240 250 248 200 200 255 150 286 (mAU) 348 (mAU) 100 100 343 50 440 420 0 0

200 300 400 200 300 400 (nm) (nm)

Figure 1: Chromatogram at 254 nm of the crude extract of the leaves of Ocotea notata byHPLC.EmphasesfortheUVspectrumofpeaks 35.57, 39.13, 39.88, 41.22, and 44.25 min.

2.7. Determination of Nitric Oxide Production by Macrophage were seeded in flat bottom 96-well tissue culture plates RAW 264.7 and Cytotoxicity. The murine macrophage cell (Corning, Inc.) in the presence or absence of four concen- line RAW 264.7 was obtained from the American Type trations of the samples (0.8, 4, 20, and 100 𝜇g/mL) and/or ∘ Culture Collection (ATCC) and grown at 37 Cand5%CO2 LPS (Escherichia coli 055:B5; Sigma-Aldrich). After a 24-hour in DMEM F-12 that was supplemented with 10% FCS and incubation period, culture supernatants were collected and 5 gentamicin (50 𝜇g/mL). RAW 264.7 cells (1 × 10 cells/well) nitrite, a stable NO metabolite, was determined by using 4 The Scientific World Journal

∗∗∗ 100 100 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗∗∗∗ ∗∗∗∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 50 50 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 0 0 ∗∗∗∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗∗∗∗∗∗∗ Inhibition (%) Inhibition Cytotoxicity (%) Cytotoxicity − + − + Control Control Control Control Crude extract [4] Crude extract [4] Crude extract [20] Crude extract [20] Crude extract [0.8] Hexane fraction [4] Hexane Crude extract [0.8] Crude extract [100] Butanol fraction [4] Butanol Hexane fraction [4] Hexane Crude extract [100] Butanol fraction [4] Butanol Aqueous fraction[4] Aqueous Aqueous fraction [4] Aqueous Butanol fraction[20] Butanol Butanol fraction [20] Butanol Hexane fraction [20] Hexane Hexane fraction [20] Hexane Hexane fraction [0.8] Hexane Butanol fraction [0.8] Butanol Butanol fraction [0.8] Butanol Hexane fraction [0.8] Hexane Hexane fraction [100] Hexane Butanol fraction [100] Butanol Butanol fraction [100] Butanol Hexane fraction [100] Hexane Aqueous fraction [20] Aqueous Aqueous fraction [20] Aqueous Aqueous fraction [0.8] Aqueous Aqueous fraction [0.8] Aqueous Aqueous fraction [100] Aqueous Aqueous fraction [100] Aqueous Ethyl acetate fraction [4] acetate Ethyl Ethyl acetate fraction [4] acetate Ethyl Ethyl acetate fraction [20] acetate Ethyl Ethyl acetate fraction [20] acetate Ethyl Ethyl acetate fraction [0.8] acetate Ethyl Ethyl acetate fraction [0.8] acetate Ethyl Ethyl acetate fraction [100] acetate Ethyl Ethyl acetate fraction [100] acetate Ethyl (a) (b)

Figure 2: (a) Inhibitory effect of Mycobacterium bovis BCGgrowthbyextractsandfractionsofleavesofOcotea notata tested at four different concentrations, 0.8, 4, 20, and 100 𝜇g/mL. The percentage of inhibition was compared with the positive control (rifampicin): 98.17±0.8%/O.D.: 0.180±0.8 and negative control (mycobacteria without treatment) 0.1±0.1%/O.D.: 0.880±1.24. (b) Cytotoxicity of the extracts and fractions of leaves of Ocotea notata tested at four different concentrations 0.8, 4, 20, and 100 𝜇g/mL in murine macrophages RAW 264.7.The percentages were compared to positive control, macrophages treated with Triton 1%: 98.88 ± 1.6/O.D.: 0.132, and negative control, macrophages without ∗ ∗∗ ∗∗∗ treatment 0.01 ± 0.1/O.D.: 0.749. 𝑃 < 0.05, 𝑃 < 0.01,and 𝑃 < 0.001, significance obtained by ANOVA and posttest Tukey (𝑛=3), when compared to the negative control (a) and positive control (b).

the Griess test [18].Asapositivecontrol,macrophages were identified with retention time of 35.57, 39.13, 39.88, untreated but stimulated with 1 𝜇g/mL LPS were used. As 41.22, and 44.25 minutes (Figure 1). UV spectrum of each a negative control, macrophages not treated and not LPS- peak revealed the flavonoid absorption profile (typical 𝜆max stimulated were used. A nitric oxide synthase inhibitor, NG- 251–271 and 335–350 nm) [20]. For the predominance of methyl-L-arginine acetate salt (L-NMMA, Sigma-Aldrich, flavonoidsinthesample,theywerequantifiedbasedonan 98% purity), was also used as a positive control at 20 𝜇g/mL area ×𝜇g calibration curve obtained using a rutin external inhibiting 59.22 ± 2.96% NO production. The release of standard.Thesumofallidentifiedpeaksinthechro- cytoplasmic enzyme lactate dehydrogenase (LDH) was deter- matogramwasassumedtorepresentthetotalflavonoidcon- mined using 50 𝜇L of culture supernatant collected at the tent in the extract, expressed as rutin equivalents, percentage end of the assay [19]. The LDH content, which represents an (w/w) g/100 g of crude extract. For this purpose, crude extract indirect indication of cytotoxicity, was determined colorimet- was analyzed in triplicate resulting in a flavonoid content of rically using a commercial kit (Doles). The specific release 2.71±0.16% w/w. Crude extract was assessed in view of verify- wascalculatedasapercentageofthecontrols(untreated ing its antimycobacterial activity. Antimycobacterial activity macrophagesasthenegativecontroland1%TritonX-100 was evaluated on Mycobacterium bovis BCG strain. This [Vetec Chem.] detergent treated macrophages as the positive strain shows a very similar genetic profile when compared to control). Final concentrations of DMSO, used as the solvent M. tuberculosis [21]. Ocotea notata crude extract exhibits, at of the samples, were tested in parallel as a control. Tests were concentration of 20 𝜇g/mL, 73.63 ± 1.86%ofmycobacterial performed in triplicate, and the mean value and standard growth inhibition and only 26.40 ± 1.50%ofcytotoxicity deviation were calculated. (Figures 2(a) and 2(b)). At concentration of 100 𝜇g/mL, it showed an inhibition of 95.75 ± 2.53%, but it was toxic when 2.8. Statistical Analysis. All experiments were performed in evaluated in RAW 264.7 macrophages culture (Figures 2(a) triplicate and results were expressed as mean ± standard and 2(b)). The inhibition extract capacity was compared deviation (M ± SD). Data was evaluated by one-way ANOVA to rifampicin, drug tested at different concentrations, and followed by Tukey test and considered statistically significant usedasapositivecontrol.Tuberculosis(TB)isoneofthe for 𝑃 < 0.05.TheIC50 (concentration able to modulate at 50% leading causes of mortality worldwide and its etiologic agent maximum activity) of the samples tested was calculated by is Mycobacterium tuberculosis bacilli but also M. bovis, M. nonlinear regression using the results of the concentration- africanum,andM. microti [22]. response curves. Microsoft Office Excel and GraphPad Prism For the promising activity observed from the crude software were used. extract, it was fractioned by liquid-liquid partition and afforded four fractions with different polarities, hexane, ethyl 3. Results and Discussion acetate, butanol, and water. The fraction that had the best performance in the inhibitory mycobacterial activity growth Ocotea notata ethanol extract (crude extract) was analyzed was the hexane fraction. At concentrations of 0.8, 4, 20, and by reversed-phase HPLC-DAD to study its chemical profile. 100 𝜇g/mL it showed, respectively, 41.63 ± 0.80%; 65.75 ± A suitable methodology was developed and five major peaks 5.30%; 90.88 ± 1.59;and102.56 ± 1.90%ofmycobacterial The Scientific World Journal 5

254 nm, 4 nm (1.00) 600 39.16/1.00 1500 1000 36.53/1.00 39.164 202 900 203 500 800 700 400 600 300 500 255 1250 354 254 400 (mAU) 200 (mAU) 300 281 348 100 294 1100 37.51/1.00 200 42.481 1000 203 100 900 0 0 1000 800 700 200 300 400 200 300 400 600 (nm) (nm) 500 255 354 400 36.532 2750 (mAU) 300 282 2500 42.81/1.00 750 200 2250 100 2000 0 1750 −100 1500 1250 263 (mAU) 200 300 400 1000 244 343 (mAU) 500 (nm) 24.255 37.548 750 280 500 250 0 32.899 34.837 −250 200 300 400 250 45.875 (nm) 44.314

0 10 20 30 40 50 60 70 (min)

(a)

175 254 nm, 4 nm (1.00) 21.943

400 150 34.286 203 34.29/1.00 21.943/1.00 300 250 214 237 125 200 200 241 255 150 (mAU) 354 100 100 (mAU) 447 50 0 100 397 422 0 200 300 400 200 300 400 (nm) (nm)

(mAU) 75

25.242 50 40.313

39.136 48.379 38.041

17.143 21.159 43.429 25 5.397 28.312

0 10 20 30 40 50 60 70 (min)

(b)

Figure 3: (a) Chromatogram at 254 nm of ethyl acetate fraction from leaves of Ocotea notata by HPLC. Emphases for the UV spectrum of peaks 36.53; 37.55; 39.16; and 42.48 min. (b) Chromatogram at 254 nm of butanol fraction from leaves of Ocotea notata by HPLC. Emphases for the UV spectrum of peaks 21.943 and 34.286 min.

growth inhibition. Hexane fraction showed activity even in The inhibitory activity of the ethyl acetate fraction was small concentrations and it was toxic for macrophages only at at concentrations of 0.8, 4, 20, and 100 𝜇g/mL, 43.63 ± 1.06, the highest concentration. This finding suggested selectivity 57.75 ± 0.46, 83.38 ± 3.54,and80.75 ± 1.15%, respectively. for antimycobacterial activity without being cytotoxic to But when the cytotoxicity to macrophages was evaluated, the macrophages at 0.8, 4 and 20 𝜇g/mL. This fraction is the most ethyl acetate fraction showed low toxicity when compared apolar and usually this kind of fraction is mainly composed to hexane fraction at the highest concentration (Figure 2). by terpenes, sterols, and fatty acids [23]. Hexane fraction was The same was observed in butanol fraction (Figure 2). followed by ethyl acetate fraction that was the second on According to Moresco and Brighente [24] fractions as inhibition of mycobacterial growth. ethyl acetate, butanol, and aqueous are rich in phenolic 6 The Scientific World Journal

R1 no antimycobacterial activity and moderated cytotoxicity. In OH theliteraturetherearefewreportsaboutflavonoidswith antimycobacterial activity. Yet some of them, especially those HO O that are less polar, could be found, as chalcones [26]and prenylated flavones [27]. However isoquercitrin and afzelin OR are glycosylated flavonoid with considerable hydrophilicity. 2 This fact complicates the permeability of these substances OH O through lipophilic bacterial wall. For the genus Ocotea there are few records about flavon-

Flavonoids R1 R2 oids isolation, and the main secondary metabolites are alka- Afzelin H 𝛼-L-Rhamnopyranose loids, lignans, and terpenoids. Flavonoids are divided into Isoquercitrin OH 𝛽-D-Glucopyranose classes according to their chemical and biosynthetic characteristics and have numerous pharmacological and biochem- Figure 4: Kaempferol 3-O-𝛼-L-rhamnopyranoside (afzelin- ical effects28 [ ]. There is only one report on the isolation 𝛽 Acet32), quercetin 3-O- -D-glucopyranoside (isoquercitrin- of the flavonoid isoquercitrin from O. notata, in addition Acet22). to a proanthocyanidin trimer and flavonoids quercitrin and reynoutrin [8]. There are reports about isoquercitrin isolation also from O. corymbosa [29]. No data describing the antimi- compounds; this can be explained by the polarity of these crobial activity of isoquercitrin were found. substances. Comparing the demonstrated results, it was Funasaki [30] reported the phytochemical study of O. noticed that butanol and ethyl acetate fractions showed an catharinensis leaves, describing the glycosylated flavonoid excellent inhibitory effect and lower cytotoxicity, especially afzelin isolation, the same that is isolated and described in the last one, so that these polar fractions were investigated this study, for O. notata.Afzelinshowednoantimicrobial by HPLC to identify the chemical constituents responsible activity described in the literature. But it presents antinoci- for this activity. HPLC profile of polar fractions pointed ceptive and anti-inflammatory activities31 [ ]andstrong the presence of secondary metabolites such as flavonoids neural protective effect and antioxidant32 [ ]. (Figures 3(a) and 3(b)). Butanol fraction presented two major peaks, 21.94 and 34.29 min at 254 nm, the second one with In addition, considering that Ocotea notata isolated UV flavonoid characteristic. Ethyl acetate fraction showed a flavonoids, isoquercitrin, and afzelin do not show significant complexprofilewithfourmajorpeaksthatwereidentifiedby antimycobacterial activity, as demonstrated in the present UV as flavonoids (36.53, 37.55, 39.16, and 42.48 min). Ethyl study, they were evaluated to verify their capacity of inhibit- acetate fraction was chosen by fractionation although butanol ing the NO production by LPS-stimulated macrophages. and hexane fractions were also selected except for future Nitric oxide is a chemical mediator with microbicide activity investigations. To compare the total flavonoid content of the that is produced by activated phagocytes during inflamma- ethyl acetate fraction and the crude extract, as reported above, tion [33]. Inflammation is strongly involved in the patho- this fraction was analyzed in triplicate resulting in a flavonoid genesis of most infectious diseases, including tuberculosis content of 37.3±1.5% w/w, rate over fifteen times higher than [34]. In general, the production of proinflammatory medi- that found in crude extract. ators by the infected macrophages, such as IL-1𝛽,TNF-𝛼, Reversed-phase chromatography of ethyl acetate fraction and NO, is essential for protection against mycobacteria afforded two isolated flavonoids codified as Acet22 and [35]. However, tissue concentrations of NO required for Acet32. These two flavonoids were analyzed by HPLC and microbicide action are toxic to the host cells and must be 1 their purity was confirmed. Mono and bidimensional Hand tightly regulated [33].InthecaseofTBmostsevereforms, 13 C NMR analyses of Acet22 allow this flavonoid characteri- additional anti-inflammatory therapy to prevent excessive zation as isoquercitrin (quercetin 3-O-𝛽-D-glucopyranoside) inflammation could be required35 [ ]. In report about treat- (Figure 4). NMR data are in accordance with the literature ment of TB together with anti-inflammatory drugs it was [25]. The flavonoid Acet32 was analyzed by NMR and MS. demonstrated that corticosteroids can be effective in reducing The molecular formula C21H20O10 was deduced from the mortality for all forms of TB [36]. The benefits of anti- + molecular ion m/z 455.2 [M + Na–H] (calculated for inflammatory treatment have also been demonstrated in C21H20O10Na). Analyzing the molecule fragmentation pat- some TB cases using nonsteroidal anti-inflammatory drugs tern can be observed in the following peaks representing the (NSAIDs). Result with infected animals and treatment with + fragments [M-Ram + Na] . m/z (309.0) relative to the loss ibuprofen (anti-inflammatory drug) showed decrease in + of rhamnose residue and [M-Kaempferol + Na] .(m/z 169.1) pulmonary infiltrates and in bacterial load and increased relative to the loss of the aglycone. These results together survival, compared to untreated animals [37]. with NMR data allow the identification of the flavonoid AscouldbeseeninFigure 7, crude extract and both afzelin (kaempferol-3-O-𝛼-L-rhamnopyranoside) (Figure 4) flavonoids, isoquercitrin (Acet22) and afzelin (Acet32), as Acet32, in accordance with literature [25]. were capable of inhibiting the NO production by As can be seen, the isolated compounds, isoquercitrin macrophages, with 𝑃 < 0.001 at concentrations of 0.8, (Figures 5(a) e 5(b)) and afzelin (Figures 6(a) e 6(b)), showed 4, 20, and 100 𝜇g/mL, when compared with positive control The Scientific World Journal 7

100 100

∗∗∗ ∗∗∗ ∗∗∗ 50 50 ∗∗∗ Inhibition (%) Inhibition Cytotoxicity (%) Cytotoxicity 0 0 + − + − Control Control Control Control Acet22 [4] Acet22 Acet22 [4] Acet22 Acet22 [20] Acet22 Acet22 [20] Acet22 Acet22 [0.8] Acet22 Acet22 [0.8] Acet22 Acet22 [100] Acet22 Acet22 [100] Acet22 (a) (b)

Figure 5: (a) Inhibitory effect of Mycobacterium bovis BCG growth by Acet22 (isoquercitrin) tested at four different concentrations, 0.8, 4, 20, and 100 𝜇g/mL. The percentage of inhibition was compared with the positive control (rifampicin), 98.17 ± 0.8%/O.D.: 0.180 ± 0.8,and negative control (medium + mycobacteria), 0.1 ± 0.1%/O.D.: 0.880 ± 1.24. (b) Cytotoxicity of Acet22 (isoquercitrin) tested at four different concentrations 0.8, 4, 20, and 100 𝜇g/mL in murine macrophages RAW 264. The percentages were compared to positive control, macrophages ∗ treatedwithTriton1%:98.88 ± 1.6/O.D.: 0.132, and negative control, macrophages without treatment 0.01 ± 0.1/O.D.: 0.749. 𝑃 < 0.05, ∗∗ ∗∗∗ 𝑃 < 0.01,and 𝑃 < 0.001, significance obtained by ANOVA and posttest Tukey (𝑛=3), when compared to the negative control (a) and positive control (b).

100 100

50 50 ∗∗∗

∗∗∗ ∗∗∗ Inhibition (%) Inhibition

Cytotoxicity (%) Cytotoxicity ∗∗∗

0 0 + − + − Control Control Control Control Acet32 [4] Acet32 [4] Acet32 Acet32 [20] Acet32 Acet32 [20] Acet32 Acet32 [0.8] Acet32 [0.8] Acet32 Acet32 [100] Acet32 Acet32 [100] Acet32 (a) (b)

Figure 6: (a) Inhibitory effect of Mycobacterium bovis BCG growth by Acet32 (afzelin) tested at four different concentrations, 0.8; 4; 20; and 100 𝜇g/mL. The percentage of inhibition was compared with the positive control (rifampicin), 98.17 ± 0.8%/O.D.: 0.180 ± 0.8, and negative control (medium + mycobacteria), 0.1 ± 0.1%/O.D.: 0.880 ± 1.24. (b) Cytotoxicity of Acet32 (afzelin) tested at four different concentrations 0.8; 4; 20; and 100 𝜇g/mL against murine macrophages RAW 264. The percentages were compared to positive control, macrophages treated ∗ ∗∗ with Triton 1%: 98.88 ± 1.6/O.D.: 0.132, and negative control, macrophages without treatment: 0.01 ± 0.1/O.D.: 0.749. 𝑃 < 0.05, 𝑃 < 0.01, ∗∗∗ and 𝑃 < 0.001, significance obtained by ANOVA and posttest Tukey (𝑛=3), when compared to the negative control (a) and positive control (b).

(LPS-stimulated RAW 264.7 macrophages). The calculated 4. Conclusion IC50 of crude extract, Acet22, and Acet32 was 3.24, 1.03, and 0.85 𝜇g/mL, respectively. Although the inhibitory effect The present study reported for the first time the antimycobac- in NO production was slightly associated with moderated terial and NO production inhibitory activities of O. notata cytotoxicity (Figures 5(b) and 6(b)), especially for isolated extract. The findings from this study reveal the potential of flavonoids, the capacity of inhibiting NO production is O. notata extract and fractions to afford bioactive compounds higher when compared to cytotoxicity. For example, at andsuggestthatafzelinandisoquercitrinisolateddonot 20 𝜇g/mL, for all tested samples, NO inhibitory activity was contribute to the ethyl acetate fraction activity. But these greater than 90%, while cytotoxicity was between 20 and compoundswereabletosignificantlysuppresstheproduc- 40%. tion of NO stimulated by LPS in RAW 264.7 macrophages. 8 The Scientific World Journal

35 duckei,” Revista Brasileira de Farmacognosia,vol.18,no.1,pp. 30 37–41, 2008. 25 ∗∗∗ ∗∗ [6]M.Funasaki,A.L.L.Lordello,A.M.Vianaetal.,“Neolignans M)

𝜇 20 and sesquiterpenes from leaves and embryogenic cultures

] ( ∗∗∗ of Ocotea catharinensis (Lauraceae),” JournaloftheBrazilian 2 15 ∗∗∗ ∗∗∗ Chemical Society,vol.20,no.5,pp.853–859,2009.

[NO 10 ∗∗∗ ∗∗∗ ∗∗∗ [7] L. E. Cuca, P. Leon, and E. D. Coy, “A bicyclo[3.2.1]octanoid 5 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ neolignan and toxicity of the ethanol extract from the fruit of 0 Ocotea heterochroma,” Chemistry of Natural Compounds,vol.45, + − no. 2, pp. 179–181, 2009. [8]R.Garrett,M.T.V.Romanos,R.M.Borges,M.G.Santos,L. Control Control Acet22 [4] Acet22 Acet32 [4] Acet32 Rocha,andA.J.R.daSilva,“Antiherpeticactivityofaflavonoid Acet22 [20] Acet22 Acet32 [20] Acet32 Acet22 [0.8] Acet22 [0.8] Acet32 Acet22 [100] Acet22 Acet32 [100] Acet32 fraction from Ocotea notata leaves,” Brazilian Journal of Phar-

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Research Article Microwave-Assisted Simultaneous Extraction of Luteolin and Apigenin from Tree Peony Pod and Evaluation of Its Antioxidant Activity

Hongzheng Wang,1,2 Lei Yang,1,2 Yuangang Zu,1,2 and Xiuhua Zhao1,2

1 Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin, Heilongjiang 150040, China 2 State Engineering Laboratory for Bioresource Eco-Utilization, Northeast Forestry University, Harbin 150040, China

Correspondence should be addressed to Lei Yang; [email protected]

Received 28 April 2014; Accepted 29 August 2014; Published 22 October 2014

Academic Editor: Fred Stevens

Copyright © 2014 Hongzheng Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

An efficient microwave-assisted extraction (MAE) technique was employed in simultaneous extraction of luteolin and apigenin from tree peony pod. The MAE procedure was optimized using response surface methodology (RSM) and compared with other conventional extraction techniques of macerate extraction (ME) and heat reflux extraction (HRE). The optimal conditions of MAE were as follows: employing 70% ethanol volume fraction as solvent, soaking time of 4 h, liquid-solid ratio of 10 (mL/g), microwave irradiation power of 265 W, microwave irradiation time of 9.6 min, and 3 extraction cycles. Under the optimal conditions, 151 𝜇g/g luteolin and 104 𝜇g/g apigenin were extracted from the tree peony pod. Compared with ME and HRE, MAE gave the highest extraction efficiency. The antioxidant activities of the extracts obtained by MAE, ME, and HRE were evaluated using a 2,2-di(4- tert-octylphenyl)-1-picrylhydrazyl (DPPH) free radical-scavenging assay, a ferric reducing antioxidant power assay (FRAP), and a reducing power assay. Meanwhile, the structural changes of the unprocessed and processed tree peony pod samples were analyzed by scanning electron microscopy.

1. Introduction landfill waste or used as low-value fuel. A growing attention to the comprehensive utilization of tree peony resource is Treepeonywaslovedasanornamentalforitsbeautifulflower. paid by people. The pod of tree peony contains valuable During its cultivation for thousands of years, many cultivars bioflavonoids. Among the flavonoids, luteolin and apigenin of tree peony have been bred. Root cortex of tree peony is also are the main flavones with better pharmacological activities used as an important traditional Chinese medicine (TCM), [4]. having been recorded in the Pharmacopoeia of the People’s Flavonoids are a group of benzo-𝛾-pyran derivatives, Republic of China [1], which is beneficial for the treatment comprising a very large class of low molecular weight of diseases related mainly to irregular menstruation and polyphenol compounds. Among the flavonoids, luteolin 󸀠 󸀠 󸀠 dysmenorrhea [2]. Peony seed oil is rich in 𝛼-linolenic acid (3 ,4 ,5,7-tetrahydroxyflavone) and apigenin (4 ,5,7,- [3], which has beneficial effects on human nutrition and trihydroxyflavone) (Figure 1) are reportedly important health. The seed oils of Fengdan (Paeonia ostii)andZiban functional components, which exhibit the pharmacological (Paeonia rockii) have been approved as new resource food effects. For example, luteolin has been found to possess by government of China in 2011. The market demand for antioxidant [5], anticancer action [6], anti-inflammatory peony tree seed oil is growing with the enhancing cognition [7], antihepatotoxic action [8], antiallergic, antiosteoporotic of its nutrition functions. The remaining pod is around 60% [9], antidiabetic [10], and antiplatelet and vasodilatory of the total bulk, and it has been either disposed of as activity [11]. In addition to antioxidant, anticancer, and 2 The Scientific World Journal

0.020 0.020 R 0.018 1 1: luteolin, R1 = R2 = OH 0.018 0.016 2: apigenin, R1 = H, R2 = OH R 1 0.016 0.014 2 0.014 0.012 0.012 0.010 HO O 0.010 0.008 2 0.008 0.006 0.006 0.004 0.004 Absorbance (AU) Absorbance 0.002 OH O (AU) Absorbance 0.002 0.000 0.000 −0.002 −0.002 024 6 8 10 12 14 16 18 20 0 2 4 6 8 101214161820 Time (min) Time (min)

(a) (b)

Figure 1: The HPLC profiles of a mixture of standards of luteolin and apigenin (a) and the two compounds in an extract obtained byMAE using 70% ethanol as extraction solvent (b). anti-inflammatory, apigenin also exhibits antihyperglycemic microstructure changes of tree peony pod samples before action [12] and antinociceptive effect [13]. Luteolin and and after extraction were characterized by scanning electron apigenin are natural food additives used extensively in the microscopy (SEM). The present study offers an alternative food and pharmaceutical industries. Flavonoids constitute a method for the highly effective utilization of a side product large part of global nutraceuticals market [14]andthecurrent of tree peony utilization. nutraceuticals market was estimated at $151 billion in 2011 and was growing by about 6.5% per annum [15]. The pod of tree peony, after flavonoids extraction, could still be used as 2. Experimental a high-polysaccharide stock feed in dry form, increasing the 2.1. Plant Materials and Chemicals. Fresh ripe fruits of the potentialreturnfortheseedoilindustryandreducingthe cultivated tree peony (Paeonia ostii) were hand-harvested pollution load on the environment. in September from Heze (Shandong, China). The pod As far as we know, luteolin and apigenin can be obtained ∘ was separated, cleaned, and dried in oven at 45 C. The from plant materials, such as Sesbania grandifolra [16], dried sample was pulverized using plant grinder (FZ102, Cajanus cajan [17], Apium graveolens [18], Platycodon gran- Taisite, Tianjin, China) and then sieved (80–120 mesh) diflorum [19], Mentha spicata [20], and Perilla frutescens [21]. before use. Apigenin (≥95%), luteolin (≥97%), gallic acid A new source of luteolin and apigenin would be provided if an (≥97.5%), rutin (≥94%), Folin-Ciocalteu reagent, 2,2-di(4- efficient extraction technology of luteolin and apigenin from tert-octylphenyl)-1-picrylhydrazyl (DPPH, 95%), 6-hydroxy- tree peony pod was developed. Meanwhile, it also could be an 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, 97%), economical utilization of the disused pod. and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, ≥98%) were pur- Manymethodshavebeendevelopedforseparationof chased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). luteolin or apigenin from plant materials, such as maceration Ethanol, sodium nitrite (NaNO2), aluminum trichloride extraction (ME) [19], Soxhlet extraction [20], and heat reflux (AlCl3) and sodium hydroxide (NaOH), sodium carbon- extraction (HRE) [21]. However, these extraction techniques ate (Na2CO3), acetic acid, hydrochloric acid (HCl), ferric are inefficient, time-consuming, and energy-consuming22 [ ]. chloride (FeCl3), sodium dihydrogen phosphate (NaH2PO4), In recent years, the development and use of environmen- disodium hydrogen phosphate (Na2HPO4), potassium ferri- tally friendly methods have become increasingly popular. cyanide, and trichloroacetic acid (TCA) were purchased from Microwave-assisted extraction (MAE) is an extraction tech- Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). nique that offers high reproducibility, short extraction time, Methanol of chromatographic grade (99.9%) was purchased simple manipulation, and low solvent consumption, temper- from J&K Scientific Ltd. (Beijing, China). All solvents and ature, and energy input [23–26]. MAE utilizes the energy chemicals except methanol were of analytical grade. Reverse of microwaves to cause dipole rotation of molecules. In the osmosis Milli-Q water (Millipore, Bedford, MA, USA) was process of MAE, the solvent is rapidly heating and the cell used for all solutions and dilutions. All of the solvents pre- wall of the plant material is quickly destroyed, accelerating paredwerefilteredthrough0.45𝜇m microporous membrane the dissolution and extraction of components. (Guangfu, Tianjin, China). In this paper, the objective is to develop an effective and environment-friendly microwave-assisted approach for theextractionofluteolinandapigeninfromthetreepeony 2.2. Apparatus. The experimental setup of the microwave pod. The influences of the conditions on yields ofthe extraction apparatus was from Wang et al. [23]andLiu luteolin and apigenin were optimized using response surface et al. [24]. Briefly, a domestic WP700 microwave-assisted methodology (RSM). Moreover, the antioxidant activities extraction unit (Glanz, Guangdong, China) with a 2450 MHz of the extracts, luteolin, and apigenin were evaluated by magnetron and power continuously adjustable was used in DPPH,FRAP,andreducingpowerassays.Furthermore,the the extraction step. The dimensions of the interior cavity of The Scientific World Journal 3

Table 1: Experimental design matrix to screen important variables for total extraction yield of luteolin and apigenin.

Factor B Factor C Response Factor A Run Microwave irradiation Microwave irradiation time Total extraction yield Liquid-solid ratio (mL/g) power (W) (min) (𝜇g/g) 1 82308228 2 8 120 10 163 3 82308231 4 81206150 5 62306179 6 61208117 7 10 120 8 175 8 10 230 10 251 9 6 230 10 187 10 82308231 11 8 385 10 182 12 8 230 8 230 13 83856175 14 10 230 6 239 15 8 230 8 230 16 63858138 17 10 385 8 204

Table2:ComparisonofMAEwithotherextractionmethods,mean± S.D (𝑛=3).

Extraction time (h) Extraction yield ± SD (𝜇g/g) Number Extraction Solvent consumption (mL/g) method Soak time (h) Energy consumption time (min) Luteolin Apigenin Total 1 ME 36 0 30 82 ± 4a 63 ± 3a 145 2 HRE 012030131± 6b 89 ± 4b 220 3 MAE 22430151± 7c 104 ± 4c 255 aValues followed by the same letter in the same assay are not significantly different (𝑃 > 0.05). the oven are 215 mm × 350 mm × 330 mm. It was modified in RSM using the Box-Behnken software in data processing. our laboratory with the addition of a water condenser whose Box-Behnken design was applied using Design-Expert 8.06 wall was coated with polytetrafluoroethylene to prevent the without any blocking. The bounds of the factors were 6– leakage of microwaves. A round-bottom flask with a capacity 10 ratio of liquid-solid, 120–385 W of microwave irradiation of 100 mL was placed in the oven and connected to a reflux power, and 6–10 minutes of microwave irradiation time. The condenser. The whole system was run at atmospheric pressure specific protocols for the experiments were shown in Table 1. and could be employed at the maximum power of 700 W.

2.3. MAE Procedure. 1.00 g of the ground dried pod sample 2.5. Traditional Reference Extraction Procedure. The ME was mixed with ethanol solution in a 100 mL round bottom and HRE experiments were operated under the optimized flask. Soaked for a certain time, then the suspension was conditions.1.00gofthegrounddriedpodsamplewasmixed extracted by MAE. The optimum ethanol volume fraction, with 10 mL 70% volume fraction of ethanol in a 100 mL round soaking time, liquid-solid ratio, microwave irradiation power, bottom flask. The main technical parameters used were listed microwave irradiation time, and number of extraction cycles in Table 2. After completion of extraction, the liquid retentate were systematically studied in this work. After MAE, it was was decanted and filtered through Whatman number 2 filter cooled to room temperature rapidly by a cold bath and paper (Whatman International Limited, Kent, England). The filtrate was concentrated in a vacuum evaporator (R206, centrifuged with 10,000 ×g for 10 minutes. The supernatant ∘ was collected for subsequent HPLC analysis. Senco Technology Co. Ltd., Shanghai, China) at 60 Cand then lyophilized in a freeze-dryer (Scientz-10N, Ningbo Sci- entz Biotechnology Co., Ltd, China) to obtain crude extract. ∘ 2.4. Optimization MAE by Response Surface Method (RSM). The cold trap temperature was −56 Candthevacuumwas In order to highlight the most influential factors and possible less than 1 Pa. The crude extract was collected and stored in ∘ interactions, the operating conditions were optimized by 4 Cuntilitwasused. 4 The Scientific World Journal

2.6. HPLC Analysis and Quantification. The HPLC sys- 517 nm, with absolute ethanol as the control, and the DPPH tem consisted of a Waters 1525 Binary HPLC Pump, 2489 radical-scavenging activity was calculated. UV/Visible Detector, and automatic column temperature FRAP was assayed according to Liu et al. method [30]. control box. Chromatographic separation was performed on Briefly, a working solution was prepared freshly by mixing Aichrom Bond-AQ C18 reversed-phase column (4.6 mm × 250mLsodiumacetatebuffer(pH3.6,300mM),25mL 250 mm, 5 𝜇m, Abel Industries, Canada). TPTZ solution (10 mM, in 40 mM HCl), and 25 mL 20 mM ∘ For HPLC analysis, the mobile phase was methanol- FeCl3 solution. The mixture was incubated at 37 Cfor30min water-phosphate acid (30 : 69.3 : 0.7, v/v/v), and the flow rate and was referred to as FRAP solution. 0.15 mL of each sample was 1 mL/min. The column temperature was maintained at at different concentration (0.05–0.25 mg/mL in 70% ethanol) ∘ 25 C. The wavelength used for luteolin and apigenin was was mixed with 2.85 mL FRAP solution and allowed standing 360 nm. 10 𝜇L example was injected and the run time was inthedarkfor30min.Thentheabsorbanceat593nm 20 min. The retention times for luteolin and apigenin were was measured. A standard curve was prepared using Trolox 11.4 and 17.1min (Figure 1), respectively. Under these condi- ranging from 37.5 to 600 𝜇M. The FRAP was expressed as tions, the two flavonoids were baseline separated. Luteolin 𝜇mol Trolox equivalents (TE)/g crude or positive control. and apigenin were identified by comparing their retention Reducing power assay was following the method of Zu time with corresponding peaks in the standard solution. et al. [29]. 0.5 mL of each sample at different concentration Corresponding calibration curves for luteolin and api- (0.015–0.25 mg/mL in 70% ethanol) was mixed with 1.5 mL 𝑌 = 63937𝑋 + 37891 (𝑅2 = 0.9999) genin were luteolin and sodium phosphate buffer (pH 6.6, 0.2 M) and 1.5 mL 1% 𝑌 = 73716𝑋 + 24357 (𝑅2 = 0.9998) potassium ferricyanide solution. The mixture was incubated apigenin . A good linearity ∘ was found for each of luteolin and apigenin in the range of in a water bath at 50 C for 20 min. Then, 1.5 mL 10% 1–150 and 0.67–85 𝜇g/mL, respectively. trichloroacetic acid (TCA) solution and 3 mL water was added. The mixture was mixed vigorously and the absorbance wasmeasuredat707nm.Increasedabsorbanceofthemixture 2.7. Determination of Total Flavonoids. The total flavonoids indicated greater reducing power. contents of crude extracts obtained by MAE, ME, and HRE were determined by the method of Guo et al. [27]withsome modifications. Sample (0.16 mg/mL; 2.5 mL) was mixed with 2.10. SEM Observation. In order to investigate the effects of 0.15 mL 5% NaNO2 solution. After 6 min, 0.15 mL 10% AlCl3 MAE, ME, and HRE on morphological alterations of the pod was added. After another 6 min, 2 mL 4% NaOH was added. material, the pod material and residues of MAE, ME, and The absorbance was measured at 510 nm using a UV-Vis HRE were scanned with a SEM system (Quanta-200, FEI spectrophotometer (Shimadzu UV-2550; Shimadzu, Kyoto, Company, USA). Samples were fixed on a specimen holder Japan) after incubation for 15 min. The determination was with aluminium tape and then sputtered with the gold and performed in triplicate. Quantification was done on the basis examined under high vacuum condition at an accelerating of the standard curve of rutin. voltage of 10 kV (20 𝜇m, 1000x magnification).

2.8. Determination of Total Phenolics. The total phenolics 2.11. Statistical Analysis. Statistical analyses were performed contents of crude extracts obtained by MAE, ME, and with SPSS Statistics (Version 19, IBM Company, USA). HRE were determined by Folin-Ciocalteu method [28]with All experimental results were the average of three parallel little modifications. Briefly, 0.5 mL sample (0.12 mg/mL) was measurements expressed as the mean ± standard deviation mixed with 2.8 mL H2O and 0.5 mL Folin-Ciocalteu reagent. (SD). The Fisher test valueF ( -value) was obtained from the < After 3 min, 1.5 mL 7.5% Na2CO3 was added. The reaction ANOVA test generated by the software. P values 0.05 were ∘ mixture was mixed thoroughly and incubated at 40 Cfor regarded as significant. 30mininthedark.Absorbancewasthenmeasuredat765nm using the spectrophotometer (UV-2550, Shimadzu, Japan). 3. Results and Discussion Gallic acid was used to calculate the standard curve (0.06– 0.3 mg/mL). The determination was performed in triplicate. 3.1. Optimization of Luteolin and Apigenin Extraction Using aFactorialDesign.The univariate method was used to 2.9. Evaluation of Antioxidant Capacity. To evaluate antiox- optimize the following parameters: ethanol volume fraction idant capacities of crude extracts obtained by MAE, ME, in the extraction solvent, pod soaking time, liquid-solid ratio, and HRE, their DPPH radical-scavenging activity, FRAR, and microwave irradiation power, microwave irradiation time, reducing power were determined, with standard compounds and extraction cycles. of luteolin and apigenin as the positive controls. DPPH radical-scavenging activity was measured accord- 3.1.1. Effect of Ethanol Volume Fraction. Species and concen- ing to the method of Zu et al. [29] with a slight modification. tration of solvent is regarded as one of the most important 0.1mLofeachsampleatdifferentconcentration(0.25– parameters for MAE, which affects the solubility of the target 1.6 mg/mL in 70% ethanol) was added to 3.9 mL 25 mg/mL component and the absorption of microwave energy [31, 32]. DPPH solution in 95% ethanol. The mixture was mixed Although methanol has a great advantage in MAE for its best vigorously and allowed to stand at room temperature in absorbance of the microwave energy, it is not recommended the dark for 30 min. Then the absorbance was measured at in food processing because of its toxicity [33]. So mixture The Scientific World Journal 5 of ethanol and water is recommended. In this study, the extraction yield of component in MAE. A higher microwave extractions were carried out with aqueous ethanol solutions power increased the temperature of the mixture of the at different concentrations (ethanol volume fraction from 0to extraction solvent and sample, leading to higher mass transfer 90%) and with other conditions of 1 h soaking time, 10 (mL/g) ratesofsubstancesfromthesample[37]. In this study, the of liquid-solid ratio, 385 W of microwave irradiation power, effect of irradiation power ranging from 120 to 700 W on 10 min of microwave irradiation time, and one extraction extraction yields of luteolin and apigenin was investigated cycle. (Figure 2(d)). It was found that the extraction yields of lute- Ethanol volume fraction significantly affected the extrac- olin and apigenin significantly increased when the microwave tion yields of luteolin and apigenin (Figure 2(a)). The extrac- irradiation power increased from 120 W to 230 W (𝑃< tion yield of luteolin significantly increased with the increase 0.05) and gradually decreased with the further increasing of ethanol volume fraction ranging from 0 to 50% and then of irradiation power, which might be due to either thermal decreased when ethanol volume fraction was higher than or oxidative degradation of luteolin and apigenin by the 80%. It was found that the higher ethanol volume fraction excessive irradiation. The results indicated that the optimum was needed to extract apigenin than luteolin from the matrix, extraction condition was the use of the microwave power of whichwaslikelyduetothesmallerpolarityofapigenin. 230 W. The most efficient ethanol volume fractions for luteolin and apigenin were 50% and 70%, respectively. Taking into account 3.1.5. Effect of Microwave Irradiation Time. The influence both of the total extraction yields and the economization on of microwave irradiation time on the extraction yields of ethanol, 70% was considered for the optimal ethanol volume the luteolin and apigenin was examined over a range of fraction. 2–12 min; other factors were fixed at 70% ethanol volume fraction as extraction solvent, soaking time 4 h, liquid-solid 3.1.2. Effect of Pod Soaking Time. Using of the technique of ratio 10 (mL/g), microwave irradiation power 230 W,and one soaking can get better extraction efficiency of target com- extraction cycle. As shown in Figure 2(e), the extraction poundinmicrowave-assistedprocessing[34]. Experiments yields of luteolin and apigenin sharply increased with the were conducted to investigate the effect of soaking time on increase of microwave irradiation time in the beginning of yields of luteolin and apigenin in MAE. Pod samples were MAE. Luteolin and apigenin could reach their optimum soakedin70%ethanolfor0,1,2,4,or8h.Thenthey extraction yields at 8 min during the extraction process. were extracted in the microwave oven for 10 min at 385 W. When microwave irradiation time was above 8 min, no Information of the effect of soaking time on the extractions of significant variation was found in extraction yields of luteolin luteolin and apigenin was given in Figure 2(b).Asubstantial and apigenin (𝑃 > 0.05). Thus, a microwave irradiation time increase in the extraction yield was obtained after soaking of 8 min was chosen as the optimal microwave irradiation the pod. The target ingredients extraction yields increased time. significantly when the soaking time was 0–4𝑃 h( < 0.05); however longer soaking time did not lead to further increases 3.1.6. Number of Extraction Cycles. Adequate extraction cycle in yield. Therefore, 4 h was chosen as the optimal soaking contributes to the full extraction of interesting substances time. from the residue. The extraction yields of luteolin and apigenin were investigated in MAE process conducted 1–4 3.1.3. Effect of Liquid-Solid Ratio. In an extraction process, cycles, with other conditions of 70% ethanol volume fraction, it is important to maximize extraction yield but also to 4 h of soaking time, 10 (mL/g) of liquid-solid ratio, 230 W minimize the consumption of solvent. Inadequate solvent of microwave irradiation power, and 8 min of microwave leads to low operational efficiency and excessive solvent leads irradiation time. The 2nd cycle significantly increased the to waste. For investigating the influence of liquid-solid ratio yields of luteolin and apigenin (𝑃 < 0.05). It was the 3rd on extraction yields of luteolin and apigenin, tests were extraction cycle, but not the 4th cycle, that significantly performed at different liquid-solid ratios ranging from 6 to enhanced the extraction yield of luteolin of MAE (𝑃 < 0.05) 30 (mL/g). The results showed that increasing liquid-solid (Figure 2(f)). For saving solvent, energy, and time, three- ratio enhanced the extraction yields of luteolin and apigenin cycle extraction was sufficient to extract luteolin and apigenin (Figure 2(c)), which was due to the increasing of driving present in tree peony pod. force generated from the gradient concentration [35]. In the tested ratio ranging from 10 to 30, no significant difference 3.2. Optimization Extraction Conditions by Response Surface was found in extraction yields of luteolin and apigenin (𝑃> 0.05 Method (RSM). In order to study the most influential factors ), which was due to the excessive swelling of the materials and the interactions between the factors, the liquid-solid caused by the large volume of solvent, according to the reports ratio, microwave irradiation power, and time were optimized of Yan et al. [33]andMaetal.[36]. Hence, a value of 10 (mL/g) by RSM. In Table 1, the total extraction yield was defined was considered the optimal liquid-solid ratio for the MAE as the sum of yields of luteolin and apigenin in an extrac- process. tion experiment. The surface response analysis for the total extraction yield indicated that the effects of all the three 3.1.4. Effect of Microwave Irradiation Power. Microwave ir- factors on the response were significant with a positive linear radiation power is the other important factor affecting relationship (𝑃 < 0.05). The empirical relationship between 6 The Scientific World Journal

100 100

d c 80 ef f 80 b b de a d c g/g) e e g/g) b b 𝜇 𝜇 a 60 c 60 d b c 40 40 b Extraction yield ( Extraction a yield ( Extraction a 20 20

0 0 10 30 50 70 80 90 01248 Ethanol volume fraction (%) Soaking time (h)

(a) (b) 100 120 c e b bc bc bc bc 80 100 cd cccd d

g/g) d

g/g) d 𝜇 a 60 𝜇 80 b b c a c b a 60 b 40 a 40 Extraction yield ( Extraction Extraction yield ( Extraction 20 20

0 0 1 : 6 1 : 81: 10 1 : 12 1 : 15 1 : 20 1 : 30 120 230 385 540 700 Liquid-solid ratio (mL/g) Microwave irradiation power (W)

(c) (d) 160 120 c d d d c c 140 b b 100 a 120 a b b g/g) d d d g/g) b 𝜇 80 c 𝜇 100 b a a 80 60 60 40 Extraction yield ( Extraction Extraction yield ( Extraction 40 20 20

0 0 246810 12 1 234 Microwave irradiation time (min) Number of extraction cycles

Luteolin Luteolin Apigenin Apigenin

(e) (f)

Figure 2: The influence conditions ((a): ethanol volume fraction; (b): soaking time; (c): liquid-solid ratio; (d): microwave irradiation power; (e): microwave irradiation time; (f): number of extraction cycles) on the extraction yields of luteolin and apigenin. The values represent means ± standard deviation. Values followed by the same letter in the same assay are not significantly different (𝑃 > 0.05, 𝑛=3). The Scientific World Journal 7

300 300

250 250 g/g) 𝜇 g/g) 𝜇 200 200

150 150

100 100 Total extraction yield ( extraction Total Total extraction yield ( extraction Total

385 10 10 10 B: microwave irradiati C: microwave irradiation time (min) 9 9 9 253 8 8 8 7 7 7 on power (W) 120 6 6 6 A: liquid-solid ratio (mL/g) A: liquid-solid ratio (mL/g)

(a) (b)

300

250 g/g) 𝜇 200

150

100 Total extraction yield ( extraction Total

10 385 C: microwave irradiation time (min) 9 8 253 7 6 120

B: microwave irradiation power (W)

(c)

Figure 3: Response surface plots showing the effects of variables on total extraction yield. (a) Interaction of liquid-solid ratio and microwave irradiation power; (b) interaction of liquid-solid ratio and microwave irradiation time; (c) interaction of microwave irradiation power and time.

the total extraction yield and the extraction parameters was the model was 0.996, which conformed with the fact that generated as follows: themodelcouldadequatelyrepresentthetruerelationships 2 between the three parameters. In this case, 𝐴, 𝐵, 𝐶, 𝐴𝐵, 𝐴 , 𝑌 = −397.079 + 61.705𝐴 + 1.880𝐵 + 15.070𝐶 +0.007𝐴𝐵 2 2 𝐵 ,and𝐶 were the significant model terms. To highlight the + 0.250𝐴𝐶 − 0.006𝐵𝐶 − 3.125𝐴2 − 0.004𝐵2 − 0.875𝐶2. interactions of the three factors on total extraction yield, 3D profiles of the model were illustrated in Figure 3, when the (1) other parameters were kept constant. It was found that there was no significance in the lack The interaction of liquid-solid ratio and microwave irra- of fit (𝑃 > 0.05). The ANOVA analysis results showed diation power was shown in Figure 3(a). In these experi- that the quadratic model was valid for the spatial influence ments, the other parameters were kept as follows: 70% ethanol 2 of variables on the response. Additionally, the 𝑅 value for volume fraction, 4 h of soaking time, 8 min of microwave 8 The Scientific World Journal irradiation time, and 3 extraction cycles. Increase of the MAE, ME, and HRE were compared, and the results were microwave irradiation power from 120 W to 265.6 W with the showninTable2. The extraction yields of luteolin and api- liquid-solid ratio increasing from 6 to 10 (mL/g) enhanced the genin were significantly higher with MAE than with HRE and total extraction yield. The further increase of the microwave ME (𝑃 < 0.05). The total extraction yield of MAE, HRE, and irradiation power decreased the total extraction yield, likely ME was 255, 220, and 145 𝜇g/g, respectively. The time of MAE due to the degradations of luteolin and apigenin under with energy consumption was also shorter, with the HRE the high irradiation power. Additionally, denser contours taking 120 min and the MAE only 24 min. The comparison of were found along the axis of microwave irradiation power MAEwithMEshowedthatthemicrowaveirradiationenergy than along the axis of liquid-solid ration, indicating that was important for increasing the extraction efficiency. MAE microwave irradiation power had more influence on the total was an efficient method for the simultaneous extraction of extraction yield than liquid-solid ratio. luteolin and apigenin from tree peony pod. Figure 3(b) represented the interaction of liquid-solid ratio and microwave irradiation time. In these experiments, 3.4. Evaluation of Antioxidant Activities the other parameters were kept as follows: 70% ethanol volume fraction, 4 h of soaking time, 230 W of microwave 3.4.1. DPPH Assay. In the present investigation, the DPPH irradiation power, and 3 extraction cycles. A steady increase assay was used to evaluate the radical-scavenging activity of of total extraction yield was found when the liquid-solid tree peony pod extracts obtained by MAE, ME, and HRE, as ratio increased from 6 to 10 (mL/g) along with the increase well as of pure luteolin and apigenin. As shown in Figure 4(a), of the microwave irradiation time from 6 to 9.6 min. A all extracts obtained by MAE, HRE, and ME possessed high comparison of contour density along axis of liquid-solid scavenging activity of DPPH radical. The IC50 values of ratio and microwave irradiation time suggested that the total the three extracts were higher than that of the standard extraction yield was more influenced by the liquid-solid ratio compound of luteolin and lower than apigenin. In the three than microwave irradiation time. extracts, the highest scavenging activity for DPPH radical The response surface for total extraction yield with was found in the extract obtained by MAE, suggesting that various microwave irradiation powers and time was showed more compounds with hydrogen donating capabilities were in Figure 3(c). In this case, the parameters of ethanol volume extracted in MAE than in ME or HRE. fraction, soaking time, liquid-solid ratio, and extraction cycleswerefixedat70%,4h,10(mL/g),and3,respectively. Being the same as that found in Figure 3(a),thetotal 3.4.2. Ferric Reducing Antioxidant Power Assay. Antioxidant extraction yield was elevated when the microwave irradiation potential of the three extracts obtained by MAE, HRE, and power increased from 120 to 265.6 W along with the increase ME was estimated from their abilities to reduce TPTZ-Fe (III) of microwave irradiation time from 6 to 9.6 min. The denser complex to TPTZ-Fe (II) complex, with pure luteolin and contours along the axis of microwave irradiation power apigenin. Among all the samples, luteolin showed the highest FRAP (10838 𝜇mol TE/mg) and apigenin showed the lowest indicated that the parameter has a greater impact on the total 𝜇 extraction yield. FRAP (163 mol TE/mg). Similar to the result in DPPH assay, From the analysis of RSM, the optimal conditions of MAE MAE showed the pronounced effect on FRAP. The FRAP of extract obtained by MAE was higher than those obtained can be summarized as follows: 70% ethanol volume fraction, ± ± 4 h soaking time, 10 (mL/g) of liquid-solid ratio, 265.6 W of by HRE and ME, 20.0 1.4% and 36.9 3.3%, respectively microwave irradiation power, 9.6 min of microwave irradi- (Figure 4(b)). ation time, and 3 extraction cycles. Under these conditions, the total extraction yields can reach 256 𝜇g/g. Taking into 3.4.3. Reducing Power Assay. The reducing powers of the account the feasibility in the actual operation, the parameter extracts obtained by MAE, HRE, and ME were also tested 3+ of microwave irradiation power was adjusted to 265 W. To in another system, in which the Fe /ferricyanide com- verify the model, MAE was done three times under these plex transformed into its ferrous form. The amount of 2+ conditions. The actual total extraction yield was 255 𝜇g/g with Fe /ferricyanide complex was monitored by measuring a deviation of −0.39%. the formation of Perl’s Prussian blue at 707 nm. Higher Under the conditions optimized by RSM, the extraction absorbance value indicated the higher reducing power [29, yields of luteolin and apigenin from tree peony pod were, 38]. As shown in Figure 4(c),thereducingpowersof respectively, 151 𝜇g/g and 104 𝜇g/g. Luteolin and apigenin all samples were concentration dependent ranging from have also been reported in some other plant materials, such 0.016 mg/mL to 0.25 mg/mL, and good linear relationships as S. grandifolra, C. cajan,andA. graveolens.Thecontents were found between the samples concentration and the ofluteolinandapigenininthemwere,respectively,inrange reducing powers. Agreeing with the DPPH and FRAP tests, of about 20–550 𝜇g/g and 40–730 𝜇g/g [16–18]. The data the reducing powers of the crude extracts and the pure indicated that the extract of tree peony pod would have compounds were arranged as follow: luteolin > MAE > HRE favorable application value. > ME > apigenin. From the results of DPPH, FRAP, and reducing power assay, it was obvious that MAE was beneficial for the extrac- 3.3. Comparison of MAE with Other Methods. The extraction tion of the antioxidant substances. Polyphenols are the most yieldsofluteolinandapigeninfromtreepeonypodwith abundant antioxidants in the plants and provide the main The Scientific World Journal 9

100 12000 e

10000 80 0.16 d c 8000 60 0.14 b

0.12 6000 mol TE/mg) mol 𝜇 (mg/mL)

40 50 0.10 IC a 4000 d FRAP ( c 0.08 NA∗ b DPPH scavenging activity (%) activity scavenging DPPH 20 0.00 2000 ME HRE MAE

Luteolin a 0 Apigenin 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Luteolin Apigenin ME HRE MAE Concentration (mg/mL) Luteolin HRE Apigenin MAE ME

(a) (b) 10 600 4 e 500 8 c 400 b a 3 6 300 nm) c d

Slope of the line of Slope b 707 4 a 200 2 0 b c 100 a Content (mg/g) Content ME HRE MAE 2.5

Absorbance ( Absorbance c Luteolin

Apigenin b 2.0 a b 1 a 1.5 a 1.0 0.5 0 0.0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 ME HRE MAE Concentration (mg/mL) Luteolin HRE Total phenolics Luteolin Apigenin MAE Total flavonoids Apigenin ME

Figure 4: Antioxidant activity and antioxidants contents of crude extracts obtained by ME, HRE, and MAE of tree peony pod. (a) DPPH scavenging activity of the crude extracts; (b) FRAP of the crude extracts; (c) reducing power of the crude extracts; (d) total phenolics, total flavonoids, luteolin, and apigenin contents of the crude extracts. The values represent means ± standard deviation. Values followed by the same letter in the same assay are not significantly different (𝑃 > 0.05, 𝑛=3). Note: ∗ means the value of IC50 cannot been obtained.

antioxidation in human diet [39]. In the three extracts, the with the results in other studies [40–42]. The contents of highest contents of total phenolics and total flavonoids were luteolin and apigenin were also determined in the three found in the extract obtained by MAE (Figure 4(d)), which extracts, and a positive relationship was found between the was in agreement with the results of antioxidant activities content of luteolin and antioxidative capacity of extract, but tests in vitro. In our study, antioxidant activities of luteolin notthecontentofapigenin.Itindicatedthatluteolinwasan were obviously higher than apigenin, which was consistent important antioxidant in the extract. 10 The Scientific World Journal

(a) (b)

Figure 5: SEM images (20.0 lm, 15.0 kV) of untreated tree peony pod sample (a), sample after ME (b), sample after HRE (c), and sample after MAE (d).

3.5. Structural Changes after Extraction. The various extrac- which built up a pressure within the cell. As a result of tion methods produced different physical changes in tree the continuous increase of the pressure, the structure of cell peony pod. To investigate these physical changes during walls of sample particles was disrupted and that helped the MAE, HRE, and ME, the pod samples were analyzed by rapid release of substances inside of the material particles to SEM. Figure 5 displayed the micrographs of the pod samples solvents [44]. before and after the different extraction methods. As shown in Figure 5(a),nubbyparenchymaandnondestructedcell walls could be observed in the untreated raw material. No 4. Conclusions obviouschangewasfoundinsamplewithMEtreatment compared to the untreated raw material, except for a few In the present study, an efficient MAE method for simulta- perforations (Figure 5(b)), which might be caused by the neous extraction of luteolin and apigenin from tree peony dissolution and denaturation of some compositions of cell pod has been developed. The optimum conditions for MAE wall. After HRE or MAE treatment, the sample became a were studied using RSM. Under the optimized conditions, crumbly texture (Figures 5(c) and 5(d)). However, a higher 151 𝜇g/g luteolin and 104 𝜇g/g apigenin were obtained. Com- degreeofdamagewasfoundinthesamplewithMAE pared to other methods, the proposed approach provides treatment. MAE treatment induced clear wrinkle, dispersion, higher extraction yields and significantly reduced energy and fragmentation of external and internal cell walls of the consumption time. Higher antioxidant activities were found particles, which might be due to the severe thermal stress and in extract obtained by MAE than those obtained by HRE localized high pressure [43].Whenthesamplewassubjected and ME, being estimated in in vitro systems of DPPH, 3+ to irradiation, rapid heating of polar molecules in material TPTZ-Fe (III), and Fe /ferricyanide complex. This indicated and solvent led to the rapid expansion of the solvent volume, that more antioxidants were extracted from the pod using The Scientific World Journal 11

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Research Article Analysis of Flavone C-Glycosides in the Leaves of Clinacanthus nutans (Burm. f.) Lindau by HPTLC and HPLC-UV/DAD

June Lee Chelyn, Maizatul Hasyima Omar, Nor Syaidatul Akmal Mohd Yousof, Ramesh Ranggasamy, Mohd Isa Wasiman, and Zakiah Ismail Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

Correspondence should be addressed to June Lee Chelyn; [email protected]

Received 3 July 2014; Accepted 7 September 2014; Published 22 October 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 June Lee Chelyn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C. nutans. The C-glycosidic flavones such as shaftoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. Therefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C. nutans leaves. The TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid:water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaftoside, orientin, 2 isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4–200 𝜇g/mL, r ≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95–105%). The concentration of shaftoside, orientin, vitexin, and isovitexin in C. nutans leave samples was 2.55–17.43, 0.00–0.86, 0.00–2.01, and 0.00–0.91 mmol/g, respectively.

1. Introduction of C. nutans in which they were shown to inhibit Herpes simplex virus-1 (HSV-1) activity [2, 4]. Other studies have Clinacanthus nutans (Burm. f.) Lindau (family Acanthaceae) also reported anti-inflammatory and immune-modulatory is a medicinal herb that is native to many tropical Asia activities in the ethanolic extracts of C. nutans leaves [6, countries including Malaysia, Thailand, Indonesia, and China 7]. However, the exact compound or phytochemicals which [1]. This herb is highly reputed in Thailand where it is known are responsible for the anti-inflammatory and immune- locally as Phaya Yo or Phaya Plong Thong [2]. This herb modulatory activities observed are unknown. is also gaining popularity in Malaysia whereby it is more From the known phytochemicals, the six C-glycosyl commonly known as Belalai Gajah or Sabah Snake Grass [3]. flavones (shown in Figure 1), shaftoside1 ( ), isomollupentin The fresh leaves of this herb have been traditionally used to 7-O-𝛽-glucopyranoside, orientin (2), isoorientin (3), vitexin treatskinrashesandbitescausedbyinsectsandsnakes[2, 4]. (4),andisovitexin(5) isolated from the methanolic extract of In addition, the ethanolic extracts from the leaves of C. nutans C. nutans leaves, were of interest due to their rare occurrence have also been used clinically in the form of topical cream to in plants [8]. The flavones C-glycosides are an important treat Herpes genitalis and Herpes zoster lesions [5]. subclass of the flavonoids group of compounds and have been Some of the bioactive components that have been isolated reported in plant from families Verbenaceae, Passifloraceae, from C. nutans include the chlorophyll phaeophytin deriva- Caryophyllaceae, and Polygonaceae [8]. They possess impor- tives isolated from the chloroform extract of C. nutans leaves tant biological activities including antimicrobial activity and a mixture of cerebrosides and monoacylmonogalactosyl- (isoorientin, vitexin), hepatoprotective activity (isoorientin), glycerol isolated from the ethanolic extract of the fresh leaves and antioxidant activity (isovitexin) [9–11]. Despite that, there 2 The Scientific World Journal

HOH C O 2 O HO OH HO OH HO HO OH OH O O HO O HOH C O 2 O HO HO HO OH HO OH OH O OH O Isomollupentin 7-O-𝛽-glucopyranoside

Shaftoside (1) OH HOH2C O HO OH HO HO O OH HOH C OH 2 O HO O HO OH HO OH OH O Isoorientin (3) OH O OH Orientin (2) HOH C 2 O HO O HO OH HOH C HO 2 O OH HO HO O HO OH OH O Isovitexin (5)

OH O Vitexin (4)

Figure 1: Chemical structures of known flavone C-glycosides shaftoside (1), isomollupentin 7-O-𝛽-glucopyranoside, orientin (2), isoorientin (3), vitexin (4), and isovitexin (5). is currently no report on the qualitative and quantitative 2.2. Chemicals and Standards. Chemical standards shafto- determination of the chemical markers including the C- side, isoorientin, orientin, isovitexin, and vitexin (all primary glycosyl flavones in the leaves of C. nutans which hinder the grades) were purchased from Chromadex (Irvine, CA, USA). development of C. nutans medicinal products. The purpose of Solvents acetonitrile and ethanol (both HPLC grade) and this study is therefore to develop a separation technique using ethyl acetate, acetic acid, and formic acid (analytical grade) high performance thin-layer chromatography (HPTLC) and were purchased from Merck (Merck Co., Darmstadt, Ger- highperformanceliquidchromatographycoupledtoaphoto- many). Water was purified with an ultrapure water system diode array detector (HPLC-UV/DAD) for the simultaneous (Sartorius, Germany). determination and quantification of flavones C-glycosides in C. nutans that could accurately identify C. nutans raw 2.3. Extract and Standard Preparation. The fresh leaves were ∘ materials and products. oven dried at 40 Cthengrindtofinepowder.Theethanolic extract was prepared by adding ethanol (5 mL) to 0.5 g of 2. Materials and Methods powdered raw material, sonicated for 30 minutes, and then filtered using Whatman no. 1 filter paper. The supernatant 2.1. Plant Materials. Fresh leaves of C. nutans were is collected and used for HPTLC and HPLC analysis. The collected from three different geographical locations in standard stock solution (1 mg/mL) of shaftoside, orientin, isovitexin, and vitexin was prepared in HPLC grade ethanol Malaysia. The locations that were selected are Taiping, ∘ Perak (L1), Kota Tinggi, Johor (L2), and Sendayan, Negeri and stored at 4 C. Working solutions of lower concentration Sembilan (L3). Voucher specimens UPM/IBS/UB/H39-12, (shaftoside: 20, 40, 60, 80, 100, 150, 175, and 200 𝜇g/mL, UPM/IBS/UB/H46-12, and SBID 019/12 were prepared orientin, isovitexin, vitexin: 0.2, 0.4, 0.6, 0.8, 10.0, 20, 40, 60, and authenticated by botanists from the University 80, and 100 ug/mL) were prepared by appropriate dilution of Putra Malaysia (UPM) and the Forest Research Institute thestocksolutionsinethanol. Malaysia (FRIM). The voucher specimens were deposited in the herbarium of FRIM and Institute for Medical 2.4. HPTLC Analysis. Thin-layer chromatography was per- Research. formed on plates precoated with silica gel 60 F254 (Merck, The Scientific World Journal 3

Germany). Samples (5 𝜇L) were applied to the plate as 8 mm width bands using Camag (Switzerland) Linomat V sample applicator. The plate is then developed in an automatic developing chamber (ADC2) (Camag, Switzerland) using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. Developing distance was 70 mm. After development, the plate was air-dried and derivatized by immersing the plate in natural product reagent (2-aminoethyl diphenylborinate) (1.0 g) in methanol (200 mL) and PEG 400 (10.0 g) in methanol (200 mL).

2.5. HPLC Analysis. HPLC-UV/DAD analysis was performed on a Waters Alliance 2695 (Millford, MA, USA) system connected to Waters 2996 photodiode array detector (DAD). The chromatographic separation was performed using a Kinetex PFP column (250 × 4.6 mm, 5 𝜇m, Phe- ∘ nomenex, USA) at 40 C. The solvent system consisted of mixtures of water with 0.8% (v/v) glacial acetic acid (solvent Figure 2: TLC profile of standards and extracts of C. nutans leaves A) and acetonitrile (solvent B). The solvents were degassed from different location. S: standard mixtures of shaftoside𝑅 ( 𝑓 = before delivering into the system. Samples for HPLC analysis 0.23), isoorientin (𝑅𝑓 = 0.48), isovitexin (𝑅𝑓 = 0.56), orientin 𝜇 were filtered through a 0.45 m membrane filter. The opti- (𝑅𝑓 = 0.60), and vitexin (𝑅𝑓 =0.66). C. nutans samples L1: Taiping, mized HPLC condition is as follows: 5–19% B (30 min), 19– Perak, L2: Kota Tinggi, Johor, and L3: Sendayan, Negeri Sembilan. 95% B (3 min), 95% B (5 min), 95–5% B (1 min), and 5% B (3 min). The flow rate was 0.7 mL/min. The injection volume was 10 𝜇L. Signal was monitored at 330 nm. acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) gave good separation of the compounds of interest. For visualiza- 2.6. Validation of HPLC Method. The optimized HPLC tion of the bands, an additional step of derivatization was method was validated in terms of linearity, limit of detection necessary using natural product reagent followed by PEG (LOD), limit of quantification (LOQ), precision and accuracy 400. The marker compounds after derivatization observed according to the International Conference on Harmonization under UV 366 nm gave fluorescent bands on the TLC plate. guidelines [12]. The calibration curves were obtained by the The flavones C-glycosides with the apigenin backbone gave external standard method on eight levels of concentration of characteristic green coloured bands at retention factors (𝑅𝑓) standard mixtures, with three injections per level. Calibration of 0.23 (shaftoside), 0.56 (isovitexin), and 0.66 (vitexin) while curve was plotted using chromatogram peak areas on 330 nm flavones C-glycosides with luteolin backbone gave distinct against the known concentrations of standard solutions. A yellow bands for isoorientin and orientin 𝑅𝑓 0.48 and 0.60, linear regression equation was calculated by least squares respectively. The retention time𝑅 ( 𝑓) and colour of the method. LOD and LOQ were estimated experimentally by bands were compared between the standard mixture and injecting a series of dilute solutions with known concen- samples from three locations to confirm the presence of tration until the signal-to-noise ratio reached 3 : 1 for LOD these markers in samples. The preliminary TLC screening and 10 : 1 for LOQ. Three different concentrations of standard results showed that shaftoside was present in all three sample mixtures (5, 10, and 20 𝜇g/mL) were used for intra- and locations. Isoorientin, isovitexin, orientin, and vitexin were interday precision testing. For intraday precision, the peak only detected in two locations (L1 and L2) but were absent in area and retention time of three different concentrations the third location (L3) as observed in Figure 2. of the standard solutions were determined in one day and expressed as relative standard deviation (RSD). For interday precision, the RSD of the standards were determined on three 3.2. Optimization of HPLC Method. Several parameters were different days. Accuracy of the HPLC method was deter- optimized to obtain the best HPLC chromatographic sep- mined by recovery tests analyzing sample extracts spiked with aration. The first parameter that was investigated was the three different standard mixture concentrations (5, 10, and use of different mobile phase combination of either water- 20 𝜇g/mL). acetonitrile or water-methanol in which the combination of water-acetonitrile gave better chromatographic resolution compared to the later. Addition of 0.8% glacial acetic 3. Results and Discussion acid to the mobile phase was found to improve peak resolution and peak shape and eliminate peak tailing of 3.1. HPTLC Screening Results. TLC chromatographic condi- the marker compounds. Besides that, a gradient elution tions were optimized to obtain a system in which the flavones method improved the resolution of compounds in sample C-glycosides would be separated in the best manner. The compared to isocratic mode. For this study a shallow gra- mobile phase containing a mixture of ethylacetate : formic dient profile from 5–19% B for 30 minutes was optimized 4 The Scientific World Journal

330 nm 100 2 4 1 75 5 3 50

(mAU) 25 0

2 4 6 8 10121416182022242628303234 (min) Mix standard solution (3) Orientin (1) Shaftoside (4) Isovitexin (2) Isoorientin (5) Vitexin

(a) 1 8 L3: Sendayan, Negeri Sembilan 330 nm 6 e 2, c 4 0 a b d −2 36 L2: Kota Tinggi, Johor 1 27 18 2, c 3 e 5

(mAU) 9 4 a b d f 0 40 L1: Taiping, Perak 1 30 e 20 2, c 3 5 10 a b d 4 f 0 246810121416182022242628303234 (min)

(b)

Figure 3: HPLC-UV/DAD chromatogram of (a) standard solution mixture and (b) ethanolic extracts of C. nutans leaves, 𝜆 = 330 nm. C. nutans samples L1: Taiping, Perak, L2: Kota Tinggi, Johor, and L3: Sendayan, Negeri Sembilan. (1) Shaftoside, (2) isoorientin, (3) orientin, (4) vitexin, and (5) isovitexin, a–f. Unknown.

and used to quantitate markers of interest. In addition, 3.3. HPLC Validation Data. The linear range, regression different chromatographic columns including Kinetex PFP equation, correlation coefficient of each marker, and LOD column (250 × 4.6 mm, 5 𝜇m, Phenomenex, USA), Sun- and LOQ values are summarized in Table 1. The eight point 2 fire C18 (250 × 4.6 mm, 5 𝜇m, Waters, USA), and Synergi calibration curve showed good linearity (𝑅 = 0.996)in × 𝜇 Polar-RP (250 4.6 mm, 4 m, Phenomenex, USA) were the given concentration ranges. The LOD and LOQ values tested to improve chromatographic resolution between the showed the adequate sensitivity of the HPLC method devel- markers. A satisfactory separation of marker compounds oped (Table 1). The interday and intraday precision range × was obtained using Kinetex PFP column (250 4.6 mm, from 0.62 to 4.54% (Table 2) showed that the method was 𝜇 5 m, Phenomenex, USA). However, investigation of the peak repeatable. The recovery values of the marker compounds purity of isoorientin in samples showed that the isoorientin are presented in Table 2. The average recoveries which peak (2) was coeluting with another compound (Peak c) as ranged from 95.33% to 105.13% showed good accuracy of the shown in Figure 3(b). Therefore, quantification of isoorientin method. However, recoveries were above 100% for certain was not carried out in this study. The HPLC separation was monitored at 330 nm according to the absorption maxima of samples possibly due to the inaccuracy of spiking at lower themarkercompounds.TheHPLC-UV/DADchromatogram ranges. The compounds that showed high recoveries were 𝜇 ofthestandardsolutionmixtureandtheethanolicextractsof orientin 105% (amount recovered 5.25 g/mL), shaftoside C. nutans leaves at 330 nm are shown in Figures 3(a) and 3(b), 102% (10.21 𝜇g/mL), isovitexin 101% (5.09 𝜇g/mL), and vitexin respectively. 102% and 101% (5.1 𝜇g/mL and 10.15 𝜇g/mL). The Scientific World Journal 5

Table 1: Linear range, regression equations, LOD, and LOQ for quantitative analysis of HPLC (𝑛=3).

a 2 Compound 𝑇𝑅 (min) 𝜆 (nm) Linear range (𝜇g/mL) Regression equation 𝑅 LOD (𝜇g/mL) LOQ (𝜇g/mL) 2 Shaftoside 22.79 271, 337 20.0–200 𝑦 = 28370𝑥 − 230484 𝑅 = 0.999 0.2 0.6 2 Orientin 25.58 255, 349 0.8–100 𝑦 = 27655𝑥 − 27061 𝑅 = 0.996 0.4 0.8 2 Isovitexin 27.16 269, 337 0.8–100 𝑦 = 34067𝑥 + 47328 𝑅 = 0.999 0.4 0.8 2 Vitexin 28.61 268, 337 0.8–100 𝑦 = 29217𝑥 + 33931 𝑅 = 0.997 0.4 0.8 a𝑦=𝑎𝑥+𝑏,where𝑥 is concentration in 𝜇g/mL and 𝑦 is area under curve at UV 330 nm wavelength.

Table 2: Intra- and interday precision, recovery, and accuracy data.

Precision (RSD % RT, AUC) Recovery Compound Concentration (𝜇g/mL) Intraday (𝑛=3)Interday(𝑛=6) Amount added (𝜇g/mL) Recovery (%) RSD (%) 5 3.24 2.80 5 95.33 2.18 Shaftoside 10 2.96 2.11 10 102.10 3.49 20 0.85 3.39 20 95.50 0.74 5 3.24 2.94 5 105.13 1.43 Orientin 10 2.96 1.92 10 99.53 1.03 20 0.85 3.08 20 98.11 0.10 5 1.55 0.96 5 101.97 2.93 Isovitexin 10 4.18 2.43 10 97.67 3.53 20 0.70 3.29 20 96.49 0.53 5 2.38 2.11 5 102.61 1.25 Vitexin 10 3.11 4.54 10 101.50 2.10 20 0.62 3.48 20 95.52 1.43 AUC: area under curve.

Table 3: Content of shaftoside, orientin, isovitexin, and vitexin in samples of C. nutans from different locations. L1: Taiping, Perak, L2: Kota Tinggi, Johor, and L3: Sendayan, Negeri Sembilan.

Contentofmarkercompounds(mmol/g) Compound L1 L2 L3 Shaftoside 17.43 ± 0.01 16.33 ± 0.03 2.55 ± 0.04 Orientin 0.86 ± 0.04 0.59 ± 0.003 ND Isovitexin 2.01 ± 0.02 1.03 ± 0.09 ND Vitexin 0.91 ± 0.03 0.43 ± 0.006 ND ND: not detected. Values are means ± SD, 𝑛=3.

3.4. Sample Analysis. The optimized and validated method quantity. The lower amount of flavones detected including was applied to determine the concentration of the marker shaftosideinL3comparedtoL1andL2couldbecausedby compounds in the samples from the three geographical differences in climate, soil regime of the different region, and locations. The content of shaftoside, orientin, isovitexin, and differences in processing such as harvest time, drying, and vitexin in samples is presented in Table 3.Theresultsshowed storage all of which may affect the composition of flavonoids. that shaftoside was the major flavone present in samples from all three locations in which concentration ranges from 4. Conclusion 2.55 mmol/g to 17.43mmol/g. The other known flavones C- glycosides were present in lower amounts, ranging from Chemical markers are essential as part of identification and the highest for isovitexin (0.00–2.01 mmol/g), followed by also quality control of herbal material. In the present study, orientin (0.00–0.86 mmol/g), to the lowest amount which HPTLC technique was used for the rapid identification of was vitexin (0.00–0.91 mmol/g). Flavones C-glycosides are five known flavones C-glycosides in C. nutans raw materials. favourable chemical markers due to their large structural In HPTLC, the unique characteristic fluorescent bands after diversity that could help to distinguish rapidly between other derivatization provided important clues for the identification plant species [13]. The results also allowed us to conclude that of the major flavone present in the samples. A second shaftoside would be best suited as chemical markers for C. step using HPLC-UV/DAD technique was employed for the nutans raw material as it is found in all locations in the highest simultaneous detection and quantification of these known 6 The Scientific World Journal flavonoids. The HPLC-UV/DAD method developed inthis apoptosis in human keratinocyte HaCat cells,” African Journal present study was effective, accurate, precise, and sensitive of Biotechnology,vol.9,no.31,pp.4999–5003,2010. for the quantification of four known flavones C-glycosides [8] K.-I. Teshima, T. Kaneko, K. Ohtani et al., “C-glycosyl flavones (shaftoside, orientin, isovitexin, and vitexin) of interest. from Clinacanthus nutans,” Natural Medicines,vol.51,no.6, The method was also applied to identify and quantify the article 557, 1997. chemical markers of interest in crude powdered leaves of C. [9]Y.Zhang,J.Jiao,C.Liu,X.Wu,andY.Zhang,“Isolationand nutans from three geographical regions in Malaysia. From purification of four flavone C-glycosides from antioxidant of the present study, the compound shaftoside could be recom- bamboo leaves by macroporous resin column chromatogra- mended as a marker for C. nutans rawmaterialasitispresent phy and preparative high-performance liquid chromatography,” in all locations and is also available as a commercial standard Food Chemistry,vol.107,no.3,pp.1326–1336,2008. for method validation. The current method developed in this [10] F. U. Afifi, E. Khalil, and S. Abdalla, “Effect of isoorientin study is also useful for the evaluation of quality of C. nutans isolated from Arum palaestinum on uterine smooth muscle of raw materials and its commercial products. rats and guinea pigs,” Journal of Ethnopharmacology,vol.65,no. 2,pp.173–177,1999. [11] Q. F. Huang, S. J. Zhang, L. Zheng et al., “Protective effect Conflict of Interests of isoorientin-2󸀠-O-𝛼-l-arabinopyranosyl isolated from Gyp- sophila elegans on alcohol induced hepatic fibrosis in rats,” Food The authors declare that there is no conflict of interests and Chemical Toxicology,vol.50,no.6,pp.1992–2001,2012. regarding the publication of this paper. [12] International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Acknowledgments Human Use, Validation of Analytical Procedures: Text and Methodology Q2(R1),2005,http://www.ich.org/fileadmin/Pub- The authors would like to thank the Director General of lic Web Site/ICH Products/Guidelines/Quality/Q2 R1/Step4/ Health, Ministry of Health, Malaysia, and Director of Insti- Q2 R1 Guideline.pdf. tute for Medical Research for giving permission to publish [13] C. D. Stalikas, “Extraction, separation, and detection methods this paper. Special record of appreciation for the support for phenolic acids and flavonoids,” Journal of Separation Science, vol.30,no.18,pp.3268–3295,2007. and funding given by the Ministry of Agriculture (MoA) under the Malaysian Herbal Monograph (MHM) Develop- ment project that aims to assist the industries in not only authenticating and confirming the raw material used but also the quality of such raw material supplied.

References

[1] I. Burkill, ADictionaryofEconomicProductsoftheMalayPenin- sula, vol. 1, Ministry of Agriculture & Cooperative Malaysia, Kuala Lumpur, Malaysia, 1966. [2] S.Sakdarat,A.Shuyprom,C.Pientong,T.Ekalaksananan,andS. Thongchai, “Bioactive constituents from the leaves of Clinacan- thus nutans Lindau,” Bioorganic & Medicinal Chemistry,vol.17, no. 5, pp. 1857–1860, 2009. [3]Y.K.Yong,J.J.Tan,S.S.Tehetal.,“Clinacanthus nutans extracts are antioxidant with antiproliferative effect on cultured human cancer cell lines,” Evidence-Based Complementary and Alternative Medicine,vol.2013,ArticleID462751,8pages,2013. [4]P.Tuntiwachwuttikul,Y.Pootaeng-On,P.Phansa,andW.C. Taylor, “Cerebrosides and a monoacylmonogalactosylglycerol from Clinacanthus nutans,” Chemical & Pharmaceutical Bul- letin,vol.52,no.1,pp.27–32,2004. [5] C. Kongkaew and N. Chaiyakunapruk, “Efficacy of Clinacan- thus nutans extracts in patients with herpes infection: systematic review and meta-analysis of randomised clinical trials,” Complementary Therapies in Medicine,vol.19,no.1,pp.47–53, 2011. [6] B.Sriwanthana,P.Chavalittumrong,andL.Chompuk,“Effectof Clinacanthus nutans on human cell-mediated immune response in vitro,” Thai Journal of Pharmaceutical Sciences,vol.20,no.4, pp. 261–267, 1996. [7] V. Thongrakard and T. Tencomnao, “Modulatory effects of Thai medicinal plant extract on proinflammatory cytokines-induced Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 306217, 10 pages http://dx.doi.org/10.1155/2014/306217

Research Article Phenolic Composition and Antioxidant Activity of Malus domestica Leaves

Mindaugas Liaudanskas,1 Pranas Viškelis,2 Raimondas Raudonis,1 Darius Kviklys,2 Norbertas Uselis,2 and Valdimaras Janulis1

1 Department of Pharmacognosy, Faculty of Pharmacy, Lithuanian University of Health Sciences, Eiveniu¸Street4, 50161 Kaunas, Lithuania 2 Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Kauno Street 30, Babtai, 54333 Kaunas, Lithuania

Correspondence should be addressed to Mindaugas Liaudanskas; [email protected]

Received 2 June 2014; Revised 11 August 2014; Accepted 25 August 2014; Published 11 September 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 Mindaugas Liaudanskas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leaves and to evaluate the antioxidant activity of these extracts. The total phenolic content was determined spectrophotometrically, as well as the total flavonoid content in the ethanol extracts of apple leaves and the antioxidant activity of these extracts, by theABTS, DPPH, and FRAP assays. The highest amount of phenolic compounds and flavonoids as well as the highest antioxidant activity was determined in the ethanol extracts obtained from the apple leaves of the cv. Aldas. The analysis by the HPLC method revealed that phloridzin was a predominant component in the ethanol extracts of the apple leaves of all cultivars investigated. The following quercetin glycosides were identified and quantified in the ethanol extracts of apple leaves: hyperoside, isoquercitrin, avicularin, rutin, and quercitrin. Quercitrin was the major compound among quercetin glycosides.

1. Introduction content of phenolic compounds would allow conducting purposeful studies leading to the usage of the raw material Studies on the chemical biodiversity of plants are recognized obtainedfromappleleavesasapotentialsourceofphenolic as being relevant and are carried out with the aim of compounds in practical medicine. Biologically active com- enriching the assortment of raw medicinal plant materials poundscouldleadtoproductionofdietarysupplements and to evaluate their potential application to the demands andcosmeticpreparationsenrichedinphenoliccompounds of practical medicine. A search for plants accumulating found in apple leaves. Small-scale studies on the chemical phenolic compounds, which have recently been considered composition of leaves have been published, where phloretin as an object of many scientific studies, is especially promising. glycosides, phenolic acids, catechins, and some quercetin gly- It is of importance to assess the composition and content of cosides were identified as the main phenolic compounds [3– phenolic compounds in plant vegetative organs, to determine 5]. Other studies on the composition and content of phenolic the patterns of their accumulation and identify new, promis- compounds in apple leaves were conducted in relation to ing sources of plant phenolic compounds. Venturia inaequalis-caused infections in the vegetative organs The domestic apple (Malus domestica Borkh.) is one of of apple trees. Phenolic compounds accumulated in fruit the most widely cultivated fruit trees. Although the chemical plants play an important role in the plant defense mechanism composition of apples has been extensively investigated [1, 2], against different fungal diseases and different stresses6 [ –9]. we have failed to find any data on the composition and Phenolic compounds, acting as natural antioxidants, content of phenolic compounds in the leaves of different scavenge free radicals and inhibit their production, stimulate apple cultivars grown in Lithuania. Comprehensive data of the synthesis of antioxidant enzymes, and thus prevent oxida- scientific research on the variation in the composition and tive stress resulting in damage to the structural molecules 2 The Scientific World Journal of the body [10–12]. The antioxidant activity of phenolic 2.2. Chemicals. All the solvents, reagents, and standards compounds is associated with other biological properties used were of analytical grade. Acetonitrile and acetic acid of these compounds, such as anti-inflammatory, antimicro- were obtained from Sigma-Aldrich GmbH (Buchs, Switzer- bial, anticancer, cardiovascular system-improving, and other land) and ethanol from Stumbras AB (Kaunas, Lithuania). activities [13–15]. Apple leaves accumulate high amounts of Hyperoside, rutin, quercitrin, phloridzin, phloretin, caf- phloridzin [16, 17], which exhibit antidiabetic activity [18]. feic acid, and chlorogenic acid standards were purchased Therefore, plant raw materials accumulating this compound from Extrasynthese (Genay, France), (+)-catechin and (– can be potentially useful for the prevention of diabetes )-epicatechin from Fluka (Buchs, Switzerland) and avicu- mellitus. larin and isoquercitrin from Chromadex (Santa Ana, USA). ∙ 󸀠 Therefore, the aim of this study was to determine the com- 1,1-Diphenyl-2-picrylhydrazyl (DPPH )radical,2,2-azino- position and content of phenolic compounds in the ethanol bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potas- extracts of apple leaves harvested from the cultivars Aldas, sium persulfate, sodium acetate trihydrate, iron (III) chloride Auksis, Ligol, and Lodel grown under Lithuanian climatic hexahydrate, and 2,4,6-tripyridyl-s-triazine (TPTZ) were conditions, to assess the variation of phenolic compounds obtained from Sigma-Aldrich (Steinheim, Germany). Folin- and to compare their antioxidant activity in the apple leaf Ciocalteu reagent, gallic acid monohydrate, sodium carbon- samples obtained from different cultivars. ate, aluminum chloride hexahydrate, and hexamethylenete- tramine were purchased from Sigma-Aldrich GmbH (Buchs, Switzerland). Deionized water, produced by the Crystal E 2. Materials and Methods high-performance liquid chromatography (HPLC, Adrona SIA, Riga, Latvia) water purification system, was used. 2.1. Plant Material. The following apple cultivars were included into this study: Auksis (early winter cv., bred in Lithuania) and Ligol (winter cv., bred in Poland) that are 2.3. Extraction. An amount of 0.25 g of lyophilized apple two main cultivars in commercial apple orchards as well leaf powder (exact weight) was weighed, added to 10 mL as Aldas (early winter cv., bred in Lithuania) and Lodel of ethanol (70%, v/v), and extracted in a Sonorex Digital 10 P ultrasonic bath (Bandelin Electronic GmbH & Co. (winter cv., bred in Poland), two cultivars resistant to apple ∘ KG, Berlin, Germany) for 40 minutes at 60 C. The extract scab (Venturia inaequalis). Cultivars Aldas and Lodel are obtained was centrifuged for 7 minutes at 6000 rpm with a recommended for organic orchards. The apple trees were Hermle Z206A centrifuge (Denville Scientific Inc., USA). The grown in the experimental orchard of the Institute of Hor- extract was collected and filtered through a membrane filter ticulture, Lithuanian Research Centre for Agriculture and ∘ 󸀠 ∘ 󸀠 with a pore size of 0.22 𝜇m(CarlRothGmbH,Karlsruhe, Forestry, Babtai, Lithuania (55 60 N, 23 48 E). Aldas: block Germany). 4, row 16, trees 5–8; Auksis: block 2, row 3, trees 25–28; Ligol: block 2, row 4, trees 21–24; Lodel: block 4, row 5, trees 12–15. The altitude of Babtai town is 57 m above sea 2.4. Spectrophotometric Studies level. Average annual precipitation is 630 mm, and average 2.4.1. Determination of Total Phenolic and Flavonoid Con- > ∘ ∘ sumofactivetemperaturesis( 10 C)–2300 .Temperatures tent. All the spectrophotometric measurements were carried during the experiment year were close to long term average. out with a Genesys-10 UV/Vis spectrophotometer (Thermo The summer of 2012 was characterised by more rainfall in Spectronic, Rochester, USA). The total phenolic content April, June, and July. Trees were trained as slender spindle. (mg GAE/g DW) in the ethanol extracts of apple leaves Pest and disease management was carried out according to was determined by the Folin-Ciocalteu method [20]. The the rules of the integrated plant protection. The experimental total amount of flavonoids in the ethanol extracts of apple orchard was not irrigated. Tree fertilization was performed leaves was determined using the described methodology [21], accordingtosoilandleafanalysis.Nitrogenwasapplied calculated from a rutin calibration curve, and expressed as before flowering at the rate of 80 kg/ha, and potassium was mg/g rutin equivalent (RE) per gram of absolutely dry weight applied after harvest at the rate of 90kg/ha. Soil conditions of (DW) (mg RE/g DW). the experimental orchard were as follows: clay loam, pH: 7.3, organic matter: 2.8%, P2O5:255mg/kg,andK2O: 230 mg/kg. 2.4.2. Determination of Antioxidant Activity Apple leaves were harvested from 10-year-old apple trees in ∙ ∙ 2012. The study sample comprised 20 healthy, fully developed (1) DPPH Free Radical Scavenging Assay. The DPPH free radical scavenging activity was determined using the method leaves collected from different places of each apple tree of ∙ each cultivar. Apple leaves were lyophilized with a ZIRBUS proposed by Brand-Williams et al. [22]. DPPH solution in 6×10−5 𝜇 sublimator 3 × 4 × 5/20 (ZIRBUS technology, Bad Grund, 96.3% v/v ethanol (3 mL, M) was mixed with 10 Lof Germany) at a pressure of 0.01 mbar (condenser temperature, the ethanol extract of apple leaves. A decrease in absorbance ∘ –85 C). The lyophilized apple leaves were ground to a fine was determined at a wavelength of 515 nm after keeping the samples for 30 minutes in the dark. powder by using a Retsch 200 mill (Haan, Germany). Loss ∙ ∙+ on drying before analysis was determined by the method of (2) ABTS + Radical Cation Decolorization Assay. An ABTS the European Pharmacopoeia [19]. Data were recalculated for radical cation decolorization assay was applied according to absolute dry lyophilizate weight. the methodology described by Re et al. [23]. A volume of 3 mL The Scientific World Journal 3

∙+ of ABTS solution (absorbance 0.800 ± 0.02) was mixed with Table 1: The total amounts of phenolic compounds (TP) and 10 𝜇L of the ethanol extract of apple leaves. A decrease in flavonoids (TFd) in the ethanol extracts of apple leaves of different absorbance was measured at a wavelength of 734 nm after cultivars. keeping the samples for 30 minutes in the dark. cv. TP (mg GAE/g DW)a TFd (mg RE/g DW)a (3) FRAP Assay. The ferric reducing antioxidant power Aldas 163.35 ± 4.36 45.02 ± 0.90 (FRAP) assay was carried out as described by Benzie and Auksis 98.81 ± 1.51 21.59 ± 0.52 Strain [24]. The working FRAP solution included TPTZ Ligol 107.93 ± 2.94 26.97 ± 0.63 (0.01 M dissolved in 0.04 M HCl), FeCl3⋅6H2O(0.02Min Lodel 159.86 ± 4.02 39.64 ± 1.31 water), and acetate buffer (0.3 M, pH 3.6) at the ratio of 𝑃 < 0.05b 𝑃 < 0.05b 1:1:10.Avolumeof3mLofafreshlypreparedFRAPreagent aValues are means ± standard deviations (𝑛=3). was mixed with 10 𝜇Loftheappleleafextract.Anincreasein bBy ANOVA test. absorbance was recorded after 30 minutes at a wavelength of 593 nm. A single factor analysis of variance (ANOVA) along with (4) Calculation of Antioxidant Activity of the Ethanol Extract the post hoc Tukey test was employed for statistical analysis. of Apple Leaves. The antioxidant activity of extracts was The Kolmogorov-Smirnov test was applied to examine the calculated from Trolox calibration curve and expressed as normality of distribution. To verify the hypothesis about 𝜇mol Trolox equivalent (TE) per gram of absolutely dry the equality of variances, Levene’s test was employed. The weight (DW). TE was calculated according to the formula: correlation was evaluated by Pearson analysis. Differences at TE =𝑐×𝑉/𝑚(𝜇mol/g); 𝑐: the concentration of Trolox 𝑃 < 0.05 were considered to be significant. established from the calibration curve (in 𝜇M); 𝑉:thevolume of leaf extract (in L); 𝑚: the weight (precise) of lyophilized leaf powder (in g). 3. Results and Discussion 3.1. Determination of Total Phenolic and Flavonoid Contents. 2.5. High-Performance Liquid Chromatography. Qualitative In order to determine the patterns of the accumulation of andquantitativeanalysisofphenoliccompoundswasper- biologically active compounds in plants, it is important to formed according to the previously validated and described identify their composition and content in separate plant high-performance liquid chromatography (HPLC) method organs. The secondary metabolites of plant metabolism, [25]. A Waters 2695 chromatograph equipped with a Waters phenolic compounds, have been detected in apple leaves 2998 photodiode array (PDA) detector (Waters, Milford, [3, 16]; therefore, this study aimed at determining the com- USA) was used for HPLC analysis. Chromatographic separa- position and content of phenolic compounds in plants and at tionswerecarriedoutbyusingaYMC-PackODS-A(5𝜇m, determining the patterns of their variation and accumulation. C18, 250 × 4.6 mm i.d.) column equipped with a YMC- In this study, the total amount of phenolic compounds Triart (5 𝜇m, C18, 10 × 3.0 mm i.d.) precolumn (YMC Europe in the ethanol extracts of apple leaves varied from 98.81 ± GmbH, Dinslaken, Germany). The column was operated at ∘ 1.51 mg GAE/g DW (cv. Auksis) to 163.35 ± 4.36 mg GAE/g a constant temperature of 25 C. The volume of the extract DW (cv. Aldas) (Table 1). In a study by Iqbal et al. [26], being investigated was 10 𝜇L. The flow rate was 1 mL/min, the total phenolic content in the ethanol extracts of apple and gradient elution was used. The mobile phase consisted leaves was 157.06 mg GAE/g DW; meanwhile one Slovenian of 2% (v/v) acetic acid in water (solvent A) and 100% (v/v) study reported a lower total phenolic content in apple leaves, acetonitrile (solvent B). The following conditions of elution ranging from 80 mg GAE/g DW to 115 mg GAE/g DW [16]. were applied: 0–30 minutes, 3%–15% B; 30–45 minutes, 15%– Both scab-resistant cultivars, Aldas and Lodel, accumulated 25% B; 45–50 minutes, 25%–50% B; and 50–55 minutes, 50%– significantly higher amounts of total phenolics and total 95% B. flavonoids. Such differences among the cultivars susceptible The identification of the chromatographic peaks was andresistanttoapplescabhavebeenestablishedinother achieved by comparing the retention times and spectral studiesaswell[27]. 𝜆 characteristics ( = 200–600 nm) of the eluting peaks with Studies investigating the total phenolic content in the leaf those of reference compounds. The compounds identified samples of other plants belonging to the Rosaceae family have were confirmed by spiking the sample with the standard been conducted as well. Methanol extracts from the leaves compound and monitoring the changes in the peak shape and of various Sorbus species were found to have lower total spectral characteristics. For quantitative analysis, a calibra- amounts of phenolics ranging from 60.6 to 90.9 mg GAE/g tion curve was obtained by injection of known concentrations DW [28]. Hua et al. [6], who investigated the samples of of different standard compounds. Dihydrochalcones and cat- pear leaves, reported a lower total phenolic content (68.1– echins were quantified at 280 nm, phenolic acids at 320 nm, 83.3 mg GAE/g DW). and flavonols at 360 nm. The biological effects of many plant raw materials depend on flavonoids; therefore, studies on the variation in their 2.6. Statistical Data Processing Methods. All the experiments content are important and relevant. This study determined were carried out in triplicate. Means and standard deviations the total flavonoids content in the ethanol extracts of apple were calculated with the SPSS 20.0 software (Chicago, USA). leaves, which ranged from 21.59 ± 0.52 mg RE/g DW (cv. 4 The Scientific World Journal

Auksis) to 45.02 ± 0.90 mg RE/g DW (cv. Aldas) (Table 1). 400 Iqbal et al., who investigated the ethanol extracts of apple 350 leaves, reported a higher total flavonoids content, reaching 300 121.86 mg RE/g DW [26]. In contrast to that, the ethanol extracts of hawthorn leaves were found to have a lower total 250 flavonoids content (9.5–13.0 mg RE/g DW) [21]. 200 mol/g DW) mol/g The data on the patterns of variation in the total phenolic 𝜇 150 and flavonoid contents of apple leaves are scarce. Therefore, TE ( 100 this study provides new knowledge about total phenolics and flavonoids content in the apple leaves of the cultivars grown 50 under Lithuanian climatic conditions, allows the comparison 0 of the results obtained with those of other studies, and is Aldas Auksis Ligol Lodel valuable to carrying out a search for promising, biologically Apple cultivar active substance-accumulating plant raw materials. DPPH FRAP ABTS 3.2. Measurements of Antioxidant Activity in Extracts. After studying the total phenolics and flavonoids content of apple Figure 1: The antioxidant activity of ethanol extracts obtained from leaves harvested from different cultivars grown under Lithua- apple leaves and determined by using DPPH, ABTS, and FRAP nian climatic conditions, it is important to examine and assess assays. Values are means and errors bars indicate standard deviations the antioxidant activity in the extracts of apple leaves. The (𝑛=3). results obtained during studies will be useful for the selection of apple cultivars in order to provide a consumer with products rich in antioxidants, will be useful for the assessment and method [37–40] probably because the DPPH method has ∙+ standardization of quality of plant raw materials and their more limitations. ABTS radical cation (ABTS )issoluble products, and will allow predicting an antioxidant effect of in water and organic solvents, which allows determining apple leaves in vivo. antiradical activity of the hydrophilic and lipophilic com- ∙ Herbal extracts are multicomponent matrices with pounds [30, 41]. DPPH radicals are only soluble in organic antioxidant activity determined by the set of different mech- solvents, which limits the evaluation of antioxidant activity of hydrophilic antioxidants [36, 42]. Wang et al. found that some anism reactions, so antioxidant effect cannot be adequately ∙+ of the compounds, which have ABTS scavenging activity, tested using only one method [29, 30]. For these reasons, it ∙ may not show DPPH scavenging activity [43]. Arts et al. is recommended to use at least two different methods for ∙+ determination of antioxidant activity in herbal extracts [31]. reported that some products of ABTS scavenging reaction may have a considerable contribution to the antioxidant In order to thoroughly evaluate the antioxidant activity of ∙+ the ethanol extracts of apple leaves, different antioxidant capacity and can continually react with ABTS [44]. capacity assays (DPPH, ABTS, and FRAP) were employed. The analysis of the antioxidant activity in apple leaves The in vitro antioxidant effect of the investigated extracts was revealed differences in the antioxidant activity among the evaluated by the DPPH and ABTS assays as a capability of ethanol extracts of the apple leaves of the cultivars inves- ∙ ∙+ DPPH and ABTS , compounds possessing an antiradical tigated (Figure 1). The ethanol extracts obtained from the activity, to scavenge free radicals [22, 23]andbytheFRAP apple leaves of the cv. Aldas showed the highest TE values. assay, as a capability of antioxidants to reduce Fe(III) to Fe(II) They reached 141.95, 280.23, and 355.54 𝜇moL/g DW for [24]. The results of in vitro antioxidant activity determined in the DPPH, ABTS, and FRAP assays, respectively. To our the ethanol extracts of apple leaves (cultivars Aldas, Auksis, knowledge, no studies on the antioxidant activity of apple Ligol, and Lodel) are summarized in Figure 1. leaves have been carried out so far. Therefore, the findings It has been previously reported that antioxidant capacity from this study on antioxidant activity were compared with determined by in vitro assays differs [32–34]. TE values, thoseobtainedinstudiesonotherplantsbelongingtothe obtained from samples of apple leaf extracts antioxidant Rosaceae family. The methanol extracts obtained from the activity studies, according to the used methods, can be leaves of different Sorbus species showed a higher antioxidant arranged in the following order: DPPH < ABTS < FRAP activity; that is, the TE values were higher than those (Figure 1). This pattern could be explained by differences of determined in the present study: 344.0–628.3 𝜇moL/g DW applied antioxidant activity determination methods. FRAP by the DPPH assay, 262.5–467.3 𝜇moL/g DW by the ABTS assay is a method for measuring total reducing power of elec- assay, and 861.6–1650.4 𝜇moL/g DW by the FRAP assay tron donating substances, whilst ABTS and DPPH assays are [28]. These results could be explained by the fact that apple methods for measuring the ability of antioxidant molecules to leaves accumulate high amounts of phloridzin, belonging to ∙+ ∙ scavenge ABTS and DPPH free radicals, respectively [35, the dihydrochalcone class [3, 16], which possesses a lower 36]. ABTS and DPPH methods have several differences from antioxidant activity than the phenolic compounds belonging one another, which causes their TE values to be different. to other classes [4, 45]. Some authors indicate that TE or VCE values determined by In order to determine the relationship between the anti- DPPH method are lower than those determined by ABTS oxidant activities of the ethanol extracts of apple leaves The Scientific World Journal 5

0.40 9 0.35 0.30 6 0.25 0.20 (AU) 0.15 7 0.10 8 10 11 0.05 2 3 1 4 5 0.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 (min)

Figure 2: Chromatogram of ethanol extract of apple leaf sample (cv. Aldas) investigated (𝜆 = 320 nm). Numbers indicate the peaks of analytes: 1: (+)-catechin, 2: chlorogenic acid, 3: caffeic acid, 4: (–)-epicatechin, 5: rutin, 6: hyperoside, 7: isoquercitrin, 8: avicularin, 9: quercitrin, 10: phloridzin, and 11: phloretin.

Table 2: Content of quercetin glycosides in ethanol extracts obtained from the apple leaves of cultivars grown in Lithuania.

Content of quercetin glycosides, mg/g (expressed for absolute dry weight)a Compound Aldas Auksis Ligol Lodel Hyperoside 8.95 ± 0.35a 5.67 ± 0.20b 4.59 ± 0.17c 7. 0 3 ± 0.20d Isoquercitrin 3.48 ± 0.12a 2.79 ± 0.11b 1.84 ± 0.07c 2.40 ± 0.08d Rutin 0.33 ± 0.01a 0.75 ± 0.03b 0.67 ± 0.02c 0.54 ± 0.02d Avicularin 2.48 ± 0.10a 2.82 ± 0.10b 2.09 ± 0.07c 2.51 ± 0.08d Quercitrin 13.36 ± 0.51a 10.29 ± 0.48b 7. 7 7 ± 0.27c 12.31 ± 0.59d aValues are means ± standard deviations (𝑛=3). The different letters indicate significant differences between the values(𝑃 < 0.05). assessed by the DPPH, ABTS, and FRAP assays and total the separation of avicularin and quercitrin with the resolution phenolic as well as flavonoid contents in these extracts, the of 2.94. The chromatograms of ethanol extracts of apple correlation analysis was carried out. There was a strong pos- leaf samples obtained from all the cultivars investigated are itive correlation between total phenolic as well as flavonoid identical regarding the number of analytes and retention contents and the antioxidant activity assessed by all the time. The example of chromatogram of the ethanol extract of methods (𝑟 = 0.84−0.98, 𝑃 < 0.05). The strongest correlation the apple leaf sample (cv. Aldas) is shown in Figure 2. was found between the antioxidant activity of the ethanol The apple leaves of the cv. Aldas contained the highest extracts of apple leaves determined by the FRAP method and total amount of the quercetin glycosides identified and total flavonoid content (𝑟 = 0.96, 𝑃 < 0.05)aswellastotal quantified. It was 1.7 times higher than their lowest amount phenolic content (𝑟 = 0.98, 𝑃 < 0.05) in these extracts. Other found in the apple leaves of the cv. Ligol (Table 2). Quercitrin studies published earlier also reported strong correlative was a predominant component among all the quercetin relationships between phenolic as well as flavonoid contents glycosides identified and quantified in the ethanol extracts and the antioxidant activity of plant extracts evaluated by of apple leaves. The apple leaves of the cv. Aldas had the different assays [46–48]. highest amount of quercitrin. The results obtained are in agreement with literature data that quercitrin is one of the 3.3. Identification and Quantification of Phenolic Compounds main flavonols found in apple leaves5 [ , 49]. The results of this by HPLC. The phenolic compounds of various groups, (+)- study are in line with those reported by the Slovenian study catechin, chlorogenic acid, caffeic acid, (–)-epicatechin, rutin, where the quercitrin content in the apple leaves collected hyperoside, isoquercitrin, avicularin, quercitrin, phloretin, in the Maribor region ranged from 6.3 mg/g to 10.8 mg/g and phloridzin, in the analyzed ethanol extracts obtained (cv. Golden Delicious) and from 3.7 mg/g to 8.5 mg/g (cv. from the apple leaves of the cultivars Aldas, Auksis, Ligol, Jonagold) [49]. The factors such as a geographical region and Lodel were identified by the HPLC method. The values and climatic-meteorological and cultivation conditions could of resolution (Rs > 2) were achieved in all the samples of influence these quantitative differences. extracts. The applied HPLC method allowed for an effective Rutinwastheminorcomponentamongallthequercetin separation of quercetin glycosides: the separation of rutin glycosides quantified in all ethanol extracts of the apple leaf and hyperoside in ethanol extracts of lyophilized apple samples analyzed. The results obtained are confirmed by leaf samples with the resolution of 2.63; the separation of previously published data on the variation in the composition hyperoside and isoquercitrin with the resolution of 3.28; and and content of quercetin glycosides in apple leaves [5]. 6 The Scientific World Journal

The highest amount of rutin was detected in the apple leaves is also characteristic for dihydrochalcone group compounds of the cv. Auksis (Table 2). However, the Slovenian study in apple leaves—glycoside phloridzin levels are significantly reported contrary findings on the flavonol content in apple higher than its aglycone phloretin levels [3, 59]. leaves. The authors of that study demonstrated that rutin Phloridzin has a wide spectrum of biological effects, along with quercitrin was one of the major compounds in inhibits the growth of cancer cells [60], improves memory apple leaves [49]. [61, 62], and is useful in the bone fracture prevention [63, 64]. When comparing quercetin glycoside content and com- One of the most important and potentially valuable phlo- position variation in apple fruits and leaves grown in Lithua- ridzin biological effects is antidiabetic activity [18, 65]. Main nian climatic conditions, it was found that apple leaves have phloridzin pharmacological mechanism of action, leading higher amounts of these compounds. For example, cv. Aldas, toitsantidiabeticeffect,istoproducerenalglycosuriaand cv. Auksis, and cv. Ligol apple fruits had hyperoside amounts block intestinal glucose absorption through inhibition of from 0.05 mg/g (cv. Auksis) to 0.19 mg/g (cv. Aldas) [25] sodium-glucose symporters in kidneys and small intestine which constitutes only 0.9–2.1% hyperoside content found [66]. The principle uses of phloridzin are associated with its inleavesofthesameapplecultivars.Similarquantitative ability to reduce plasma glucose, without changing insulin differences between apple fruit and leaf grown in Lithuania levels [67, 68]. Phloridzin ability to reverse glucotoxicity and are also typical in other quercetin group compounds. reduce blood glucose levels without increasing body weight All the quercetin glycosides identified and quantified in determines its benefits in prophylaxis and treatment of type 2 the ethanol extracts of apple leaf samples can be ranked diabetes [69–71]. Phloridzin reduces body weight by blocking in the following ascending order by their content: rutin < the absorption and resorption of glucose [72, 73], and the isoquercitrin< avicularin < hyperoside < quercitrin. This weight loss is one of the most important type 2 diabetes order was characteristic of the apple leaves of the cultivars prevention methods [74, 75]. Phloridzin consumption to Auksis,Ligol,andLodel.Intheappleleavesofthecv. reduce glucose concentration in blood plasma does not cause Aldas, the amount of isoquercitrin was higher than that of body fluid loss and hypoglycemia risk66 [ , 76]. For the avicularin. reasons stated above, it is purposeful to investigate plant Phloridzin and phloretin, which belong to the dihy- materialsandextractsthataccumulatephloridzin.Webelieve drochalcone class, were identified in the ethanol extracts it is a promising research in developing medicines and food of apple leaf samples. Phloridzin was the major phenolic supplements for body weight reduction in the prevention of compound in the apple leaves. It accounted for 76.9% to diabetes. Our assumption is supported by other scientist’s 84.2% of all phenolic compounds identified and quantified research, which offers to use apple leaves to enrich phenolic in the extracts of apple leaves by the HPLC method. The compounds composition with phloridzin in apple juice [77]. results obtained confirm literature data that phloridzin is a Theappleleavesofthecv.Ligolcontainedthehighestcon- predominant component of phenolic compounds in apple centration of phloridzin. The identified amounts of phloretin leaves [16, 17]. Determined phloretin levels were significantly were lower than those of phloridzin (Table 3). Such trends in lower. They make up 1.0–1.8% of all identified and quan- the content of compounds belonging to this dihydrochalcone tifiedphenoliccompounds.Thiscouldbeinterpretedby class have been demonstrated by other studies as well [3]. In phloretin and phloridzin molecules structural differences. contrast to the leaves of apple cultivars grown in Lithuania, Phloridzin is phloretin glycoside, which has glucose linked 󸀠 phloridzin was not the dominant compound in apple fruits. at hydroxy group in molecule’s 2 position. Gosch et al. Determined phloridzin levels were 0.06–0.14 mg/g [25], and researched dihydrochalcone synthesis processes in apple it was only 3.9–5.5% of identified and quantified phenolic leaves and found that phloridzin is formed from phloretin compounds, tested in varieties cv. Aldas, cv. Auksis, and cv. by glycosylation reaction, affected by enzyme dihydrochal- 󸀠 Ligol. There was also a distinction of composition of phe- cone 2 -O-glucosyltransferase [17]. Sugar molecule link to noliccompoundsbetweentheapplefruitandtheappleleaf the aglycone influences the physical-chemical properties samples. Phloretin was identified only in apple leaf samples of flavonoid. Commonly flavonoid glycosides have lower but not in apple fruit samples [25, 78]. antioxidant activity and are more hydrophilic than their The highest total amount of phenolic acids identified and respective aglycones [50, 51]. These characteristics relate to ± thefactthattheflavonoidaglyconehasmorefreehydroxy quantified by the HPLC method (1.61 0.07 mg/g) was found groups than their corresponding glycosides, which have one in the apple leaves of the cv. Auksis (Table 3). The ethanol or several hydroxy groups with bonded sugar [52, 53]. Rezk extracts of apple leaves contained caffeic acid; its amount was et al. indicates that formation of phloridzin by glycosylation lower than that of chlorogenic acid. The levels of chlorogenic 󸀠 of hydroxy group at phloretin molecule’s 2 position reduces acidweresimilarorslightlyhigherthanthosereported its antioxidant activity 18 times compared to phloretin by the earlier studies [49, 79]. Fruit and leaf composition [54]. Other researchers who conducted comparative studies and content of apples cultivated in Lithuanian climatic of phloretin and phloridzin anti-inflammatory effects and conditions differed. In cv. Aldas, cv. Auksis, and cv. Ligol their influence on lipid oxidation report similar patterns. apples, chlorogenic acid was the predominant compound; Phloretin has significantly stronger anti-inflammatory [55] determined amounts (0.69–2.23 mg/g) were 54.8–69.6% of and lipid oxidation inhibitory effect [56]thanphloridzin. all identified and quantified phenolic compounds25 [ ]. In The flavonoid aglycone reactivity explains why glycosides are apple leaf extracts, phloridzin was dominant and chlorogenic the most common flavonoid in plants [57, 58]. This pattern acid was only 0.3–1.0% of all identified phenolic compounds. The Scientific World Journal 7

Table 3: Content of dihydrochalcones, phenolic acids, and catechins in ethanol extracts obtained from the apple leaves of cultivars grown in Lithuania. Content of dihydrochalcones, phenolic acids, and catechins, mg/g (expressed for absolute dry weight)a Compound Aldas Auksis Ligol Lodel Phloridzin 106.01 ± 4.23a 108.9 ± 4.32b 114.43 ± 4.72c 109.51 ± 4.62d Phloretin 1.81 ± 0.07a 1.52 ± 0.06b 2.40 ± 0.09c 1.40 ± 0.06d Chlorogenic acid 0.48 ± 0.02a 1.38 ± 0.06b 1.12 ± 0.05c 0.86 ± 0.03d Caffeic acid 0.26 ± 0.02a 0.23 ± 0.03b 0.15 ± 0.01c 0.14 ± 0.01d (+)-Catechin 0.05 ± 0.01a 0.27 ± 0.02b 0.09 ± 0.01c 0.17 ± 0.01d (−)-Epicatechin 0.72 ± 0.02a 0.38 ± 0.02b 0.76 ± 0.02c 0.39 ± 0.01d aValues are means ± standard deviations (𝑛=3). The different letters indicate significant differences between the values(𝑃 < 0.05).

Composition differences were also determined; apple leaves identifiedandquantifiedinethanolextracts,anditsamount contained caffeic acid, while apple fruits did not. in the apple leaves of different cultivars ranged from 7.77 to The chemical composition of catechins (monomeric 13.36 mg/g DW. flavan-3-ols) in ethanol extracts obtained from the apple The preliminary in vitro experiments examining the leavesofthecultivarsAldas,Auksis,Ligol,andLodelwas antioxidant activity of apple leaf extracts by the ABTS, DPPH, studied. The highest and lowest total amounts of the cat- andFRAPassayshaveshownthattheseextractspossessa echins identified were found in the apple leaves of the strong antioxidant activity, which positively correlated with cultivars Ligol and Lodel, respectively (0.85 ± 0.04 mg/g 𝑟 = 0.84 0.98 ± the total phenolic and flavonoid contents ( – , versus 0.56 0.02 mg/g) (Table 3). The amounts of (–)- 𝑃 < 0.05). The ethanol extracts obtained from the apple leaves epicatechin in the extracts studied were higher than those of the cv. Aldas showed the highest TE values: 141.95 𝜇mol/g of (+)-catechin. Similar amounts of (–)-epicatechin were DW by the DPPH assay, 280.23 𝜇mol/g DW by the ABTS reported by other authors [3]. (+)-Catechin (0.05–0.15 mg/g) assay, and 355.54 𝜇mol/g DW by the FRAP assay. and (–)-epicatechin (0.24–0.45 mg/g) levels determined in apple fruits of Lithuanian cultivated cv. Aldas, cv. Auksis, The results reported in this study prompt further research and cv. Ligol were similar to those determined in apple on the chemical composition and biological effect of apple leaves. Composition differences were also determined— leaves by evaluating the antioxidant activity of individual phe- procyanidins (oligomeric flavan-3-ols) group compounds; nolic compounds in vitro and in vivo and confirm a potential procyanidin B1 and procyanidin B2 were identified in apple oftheseplantsasarawmaterialinmedicalpracticeaswellas fruits [25, 78], while in apple leaves they were not. the development and production of dietary supplements and The HPLC analysis of ethanol extracts obtained from cosmetic preparations rich in biologically active compounds. appleleavesrevealedthatphloridzinwasthemajorcom- pound in the samples investigated, and its amounts were considerably higher than those of other phenolic compounds. Abbreviations Quercitrin was a predominant component among quercetin ANOVA: A single factor analysis of variance glycosides. The results of the HPLC analysis show that apple 󸀠 leaves are a valuable, natural source of dihydrochalcones and ABTS: 2,2 -azino-bis(3-ethylbenzothiazoline-6- quercetin glycosides. This encourages further research on this sulphonic acid) plant as a raw material for use in pharmacy. cv.: Cultivar DPPH: 2,2-diphenyl-1-picrylhydrazyl DW: Dry weight 4. Conclusions FRAP: Ferric reducing antioxidant power GAE: Gallic acid equivalent In conclusion, the results of this study will provide new HPLC: High-performance liquid chromatography knowledge about the composition and content of phenolic Rs: Resolution compounds in apple leaves and the antioxidant activity of TE: Trolox equivalent their extracts, which will give a wide range of possibilities to TFd: Total amount of flavonoids employ these plants as the source of phenolic compounds. TP: Total amount of phenolic compounds The highest total amounts of phenolic compounds and TPTZ: 2,4,6-tripyridyl-s-triazine flavonoids were determined in the apple leaves of the cv. VCE: Vitamin C equivalent. Aldas (163.35 ± 4.36 mg GAE/g DW and 45.02 ± 0.90 mg RE/g DW, resp.). Phloridzin was the major compound in the ethanol extracts of apple leaves of all the cultivars investi- Conflict of Interests gated. The apple leaves of the cv. Ligol had the highest amount of phloridzin (114.43 ± 4.72 mg/g DW). Quercitrin was the The authors declare that there is no conflict of interests predominant component among the quercetin glycosides regarding the publication of this paper. 8 The Scientific World Journal

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Research Article Optimization of Ionic Liquid Based Simultaneous Ultrasonic- and Microwave-Assisted Extraction of Rutin and Quercetin from Leaves of Velvetleaf (Abutilon theophrasti) by Response Surface Methodology

Chunjian Zhao,1,2 Zhicheng Lu,2 Chunying Li,2 Xin He,2 Zhao Li,2 Kunming Shi,2 Lei Yang,2 Yujie Fu,2 and Yuangang Zu2

1 College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China 2 Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China

Correspondence should be addressed to Chunying Li; [email protected]

Received 3 July 2014; Accepted 5 August 2014; Published 27 August 2014

Academic Editor: Fred Stevens

Copyright © 2014 Chunjian Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

An ionic liquids based simultaneous ultrasonic and microwave assisted extraction (ILs-UMAE) method has been proposed for the extraction of rutin (RU), quercetin (QU), from velvetleaf leaves. The influential parameters of the ILs-UMAE were optimized by the single factor and the central composite design (CCD) experiments. A 2.00 M 1-butyl-3-methylimidazolium bromide ∘ ([C4mim]Br) was used as the experimental ionic liquid, extraction temperature 60 C, extraction time 12 min, liquid-solid ratio 32 mL/g, microwave power of 534 W, and a fixed ultrasonic power of 50 W. Compared to conventional heating reflux extraction (HRE), the RU and QU extraction yields obtained by ILs-UMAE were, respectively, 5.49 mg/g and 0.27 mg/g, which increased, respectively, 2.01-fold and 2.34-fold with the recoveries that were in the range of 97.62–102.36% for RU and 97.33–102.21% for QU with RSDs lower than 3.2% under the optimized UMAE conditions. In addition, the shorter extraction time was used in ILs-UMAE, compared with HRE. Therefore, ILs-UMAE was a rapid and an efficient method for the extraction of RU and QU from the leaves of velvetleaf.

1. Introduction antioxidant [10–12], anti-inflammatory [13, 14], antimicrobial [15], antitumor [16], and antiasthma [17]. In virtue of the Velvetleaf (Abutilon theophrasti Medik) is one of the main above important activities of RU and QU and their plentiful members in Malvaceae and it is a major annual weed in content in velvetleaf, therefore, it is very important to develop cropland [1]. There are lots of biological activity components a method for the extraction and determination of RU and in leaves and seeds from velvetleaf [2, 3]. However, there QU from velvetleaf in order to utilize the abundant velvetleaf is less information about velvetleaf flavonoids components resources in China. [4, 5]. It was reported that there were flavonoids in velvetleaf Conventional heating reflux extraction (HRE) and ultra- and the main three components were rutin (RU), quercetin sonic extraction (UE) were once applied in the extraction (QU), and kaempferol (KA) [4, 6]. Our preliminary study of flavonoids [18]. However, these extraction processes are showed that there were rich RU and QU, but a little KA from connected with long extraction time and unsatisfactory Chinese velvetleaf. recovery. Thus, it is desirable to develop a rapid and efficient RU (3,4,5,7-tetrahydroxyflavone-3-d-rutinoside) and extraction method to improve the limitations of conventional 󸀠 󸀠 QU (3,3 ,4 ,5,7-pentahydroxyflavone), whose structures are extraction of flavonoids [19]. shown in Figure 1, are two effective compounds for curing Ionic liquids (ILs) are a kind of salts that display an hypertension, diabetes, and cardiac and cerebral vascular amazingly lower melting temperature than the boiling point diseases [7–9]. In addition, they display the activities of ofwaterandtheyareoftenliquidatroomtemperature. 2 The Scientific World Journal

OH RU and QU yields were investigated and further optimized OH by a central composite design (CCD) and response surface HO O methodology (RSM).

O OH 2. Experimental OH O HO O OH OO O OH 2.1. Chemicals and Reagents. RU and QU standards were CH3 OH bought from J & K Chemical Ltd. (Beijing, China). All OH OH ionic liquids ([C2mim]Br, 1-ethyl-3-methylimidazolium OH OH OH OH O bromide; [C4mim]Br, 1-butyl-3-methylimidazolium bro- Quercetin Rutin mide; [C6mim]Br, 1-hexyl-3-methylimidazolium bromide; [C8mim]Br, 1-octyl-3-methylimidazolium bromide; Figure 1: Chemical structures of rutin (RU) and quercetin (QU). [C4mim]Cl, [C4mim]NO3, [C4mim]HSO4, [C4mim]BF4, [C4mim], that is 1-octyl-3-methylimidazolium bromide) were purchased from J & K Chemical Ltd. (Beijing, China). Deionized water for HPLC was purified using a Milli-Q ILs constituted of relatively large asymmetric organic cations Water Purification System (Millipore, MA, USA). Other and smaller inorganic or organic anions [20]. Due to their analytical reagents were purchased from the Tianjin Kermel negligible vapor pressure, good thermal sensibility, low or Chemical Reagent Co. Ltd. (Tianjin, China). virtually no volatility, good miscibility with water and organic solvents, and extractability for various organic compounds, they have recently been widely applied in the extraction and 2.2. Materials. The leaves of velvetleaf were collected in separation from natural product [21–23]. For example, dif- autumn from Shuyang County, Jiangsu, China. Voucher ferent types of compounds such as essential oils, flavonoids, specimens were deposited in the herbarium of our laboratory. suberin, polyphenolic compounds, and alkaloids were all The materials were dried in the shade at room temperature, extracted by many ILs-based extraction technologies [24– powdered by a disintegrator (HX-200A, Yongkang Hardware 26]. and Medical Instrument Plant, China), and passed through a stainless steel sieve (40–60 mesh) and stored in closed In recent times, microwave-assisted extraction (MAE) ∘ has been rapidly developed and proposed as a prospective and desiccators at 4 Cuntiluse. influential technique to replace conventional extraction techniques in the extraction of bioactive constituents from plant 2.3. Apparatus. Simultaneous ultrasonic and microwave materials because of its special heating mechanism, moderate extracting apparatus (CW-2000, Shanghai Xintuo analytical capital cost, rapid extraction, and excellent performance [27, instrument technology Co. Ltd., China, the maximum power 28]. It has been reported that microwave energy can be of 700 W and a fixed ultrasonic power of 50 W) and ultrasonic efficiently absorbed under the ILs as solvents and cosolvents extraction device (A KQ-250DB, Kunshan, China) with a conditions [29]. Considering that ILs can efficiently absorb maximum power of 250 W were used for the extraction of microwave energy, it is rather an interesting challenge to targets compounds. The HPLC system consisted of a Waters use ILs as solvent for the MAE of various biomolecules 717 automatic sample handling system series HPLC system from solid samples. Comparing with conventional organic equipped with a 1525 pump, a 717 automatic column tem- solvents, ILs are green solvents because their vapour pressure perature control box, and a 2487 UV-detector (Waters, USA) was so lower that ILs are very difficult to evaporate into the that was used for the determination of targets compounds. environment. In some cases, they could even be well recycled. Chromatographic separation was performed on a HiQ sil- They can effectively improve the selectivity and the extraction C18 reversed-phase column (4.6 mm × 250 mm, 5 m, KYA efficiency of the being investigated compounds from plant TECH). samples [29]. Ultrasonic is one of the most industrially used meth- odstoenhancetheextractioneffectsduetoitsmass 2.4. Extraction Methods transfer phenomena [30–32]. Recently, simultaneous ultrasonic/microwave assisted extraction (UMAE) coupled the 2.4.1. Heating Reflux Extraction (HRE). A1.0gofdried advantageofmicrowaveandultrasonic,presentingmany sample powders was put into a round-bottomed flask by advantages [33, 34]. However, there are no reports on adding 20 mL of methanol or 2 M [C4mim]Br; then the flask ILs-based simultaneous ultrasonic and microwave assisted was placed into oil bath with a reflux device, followed by extraction (ILs-UMAE) of RU and QU from leaves of vel- extracting at 6 h. vetleaf. In the present study, a fast and efficient method of ILs- 2.4.2. Ultrasonic-Assisted Extraction (UAE). A1.0gofdried UMAE separation and determination of two major flavones sample powders was put into a conical flask by adding 20 mL (RU and QU) from leaves of velvetleaf was developed and of methanol or 2 M [C4mim]Br; then the conical flask was the effects of extraction time, temperature, ionic liquids placed into the ultrasonic extraction device, followed by concentration, solid-liquid ratio, and microwave power on sonication for 1 h at room temperature. The Scientific World Journal 3

Water outlet 2.6. Experimental Design. First, the influencing factors of ILs-UMAE, namely, extraction time, temperature, ionic liq- Condenser uids concentration, solid-liquid ratio, and microwave power on the yields of RU and QU were investigated. On the above Program display Water inlet single factor experiments, the three dominating parameters, Control panel that is, microwave power, extraction time, and liquid-solid Video display Power ratioontheyieldsofRUandQU,wereoptimizedbyRSM. Microwave generator In detail, the effects of three independent variables including Extraction solvent Sample extraction time (X1: 6–14 min), liquid-solid ratio (X2: 20– Ultrasonic transducer 40 mL/g), and microwave power (X3: 300–700 W) at five levels (–1.68, –1, 0, +1, +1.68) were investigated using a central Figure 2: Schematic representation of the UMAE device. composite design (CCD) with RSM (Table 1). A total of 20 experiments consisting of 8 factorial points, 6 axial points, and 6 replicates at the central points were performed. Experimental data collected from the designed 2.4.3. Microwave-Assisted Extraction (MAE). A1.0gofdried experiment were analyzed by a response surface regression samplepowderswasputintoaspecialround-bottomed model using the following second-order polynomial: flask by adding 20 mL of methanol or 2 M [C4mim]Br; then the round-bottomed flask was placed into the pressure 3 3 2 3 self-control microwave decomposition system followed by 𝑌=𝛽 + ∑ 𝛽 𝑋 + ∑ 𝛽 𝑋2 + ∑ ∑ 𝛽 𝑋 𝑋 , 0 𝑖 𝑖 𝑖𝑖 𝑖 𝑖𝑗 𝑖 𝑗 (2) microwave irradiation. 𝑖=1 𝑖=1 𝑖=1 𝑗=2 𝑖<𝑗

2.4.4. Ultrasonic- and Microwave-Assisted Extraction (IL- where 𝑌 represented the response variable; 𝛽0, 𝛽𝑖, 𝛽𝑖𝑖,and UMAE). UMAE device was used to separate RU and QU 𝛽𝑖𝑗 were the regression coefficients of variables for intercept, fromtheleavesofvelvetleaf.Theapparatusisshownschemat- linearity, square, and interaction terms, respectively; 𝑋𝑖 and ically in Figure 2. With cold water running through the 𝑋𝑗 were the independent coded variables influencing the condenser of the UMAE system, sample was mixed with response variable 𝑌;and𝑘 represents the number of variables. methanol or different concentrations of IL solutions, and then the suspensions were irradiated under microwave heating and a fixed ultrasonic power of 50 W. After each irradiation, 2.7. Statistical Analysis. Design Expert (DE) software (Trial ∘ the obtained extracts were cooled to 25 C, then diluted to version 7.0.0, STAT-EASE Inc., Minneapolis, MN, USA) was 50 mL with water, and filtrated through a 0.45-𝜇mfilterfor used to analyze the experimental data and to find the response subsequent HPLC analysis. surfaces of the response models and it was used to decide and assess the statistical significance of the equations. The lack of 2 fit and coefficient of determination (𝑅 )wereusedtoevaluate 2.5. Determination of RU and QU by HPLC. The diluted theadequacyofmodel.TheFishertestvalue(𝐹-value) and extracts were directly injected into the system which was a their interactions were estimated by the analysis of variance Waters 717 automatic sample handling system series HPLC (ANOVA). Finally, in order to decide the adequacy of the system. The conditions of HPLC analysis were as follows: the fittedmodel,theactualandpredictedvalueswerecompared. mobile phase was methanol-acetonitrile-water (40 : 15 : 45, The optimum condition for three variables (extrac- v/v/v) adding 1.0% acetic acid. This mobile phase was filtered tion time: 6–14 min, liquid-solid ratio: 20–40 mL/g, and 𝜇 through a 0.45 m membrane filter and then deaerated microwave power: 300–700 W) was acquired by statistical ultrasonically prior to use. The injection volume was 10 𝜇L ∘ analysis (DE software) [35]. andthecolumntemperaturewassetat25 C. The flow rate was 1 mL/min. The UV detection wavelength applied was 360 nm. PeakareasofRUandQUwereusedforquantificationwith 3. Results and Discussion external standard method. The extraction yield of target analyte was determined as 3.1. Selection of ILs for UMAE follows: 3.1.1. Effect of Anion. The anion identity is an important factor ( / ) to impact the properties of ILs [36]. Thus, the 1-butyl-3- Yield mg g methylimidazolium ILs with five kinds of different anions − − − − − ( ) (Br ,Cl ,NO3 ,HSO4 ,andBF4 )wereselectedinUMAE. = mean mass of target analytes in sample mg . ( ) As shown in Figure 3(a), compared with the extraction results mass of samples g usingfivedifferenttypesof1MILssolutionswiththesame (1) cations but different anions, it was apparently found that [C4mim]Br was more efficient than others. The mean mass of target analytes in samples was calcu- It was probably due to the stronger multi-interactions lated after 3 repeated determinations under the optimized including 𝜋-𝜋, ionic/charge - charge, and hydrogen bonding conditions. between the ILs ([C4MIM]Br) and flavonoids [33]. 4 The Scientific World Journal

Table 1: Central composite design (CCD) of independent variables for process optimization.

Coded Actual Run Factor A Factor B Factor C Time, Liquid-solid ratio, Microwave power, (𝑋1) (𝑋2) (𝑋3) min (𝑋1) mL/g (𝑋2) W (𝑋3) 1 1 −111420700 2 −1 −11 6 20700 3 0 0 0 10 30 500 4 −11 1 6 40700 5 0 0 1.68 10 30 836.36 6 1.68 0 0 16.73 30 500 7 1111440700 8 0 0 0 10 30 500 9 −1 −1 −16 20300 10 11−11440300 11 0 0 0 10 30 500 12 00−1.68 10 30 163.64 13 −11−16 40300 14 1 −1 −11420300 15 0 0 0 10 30 500 16 0 0 0 10 30 500 17 0 −1.68 0 10 13.18 500 18 0 0 0 10 30 500 19 0 1.68 0 10 46.82 500 20 −1.68 0 0 3.27 30 500

3.1.2. Effect of the Alkyl Chain Length. The alkyl chain length ILs-UMAE process. Therefore, [C4mim]Br was selected for of the imidazolium ring of ILs has a significant influence the further experiments. on their physical and chemical properties [37, 38]; thus, the alkyl chain length of ILs will consequently influence the extraction yields of the analytes. In order to investigate 3.2. Optimization of IL-UMAE Conditions the effect of 1-alkyl-3-methylimidazolium-type ILs, ILs with different alkyl chain lengths of cation on the extraction yields 3.2.1. Single Factor Experiments. Single factor experiment of RU and QU were studied in UMAE process. The four was performed by one factor varied with different levels, kinds of ILs were investigated in UMAE, that is, [C2mim]Br, while other factors were fixed. There are many factors affect- 1-ethyl-3-methylimidazolium bromide; [C4mim]Br, 1- ing the extraction yields of target compounds, which involved butyl-3-methylimidazolium bromide; [C6mim]Br, 1-hexyl- the concentration of [C4mim]Br solution, extraction time, 3-methylimidazolium bromide; [C8mim]Br, 1-octyl-3- liquid-solid ratio, extraction temperature, and microwave methylimidazolium bromide. Water and different types of power. All results of single factor experiments were shown in 1 M ILs solution were used for the extraction solvents for Figure 4. assessing the extraction yield of RU and QU in UMAE In Figure 4, it can be apparently observed that when the procedure. The results are shown in Figure 3(b),ILswith concentration of [C4mim]Br solution was 2 M, the extraction different alkyl chain lengths of cation significantly influenced yield of RU and QU was the highest. While the concentration the extraction yields of target compounds, and obviously, of [C4mim]Br solution was less than 2 M, the extraction while [C4mim]Br was used as extraction solvent, the higher yields of target compounds increased rapidly, which might extraction yields of RU and QU were obtained than the be the reason, with the increasing of [C4mim]Br, both the other three ILs. Compared with the other ILs solutions, solubility and the extracting capacity of the solvent were water as the solvent, the extraction yields of RU and QU enhanced. At the same time, the capabilities of microwave were the most poor. The possible reason was related to the absorption and microwave conversion were both increased. solubility of flavonoids compounds in extraction solvent. However, when the concentration of [C4mim]Br solution The addition of ILs solution improved the extraction yields was more than 2 M, the extraction yields declined. The major of target compounds; it may be the reason that the strong cause was that the greater the [C4mim]Br concentration can dissolvable ability of ILs on target compounds [39–41]. severely influence the viscosity and the diffusion capacity When, the alkyl chain length was more than 4 carbons, of solutions. So 2 M of [C4mim]Br was selected for further the extraction yield of RU and QU distinctly decreased in experiments. The Scientific World Journal 5

3.5 6.0 3.0 2.72 5.5 2.5 2.26 2.10

2.0 1.74 5.0 1.5 1.24 4.5 1.0 0.5 4.0 Extraction yields (mg/g) yields (mg/g) Extraction 0.125 0.105 0.0 0.117 0.109 0.082 3 4 3.5 Extraction yield of RU (mg/g) (mg/g) RU yield of Extraction NO ] HSO ] mim]Cl mim]Br 3.0 4 4 mim]BF4

4 12345 [C [C [C C3mim [ C4mim

[ (a) Extraction solvents

Rutin 0.27 Quercetin

(a) 0.22 4.5 4.09 4.0

3.5 3.20

3.0 2.72 0.17

2.5 2.28

2.0 (mg/g) QU yield of Extraction 1.5 1.12 1.0 0.12

Extraction yields (mg/g) Extraction 0.5 0.125 0.146 0.133

0.015 0.109 12345 0.0 Factor Level

Water A (M) 0.5 1 1.5 2 2.5 mim]Br mim]Br mim]Br mim]Br 8 4 6 2 [C [C [C [C B (°C) 30 40 50 60 70 Extraction solvents C (min) 6 8 10 12 14

Rutin D (mL/g) 20 : 1 25 : 130: 135: 140: 1 Quercetin E (W) 300 400 500 600 700 (b)

Figure 3: Effect of ionic liquids anions (a) and cations (b) on Concentration Temperature the extraction yields of RU and QU. Error bars indicate standard Time Liquid-solid ratio deviation (𝑛=3). Power

(b) The extraction time is another crucial factor that should Figure 4: The effect of the concentrations of [C4mim]Br (A), be studied to increase the extraction yields of RU and QU. As extraction temperature (B), extraction time (C), liquid-solid ratio shown in Figure 4, when the extraction time increased from (D), and microwave power (E) on the extraction yields of RU and 6 to 10 min, the extraction yields of the two target compounds QU. increased dramatically. When the time variable was changed from 10 min to 14 min, the extraction yields of the two target compounds reduced slightly. Therefore, 10 min was selected for further experiments. liquid-solid ratio from 30 : 1 to 40 : 1 mL/g, the extraction As for liquid-solid ratio, Figure 4 shows that the extrac- yields of RU and QU no longer increased. Hence, 30 : 1 mL/g tion yield of two target compounds increased significantly of liquid-solid ratio was selected for further experiments. when the liquid-solid ratio increased from 20 : 1 to 30 : 1 mL/g. As shown in Figure 4, it can be seen that the extrac- In certain range, raising the liquid-solid ratio can make tion yields of RU and QU increased with the increase ∘ sample completely immerse into solvent and increase the of the extracting temperature from 30–60 C, which might mass transfer, and it results in the higher extraction yields be because the increasing of the extracting temperature of target compounds. Furthermore, with the increase of contributes to reducing the viscosity of ILs and enhancing 6 The Scientific World Journal thespreadabilityandsolubilityofILs,whichwasbeneficial The extraction yield of RU (mg/g) to dissolve and extract target compounds. However, when 5.40 ∘ R2 = 0.9790 the temperature was higher than 60 C, the extraction yield of RU and QU decreased slightly. The main reason may be 4.90 the decomposition of some RU and QU at high temperature. ∘ Thus 60 C of temperature was selected for further experi- 4.40 ments. It can be shown from Figure 4 that with the increase of 3.90 microwave power from 300 to 600 W,the extraction yields of Predicted RU and QU increased rapidly. When the microwave power 3.40 was higher than 600 W, the extraction yields of RU and QU 2.90 began to gradually decrease. Therefore, 600 W of power was 2.90 3.40 3.90 4.40 4.90 5.40 selected for further experiments. Actual

3.2.2. Statistical Analysis. The extraction time, liquid-solid (a) The extraction yield of QU (mg/g) ratio, and microwave power were selected as three indepen- 0.28 dent factors, and the dependent variable (response, yields R2 = 0.9787 of RU and QU of each run of the experimental design) 0.26 were investigated by RSM. In order to minimize the effects 0.24 of the uncontrolled factors, the experimental sequence was randomized (see Table 1). The experimental results obtained 0.22 at each point are shown in Table 2.Thesevaluesofthe 0.20 Predicted significance of each experimental variable can be justified, 0.18 which were made of the model fitted, the software gener- 2 ated model coefficients, 𝑅 -values, Fit-values (𝐹-values), and 0.16 significant probabilities. The response and variables were 0.14 mutually fitted by multiple regressions. Regression analysis 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 is the general approach to fit the empirical model with Actual the collected response variable data [42]. The second-order (b) polynomial model was generated to describe the empirical relationship between the yields of target compounds and Figure 5: Actual versus predicted values obtained from estimated operational conditions (𝑋1: extraction time, 𝑋2:liquid-solid model. ratio, and 𝑋3: microwave power) in terms of coded values:

𝑌1 = 5.32 + 0.58𝑋1 + 0.16𝑋2 + 0.34𝑋3 − 0.058𝑋1𝑋2 all responses are tabulated in Table 3 for the extraction 2 2 2 + 0.029𝑋1𝑋3 − 0.1𝑋2𝑋3 − 0.48𝑋1 − 0.24𝑋2 − 0.27𝑋3, yield of RU and Table 4 for the extraction yield of QU, −3 respectively. 𝑌2 = 0.26 + 0.029𝑋1 + 0.016𝑋2 + 8.322 × 10 𝑋3 In Table 3,themodel𝐹-value of 51.71 with a very low 𝑃 𝑃 < 0.0001 −3 −3 value ( ) displayed that the generated model was − 6.436 × 10 𝑋1𝑋2 + 2.864 × 10 𝑋1𝑋3 statistically significant and indicated that the extraction yields of RU by IL-UMAE could be well described with this model. − 2.561𝑋 𝑋 − 0.026𝑋2 − 0.024𝑋2 − 0.010𝑋2. 2 3 1 2 3 It was also observed that the linear term of extraction time (3) (𝑋1) and microwave power (𝑋3)havelargesignificanteffect 𝐹 The predicted values for extraction yield of RU and QU on the yield of RU because of the high -value of 191.7 and 𝑋2 obtained using above model were seen in Table 2.Theplot 65.53, respectively. The quadratic term of extraction time ( 1) 𝑋2 𝐹 of actual values versus predicted values for the estimated and microwave power ( 3) are also significant with -value model is shown Figure 5. The relationship between the of 143.34 and 43.04, respectively. According to the software actual and predicted values showed that the plotted points analysis, the lack of fit 𝐹-value was 3.33, and the 𝑃 value 2 cluster around the diagonal line. Predicted 𝑅 are 0.9790 for (0.1065) was greater than 0.05 indicating that the lack of fit extraction yield of RU and 0.9787 for extraction yield of QU, was not significant relative to the pure error43 [ ]. respectively; residual standard deviation (RSD) is 3.34% for From Table 4,themodel𝐹-value of 51.13 with a very low extraction yield of RU and 3.92% for extraction yield of QU, 𝑃 value (𝑃 < 0.0001) implies that the model is significant. 𝑋 respectively. It was clearly observed that extraction time ( 1) and liquid- 𝑋 Positive sign in model of each term represents syn- solid ratio ( 2)havelargesignificanteffectontheyieldofQU due to the high 𝐹-value of 158.99 and 45.26, respectively. The ergistic effect, while antagonistic effect is represented by 2 negative sign. Analysis of variance (ANOVA) was then quadratic term of extraction time (𝑋1) and microwave power 2 used to assess the goodness of fit. The significant quadratic (𝑋2)havealsosignificantwith𝐹-value of 135.98 and 116.53, models and the corresponding significant model term for respectively. According to the software analysis, the lack of fit The Scientific World Journal 7

6 6

5 5 rutin (mg/g) rutin 4 4 3 3 2 2

1 1 Extraction yield of of yield Extraction 50 (mg/g) rutin of yield Extraction 900 18 Liquid-solid40 ratio (mL/g) 18 16 700 16 14 Microwave power 14 30 12 12 10 500 10 20 8 8 6 300 6 4 Time (min) 4 Time (min) 10 2 100 2 (W)

(a) (b)

6 0.28 0.24 5 0.20 4 0.16 3 0.12 2 0.08 1 0.04

Extraction yield of rutin (mg/g) rutin of yield Extraction 50 900 Extraction yield of quercetin (mg/g) quercetin of yield Extraction 18 50 40 16 700 Liquid-solid ratio (mL/g) 14 Microwave power (W) 40 30 12 500 10 30 8 20 6 300 20 4 10 2 Time (min) 100 10 Liquid-solid ratio (mL/g)

0.28 0.28 0.24 0.24 0.20 0.20 0.16 0.16 0.12 0.12 0.08 0.08 0.04 0.04 900 900

18 700 50

Extraction yield of quercetin (mg/g) quercetin of yield Extraction 700 Microwave power (W) 16 (mg/g) quercetin of yield Extraction 14 Microwave power (W) 40 /g) 12 500 500 10 30 8 300 300 6 20 lid ratio (mL 4 100 2 Time (min) 100 10 Liquid-so

(e) (f)

Figure 6: Response surface plots for extracting RU and QU by UMAE: (a) liquid-solid ratio and extraction time, (b) irradiation power and time, (c) irradiation power and liquid-solid ratio on yield of RU, (d) liquid-solid ratio and extraction time, (e) irradiation power and time, and (f) irradiation power and liquid-solid ratio on yield of QU. 8 The Scientific World Journal

Table 2: Actual and predicted results for the extraction yields of RU and QU.

Actual values Predicted values Run The extraction yield of RU The extraction yield of QU The extraction yield of RU The extraction yield of QU (mg/g) (mg/g) (mg/g) (mg/g) 1 5.22 0.23 5.27 0.23 2 3.90 0.16 3.93 0.16 3 5.31 0.26 5.32 0.26 4 4.10 0.19 4.17 0.20 5 5.31 0.25 5.13 0.24 6 4.91 0.24 4.93 0.24 7 5.18 0.24 5.28 0.25 8 5.41 0.26 5.32 0.26 9 3.26 0.14 3.11 0.14 10 4.83 0.22 4.74 0.23 11 5.45 0.27 5.32 0.26 12 3.74 0.21 3.99 0.22 13 3.86 0.19 3.75 0.19 14 4.46 0.21 4.33 0.21 15 5.22 0.25 5.32 0.26 16 5.18 0.26 5.32 0.26 17 4.27 0.16 4.36 0.17 18 5.36 0.25 5.32 0.26 19 4.92 0.23 4.91 0.22 20 2.91 0.14 2.97 0.14

Table 3: ANOVA of the fitted quadratic polynomial model for the extraction yield of RU.

Source Sumofsquares Degreesoffreedom(df) Meansquares 𝐹 value 𝑃 value Remarks Model 11.20 9 1.24 51.71 <0.0001 Significant

𝑋1 4.61 1 4.61 191.70 <0.0001 Significant

𝑋2 0.36 1 0.36 15.05 0.0031

𝑋3 1.58 1 1.58 65.53 <0.0001 Significant

𝑋1𝑋2 0.027 1 0.027 1.13 0.3129

𝑋1𝑋3 0.0068 1 0.0068 0.28 0.6071

𝑋2𝑋3 0.081 1 0.081 3.38 0.0959 2 𝑋1 3.38 1 3.38 140.34 <0.0001 Significant 2 𝑋2 0.85 1 0.85 35.40 0.0001 2 𝑋3 1.04 1 1.04 43.04 <0.0001 Significant Residual 0.24 10 0.024 Lack of fit 0.19 5 0.037 3.33 0.1065 Not significant Pure error 0.056 5 0.011 Cor. total 11.44 19

𝐹-value was 2.48, and the 𝑃 value (0.1711) was greater than microwave power and an extraction time at a constant ratio 0.05 indicating that the lack of fit was not significant relative of plant material to solvent, the extraction yield was maximal. to the pure error. The optimum extraction conditions (independent variables) proposed by DE software were as follows: extraction time of 12.27 min, liquid-solid ratio of 31.78 mL/g, and microwave 3.3. Optimization Analysis. The interaction between the vari- power of 533.87 W. The predicted extraction yield under the ables was shown in Figure 6 and it can be seen from Figure 6 above conditions was computed as 5.58 mg RU and 0.27 mg that the 3D response surfaces show that, at high levels of QU per 1 g of dried plant material. Considering the precision The Scientific World Journal 9

Table 4: ANOVA of the fitted quadratic polynomial model for the extraction yield of QU.

Source Sumofsquares Degreesoffreedom(df) Meansquares 𝐹 value 𝑃 value Remarks Model 0.034 9 0.037 51.13 <0.0001 Significant

𝑋1 0.012 1 0.012 158.99 <0.0001 Significant −3 −3 𝑋2 3.312 × 10 1 3.312 × 10 45.26 <0.0001 Significant −4 −4 𝑋3 9.458 × 10 1 9.458 × 10 12.92 0.0049 −4 −4 𝑋1𝑋2 3.314 × 10 1 3.314 × 10 4.53 0.0592 −5 −5 𝑋1𝑋3 6.561 × 10 1 6.561 × 10 0.90 0.3660 −5 −5 𝑋2𝑋3 5.246 × 10 1 5.246 × 10 0.72 0.4170 2 −3 −3 𝑋1 9.950 × 10 1 9.950 × 10 135.98 <0.0001 Significant 2 −3 −3 𝑋2 8.527 × 10 1 8.527 × 10 116.53 <0.0001 Significant 2 −3 −3 𝑋3 1.588 × 10 1 1.588 × 10 21.70 0.0009 −4 −5 Residual 7.317 × 10 10 7.317 × 10 −4 −4 Lack of fit 5.213 × 10 5 1.043 × 10 2.48 0.1711 Not significant −4 −5 Pure error 2.105 × 10 5 4.209 × 10 Cor. total 0.034 19

6.0 the extraction yields of two target compounds. However, 5.49 5.45 methanol was volatile, flammable, and harmful to human 4.79 5.0 4.68 and environment. Therefore, [C4mim]Br was chosen as 3.74

4.0 3.62 extraction solvent in this study.

2.82 ItcanbeseenfromthattheextractionyieldofRUandQU 3.0 2.73 obtained by ILs-UMAE was, respectively, 5.49 mg/g for RU 2.0 and 0.27 mg/g for QU, which increased, respectively, 2.01-fold 1.0 and2.34-foldcomparedtoconventionalmethanol-HRE.In 0.269 0.263 0.226 0.212 Extraction yield (mg/g) Extraction 0.164 0.154 0.109 0.0 0.102 conclusion, compared with other three extraction methods, ILs-UMAE had the highest extraction yield of RU and QU from the leaves of velvetleaf with the shortest extracting time. For UMAE, cavitation and microwave irradiation resulted in high effective temperatures and pressures at the interphase mim]Br-HRE mim] Br-UAE mim]Br-MRE 4 4 4 Methanol-UAE Methanol-RHE Methanol-MAE between solvent and solid matrix; moreover, microwaves [C [C [C Comparison of different extraction methods raise the temperature suddenly and disrupt the structure of Rutin vegetalcell[34]. Quercetin 3.5. Quantitative Analysis by HPLC. Under the chromato- Figure 7: Effect of different extraction methods on yield of RUand graphic conditions of 2.5, the peaks of RU and QU were QU. Error bars indicate standard deviation (𝑛=3). observed with an acceptable resolution from the peaks of neighboring compounds. The HPLC chromatograms of theanalyzedextractsareshowninFigure 8.Furthermore, of extraction device, the optimal condition for extracting RU recoveries were evaluated by standard-addition method, and and QU was selected as extraction time 12 min, liquid-solid the extracts were spiked with known quantities of standards. ratio 32 mL/g, and microwave power of 534 W. Under the Results showed that the recoveries were in the range of 97.62– above optimized condition, sample was repeatedly extracted 102.36% for the RU and 97.33–102.21% for QU with RSDs for 6 times, the extraction yields were 5.67 ± 0.12 mg/g for lower than 3.2% under the optimized UMAE conditions. RU and 0.29 ± 0.01 mg/g for QU (the extraction yield values The reproducibility and recovery proved that the present corresponded to the 100% yield values); sample was extracted method was credible. once, the extraction yields were 5.49 ± 0.16 mg/g for RU and 0.27 ± 0.01 mg/g for QU, and the yields of RU and QU were, respectively, 96.8% and 94.2%. 4. Conclusion In the present study, the ILs-UMAE was used to extract 3.4. Comparison of Different Extraction Procedures. Acom- the two objective compounds (RU and QU) from leaves of parison between UMAE and the HRE, UAE, and MAE was velvetleaf. According to the single factors experiments and performed and the results were seen in Figure 7. CCD test, we concluded the optimized extraction solvent ∘ Figure 7 showsthat,foranymethod,itisworthnoting 2.00 M [C4mim]Br, extraction temperature 60 C, extraction that [C4mim]Br and methanol were almost the same as time 12 min, liquid-solid ratio 32 mL/g, microwave power 10 The Scientific World Journal

0.28 0.28 Acknowledgments This work was financially supported by the Fundamen- 0.22 0.22 tal Research Fund for Central Universities (DL12CA05, 0.16 1 2 0.16 DL12EA01-04, and DL13CA06), the National Natural Science

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Research Article Phytochemical Profiles and Antioxidant and Antimicrobial Activities of the Leaves of Zanthoxylum bungeanum

Yujuan Zhang, Ziwen Luo, Dongmei Wang, Fengyuan He, and Dengwu Li

College of Forestry, Northwest A&F University, Yangling 712100, China

Correspondence should be addressed to Dongmei Wang; [email protected]

Received 5 May 2014; Revised 3 July 2014; Accepted 8 July 2014; Published 24 July 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 Yujuan Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The ethanol crude extracts (ECE) and their subfractions from Zanthoxylum bungeanum leaves were prepared and their phytochemical profiles and antioxidant and antimicrobial activities were investigated. Moreover, the effective HPLC procedure for simultaneous quantification of twelve compounds in Z. bungeanum leaves was established. The correlation between the phytochemicals and antioxidant activity was also discussed. The ethyl acetate fraction (EAF) had the highest total phenolic (97.29mmol GAE/100g) and flavonoid content (67.93 mmol QE/100 g), while the greatest total alkaloid content (4.39 mmol GAE/100 g) was observed inthe chloroform fraction (CF). Twelve compounds were quantified by RP-HPLC assay. EAF exhibited the highest content of quercitrin, kaempferol-3-rhamnoside, quercetin, sesamin, and nitidine chloride (125.21, 54.95, 24.36, 26.24, and 0.20 mg/g); acetone fraction (AF) contained the highest content of chlorogenic acid, rutin, hyperoside, and trifolin (5.87, 29.94, 98.33, and 31.24 mg/g), while kaempferol-3-rhamnoside, xanthyletin, and sesamin were rich in CF. EAF and AF exhibited significant DPPH, ABTS radical scavenging abilities and reducing power (FRAP), whereas CF exhibited significant antifungal activity. Moreover, EAF also showed stronger antibacterial activity. In conclusion, Z. bungeanum leaves have health benefits when consumed and could be served as an accessible source for production of functional food ingredients and medicinal exploration.

1. Introduction eczema, and snakebites [3–7]. Recent experimental studies have shown that the pericarp of Z. bungeanum possesses Zanthoxylum bungeanum, known as the Da Hongpao Hua- cardiovascular activity [8]; it also can be used as an ingredient jiao, which belongs to the Zanthoxylum genus of the family in cosmetic products [9]; methanol extracts of Z. bungeanum Rutaceae, is widely distributed in Hebei, Shanxi, Sichuan, have anti-inflammatory activity [10]; the essential oil of seed Gansu, and Shandong provinces of China and some Southern and fruit exhibits marked antioxidant activity as well as Asian countries [1]. Just like other species of this genus, antimicrobial activity [11–13]. Z. bungeanum has a distinctive tingling taste. Due to its The leaves of Z. bungeanum are edible; they taste acrid fresh aroma and taste, the dried fruits are used ground andinnocuous.Insomeruralareas,localpeopleeatthenew or whole as a spice in local cuisines, which can stimulate leaves as vegetables in spring seasons [14]. Furthermore, it salivaproductionandincreaseappetite[2]. Consisting of salt is also commonly used as condiments in Chinese cuisine and Sichuan pepper (Z. bungeanum), hua jiao yen is often and in the preparation of refreshments to add flavor11 [ ]. In used as a condiment in barbecue foods, such as chicken spite of its long history of consumption, only a few people tikka or roast duck. Apart from its common application as pay attention to the chemical work on this material. Fan and a condiment to make foods more flavoring, each part of Z. coworkers did a study on the ultrasonic-assisted extraction bungeanum has numerous medicinal virtues. In traditional of total flavonoids from Z. bungeanum leaves [15]. Yang and Chinese medicine, the pericarp can be used for gastralgia coworkers identified 13 polyphenolics from the leaves of Z. and dyspepsia; the seed is reported to be antiphlogistic and bungeanum grown in Hebei, China, by HPLC/MS, among diuretic; the leaves are considered carminative, stimulant, and which chlorogenic acid, hyperoside, and quercitrin were the sudorific; the root can cure epigastric pains and treat bruises, major constituents [1]. But, there are still some unclear points 2 The Scientific World Journal for consumers on the phytochemical profiles and physiologi- reduced pressure. 15.56 g of ECE was stored for further anal- cal effects of this edible material. In order to fully investigate, ysis. The remaining ECE (1824.40 g) was further fractioned utilize, and develop this material, we designed an experiment: by column chromatography on silica gel (silica gel 200– (1) to evaluate the contents of total flavonoid, phenol, and 300 mesh, 120∗10 cm i.d., flow rate 10 mL/min), successively alkaloid in the different polarity fractions of Z. bungeanum eluting with petroleum ether, chloroform, ethyl acetate, ace- leaves; (2) to measure the antioxidant and antimicrobial tone, and methanol. The eluents of the five different polarity activities of the different polarity fractions; (3) to quantify the solvents were collected separately and evaporated to dryness ∘ content of twelve natural compounds (chlorogenic acid, epi- by rotary evaporation at 45 C under reduced pressure. Thus, catechin, rutin, hyperoside, trifolin, quercitrin, kaempferol- the different polarity fractions (PEF, 105.98 g; CF, 112.76 g; 3-rhamnoside, quercetin, nitidine chloride, chelerythrine, EAF, 40.70g; AF, 124.93g; and MF, 624.35g) were obtained ∘ xanthyletin, and sesamin) in the different polarity fractions and carefully stored at −20 Candprotectedfromlightuntil by RP-HPLC analysis; and (4) to compare the similarities and further analysis [16, 17]. differences of the phytochemical composition in the different polarity fractions. Based on these results, the most bioactive 2.3. Determination of Total Flavonoid Content (SBC Method). fraction could be selected as a potential source of natural The total flavonoid content (TFC) was determined based antioxidants and antiseptics. In addition, phytochemicals on a SBC assay using sodium borohydride/chloranil as might be responsible for their profound bioproperties which described previously [18–20]. This assay allows the detec- will be screened out. The results also could explain its tion of numerous flavonoid varieties, including flavones, frequent addition to the Chinese diet for promoting human flavonols, flavonones, flavononols, isoflavonoids, and antho- health and for disease prevention. cyanins [20]. A calibration curve was constructed to create a standard using different concentrations of quercetin (0.1– 2. Materials and Methods 10.0 mM). TFC of extracts and different polarity fractions from Z. bungeanum leaves were expressed as mmol quercetin 2.1. Plant Material and Chemicals. Z. bungeanum leaves were equivalentper100gandallsampleswereevaluatedin collected from Taibai Mountains of Shaanxi province, China, triplicate. in September, 2012, and authenticated by the Herbarium of the Northwest A&F University, Yangling, China. 2.4. Determination of Total Phenolic Content. The total The following were obtained: Folin-Ciocalteu reagent phenolic content (TPC) was determined using the Folin- (Shanghai Solarbio Bioscience & Technology Co., Ltd., Ciocalteu colorimetric method as described previously [21, China); 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino- 22]. TPC was calculated by gallic acid equivalent from the bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium calibration curve from the gallic acid standard solutions (20– salt (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), and 6- 300 𝜇g/mL). TPC of extracts and different polarity fractions hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid from Z. bungeanum leaves were expressed as mmol gallic acid (Trolox) (Sigma-Aldrich Co., St. Louis, USA); vanillin, equivalentper100g,andallthesamplesweremeasuredin bromocresol green, tetrahydrofuran (THF), sodium triplicate. borohydride, and trifluoroacetic acid (Chengdu Kelong Chemical Co., Ltd., China); chloranil (Aladdin Industrial 2.5. Determination of Total Alkaloid Content. The total alka- Corporation, Shanghai, China); gallic acid, chlorogenic loid content (TAC) was determined using the acid dye acid, epicatechin, rutin, hyperoside, trifolin, quercitrin, colorimetric method with the following modifications23 [ ]. kaempferol-3-rhamnoside, quercetin, nitidine chloride, Chelerythrine (0.2–1.0 mg/mL) was used as a reference for chelerythrine, xanthyletin, and sesamin (Shanghai the calibration curve. TAC of extracts and different polarity Winherb Medical Science Co., Ltd.); and amphotericin fractions from Z. bungeanum leaves were expressed as mmol and benzylpenicillin (Shanghai Sunny Biotechnology Co. of chelerythrine equivalent per 100 g, and all of the samples Ltd., China). All solvents used were of AR-grade. Deionized were analyzed in triplicate. water (18 MΩ cm) was used to prepare aqueous solutions. Twenty fungi (Botrytis cinerea, Piricularia oryzae, Physa- 2.6. Assessment of the Twelve Compounds by HPLC. The lospora piricola, Glomerella cingulata, and Venturia pyrina, content of twelve compounds (chlorogenic acid, epicat- etc.) and two Gram-positive (Staphylococcus aureus and echin, rutin, hyperoside, trifolin, quercitrin, kaempferol- Bacillus subtilis) and one Gram-negative (Escherichia coli) 3-rhamnoside, quercetin, nitidine chloride, chelerythrine, bacteria were provided by the College of Resources and xanthyletin, and sesamin) was assayed using an Agilent Environment, Northwest A&F University, China. Technologies 1260 series liquid chromatograph (RP-HPLC) coupled with a variable wavelength detector. The quantifi- 2.2. Preparation of the Ethanol Crude Extracts and Fractions. cation was carried out on a SB-C18 reversed phase column The air-dried and powdered leaves of Z. bungeanum (9.40 Kg) (5 𝜇m, 4.6∗250 mm) at ambient temperature [24, 25]. The were extracted using 95% ethanol at room temperature for mobile phase consisted of water with 0.5% trifluoroacetic 24 h, with solid to liquid ratio of 1 : 5, repeating 6 times. acid (solvent A) and acetonitrile with 0.5% trifluoroacetic The ethanol crude extracts (ECE, 1839.96 g) were filtered and acid (solvent B). The flow rate was 0.8 mL/min. The gradient ∘ evaporated to dryness by rotary evaporation at 45 C under program was set as follows: from 0 to 30 min, eluent B was The Scientific World Journal 3

increased from 15% to 35%; from 30 to 35 min, eluent B was sample and 2 mL DPPH⋅), and 𝐴𝑗 istheabsorbanceofthe increased from 35% to 65%; and from 35 to 55 min, eluent blank (2 mL sample and 2 mL ethanol). B was increased from 65% to 100% and then maintained at 𝜇 100% for 10–20 min. The injection volume was 20 Land 2.9. ABTS Radical Cation Decolorization Assay. Antioxidant the detection wavelength was 254 nm. Samples were filtered activity was determined according to the decolorizing free 𝜇 + through a 0.22 m membrane filter prior to injection. The radical ABTS⋅ method [29] as described previously [30– major constituents in the ECE and its five different polarity 32]. For each analysis, 100 𝜇L of sample (1 mg/mL) and fractions were identified by comparing their retention times the positive controls (rutin and quercetin, 0.05 mg/mL) was + and the spectral characteristics of their peaks with those of added to 3.9 mL of the ABTS⋅ solution, and the decrease in the standards. The analyses were all performed in triplicate. absorbance at 734 nm was recorded within 6 min. The results were expressed as micromoles of trolox equivalent per g. All 2.7. Validation of the HPLC Method. The linear calibration determinations were carried out in triplicate. curves contained six different concentrations of each standard compound by a series of appropriate dilutions with 2.10. Ferric Reducing Antioxidant Power (FRAP) Assay. The mobile phase. All calibration curves were constructed by FRAP assay [33] was performed with some modifications plotting the peak areas of the standard substances versus [31]. For each analysis, 400 𝜇L of the sample (1 mg/mL) the corresponding concentrations of the injected standard and the positive controls (rutin and quercetin, 0.05 mg/mL) solutions. was added to 3 mL of the FRAP solution. The increase in The HPLC procedure was also validated for its precision, absorbance at 593 nm was recorded in 15 s intervals over the ∘ reproducibility, and recovery test [26]. To determine the pre- course of 30 min at 37 C. The FRAP results were expressed cision of the procedure, the standard compounds solutions as micromoles of trolox equivalent per g. All determinations were analyzed in triplicate for three times within one day, were carried out in triplicate. whileforinterdayvariability,thesampleswereexaminedin triplicate for three consecutive days. To determine the repro- 2.11. Antifungal Activity. Antifungal assays [34] were per- ducibility, six working solutions were prepared using the formed with some modifications as described by Ai et al.[35], ethanol crude extractions (ECE, 5 mg/mL). The recovery test Wang et al. [17], Hsu et al. [36], and Tian et al. [37]. Each wasusedtoevaluatetheaccuracyoftheproposedmethod. extract and fraction was dissolved in different proportions Accuracy was determined by adding different concentrations of acetone and water, that is, 100% acetone for PEF, CF, of the mixed standard solutions into the known amounts EAF, and AF and 50% acetone for ECE and MF. The treated ∘ of sample solutions of ECE. Then the compounds in the dishes were incubated in the dark at 27.5–28.5 Cfor72h resultant samples were analyzed with the proposed method. at moderate humidity. The relative growth inhibition (%) of The recovery was calculated as follows: thetestsamplecomparedwiththecontrolwascalculatedas Recovery (%) follows: (𝐶−𝑇) total detected amount − original amount Inhibitory activity (%) =[ ] × 100%, (3) =( ) ∗ 100. (𝐶−4mm) added amount (1) where 𝐶 is the colony diameter of the mycelium on the control plate (mm) and 𝑇 isthecolonydiameterofthemyceliumon The RSD values were taken as measurements for preci- the test petri plate (mm). sion, reproducibility, and recovery tests. B. cinerea, P. or y z ae , P. pir i col a , G. cingulata,andV. py rina were chosen for growth kinetics assays. The solutions of ECE, 2.8. DPPH Radical Scavenging Activity. DPPH radical scav- PEF,CF,EAF,AF,andMFwereseriallydilutedbythetwofold enging activity was evaluated using the method described by serial dilution method and added to PDA with concentrations Yen and Chen [27]andSultanaetal.[28]withsomemod- ranging from 6.25 to 100 mg/mL. Amphotericin was used ifications17 [ ]. A 2 mL volume of the sample solutions (20– as standard. And the concentration of the sample required 1000 𝜇g/mL)orthepositivecontrolsrutinandquercetin(1– for 50% inhibitory activity (EC50) was calculated using 200 𝜇g/mL) was added to 2 mL of DPPH solution (100 𝜇M); linear regression analysis. All experiments were conducted in and the absorbance was measured with a spectrophotometer triplicate. (Shimadzu UV-1800) at 517 nm after standing in the dark for 30 min. All the tests and the controls were repeated in 2.12. Antibacterial Activity. The paper disc diffusion method, triplicate. The DPPH free radical scavenging activity was also known as the agar diffusion method, was used to calculated using the following equation: detect the antibacterial activity of the extracts and fractions of the leaves of Z. bungeanum [38, 39]. The beef extract 1−(𝐴 −𝐴 ) 𝑖 𝑗 peptone medium was inoculated with 3 𝜇L aliquots of cul- Scavenging (%) =[ ] × 100%, (2) 5 𝐴𝑜 ture containing approximately 10 cfu/mL of each organism. Sterilized filter paper discs (5 mm) were soaked in 5mL where 𝐴𝑜 is the absorbance of ethanol (2 mL) and DPPH⋅ of various concentrations (6.25 to 100 mg/mL) of samples. (2 mL), 𝐴𝑖 istheabsorbanceofthetestedsample(2mL Benzylpenicillin was used as standard (0.01 to 10 mg/mL). 4 The Scientific World Journal

The paper discs soaked in the solvent without extracts quantities of standard compounds. The average percentages or fractions (80% acetone) served as black control. The of recovery of the twelve standard compounds ranged from MIC values were determined as the lowest concentration of 98.37 to 103.76%. In addition, the RSDs varied from 0.35 extracts inhibiting visible growth of each organism on the to 1.66% (𝑛=6). All the results demonstrated that the agar plate. The soaked discs were placed in the plates and conditions of the analysis were repeatable and accurate. ∘ incubated for 24 h at 28 C. Following the incubation period, The content of twelve compounds in the extracts the inhibition zones formed in the medium were measured in and its five fractions from Z. bungeanum leaves were millimeters (mm). All the tests were performed in triplicate determined by matching their retention times against and the MIC values were calculated. those of the standards (Table 3). Good correlation was observed between the peak area and the content. We 2.13. Statistical Analysis. All results were expressed as the established a standard HPLC method to determine 12 mean ± standard deviation (SD). The significant difference phytochemicals from the leaves of Z. bungeanum simul- was calculated by SPSS one-way ANOVA followed by Dun- taneously. Epicatechin (27.45mg/g), rutin (16.86 mg/g), can’s test; values <0.05 were considered to be significant hyperoside (19.25 mg/g), quercitrin (16.73 mg/g), chlorogenic (SPSS Inc., Chicago). The linear correlations among the acid (3.78 mg/g), kaempferol-3-rhamnoside (3.75 mg/g), various parameters were also investigated using the SPSS 18.0 trifolin (4.53 mg/g), and sesamin (5.13 mg/g) were the major software. phenolic components in ECE, other compounds (quercetin, nitidine chloride, chelerythrine, and xanthyletin) had a lower level of content (less than 1 mg/g). Among the 5 subfractions, 3. Results and Discussion the EAF exhibited the highest content of quercitrin (125.21 mg/g), kaempferol-3-rhamnoside (54.95 mg/g), 3.1. Total Phenolic, Flavonoid, and Alkaloid Content. The quercetin (24.36 mg/g), nitidine chloride (0.20 mg/g), and total phenolic, flavonoid, and alkaloid content of ethanol sesamin (26.24 mg/g). The AF exhibited the highest crude extracts (ECE) and its five different polarity fractions content of chlorogenic acid (5.87 mg/g), rutin (29.94 mg/g), (PEF, CF, EAF, AF, and MF) from Z. bungeanum leaves were hyperoside (98.33 mg/g), and trifolin (31.24 mg/g). The CF screened and compared. According to the results presented exhibited the highest content of xanthyletin (0.09 mg/g), in Table 1, there was a statistically significant difference𝑃< ( 0.05 whiletheMFhadthehighestcontentofepicatechin ) among all the samples investigated. EAF exhibited the (39.32 mg/g) and chelerythrine (0.09 mg/g). It is indicated highest TFC (67.93 mmol/100 g) and TPC (97.29 mmol/100 g) that the major phytochemicals, especially phenolic followed by AF (47.62 mmol/100 g for TFC; 62.87 mmol/100 g compounds, were concentrated in EAF and AF, which may for TPC), which were much higher than ECE and other result from the enrichment effects during chromatographic fractions. Compared with the high flavonoid and phenolic fractionation.Andwehypothesizedthatthesebioactive content, alkaloid yields were the lowest, since this group phytochemicals might be responsible for their profound of compounds is sparsely distributed and more specific of bioproperties. genera and species [40]. Among the extracts and fractions ChromatographyoftheECEanditsfivedifferentpolarity of Z. bungeanum leaves, CF exhibited the greatest TAC fractions revealed that there are significant differences among (4.39 mmol/100 g) followed by PEF (1.71 mmol/100 g). Chen the tested samples (Figure 1(b)). Figure 1(a) was the HPLC et al. reported that 23 alkaloids were isolated from the chromatograms of the 12 standard compounds. ECE con- CHCl3 and MeOH extracts of the root bark of Z. simulans tained all the phytochemicals detected and exhibited the rich- [41]. Ren and Xie reported 6 alkaloids from the root of Z. est peaks. But further fractionation of ECE by column chro- bungeanum [42]. We can hypothesize that CF from the leaves matography on silica gel produced five subfractions with even of Z. bungeanum can be further fractioned to gain bioactive higher content of all the detected phytochemicals. Among alkaloids. the five subfractions, peaks 1–9 (chlorogenic acid, epicatechin, rutin, hyperoside, trifolin, quercitrin, kaempferol-3- 3.2. HPLC Analysis of the Extracts and Fractions. As seen in rhamnoside, quercetin, and nitidine chloride) were common Table 2, the linear regression results indicated good linear peaks in EAF and AF. Peak 7 (kaempferol-3-rhamnoside), correlation by the correlation coefficients between 0.9991 peak 11 (xanthyletin), and peaks 12–15 were common peaks and 0.9999 for all of the standard compounds within the detectedinPEFandCF.Thepeakareaofpeaks11–14was appropriate concentration ranges. The precision of the ana- the greatest in CF. Finally, peaks 1–6 (chlorogenic acid, lytical method was analyzed in triplicate for three times epicatechin, rutin, hyperoside, trifolin, and quercitrin) and within one day, while for interday variability, the samples peak 10 (chelerythrine) were detected in MF at low levels, were examined in triplicate for three consecutive days, and except for peak 2. the RSDs of the peak areas were estimated to be 0.71–1.51% The present results exhibited significant differences with (𝑛=6). The repeatability of the method was determined the reported study on the phytochemical composition of the by injecting the ECE for six times, while the peak area Z. bungeanum leaves of Hebei, China [1]. Three major com- of the twelve detected compounds was recorded, and the pounds (trifolin, kaempferol-3-rhamnoside, and quercetin) RSDs of their peak area varied from 0.16 to 2.94%. To detected in the Z. bungeanum leaves of Taibai were not iden- confirm the accuracy of the method, a recovery experiment tified in that of Hebei. Nevertheless, other major phytochem- was performed by mixing quantified samples with specific icals, such as chlorogenic acid, epicatechin, rutin, hyperoside, The Scientific World Journal 5

Table 1: Content of total flavonoid, phenolic, and alkaloid and antioxidant capacity of ethanol extracts and its five fractions from Z. bungeanum leaves. Content Antioxidant capacity Samples TFC TPC TAC FRAP ABTS DPPH (𝜇g/mL) (mmol QE/100 g) (mmol GAE/100 g) (mmol CE/100 g) IC50 (𝜇mol Trolox/g) (𝜇mol Trolox/g) ECE 60.96 ± 8.87a 58.78 ± 9.37b 0.47 ± 0.28c 40.75 ± 0.21c 317.11 ± 9.71b 1122.91 ± 34.62c PEF 4.01 ± 3.42d 4.43 ± 2.17c 1.71 ± 0.07b 377.95 ± 39.39f 81.56 ± 7.41e 264.20 ± 37.27f CF 2.29 ± 2.03d 7.07 ± 2.36c 4.39 ± 0.05a 169.15 ± 4.60e 170.44 ± 10.45d 563.86 ± 22.66e EAF 67.93 ± 9.29a 97.29 ± 17.53a 0.25 ± 0.02c 13.20 ± 0.85b 615.88 ± 1.86b 2147.83 ± 23.08b AF 47.62 ± 6.34bc 62.87 ± 10.80b 0.06 ± 0.01c 18.55 ± 0.35b 594.15 ± 8.89b 2044.58 ± 19.99b MF 31.63 ± 5.30c 8.35 ± 0.30c 0.10 ± 0c 85.85 ± 2.19d 191.93 ± 2.22d 747.69 ± 38.77d Quercetin — — — 2.60 ± 0.10a 1865.80 ± 33.40a 20113.58 ± 23.20a Rutin — — — 10.42 ± 0.14ab 1722.59 ± 6.42a 20153.46 ± 46.05a TFC expressed as mmol quercetin equivalent per 100 g. TPC expressed as mmol gallic acid equivalent per 100 g. TAC expressed as mmol chelerythrine equivalent per 100 g. DPPHIC50 values were the effective concentrations at which DPPH radicals were scavenged by 50%. FRAP and ABTS results expressed as micromoles of trolox equivalent per g. Values are the mean of three replicates ± SD. Means with different letters within a column were significantly different𝑃 ( < 0.05). and quercitrin, were both detected in the Z. bungeanum leaves scavenging ability of the extracts and fractions from Z. of Taibai and in that of Hebei, though the contents were bungeanum leaves compared to rutin and quercetin has been significantly different. The main reasons for these variations depicted in Table 1; we also found that EAF and AF were maybesomegeographicaldifferencesandgenemutation the most effective fractions with the highest ABTS radical [43, 44]. Further isolation and purification of the fractions scavenging abilities. The ABTS radical scavenging ability of (EAF, AF, and CF) with the richest phytochemicals ought EAF was 2147.83 𝜇mol Trolox/g, which was not significantly to be conducted, and later chemical structures of the new different from that of AF (2044.58 𝜇mol Trolox/g, 𝑃 < 0.05) bioactive compounds need to be analyzed. but was 1.9-, 2.9-, 3.8-, and 8.1-fold higher than that of ECE, MF, CF, and PEF, respectively (𝑃 < 0.05). 3.3. DPPH Radical Scavenging Activity. In the present study, the ethanol crude extracts (ECE) and its five different polarity 3.5. Ferric Reducing Antioxidant Power (FRAP). The FRAP fractions showed DPPH radical scavenging activity in a values of the extracts and fractions from Z. bungeanum dose dependent manner at concentration of 20–1000 𝜇g/mL leaves have been depicted in Table 1.EAFandAFwerealso (Figure 2). The EAF and AF exhibited higher DPPH radical screened as the most effective fractions with the highest scavenging activity than ECE and other fractions. In order reducing values, which were rather consistent with the results to further quantify the DPPH radical scavenging activity, the of the scavenging capacity on DPPH and ABTS radical. It is IC50 values of the extracts and five fractions were determined exhibited that the reducing ability of EAF was 615.88 𝜇mol and shown in Table 1. All the extracts and fractions showed Trolox/g, which was not significantly different from that of significant𝑃 ( < 0.05) differences in their ability to reduce the AF (594.15 𝜇mol Trolox/g, 𝑃 < 0.05) but was 1.9-, 3.2-, 3.6-, DPPH radical; EAF and AF were selected as the most effective and 7.6-fold higher (𝑃 < 0.05) than that of ECE, MF, CF, and fractions with the highest DPPH radical scavenging ability, PEF, respectively. which were not significantly different with the reference 𝜇 compound (rutin, IC50 =10.42 g/mL). As seen in Table 1, 3.6. Correlation between the Total Phenolic and Flavonoid 𝜇 the IC50 value of EAF was 13.20 g/mL, which was not Content and the Antioxidant Assays. Basedonthecorrelation 𝜇 significantly different from that of AF (18.55 g/mL) but was matrix (Table 4), each coefficient was assessed to establish 3.1-, 6.5-, 12.8-, and 28.6-fold lower than that of ECE, MF, CF, the correlations between different assays. As displayed in 𝑃 < 0.05 and PEF, respectively ( ). Because the antioxidant Table 4,ahighcorrelationwasobservedamongthethree activities were inversely correlated with the IC50 values, the methods for antioxidant activity measurement (−0.777 ≤𝑟≤ DPPH radical-scavenging activity, in decreasing order, was 0.999, 𝑃 < 0.01), indicating a great degree of equivalence > > > > EAF AF MF CF PEF. among the measurements. Okonogi et al. demonstrated that the relationship between ABTS radical scavenging activities 𝑟 = −0.797 3.4. ABTS Radical Cation Decolorization Activity. Another and the DPPHIC50 of the samples was nonlinear ( ). effective method to measure radical scavenging activity is However, the logarithmic values of DPPHIC50 against ABTS the ABTS radical cation decolorization assay, which showed radical scavenging activities gave good linearity (𝑟 = −0.968) similar results to those obtained in the DPPH reaction. The [45]. Thus, the ABTS result was in good agreement with scavenging activity of the extracts on free radical ABTS that of the DPPH assay; among the evaluated extracts and generated by potassium persulfate was compared with a fractions, EAF and AF were selected as two fractions with standard amount of trolox. The result was calculated as the highest free radical and hydroxyl radical-scavenging micromoles of trolox equivalent per g. The ABTS radical activities. The FRAP values exhibited a significant linear 6 The Scientific World Journal (%) RSD 1.51 1.45 0.91 0.91 1.02 1.01 1.32 1.75 0.31 0.31 0.88 0.86 0.89 0.88 1.65 1.66 1.02 1.02 1.04 1.06 0.93 0.94 0.35 0.35 ± ± ± ± ± ± ± ± ± ± ± ± rate (%) Average recovery (%) RSD 7.01 0.81 100.81 11.06 0.16 99.90 1.65 2.38 99.87 21.67 0.98 102.13 0.81 2.94 101.52 3.32 1.65 99.38 36.75 1.03 98.84 4.83 0.92 101.35 5.52 1.11 99.75 10.68 1.42 100.60 6.22 1.56 103.76 min) 13.07 1.03 98.37 ± ± ± ± ± ± ± ± ± ∗ ± ± ± Area of peak (mAU (%) RSD 7.06 0.71 1125.80 7.69 1.07 1944.37 27.59 1.08 5210.20 7. 31 1 . 5 1 8 5 . 1 6 35.73 0.74 753.97 2.41 1.11 265.33 9.38 0.76 3573.98 8.03 1.32 215.04 8.62 1.50 150.20 8.35 1.12 93.52 6.35 1.06 100.10 6.47 1.17 130.11 min) ± ± ± ± ± ± ± ± ± ± ± ± ∗ Precision experiment Repeatability Recovery experiment 114.54 110.68 182.53 Area of peak 140.71 160.80 (mAU 377.97 865.77 223.05 1042.93 1000.50 3641.90 2350.82 = 0.9999) = 0.9998) = 0.9994) = 0.9997) = 0.9993) = 0.9993) = 0.9995) = 0.9991) = 0.9998) = 0.9999) = 0.9999) = 0.9999) 2 2 2 2 2 2 2 2 2 2 2 2 Regression equation 𝑦 = 7.747𝑥 − 1.0908 (𝑅 𝑦 = 1.878𝑥 − 1.3776 (𝑅 𝑦 = 41.826𝑥 + 1.308 (𝑅 𝑦 = 49.53𝑥 − 0.2025 (𝑅 𝑦 = 4.1445𝑥 + 107.11 (𝑅 𝑦 = 63.459𝑥 − 171.41 (𝑅 𝑦 = 14.392𝑥 + 15.374 (𝑅 𝑦 = 98.498𝑥 + 67.619 (𝑅 𝑦 = 22.356𝑥 + 55.311 (𝑅 𝑦 = 175.41𝑥 + 0.7376 (𝑅 𝑦 = 166.49𝑥 + 33.656 (𝑅 𝑦 = 41.138𝑥 − 436.10 (𝑅 5 5 5 ∼ ∼ ∼ 50 50 500 500 500 500 1000 100 100 ∼ ∼ ∼ ∼ ∼ ∼ ∼ ∼ ∼ g/mL) 𝜇 Test range ( Table 2: Validation for the quantitative determination of twelve standard compounds using RP-HPLC. ). 𝑛=6 SD ( ± Compound Peak no. 1 Chlorogenic acid 1 6 Quercitrin 10 15 Sesamin 10 28 Epicatechin 10 Quercetin 1 11 Xanthyletin 0.1 7 Kaempferol-3-rhamnoside 1 3Rutin10 5Trifolin1 10 Chelerythrine 0.1 49 Hyperoside Nitidine chloride 50 0.1 Values were expressed as mean The Scientific World Journal 7

Table 3: Content of twelve compounds in extracts and five fractions from Z. bungeanum leaves.

Content (mg/g) Peak no. Compounds ECE PEF CF EAF AF MF 1 Chlorogenic acid 3.78 ± 0.12b ND ND 2.96 ± 0.06b 5.87 ± 0.10a 2.40 ± 0.11d 2 Epicatechin 27.45 ± 0.93c ND ND 33.12 ± 0.61b 27.42 ± 0.17c 39.32 ± 1.30a 3 Rutin 16.86 ± 0.26b ND ND 4.79 ± 0.03d 29.94 ± 0.01a 14.72 ± 0.09c 4 Hyperoside 19.25 ± 0.55c ND ND 24.17 ± 0.90b 98.33 ± 1.14a 11.97 ± 0.10d 5 Trifolin 4.53 ± 0.07c ND ND 21.22 ± 0.12b 31.24 ± 0.78a 1.03 ± 0.01d 6 Quercitrin 16.73 ± 0.97c ND ND 125.21 ± 0.90a 116.63 ± 1.42b 2.49 ± 0.09d 7 Kaempferol-3-rhamnoside 3.75 ± 0.02c 0.48 ± 0.01e 1.23 ± 0.02d 54.95 ± 0.95a 16.71 ± 0.49b ND 8 Quercetin 0.76 ± 0.03b ND ND 24.36 ± 0.71a 0.90 ± 0.05b ND 9 Nitidine chloride 0.18 ± 0.003a ND ND 0.20 ± 0.004a 0.06 ± 0.003b ND 10 Chelerythrine 0.09 ± 0.002a ND ND ND 0.08 ± 0.005a 0.09 ± 0.003a 11 Xanthyletin 0.06 ± 0.01b 0.08 ± 0.01b 0.09 ± 0.01a 0.07 ± 0.005c ND ND 15 Sesamin 5.13 ± 0.11c 1.20 ± 0.14d 8.09 ± 1.07b 26.24 ± 0.87a ND ND Values are the mean of three replicates ± SD. Means with different letters within a row were significantly different𝑃 ( < 0.05). ND: not detectable.

Table 4: Correlation matrix between the results of the total phenolic compounds with good reducing capacity and good electron and flavonoid content and the FRAP, ABTS, and DPPH activities. donors. log DPPH DPPH ABTS FRAP TPC TFC log DPPH 1 3.7. Antifungal Activity. The antifungal activity of ECE, PEF, ∗ CF, EAF, AF, and MF against 20 varieties of plant pathogenic DPPH −0.909 1 ∗∗ fungi was studied using the mycelial growth method. The ABTS −0.968 −0.797 1 ∗∗ ∗∗ inhibition of ECE ranged between 6.00 and 65.22%, while FRAP −0.958 −0.777 0.999 1 ∗ ∗ ∗ those of PEF, CF, EAF, AF, and MF were between 10.00 TPC −0.916 −0.829 0.827 0.808 1 and 70.00% at a concentration of 50 mg/mL. Five plant ∗∗ ∗∗ ∗∗ ∗ TFC −0.922 −0.724 0.924 0.923 0.908 1 pathogenic fungi (B. cinerea, P. or y z ae , P. pir i col a , G. cingu- ∗ ∗∗ Correlation is significant at the 0.05 level; correlation is significant at the lata, and V. py rina) with higher antifungal activity (over 50%) 0.01 level. were chosen for further growth kinetics assays (Table 5). The antifungal kinetics of extracts and fractions of Z. bungeanum leaves were tested on the five selected fungi. correlation with ABTS result (𝑟 = 0.999, 𝑃 < 0.01)and The phytochemicals of Z. bungeanum leaves inhibited fungal 𝑟 = −0.958, 𝑃 < 0.01 log values of DPPHIC50 ( ), indicating growth (2.32–92.10%) at concentrations of 6.25–100 mg/mL that the phytochemicals with radical scavenging abilities also (Figure 3). The growth inhibition of each sample increased possessed reducing abilities. Log DPPHIC50,ABTS,andFRAP with concentration and then plateaued, notwithstanding the results were significantly correlated with the TFC (𝑟 = −0.922 increases in concentration. At 100 mg/mL concentrations of 𝑃 < 0.01 𝑟 = 0.924 𝑃 < 0.01 for log DPPHIC50, ; for ABTS, ; CF and EAF, the inhibitory activity of G. cingulata was 𝑟 = 0.923 for FRAP, 𝑃 < 0.01). According to Prior and others 90.79% and 92.10%, respectively (Figures 3(c) and 3(d)). [46] and Huang and others [47], the Folin-Ciocalteu method At 50 mg/mL concentrations, significant inhibitory activity (used for determination of the total phenolic content) is based (above 50%) was also observed for ECE, PEF, CF, EAF, on oxidation-reduction reactions (single electron transfer and AF (Figures 3(a), 3(b), 3(c), 3(d),and3(e)), indicating (SET)) and can thus be considered as one of the methods for that the phytochemicals of Z. bungeanum leaves possessed the determination of antioxidant activity. In addition, in our broad-spectrum antifungal property; yet the MF (Figure 3(f)) present study, good correlations were also observed between exhibited less inhibitory activity (less than 50%) than the 𝑟 = −0.916 theTPCandlogDPPHIC50 or ABTS results ( for fiveselectedpathogenicfungi.Amphotericinwasusedasthe 𝑃 < 0.05 𝑟 = 0.827 𝑃 < 0.05 log DPPHIC50, ; for ABTS, ). positive control (Figure 3(g)). In our present study, it can be inferred that EAF and As seen in Table 6, the CF, with the lowest EC50 values of AF contained the highest total polyphenol and flavonoid 0.83, 9.39, 4.18, 10.89, and 5.35 mg/mL against the growth of levels and exhibited the highest antioxidant capacity among G. cingulata, B. cinerea, P. or y z ae , P. pir i col a ,andV. py rina the five different polarity fractions. Since the antioxidant separately, exhibited the greatest inhibitory activity closely activity of plants depend on the amount and type of phenolic followed by EAF, with EC50 values of 9.25, 24.39, 17.81, 13.73, compounds that occur in them [48], we hypothesized that the and 8.11 mg/mL, respectively (𝑃 < 0.05). CF and EAF phenolic compounds of the analyzed extracts and fractions exhibited greater antifungal activities, which might be further were responsible for the profound antioxidant effects. Due studied to determine whether this activity can be retained to the enrichment effects during the chromatography frac- in vivo. The fractions (CF and EAF) with low and medium tionation, EAF and AF were effective in the recuperation of polarity phytochemicals exhibited the highest antifungal 8 The Scientific World Journal

Table 5: Preliminary antifungal activity of ethanol extracts and its five fractions from Z. bungeanum leaves tested at 50 mg/mL against 20 plant pathogenic fungi.

Inhibitory activity (%) Species ECE PEF CF EAF AF MF Alternaria alternata 30.91 ± 1.57c 28.18 ± 1.57c 34.55 ± 0.00b 43.64 ± 1.57a 36.36 ± 3.15b 17.27 ± 1.57d Alternaria brassicae 65.22 ± 3.77ab 60.87 ± 5.65bc 69.57 ± 1.88a 63.04 ± 3.77abc 57.61 ± 3.26c 60.87 ± 3.26bc Alternaria solani 29.51 ± 1.42d 34.43 ± 1.42c 41.80 ± 1.42b 46.72 ± 1.42a 40.98 ± 2.46b 23.77 ± 2.46e Bipolaris sorokiniana 24.55 ± 1.57c 27.27 ± 4.17c 35.45 ± 1.57b 50.91 ± 2.73a 28.18 ± 1.57c 24.55 ± 3.15c Botrytis cinerea 38.76 ± 1.34cd 42.64 ± 2.69c 60.47 ± 2.33a 62.79 ± 2.33a 49.61 ± 5.85b 34.88 ± 6.15d Cladosporium fulvum 29.03 ± 5.59c 41.94 ± 2.42b 50.81 ± 1.40a 50.81 ± 1.40a 43.55 ± 1.40b 25.00 ± 0.00c Colletotrichum gloeosporioides 28.18 ± 4.17c 29.09 ± 5.45c 37.27 ± 2.73b 52.73 ± 4.17a 29.09 ± 2.73c 16.36 ± 1.57d Cucumis dahlia 25.47 ± 1.63c 35.85 ± 1.63b 43.40 ± 2.83a 34.91 ± 2.83b 34.91 ± 2.83b 23.58 ± 2.83c Dothiorella gregaria 45.38 ± 1.33d 52.31 ± 1.33c 70.00 ± 2.31a 62.31 ± 3.53b 46.15 ± 1.33d 44.62 ± 6.11d Fusarium oxysporum 36.84 ± 2.63c 25.44 ± 1.52d 35.09 ± 1.52c 58.77 ± 3.04a 42.11 ± 0.11b 27.19 ± 1.52d Glomerella cingnlata 50.00 ± 2.63c 53.51 ± 5.48bc 55.26 ± 0.00b 64.04 ± 1.52a 49.12 ± 1.52c 37.72 ± 1.52d Phacidiopycnis washingtonensis 24.14 ± 2.99c 25.00 ± 2.59c 35.34 ± 2.59b 50.00 ± 1.49a 37.07 ± 1.49b 35.34 ± 2.59b Physalospora piricola 43.75 ± 8.33bc 45.83 ± 5.51bc 51.39 ± 1.20b 60.42 ± 2.08a 51.39 ± 3.18b 40.97 ± 1.20c Piricularia oryzae 42.22 ± 4.44d 51.85 ± 2.57c 68.15 ± 1.28a 60.74 ± 1.28b 49.63 ± 1.28c 43.70 ± 1.28d Rhizoctonia cerealis 6.00 ± 3.46b 10.00 ± 6.00b 28.00 ± 6.00a 34.00 ± 6.00a 24.00 ± 3.46a 26.00 ± 9.17a Sclerotinia sclerotiorum 19.89 ± 1.70d 56.82 ± 2.60b 59.66 ± 2.60ab 64.20 ± 1.70a 29.55 ± 4.92c 26.70 ± 2.95c Thanatephorus cucumeris 31.03 ± 3.95d 37.93 ± 2.59bc 34.48 ± 3.95bcd 40.52 ± 4.48a 33.62 ± 2.99cd 32.76 ± 2.59cd Valsa mali 23.64 ± 2.73c 26.36 ± 2.73c 42.73 ± 2.73b 50.91 ± 2.73a 39.09 ± 4.17b 15.45 ± 2.73d Venturia pyrina 54.62 ± 2.66a 47.69 ± 1.33b 56.92 ± 3.53a 58.46 ± 0.00a 54.62 ± 1.33a 37.69 ± 2.31c Verticillium dahliae 24.07 ± 3.21b 21.30 ± 5.78b 35.19 ± 1.60a 37.96 ± 3.21a 34.26 ± 1.60a 23.15 ± 1.60b Values are the mean of three replicates ± SD. Means with different letters within a row were significantly different𝑃 ( < 0.05).

Table 6: EC 50 values of ethanol extracts and its five fractions from Z. bungeanum leaves against 5 selected plant pathogenic fungi. EC (mg/mL) Sample 50 Botrytis cinerea Piricularia oryzae Physalospora piricola Glomerella cingulata Venturia pyrina ECE 11.82 ± 1.15ab 12.31 ± 0.45a 39.48 ± 2.25ab 13.00 ± 1.34bc 33.22 ± 3.61d PEF 69.34 ± 3.99d 30.02 ± 3.25ab 65.32 ± 2.39b 32.83 ± 4.61d 31.77 ± 0.77cd CF 9.39 ± 0.17ab 4.18 ± 0.08a 10.89 ± 1.62ab 0.83 ± 0.24a 5.35 ± 0.34ab EAF 24.39 ± 2.38b 17.81 ± 0.19a 13.73 ± 0.69ab 9.25 ± 0.11b 8.11 ± 0.74b AF 51.16 ± 3.54c 75.63 ± 18.53b 46.69 ± 11.08ab 14.96 ± 1.11c 26.44 ± 3.11c MF 598.31 ± 18.91e 625.81 ± 49.58c 646.04 ± 56.20c 227.90 ± 2.64e 110.18 ± 4.15e Amphotericin 0.03a 0.01a 0.08a 0.01a 0.37a Values are the mean of three replicates ± SD. Means with different letters within a column were significantly different𝑃 ( < 0.05). activities. These results have shown that CF and EAF from Z. pathogenic strains and the adverse side effects due to the use bungeanum leaves might be an attractive alternative for the of conventional antibiotics, the discovery of new antimicro- use of a natural product for control of fungi that attack food bial agents is a vital aspect of research and development in the and crops, avoiding fungicides application. realm of public health [49]. It is promising that EAF from Z. bungeanum leaves may harbor therapeutic compounds with significant antibacterial activity. 3.8. Antibacterial Activity. MIC values of extracts and fractions of Z. bungeanum leaves were shown in Table 7.The control (80% acetone) did not inhibit any of microorgan- 4. Conclusions isms tested. The EAF showed the best antibacterial activity against both Gram-positive and Gram-negative bacteria, S. Phytochemical profiles and bioactivities of extracts and aureus (2.38 mg/mL), E. coli (2.32 mg/mL), and B. subtilis fractions from the leaves of Z. bungeanum were studied. (4.24 mg/mL), and were not significantly different from those Basedonourresults,EAF,AF,andCFwereselectedasthe of MF (2.65, 3.10, and 4.10 mg/mL, resp.). Benzylpenicillin most effective fractions due to higher phytochemical contents was only effective in the inhibition of Gram-positive bac- and significant bioactivities. Moreover, a simple, rapid, and teria. With the rapid emergence of multiple drug resistant effective HPLC procedure for simultaneous quantification of The Scientific World Journal 9

Table 7: MIC values of ethanol extracts and its five fractions from Z. bungeanum leaves against 3 selected bacteria.

MIC (mg/mL) Bacteria ECE PEF CF EAF AF MF Benzylpenicillin Staphylococcus aureus 4.93 ± 0.69c 2.45 ± 1.25b 4.76 ± 0.17c 2.38 ± 0.82b 5.15 ± 0.16c 2.65 ± 0.97b 1.31 ± 0.01a Escherichia coli 4.61 ± 2.11bc 4.34 ± 1.78bc 3.15 ± 0.72ab 2.32 ± 0.83a 5.70 ± 0.31c 3.10 ± 0.43ab >10 Bacillus subtilis 8.29 ± 0.98c 4.78 ± 0.42b 4.48 ± 0.79b 4.24 ± 0.54b 5.42 ± 1.04b 4.10 ± 0.23b 0.01 ± 0a Values are the mean of three replicates ± SD. Means with different letters within a row were significantly different𝑃 ( < 0.05). Benzylpenicillin was used as the positive control.

6 (quercitrin) 4000

3000 7 (kaempferol-3-rhamnoside)

2000 1 (chlorogenic acid)

Absorbance (mAU) Absorbance 4 (hyperoside) 10 (chelerythrine) 1000 3 (rutin) 5 (trifolin) 9 (nitidine chloride) 11 (xanthyletin)

2 (epicatechin) 8 (quercetin) 15 (sesamin) 0 0 5 1015202530354045 Retention time (min)

(a) 6

2500 Figure 2: Scavenging effect on DPPH radical of extracts and 4 fractions from Z. bungeanum leaves. Rutin and quercetin were used 2000 as the positive controls.

1500 12 5 14 15 1 2 3 7 13 twelve compounds in Z. bungeanum leaves was established. 1000 MF 8 10 ECE Our current work has shown that the phytochemicals present Absorbance (mAU) Absorbance AF in Z. bungeanum leaves have potent bioproperties and that the 500 9 EAF antioxidant properties are positively correlated with the total 11 CF flavonoid and phenolic content. HPLC analysis indicated PEF 0 that the major phytochemicals (chlorogenic acid, epicate- 5 10 15 20 25 30 35 40 45 50 chin, rutin, hyperoside, trifolin, quercitrin, kaempferol-3- Retention time (min) rhamnoside, quercetin, sesamin, and nitidine chloride) were concentratedintheEAFandAF,whichmaybedueto (b) the enrichment effects during chromatographic fractionation. These bioactive phytochemicals might be responsible Figure 1: HPLC analysis of extracts and fractions from Z. for their profound bioproperties. Furthermore, some lower bungeanum leaves. (a) Chromatography of the twelve standard polarity phytochemicals, such as kaempferol-3-rhamnoside, compounds. (b) Chromatography of the ethanol extracts and their xanthyletin, sesamin, and other unknown compounds, might fivefractions(ECE,PEF,CF,EAF,AF,andMF)monitoredat254nm be responsible for the significant antifungal activity of CF. and identified by their retention time (min): chlorogenic acid (6.62, These results clearly demonstrated that the crude extracts peak 1), epicatechin (8.31, peak 2), rutin (12.12, peak 3), hyperoside and subfractions from the leaves of Z. bungeanum could be (13.61, peak 4), trifolin (15.73, peak 5), quercitrin (16.82, peak 6), served as an accessible potential source for the production of Kaempferol-3-rhamnoside (20.61, peak 7), quercetin (28.32, peak functional food ingredients and medicinal exploration. This 8), nitidine chloride (35.61, peak 9), chelerythrine (36.95, peak 10), xanthyletin (37.20,peak 11), and sesamin (46.44, peak 15). Peaks 9–11 also could explain its frequent addition to the Chinese diet for were very weak. promoting human health and for disease prevention. Further study is required to identify and quantify new bioactive 10 The Scientific World Journal

80 90

80 70

70 60

60 50

50 40

40 30

30 20 Growth inhibition (%control) inhibition Growth 20 (%control) inhibition Growth 10

10 0 0 20 40 60 80 100 0 20406080100 Concentration of ECE (mg/mL) Concentration of PEF (mg/mL) Botrytis cinerrea Physalospora piricola Botrytis cinerrea Physalospora piricola Venturia pyrina Piricularia oryzae Venturia pyrina Piricularia oryzae Glomerella cingnlata Glomerella cingnlata

(a) (b)

90 90 80 80 70 70 60 60 50 50 40 40 30 30 Growth inhibition (%control) inhibition Growth 20 (%control) inhibition Growth 20 10 10 020406080100 0 20406080100 Concentration of CF (mg/mL) Concentration of EAF (mg/mL) Botrytis cinerrea Physalospora piricola Botrytis cinerrea Physalospora piricola Venturia pyrina Piricularia oryzae Venturia pyrina Piricularia oryzae Glomerella cingnlata Glomerella cingnlata

40 50

30 40

20 30 Growth inhibition (%control) inhibition Growth 20 (%control) inhibition Growth 10

10 0 02040 60 80 100 0 20 40 60 80 100 Concentration of AF (mg/mL) Concentration of MF (mg/mL) Botrytis cinerrea Physalospora piricola Botrytis cinerrea Physalospora piricola Venturia pyrina Piricularia oryzae Venturia pyrina Piricularia oryzae Glomerella cingnlata Glomerella cingnlata

(e) (f)

Figure 3: Continued. The Scientific World Journal 11

100

30 Growth inhibition (%control) inhibition Growth 20

10 0 0.2 0.4 0.6 0.8 1 Concentration of amphotericin (mg/mL) Botrytis cinerrea Physalospora piricola Venturia pyrina Piricularia oryzae Glomerella cingnlata

(g)

Figure 3: Inhibitory activity of extracts and fractions from Z. bungeanum leaves against 5 plant pathogenic fungi. Amphotericin was used as the positive control.

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Research Article Optimization of Reflux Conditions for Total Flavonoid and Total Phenolic Extraction and Enhanced Antioxidant Capacity in Pandan (Pandanus amaryllifolius Roxb.) Using Response Surface Methodology

Ali Ghasemzadeh and Hawa Z. E. Jaafar

Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Ali Ghasemzadeh; [email protected]

Received 11 April 2014; Revised 6 June 2014; Accepted 3 July 2014; Published 23 July 2014

Academic Editor: Fred Stevens

Copyright © 2014 A. Ghasemzadeh and H. Z. E. Jaafar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Response surface methodology was applied to optimization of the conditions for reflux extraction of Pandan (Pandanus amaryllifolius Roxb.) in order to achieve a high content of total flavonoids (TF), total phenolics (TP), and high antioxidant capacity (AC) in the extracts. Central composite experimental design with three factors and three levels was employed to consider the ∘ effects of the operation parameters, including the methanol concentration (MC, 40%–80%), extraction temperature (ET, 40–70 C), and liquid-to-solid ratio (LS ratio, 20–40 mL/g) on the properties of the extracts. Response surface plots showed that increasing these operation parameters induced the responses significantly. The TF content and AC could be maximized when the extraction ∘ conditions (MC, ET, and LS ratio) were 78.8%, 69.5 C, and 32.4 mL/g, respectively, whereas the TP content was optimal when these ∘ variables were 75.1%, 70 C, and 31.8 mL/g, respectively. Under these optimum conditions, the experimental TF and TP content and AC were 1.78, 6.601 mg/g DW, and 87.38%, respectively. The optimized model was validated by a comparison of the predicted and experimental values. The experimental values were found to be in agreement with the predicted values, indicating the suitability of the model for optimizing the conditions for the reflux extraction of Pandan.

1. Introduction most suitable protocols for a specific group of phytochemicals or plant species. Traditionally, the extraction of phenolic Phytochemicals are important compounds found in medic- acids and flavonoid compounds is accomplished by reflux inal plants that exert positive effects on human health or or Soxhlet extraction [4]. However, prolonged extraction at in amelioration of diseases. Though many phytochemicals high temperature may degrade flavonoid and phenolic acid have been identified, a great many are yet to be identified compounds [5] and involves a high energy cost. A model [1]. According to a report by the World Health Organization, for optimizing the most relevant operational parameters is 80% of the population in developing countries depends required in order to achieve higher extraction yield. Response on traditional medicine for primary health care and 85% surface methodology (RSM) is a collection of statistical and of traditional medicine is derived from plant extracts [2]. mathematical techniques that is used to optimize the range Extraction and analysis of plant matrices are primary and of variables in various experimental processes with reducing important processes for the quality control, modernization, the number of experimental runs, cost, and time compared and development of herbal formulations [3]. In general, to other methods [6, 7]. Pandan (Pandanus amaryllifolius the first step of complete extraction is the selection of Roxb.) is a tropical plant of the family Pandanaceae. Pandan plant parts and careful preparation of plant extracts and a has narrow and strap-shaped green leaves with a spiral thorough review of the existing literature to determine the arrangement [8]. Pandan is characterized by a sweet and 2 The Scientific World Journal

Table 1: Independent variables and their coded and actual values used for optimization.

Coded level Independent variable Unit Symbol −10+1Axial(−𝛼)Axial(+𝛼)

MC % 𝑋1 10.52 50 89.48 20 80 ∘ ET C 𝑋2 23.68 50 76.32 30 70

LS ratio % 𝑋3 16.84 30 43.16 20 40 MC: methanol concentration; ET: extraction temperature; LS ratio: liquid to solid ratio.

Table 2: The result of experimental and predicted values of reflux extraction of TF, TP, and AC.

TF (mg/g DW) TP (mg/g DW) AC% Run 𝑋1 𝑋2 𝑋3 Experimental Predicted Experimental Predicted Experimental Predicted values values values values values values 1 10.52 50 30 0.55 0.56 3.12 3.18 44.70 44.80 2807020 1.52 1.50 6.24 6.20 83.20 83.37 3 50 50 16.84 1.16 1.15 5.55 5.50 70.40 70.42 4 (C) 50 50 30 1.33 1.26 5.94 5.90 75.80 74.48 5807040 1.74 1.70 6.58 6.55 87.50 87.36 6203020 0.67 0.68 3.79 3.82 52.00 52.19 7 50 76.32 30 1.40 1.39 6.06 6.06 80.00 80.10 8 (C) 50 50 30 1.22 1.26 5.84 5.90 73.00 74.48 9803040 1.44 1.43 6.10 6.16 79.40 79.81 10 20 30 40 0.71 0.71 3.82 3.85 53.10 52.98 11 (C) 50 50 30 1.31 1.26 5.89 5.90 75.30 74.48 12 50 50 43.16 1.35 1.33 5.95 5.91 75.80 75.60 13 (C) 50 50 30 1.27 1.26 6.02 5.97 74.20 74.48 14 50 23.68 30 1.15 1.17 5.89 5.83 72.70 73.10 15 20 70 20 0.81 0.79 3.99 3.93 53.80 53.43 16 (C) 50 50 30 1.28 1.26 5.94 5.90 74.50 74.48 17 89.48 50 30 1.38 1.40 6.11 6.14 80.50 80.61 18 (C) 50 50 30 1.21 1.26 5.85 5.90 73.80 74.48 19 20 70 40 0.88 0.90 4.10 4.12 54.90 55.82 20 80 30 20 1.37 1.32 6.00 5.98 78.80 77.49

(C): central point; 𝑋1:MC;𝑋2:ET;𝑋3:LSratio. delightful flavor and is widely used as a natural flavor in 2. Material and Methods East Asian countries including Indonesia, Thailand, India, and Malaysia. In Malaysia, Pandan leaves are also used in the 2.1. Plant Material. Plant samples (P. amaryllifolius) were production of coconut jam, sweets, desserts, and ice cream. A collected from the North of Malaysia, Bachok, Kelantan number of studies have demonstrated that Pandan leaves are a province. The samples were identified by the Malaysian valuable source of phenolic compounds [9–11]. However, far Agriculture Research and Development Institute (MARDI) too little attention has been paid to optimization of Pandan with voucher specimens of MTP008/1. The leaves were shade extract in folk medicine. To the best of our knowledge, dried and were powdered using a mechanical grinder. This there are no studies and reports undertaken to optimize powered material was used for further analysis. the flavonoid and phenolic extraction from Pandan leaf, following that improving of antioxidant activity using RSM. 2.2. Extraction. The optimization procedure for the extrac- ∘ The aim of this study is to optimize the extraction conditions tionprocessfocusingontheMC(20–80%),ET(30–70C), of a Malaysian Pandanus amaryllifolius variety to achieve andLSratio(20–40mL/g)wasdevisedbasedonthree- high TF and TP content and thus to enhance the AC based factor central composite design, as summarized in Table 1. on the use of RSM method with a central composite design Dissolving of different LS ratio in different MC was designed (CCD) for optimization of the reflux extraction conditions. by RSM software (Table 2) with a total of 20 extraction The Scientific World Journal 3 running sets. Solutions were refluxed at various temperatures were dissolved in HPLC grade methanol. The linear regres- ∘ (30–70 C) for 2 h. After reflux, the solutions were cooled at sion equation was calculated with 𝑌=𝑎𝑋±𝑏,where𝑋 room temperature and were filtered with a Whatman number was concentration of flavonoid and 𝑌 was the peak area of 1 filter paper and used for future analysis. flavonoids obtained from UHPLC [15]. Compounds were tentatively identified by comparison of retention times of 2.3.DeterminationofTotalFlavonoids. After extraction, 1 mL standards. All flavonoids and phenolic acids standards were of extracts was diluted with distilled water (4 mL). Initially, purchased from Sigma-Aldrich (Malaysia). NaNO2 solution (5%, 0.3 mL) was added to each sample. After 5 min AlCl3 solution (10%) and at 6 min NaOH (1.0 M, 2.7. Experimental Design. RSM was used to determine the 2 mL) were added. Absorbance of the solutions was read at optimal extraction conditions for maximizing the TF (𝑌1), TP 430 nm [12]. (𝑌2), and the AC (𝑌3). The central composite experimental design with 3 levels and 3 factors was used to examine the 2.4. Determination of Total Phenolic Content. Leaf extracts extraction variables. Design-Expert software (Version 7.0.0) (1 mL) were diluted with distilled water (10 mL), and 1 mL was used for data analysis, model building, and experimental of Folin-Ciocalteu reagent was then added. Solutions were design. The statistical significance of the model and model variables was determined at the 5% probability level (𝑃< allowed to stand for 5 min. Sodium carbonate (2 mL, 20%) 0.05 was added to the solutions, which were then stored under ). ∘ completely dark conditions at room temperature (25 C) for 90 min. The absorbance of the mixtures was read at 750 nm 2.8. Statistical Analysis. The Design-Expert software (Version [13]. 7.0.0) was used for data analysis, model building, and experimental design. Analysis of variance and response surface analysis were employed to determine the regression coeffi- 2.5.DeterminationofAntioxidantCapacity cients and statistical significance of the model terms and to fit 2.5.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Assay. The free the mathematical models of the experimental data that aimed radical scavenging activity of extracts was determined to optimize the overall region for both response variables. A according to the Mensor et al. [14] with some modifications. model was applied to predict the response variables as given DPPH was dissolved in methanol to give final concentration below: 2 2 2 2 of 2 mM. Following that, 1 mL of DPPH solution was added 𝑌=𝑏0 +𝑏1𝑋1 +𝑏2𝑋2 +𝑏3𝑋3 +𝑏1 𝑋1 +𝑏2 𝑋2 to different concentration of Pandan extracts (20, 40, 60, (2) 80,and100mg/mL).Themixturewasshakengentlyand +𝑏2𝑋 2 +𝑏𝑏 𝑋 𝑋 +𝑏𝑏 𝑋 𝑋 +𝑏𝑏 𝑋 𝑋 , ∘ 3 3 1 2 1 2 1 3 1 3 2 3 2 3 incubated at 28 C in a dark room for 40 min. For the control, methanol was used as a blank. The absorbance of where 𝑌 is the predicted dependent variable, 𝑏0 is a constant the samples was read at 517 nm using spectrophotometer. that fixes the response at the central point of the experiment, BHT (butylhydroxytoluene) and 𝛼-tocopherol were used 𝑏1, 𝑏2,and𝑏3 are the regression coefficients for the linear effect as positive controls. The scavenging activity was calculated terms, 𝑏1𝑏2, 𝑏1𝑏3,and𝑏2𝑏3 are the interaction effect terms, and 2 2 2 using the following formula: 𝑏1 , 𝑏2 ,and𝑏3 are the quadratic effect terms, respectively. The relationship between the independent variables (MC: %inhibition 𝑋1;temperature:𝑋2, and LS ratio: 𝑋3)andtheresponse variables (TF: 𝑌1;TP:𝑌2,andAC:𝑌3) was demonstrated ( − ) =[ absorbance of control absorbance of sample ] by the response surface plots. Table 1 shows information absorbance of control about extraction temperature, MC, and LS ratio of the 20 experiments. × 100. (1) 3. Result and Discussion 2.6. Separation and Analysis of Flavonoids by Ultrahigh 3.1. Model Fitting, Statistical Significance Analysis, and Performance Liquid Chromatography (UHPLC). The UHPLC Response Surface of Reflux Extraction of Total Flavonoid and ∘ system (Agilent, Model 1200) with Agilent C18 (4.6 × Phenolics. In this study, extraction temperatures below 80 C 250 mm, 5 𝜇m) column was used for flavonoid separation and were used for the extraction to minimize the possibility identification. In this system two mobile phases including of degradation of the flavonoid and phenolic compounds, 0.03 M orthophosphoric acid (A) and methanol HPLC grade which has been observed to occur with the application of (B) were used. The column temperature, flow rate, and high temperatures [5, 16]. In addition, the Maillard reaction ∘ injection volume were adjusted at 35 C, 20 𝜇L, and 1 mL/min, may occur at high temperatures, resulting in undesired respectively. The range of detecting wavelength was between compounds [17]. The results of the experiment and the 260 and 360 nm. Gradient elution was performed as follows: extraction conditions are shown in Table 2.Significant(𝑃< 0–10 min 40–100% B, 10–15 min 100% B, and 15–20 min 100– 0.05) regression relationships between the response and inde- 40%Bandfinallywashingofthecolumn.Topreparethe pendent variables were observed. The TF and TP contents standard solution all flavonoid and phenolic acid standards of the extract were more significantly affected by the MC 4 The Scientific World Journal

Table 3: Predicted models and statistical parameters calculated after implementation of three-factor central composite design.

Measured 𝑅2 Regression Lack of fit Predicted models 𝑅2 parameters (adjusted) (𝑃 value) (𝑃 value)

+1.26 + 0.36𝑋1 + 0.095𝑋2 + 0.057𝑋3 + 0.018𝑋1𝑋2 + 0.023𝑋1𝑋3 TF 2 2 2 0.98 0.97 0.0001 0.366 + 0.023𝑋2𝑋3 − 0.16𝑋1 + 0.021𝑋2 + 0.00938𝑋3

+5.92 + 1.15𝑋1 + 0.12𝑋2 + 0.097𝑋3 + 0.030𝑋1𝑋2 + 0.038𝑋1𝑋3 TP 2 2 2 0.99 0.99 0.0001 0.295 + 0.040𝑋2𝑋3 − 0.76𝑋1 + 0.026𝑋2 − 0.10𝑋3

+74.46 + 14.19𝑋1 + 2.20𝑋2 + 1.20𝑋3 + 1.18𝑋1𝑋2 + 0.40𝑋1𝑋3 DPPH 2 2 2 0.99 0.99 0.0001 0.266 + 0.40𝑋2𝑋3 − 6.89𝑋1 + 1.05𝑋2 − 0.82𝑋3

∘ (20–80%), ET (30–70 C), and LS ratio (20–40 mL/g). High of 40 : 1 (mL/g). The results of another study showed that a TFandTPcontentsof1.74and6.58mg/gDW,respectively, high total phenolics value was obtained from Parkia speciosa were observed in the Pandan extracts using treatment run 5 attheLSratioof20mL/g,whereasthetotalphenolicscontent (Table 2). The predicted TF and TP contents for treatment 5 was unaffected by the ET [20]. In current study, the predicted were 1.7 and 6.55 mg/g DW, which were consistent with the model obtained for TP (𝑌2) extraction was as follows: experimental values. The most striking observation from the 𝑌 = + 5.92 + 1.15𝑋 + 0.12𝑋 + 0.097𝑋 data is that when the MC (𝑋1)increasedfrom20%to80% 2 1 2 3 𝑋 𝑋 (at 2:30and 3:40),theTFandTPcontentincreased + 0.030𝑋 𝑋 + 0.038𝑋 𝑋 + 0.040𝑋 𝑋 from 0.71 to 1.44 mg/g DW and from 3.82 to 6.10 mg/g DW, 1 2 1 3 2 3 (4) respectively. Increasing the LS ratio resulted in an increment − 0.76𝑋 2 + 0.026𝑋 2 − 0.10𝑋 2. of the TF content of the extracts. In addition, a lower TF 1 2 3 content was recorded at lower LS ratio (20:1). Increasing the The model 𝑃 value of 0.0001 implies that the model is LS ratio from 20 to 40% increased the TF content by 14.4% 𝑋 𝑋 ∘ significantTable ( 3). There is only a 0.01% chance that such (at 1:80%and 2:70 C). In the analysis of the TF content, a as large “model 𝑃 value” could be due to noise. The “lack of fit 𝑅2 = 0.98 good coefficient of determination ( )wasobtained, 𝑃 value” of 0.295 implies that the lack of fit is not significant, where the model explained most of the observed variations relativetothepureerror.Theeffectofthevariablesandthe 𝑃 < 0.05 (Table 3). Significant ( ) linear and quadratic effects interaction of these variables on the responses can be seen in 𝑌 of the ET, MC, and LS ratio on the TF content ( 1)were Figures 1 and 2. Figure 1(a) shows the effect of interaction of 𝑃 observed. The model value of 0.0001 implies that the model the MC and the ET on the TF content of the extract at a fixed 𝑃 is significant, with only a 0.01% chance that a “model LS ratio of 30%. The minimum TF value was obtained at the value”thislargecouldbeduetonoise.Thelackoffittest lowestMCandthemaximumTFvaluewasobtainedat80% ∘ for the model describes the variation in the data around methanol at the fixed ET of 70 C. Figure 1(b) shows the effect the fitted model. If the model does not fit the data well, the of the interaction of MC and the LS ratio on the TF content at ∘ lack of fit value will be significant; consequently, proceeding afixedETof50C. The minimum TF value was also obtained with investigation and optimization of the fitted response at the lowest MC and the maximum TF value was obtained at surface is likely to give misleading results. The “lack of fit 80% methanol at the fixed LS ratio of 40%. Figure 1(c) shows 𝑃 value” of 0.366 obtained herein implies that the lack of the effect of interaction of the ET and the LS ratio on theTF fitisnotsignificantrelativetothepureerror.However,no content at a fixed MC of 50%. The minimum TF content was interactive effect of the independent variables was observed. observed at the lowest LS ratio (20%) and the maximum TF 𝑌 The predicted model obtained for TF ( 1) was as follows: value was obtained at a LS ratio of 40% using the fixed ET ∘ of 70 C. Moreover, the results indicated that the MC was the 𝑌1 = + 1.26 + 0.36𝑋1 + 0.095𝑋2 + 0.057𝑋3 + 0.018𝑋1𝑋2 most significant factor affecting the responses at the 𝑃 < 0.01 2 2 + 0.023𝑋1𝑋3 + 0.023𝑋2𝑋3 − 0.16𝑋1 + 0.021𝑋2 level. Figure 2(a) showstheeffectofinteractionoftheMCand theETontheTPcontentatafixedLSratioof30%.Asshown 2 + 0.00938𝑋3 . in Figure 2, the TP content increased significantly to ca. (3) 84.5% as the MC increased (from 20 to 80%). Furthermore, the TP content increased slightly as the LS ratio increased The data in Table 2 demonstrate that when the LS ratio from 20 to 40% (Figure 2(b)). Figure 2(c) shows the effect of increased from 20 : 1 to 40 : 1, the TP content also increased by interaction of the ET and the LS ratio on the TP content at ∘ about 5.4% (at 𝑋1:80%and𝑋2:70 C). It is plausible that these 50% methanol. The TP content increased significantly as the results are due to the fact that more solvent could enter the LS ratio increased up to 32%, but beyond a LS ratio of ∼32% cells while more phenolic compounds could permeate into the TP content decreased. These findings suggest that the the solvent at higher LS ratios [18]. The results of previous extraction yield of TF and TP was influenced primarily by the study showed that as the liquid/solid ratio increased, the MC rather than the ET. It is difficult to explain this result, but content of phenolic compounds in the extract of E. oleracea thistrendmightberelatedtotheincreasedsolubilityofthe was enhanced [19]. The highest content of phenolics was flavonoid compounds in the mixture of methanol and water reportedly obtained from fruits of E. oleracea at a LS ratio [21]. The findings of the current study are consistent with The Scientific World Journal 5

1.6 1.6 1.325 1.325 1.05 1.05 0.775

0.775 TF (mg/g DW) (mg/g TF

0.5 DW) TF (mg/g 0.5

70.00 80.00 40.00 80.00 60.00 65.00 35.00 65.00 B: extraction5 temperature0.00 50.00 C: liquid to solid ratio 40.00 30.00 50.00 35.00 25.00 35.00 30.00 20.00 20.00 20.00 A: methanol concentration A: methanol concentration

(a) (b)

1.47

1.3875

1.305

1.2225 TF (mg/g DW) TF (mg/g 1.14

40.00 70.00 35.00 60.00 C: liquid to 30.00solid ratio 50.00 25.00 40.00 n temperat 20.00 30.00 B: extraction temperature

(c)

∘ Figure 1: Response surface plots for the effects of MC (20–80%), ET (30–70 C), and LS ratio (20–40 mL/g) on the TF content of Pandan extract. MC and ET (a), MC and LS ratio (b), and ET and LS ratio (c). those of Liyana-Pathirana and Shahidi [22], who found that Gan and Latiff [20] reported that, in the extraction of Parkia the TF content of wheat increased with increasing ethanol speciosa, the highest phenolic concentration was achieved at ∘ concentration. an ET of 35 C and the extraction rate could be increased by The result of our study showed that TF and TP con- reducing the extraction time as well as increasing the ET [28]. ∘ tent increased with increasing of the temperature till 70 C, but we cannot say that this trend will continue even for 3.2. Model Fitting, Statistical Significance Analysis, and high temperatures. Previous studies have shown that the ∘ Response Surface of Reflux Extraction of Antioxidant Capacity application of very high temperatures (⩾95 C) may alter (AC). The AC of the extract was significantly affected by the the concentration and composition of phenolic compounds temperature, solvent concentration, and LS ratio (𝑃 < 0.05) [23]. Some of the authors reported that critical temperature with three linear effects𝑋 ( 1, 𝑋2,and𝑋3), two quadratic ∘ 2 2 for flavonoids is below 80 C[24]. In different plants and effects (𝑋1 and 𝑋2 ), and three interactive effects𝑋 ( 1𝑋2, organsthistemperaturewillbevariableandaccordingtothe 𝑋1𝑋3,and𝑋2𝑋3). The DPPH capacity of the extract ranged previous studies this changes in phenolic acids and flavonoids from 44.7 to 87.5%when treatments 1 and 5 were, respectively, at high temperature could be related to PAL or CHS enzymes employed. The predicted AC values for treatments 1 and 5 activity at high or low temperature [25]. At high temperature, were, respectively, 44.8 and 87.36%, which were close to the the flavonoid and phenolic content can be increased asa experimental values. The effect of the variables and their result of enhancement of their solubility, extraction rate, interaction on the AC of the Pandan extracts is shown in diffusion rate, and the reduced surface tension and solvent Figure 3. The AC increased in positive proportion to the MC viscosity [26]. However, further increment of the ET may in the range of 20–80% for the extraction medium. Thus, the degrade flavonoids and phenolics due to destabilization of MC of the extraction medium had a significant influence on the compounds by reaction with other plant components the antioxidant properties of the Pandan extracts. Liyana- or enzymatic and chemical degradation, thus reducing the Pathirana and Shahidi [22] reported that a higher AC was extraction efficiency [27]. In contrast with the current results, obtained in the extraction of wheat by using 50% ethanol 6 The Scientific World Journal

6.6 6.4

5.725 5.575

4.85 4.75

3.975 3.925 TP (mg/g DW) TP (mg/g TP (mg/g DW) TP (mg/g 3.1 3.1

70.00 80.00 40.00 80.00 60.00 65.00 B: extraction temperature 35.00 65.00 50.00 50.00 C: liquid to solid30.00 ratio 50.00 40.00 35.00 25.00 35.00 30.00 20.00 20.00 20.00 A: methanol concentration A: methanol concentration

(a) (b)

6.12

5.9775

5.835

5.6925 TP (mg/g DW) TP (mg/g 5.55

40.00 70.00 35.00 60.00 C: liquid to solid30.00 ratio 50.00 25.00 40.00 20.00 30.00 B: extraction temperature

(c)

∘ Figure 2: Response surface plots for the effects of MC (20–80%), ET (30–70 C), and LS ratio (20–40 mL/g) on the TP content of Pandan extract. MC and ET (a), MC and LS ratio (b), and ET and LS ratio (c). compared to other aqueous solvents. The current finding is AC. In another study, high AC of Parkia speciosa extract in agreement with those of Pompeu et al. [19]andKiassoset was achieved using a liquid/solid ratio of 20 mL/g [20]. The al. [29], who demonstrated that the concentration of ethanol regression equation obtained for the AC (𝑌3)astheresponse had a significant influence on the AC of onion extract. variable also showed significant𝑃 ( < 0.05) dependence of 𝑌3 The ET caused a linear increase in the AC of wheat extract, on the variation of the independent variables. The predicted andincreasingthetemperatureincreasedthetotalAC.It model obtained for 𝑌3 is given below: was confirmed that the rate of extraction of thermally stable 𝑌3 = + 74.46 + 14.19𝑋1 + 2.20𝑋2 + 1.20𝑋3 antioxidants at elevated temperature was higher than the rate of decomposition of less soluble antioxidants [22]. The + 1.18𝑋1𝑋2 + 0.40𝑋1𝑋3 + 0.40𝑋2𝑋3 (5) temperature utilized during extraction generally influences 2 2 2 thecompoundstabilityduetochemicalandenzymaticdegra- − 6.89𝑋1 + 1.05𝑋2 − 0.82𝑋3 . dation and losses by thermal decomposition; these factors The model 𝑃 value of 0.0001 obtained for the AC implies that have been suggested to be the main mechanisms underlying the model is significantTable ( 3). The “lack of fit 𝑃 value” of reduction of the polyphenol content in the extraction of grape 0.266 implies that the lack of fit is not significant, relative to [30]. Pompeu et al. [19] obtained high AC in the extraction ∘ thepureerror.Thereisa26.65%chancethata“lackoffit of Euterpe oleracea at a temperature of 58 C. Similarly, high 𝐹 value” this large could occur due to noise. One question AC of wheat extracts was observed when a temperature of ∘ that needs to be asked, however, is why does software take 61 Cwasutilized[22] and high AC was achieved (83.37%) about five or six center points in the design? The reason is when the LS ratio was low (20 mL/g). The latter is plausibly also related to the variance of a predicted value. When fitting due to increased probability of the antioxidant components a response surface we want to estimate the response function coming into contact with the extraction solvent as the amount in this design region where we are trying to find the optimum. of solvent increased. However, further increase of the LS Wewantthepredictiontobereliablethroughouttheregion ratio may dilute the extraction solution thereby lowering the and especially near the center since we hope the optimum The Scientific World Journal 7

87 83

) 76.25

) 73.25 % % 65.5 63.5

DPPH ( 54.75 DPPH ( 53.75

44 44

70.00 80.00 40.00 80.00 B: extraction60.00 temperature 65.00 35.00 65.00 50.00 50.00 C: liquid to30.00 solid ratio 50.00 40.00 35.00 25.00 35.00 30.00 20.00 20.00 20.00 A: methanol concentration A: methanol concentration

(a) (b)

) 77.6 % 75.2

DPPH ( 72.8

70.4

40.00 70.00 35.00 60.00 C: liquid to solid30.00 ratio 50.00 25.00 40.00 20.00 30.00 B: extraction temperature

(c) ∘ Figure 3: Response surface plots for the effects of MC (20–80%), ET (30–70 C), and LS ratio (20–40 mL/g) on the AC of Pandan extract. MC andET(a),MCandLSratio(b),andETandLSratio(c). is in the central region. By picking five to six center points, conditions for the three responses to evaluate the adequacy the variance in the middle is approximately the same as the of the response surface models for predicting the optimum variance at the edge. If we only had one or two center points, response values. As shown in Table 4,theobserved thenwewouldhavelessprecisioninthemiddlethanwe values of the TF and TP content and AC were 1.78, wouldhaveattheedge.Aswegofartheroutbeyondadistance 6.601 mg/g DW, and 87.38%, respectively. The response of 1 in coded units, we get more variance and less precision. surface models of TF, TP, and antioxidant activity were What we are trying to do is to balance the precision at the verified using the experimental and predicted values. The edge of the design relative to the middle. obtained results from verification experiment were in consent with the predicted values, because nonsignificant 3.3. Optimization of Reflux Extraction Condition for TF (𝑃 > 0.05) difference was observed between the verification and TP Content and AC. The optimum reflux extraction 𝐸 = 0.069 experimental and the predicted values ( TF,TP %; conditions for maximizing the TF and TP and for achiev- 𝐸 = 0.48%). ing high AC of Pandan extracts were predicted using the DPPH Design-Expert software. Multiple graphical and numerical optimizations were carried out to determine the optimum 3.5. Identification of Flavonoids and Phenolic Acids. Figure 5 level of independent variables with desirable response goals. shows the UHPLC chromatogram of the identified flavonoid Two optimal conditions were developed for the responses: and phenolic acids of the Pandan extract. Gallic acid, (+)- the TF content and AC were maximized using a MC of catechin, caffeic acid, myricetin, luteolin, and quercetin were ∘ 78.8%, ET of 69.5 C, and LS ratio of 32.4 mL/g, whereas the identified in the Pandan extract at concentrations of 0.489, corresponding conditions for maximizing TP were 75.1%, 0.594, 0.856, 0.076, 0.08 and 0.112 mg/g DW, respectively. ∘ 70 C, and 31.8 mL/g, respectively (Table 4, Figure 4). Caffeic acid was the most abundant compound of the identified compounds, and the concentration of phenolic acids was 3.4.VerificationoftheModels.The experiment was per- higher than that of the flavonoid compounds in the Pandan formed using the recommended optimum treatment extract. 8 The Scientific World Journal

20.00 80.00 30.00 70.00 20.00 40.00

Methanol concentration = 78.80 Extraction temperature = 69.50 Liquid to solid ratio = 32.40

0.55 1.80 3.12 6.58 44.7 87.5

TF = 1.78125 TP = 6.52991 DPPH =87.38

Desirability = 0.949

(a)

20.00 80.00 30.00 70.00 20.00 40.00

Methanol concentration = 75.10 Extraction temperature = 70.00 Liquid to solid ratio = 31.80

0.55 1.74 3.12 6.65 44.7 87.5

TF = 1.59726 TP = 6.6010 DPPH = 86.0711

Desirability = 0.943

(b)

Figure 4: Predicted value of TF, TP, and DPPH activity of Pandan leaf from optimized extraction condition using RSM ((a) optimized condition for TF and DPPH activity; (b) optimized condition for TP).

Table 4: Optimum conditions and experimental value of responses at the optimum conditions.

Optimum conditions TF content Optimum conditions TP content Optimum conditions AC (DPPH assay) (predicted) (predicted) (predicted) MC (%) 78.8 75.1 78.8 ∘ ET ( C) 69.51.78 706.601 69.5 87.38 LS ratio (mL/g) 32.4 31.8 32.4 TF and TP: mg/g DW; AC: %.

3 enhancement of the AC of the extracts. The results can be 600 1 2 easily explained on the basis that both the ET and the MC 400 have a positive effect on the solubility of flavonoids in the 200 4 56 (mAU) extraction solution. The most efficient set of reflux conditions 0 ∘ for Pandan leaf extraction are at MC 78.8% (𝑋1), ET 69.5 C 0 24681012 14 16 18 (𝑋2), and LS ratio of 32.4 mL/g (𝑋3)formaximizingtheTF (min) ∘ content and AC and 75.1% (𝑋1), 70 C(𝑋2), and 31.8 mL/g 𝑋 Figure 5: UHPLC chromatogram of Pandan extract. Identified ( 3) for maximizing the TP content with consequently high compounds are gallic acid (1), catechin (2), caffeic acid (3), myricetin AC.Moreover,themodelsusedtofittheresponsevariables (4), luteolin (5), and quercetin (6). were significant (𝑃 < 0.01), and the “lack of fit” was not significant𝑃 ( > 0.05) for all responses, indicating that the models used to fit the response variables were adequate for representing the relationship between the response values 4. Conclusion and the independent variables.

The reflux extraction of TF and TP and the AC of Pandan Conflict of Interests extract were successfully optimized using RSM. The results indicate that the MC, ET, and LS ratio had a significant The authors declare that they have no conflict of interests effect on the TF and TP extraction yields with consequent regarding the publication of this paper. The Scientific World Journal 9

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Research Article HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora

Fang-Ju Lin,1 Feng-Lin Yen,1,2 Pei-Chun Chen,1 Moo-Chin Wang,1 Chun-Nan Lin,1 Chiang-Wen Lee,3,4 and Horng-Huey Ko1

1 Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan 2 Institute of Biomedical Sciences, Sun Yat-Sen University, Kaohsiung 804, Taiwan 3 Graduate School of Nursing, Division of Basic Medical Sciences and Chronic Disease and Health Promotion Research Center, Chang Gung University of Science and Technology, Chiayi 613, Taiwan 4 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan

Correspondence should be addressed to Horng-Huey Ko; [email protected]

Received 28 March 2014; Revised 15 June 2014; Accepted 24 June 2014; Published 20 July 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 Fang-Ju Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Phyla nodiflora is a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of 󸀠 󸀠 󸀠 this plant is necessary. In the present study, ten compounds, namely, 3,7,4 ,5 -tetrahydroxy-3 -methoxyflavone (1), nodifloretin 󸀠 󸀠 󸀠 (2), 4 -hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4 -tetrahydroxy-3 -methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), and 𝛽-sitosterol were isolated from the methanolic extract of the aerial part of P. no difl ora (PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds 4, 5,and 7 showed strong antioxidant activity. To control the quality of P. no difl ora , a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4)andeupafolin(7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM.

1. Introduction aerial part of P. no difl ora (PNM) exerted an antimelanogenesis effect by downregulating the microphthalmia-associated Phyla nodiflora (L.) Greene (syn. Lippia nodiflora (L.) Michx, transcription factor (MITF) expression level and decreasing Verbenaceae), a creeping perennial herb with small white the tyrosinase activity and melanin production [8]. Abbasi et flowers, is distributed throughout India, Ceylon, Bangladesh, al. [9] also mentioned the ethnopharmacological application Baluchistan, Africa, and most tropical and subtropical of P. no difl ora for skin diseases and in folk cosmetics, for the regions [1]. In Taiwan, P. no difl ora hasbeenusedasafolk treatment of pimples, carbuncles, and skin burns. medicine in the form of a herbal drink, nourishing agent, Previous phytochemical studies on this plant have immunomodulator, and as an anti-inflammatory agent2 [ ]. P. afforded flavonoids, quinols and quinol glucosides, steroids, nodiflora possesses many pharmacological activities such as phenylpropanoids, alkaloids, resin, tannins, terpenoids, and anti-inflammatory, analgesic, antipyretic, antiatherosclerotic, volatiles [10–13]. It is well known that the majority of phar- antidandruff, antibacterial, hepatoprotective, antiurolithiatic, macological effects of medicinal herbs could be attributed antimicrobial, and antioxidant abilities [3–7]. In a previous to their secondary metabolites. However, various factors, study, we demonstrated that the methanolic extract of the such as different cultivation areas, climatic conditions, and 2 The Scientific World Journal harvestable seasons, may significantly affect the level of these mg)wereisolatedandpurifiedbysilicagelwithn- components. Thus, a systematic quality standard for quality hexane : EtOAC : MeOH (6 : 69 : 25), followed by RP18 gel assessment is imperative. In fact, no HPLC method was with MeOH : H2O(1:1),acetone:H2O (3 : 1), and Sephadex established for analysis of this herbal medicine; therefore, LH-20 CC from Fr. D6.Compounds1–9 were recrystallized developing a suitable quality control method for it is required. andidentifiedbyNMRandotherspectroscopicdatafollow- Based on preliminary screening data, PNM showed a ing comparison with values in the literature [11, 13, 14]. strong radical scavenging activity and antimelanogenesis Another two batches of P. no difl ora were also collected effect. These findings led us to focus on the isolation of at the same place in different years and seasons as shown in active components in PNM; meanwhile, a method combined specimen numbers 2007-02-PNM and 2009-06-PNM. 2007- with high-performance liquid chromatography (HPLC) with 02-PNM was collected in February (spring) 2007 and 2009- ultraviolet (UV) detector was developed for the simulta- 06-PNM was collected in June (summer) 2009, respectively, neous chemical fingerprint and quantification of the active and they were kept at our laboratory for future reference. components. The results indicated that PNM possesses good Together with 2010-06-PNM, the air-dried samples of these antioxidant and antityrosinase potentials and the developed three batches were lyophilized and stored at low temperature fingerprint could further serve for quality and quantity until analysis. analysis of PNM added in cosmetic industry and herbal 󸀠 medicines. 2.3. Chemicals and Reagents. 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2- 2. Materials and Methods diphenyl-1-picrylhydrazyl (DPPH), ethylenediaminetetra- acetic acid disodium salt dehydrate (EDTA∙2Na), nitro- 2.1. General. Melting points were recorded on an electrother- tetrazolium blue chloride (NBT), L-tyrosine disodium salts, mal MEL-TEMP 3.0 apparatus. UV spectra were measured tyrosinase (from mushroom, 5370 U/mg), xanthine, and in methanol on a Beckman Coulter-DU 800 UV-visible xanthine oxidase (from buttermilk, 0.07 U/mg) were all spectrophotometer. IR spectra were recorded on a Perkin purchased from Sigma-Aldrich (St. Louis, MO, USA). 1 13 Elmer system 2000 FT-IR spectrophotometer. Hand C L-Ascorbic acid was obtained from J. T. Baker (Austin, USA). NMR spectra were measured and recorded on a Bruker- All chemicals and solvents were of high-performance liquid 400 MHz FT-NMR spectrometer and a Mercury-400 MHz chromatography (HPLC) or analytical grade. Ethanol (99.8%) FT-NMR spectrometer. ESI-MS were recorded on a Bruker was purchased from Panreac (Barcelona, Spain). HPLC- DaltonicsApexII30e.Theabsorbancesinbioassayswere grade methanol was obtained from ECHO (Miaoli, Taiwan). measured and recorded on a multiplate spectrophotometer Sodium phosphate buffer (PBS) was freshly prepared in our (𝜇Quant, BioTek, USA). laboratory at different concentrations and adjusted to the appropriate pH value before use. Ultrapure water was used in the experiments (Milli-Q system, Millipore, Massachusetts, 2.2. Plant Material, Extraction, and Isolation. P. nodiflora Molsheim, France). was collected in June 2010 in Tainan, Taiwan, and identified by Professor I. S. Chen, School of Pharmacy of Kaohsiung ∙ 2.4. DPPH Radical (DPPH ) and ABTS Cation Radical Medical University, Kaohsiung, Taiwan. A voucher specimen ∙+ ∙ (ABTS ) Scavenging Assay. In the DPPH radical (DPPH ) (2010-06-PNM) was deposited at the Herbarium of the scavenging assay [15], fifty microliters of samples of various Department of Fragrance and Cosmetic Science, Kaohsiung ∙ concentrations were mixed with 150 𝜇lofDPPH solution Medical University, Kaohsiung, Taiwan. The dried aerial (0.1 mM) dissolved in methanol. Each mixture was allowed to part of P. no difl ora (4.6 kg) was chopped and immersed in standfor30minandmeasuredat517nmusingamultiplate methanol for three times at room temperature. The mixtures spectrophotometer (𝜇Quant, Bio-Tek, USA). The scavenging were filtered and concentrated to dryness under reduced ability was calculated using the following formula: pressure, producing a methanolic extract (PNM, 525 g). The PNM (160 g) was taken and further purified with n-hexane Scavenging ability (%) and an increasing polarity of EtOAc and MeOH, yielding 8 󸀠 󸀠 fractions (Fr. D1–D8). 𝛽-Sitosterol and 3,7,4 ,5 -tetrahydroxy- sample absorbance (1) 󸀠 1 =(1− ) × 100%. 3 -methoxyflavone ( )(3.9mg)werepurifiedbysilicacol- control absorbance umn chromatography (CC) with n-hexane : acetone (10 : 1), followed by n-hexane : CH2Cl2 :EtOAc(5:2:1)fromFr.D2. The ABTS decolorization assay was performed as Hispidulin (8) (4.3 mg) and larycitrin (9)(1.2mg)were described [16], with slight modification. The ABTS cation ∙+ purified by silica CC with n-hexane : EtOAC (2 : 3), followed radical (ABTS )wasproducedbyreacting7mMstock by RP18 silica gel with acetone : H2O (3:2) from Fr.D4. solution of ABTS with 2.45 mM potassium persulfate (final Nodifloretin (2) (9.5 mg) and eupafolin (7)(13.5mg)wereiso- concentration) and allowing the mixture to stand in the ∙+ latedandpurifiedbysilicagelwithn-hexane : EtOAC : MeOH dark for 6–8 h at room temperature before use. The ABTS (7 : 80 : 13), followed by RP18 silica with acetone : H2O(1:1) solution was diluted to an absorbance of 0.7 ± 0.02 at 734 nm. 󸀠 and Sephadex LH-20 CC from Fr. D5.4-Hydroxywogonin Thirty microliters of samples of different concentrations ∙+ (3) (10.4 mg), onopordin (4) (21.8 mg), cirsiliol (5) (11.8 were mixed with 170 𝜇lofABTS working solution under 󸀠 󸀠 mg), and 5,7,8,4 -tetrahydroxy-3 -methoxyflavone (6)(10.3 a dark condition and shaken for 15 s. The absorbance was The Scientific World Journal 3 measured at 734 nm after 20 min. The scavenging capacity 2.9. Sample Preparation. The different tested samples were wascalculatedusingthesameformulaasfortheDPPH weighted and dissolved with M-FA (50 : 50, v/v) to a final radical scavenging assay. concentration of 1 mg/ml. The sample solution was filtered through a 0.45 𝜇m membrane filter prior to injection into the ∙− HPLC system. 2.5. Superoxide Anion Radical (O2 )ScavengingAssay and Mushroom Tyrosinase Inhibition Assay. The proce- ∙− 2.10. Method Validation. The analytical method was validated dures of O2 scavenging activity and mushroom tyrosinase inhibitory effect were performed as previous report17 [ ]. In for linearity, limit of detection and quantification (LOD ∙− and LOQ), precision (interday and intraday), repeatability, the O2 scavenging assay, xanthine (0.1 mM, 15 𝜇l) in 50 mM phosphate buffer (pH 7.4), NBT (25 𝜇M, 16 𝜇l), EDTA and recovery test, following the reports on quantitative 𝜇 𝜇 𝜇 determination [18, 19]. Standard stock solutions of onopordin (0.1 M, 14 l), and 100 l of various concentrations of test 4 7 compounds were mixed in the 96-well plates. Reactions were ( )andeupafolin() were prepared as shown in section 2.8 and diluted into 0.5, 1.0, 5.0, 10.0, and 15.0 𝜇g/ml of initiated by adding 0.5 U xanthine oxidase. After 30 min, the 4 𝜇 7 absorbance of each mixture was measured at 540 nm. In ,and1.0,3.0,5.0,7.0,and10.0 g/ml of ,respectively. the mushroom tyrosinase inhibitory assay, L-tyrosine (2 mM, The calibration curve was performed by analyzing the two 80 𝜇l) in 50 mM phosphate buffer (pH 6.5) and various con- reference solutions in triplicate at five concentrations and centrations of test compounds (100 𝜇l)wereaddedto96-well drawn by plotting the peak areas versus the injection quantity plates. After 10 min incubation, 20 U mushroom tyrosinase ofeachcompound.TheLODandLOQunderthepresent was added to start the reaction. The absorbance of each chromatographic conditions were evaluated at S/N of 3 and mixture was measured at 490 nm after 30 min incubation. 10, respectively. ∙− The O2 scavenging capacity and the percentage inhibition of mushroom tyrosinase were calculated using the same 2.11. Data Analysis. In the bioassay, the average values of formula as for the DPPH radical scavenging assay. three independent analyses were presented as means ± S.D. In the chromatographic fingerprint, data analysis calculated the correlative coefficient for samples and compared the 2.6. Lineweaver-Burk Plots. This study used Lineweaver-Burk similarities of different chromatograms with the mean chro- plots to determine the inhibitory mode of test samples [15]. matogram among the samples tested. The kinetic study was performed in the presence or absence of test samples with various concentrations of tyrosine or xanthine as the substrate. 3. Results and Discussion 3.1. Free Radicals Scavenging and Tyrosinase Inhibitory Activ- ities. Bioassay-guided fractionation of the PNM led to 2.7. HPLC Conditions. Chromatographic analysis was per- 󸀠 󸀠 󸀠 the isolation of nine flavonoids, 3,7,4 ,5 -tetrahydroxy-3 - formed using an ELITE LaChrom high- performance liquid 󸀠 methoxyflavone (1), nodifloretin (2), 4 -hydroxywogonin chromatography (HPLC) system coupled with a L-2420 UV- 3 4 5 󸀠 󸀠 Vis detector, a L-2200 autosampler, and a L-2130 pump. ( ), onopordin ( ), cirsiliol ( ), 5,7,8,4 -tetrahydroxy-3 - methoxyflavone (6), eupafolin (7), hispidulin (8), and laryc- Chromatographic data were processed by Hitachi Model 9 D-2000 Elite chromatography data station software. The itrin ( )(Table 1). Table 2 showed the results of these com- chromatographic separations were performed on a Hypersil pounds on radicals scavenging and tyrosinase inhibitory ODS-C18 column (250 mm × 4.6 mm i.d., 5 𝜇m). The mobile effects. When compared to apigenin, a common flavone phase consisted of 0.1% formic acid (FA) and MeOH (M), and in plants, and vitamin C, a well-known antioxidant and whitening agent used in cosmetic products, eupafolin (7) the flow rate was 0.5 ml/min. The gradient elution program ∙− ∙+− ∙− was conducted as follows: a gradual increase of M 0–2% exhibited potent effects in DPPH ,ABTS ,andO2 - (0–5 min), M 2–6% (5–9 min), M 6–8% (9–12 min), M 8– scavenging assays and with a concentration-dependent scavenging manner (data are not shown). Compounds 4 and 5 12% (12–14 min), M 12–17% (14–20 min), M 17–22% (20– ∙− ∙− 30 min), M 22–27% (30–35 min), M 27–31% (35–41 min), M showed significant DPPH and O2 -scavenging activities. 31–40%(41–60min),M40–43%(60–75min),M43–45% Compounds with a catechol moiety in the B ring and a 2,3- (75–80 min), and M 45–80% (80–120 min). The wavelength double bond in conjugation with a 4-carbonyl group in the ring C show potent radical scavenging activity [20]. ofthedetectorwassetat326nmandthesampleinjection ∙− 7 volume was 20 𝜇l. In the O2 -scavenging assay, eupafolin ( )showedstrong scavenging activity with a SC50 value of 8.3 ± 0.4 𝜇M (Table 2). Figure 1 showed the Lineweaver-Burk transforma- 2.8. Preparation of Standard Stock Solutions. The reference tion of data, indicating that compound 7 was a mixed-type 2 4 inhibitor of xanthine oxidase (XO), with Ki values of 4.48, standards stock solutions of nodifloretin ( ), onopordin ( ), −2 and eupafolin (7) were accurately weighted and dissolved 5.05, and 8.93 𝜇Mand𝑉max (ΔO.D./min) of 3.6 × 10 ,3.2 −2 −2 in methanol and then diluted to appropriate concentration × 10 , and 3.1 × 10 for 7 at 5, 10, and 20 𝜇M, respectively. ranges for the establishment of calibration curves. All of the Biochemically, this enzyme inhibitor is associated with the ∘ stock and working standard solutions were stored at 4 Cuntil hydrogen binding of phenolic hydroxyls or carbonyls of being used for analysis. the substrate with the amide carbonyls or amino group in 4 The Scientific World Journal

Table 1: Structure of compounds 1–9 of P. no difl ora .

R3󳰀

R4󳰀 R8 R7 O R5󳰀

R 6 R3 O R5

󸀠 󸀠 󸀠 Compounds R3 R5 R6 R7 R8 R3 R4 R5 1 OH H H OH H OMe OH OH 2 HOHOHOHH OMeOHH 3 H OH H OH OMe H OH H 4 HOHH OHOMeOHOHH 5 H OH OMe OMe H OH OH H 6 H OH H OH OH OMe OH H 7 HOHOMeOHH OHOHH 8 H OH OMe OH H H OH H 9 OH OH H OH H OMe OH OH

Table 2: Antioxidant and tyrosinase inhibitory activities of compounds 1–9 isolated from P. no difl ora a.

∙ ∙+ ∙− DPPH ABTS O2 Tyrosinase Samples c d SC50 (𝜇M) IC50 (𝜇M) 1 —e —e —e —e 2 198.2 ± 3.8 62.2 ± 2.5 42.3 ± 1.5 127.6 ± 1.3 3 >300 >300 59.9 ± 2.1 169.9 ± 3.8 4 24.7 ± 1.2 53.7 ± 3.9 23.5 ± 0.5 65.8 ± 3.2 5 21.8 ± 1.2 58.4 ± 2.7 10.7 ± 1.3 121.8 ± 4.8 6 >300 >300 34.6 ± 2.4 152.8 ± 4.4 7 24.1 ± 0.4 30.5 ± 1.2 8.3 ± 0.4 56.0 ± 5.0 8 57.5 ± 2.7 55.3 ± 3.8 146.0 ± 2.2 9 —e —e —e —e Apigeninb,+ >300 159.6 ± 4.4 81.3 ± 2.0 304.8 ± 3.1 Vit. Cb 64.6 ± 4.4 49.9 ± 2.1 23.5 ± 1.7 —e Kojic acidb —e —e —e 88.1 ± 1.8 aValues are presented as means ± SD (𝑛=3). bApigenin, Vit. C, and Kojic acid are used as positive controls. +The data of apigenin are referred to previous c d report (Lan et al. 2013) [15]. SC50 is the concentration of test samples that scavenges 50% radicals. IC50 is the concentration of test samples that inhibits 50% tyrosinase. eNot tested. the peptide chain of the enzyme [21]. Compound 7,with antibrowning,buttheyhavebeenallegedtohaveseriousside acatecholmoietyandaconjugatedcarbonylgroup,may effects. For example, vitamin C is unstable and photosensi- beboundtofreeenzymesandformanenzymesubstrate tive. High doses of arbutin induce chronic poisoning of the complex. Thus, let compound 7 exhibit a potent inhibitory skin, resulting in ochronosis [24].Kojicacidlossfunction ∙− effect on XO activity, decreasing O2 generation. The gener- after exposure to sunlight can cause contact allergy after ation of free radicals and reactive oxygen species (ROS) are usage [25]. Seeking new and safe depigmentation agents from involved in various diseases such as hepatitis, inflammation, natural products has been noticed in the past few years. carcinogenesis, and aging [22]. ROS may induce melanocyte Numerousflavonoidshavebeenreportedtoberelativelymild proliferation and the release of 𝛼-melanocyte-stimulating and safe tyrosinase or melanogenesis inhibitors, whereas they hormone (𝛼-MSH). In this case, an excess of melanogenesis exhibit antioxidant, anti-inflammatory, and other biological canleadtoabnormalpigmentation[23]. Hence, the use of activities [19, 26, 27]. Since onopordin (4), cirsiliol (5), radical scavengers or antioxidants to prevent pigmentation and eupafolin (7)displayedapotentantioxidantability, disorders and skin aging in cosmetic and medicinal industries they probably also possess good tyrosinase inhibitory effect. is becoming increasing tendency. Thus, this study also evaluated the antityrosinase activity Many antioxidants and tyrosinase inhibitors are used of the isolates of PNM. Figure 2 and Table 2 showed that in cosmetics and food products for depigmentation or onopordin (4), with an IC50 value of 65.8 ± 3.2 𝜇M, acted The Scientific World Journal 5

400 350 300 250 200 150

(min/O.D.) 100 V /

1 50 0 −12 −4 4 1220283644 −50 1/[xanthine] (mM)−1 −100

Figure 1: Lineweaver-Burk plot of xanthine oxidase inhibition of eupafolin (7) with various concentrations of xanthine. (×) Without eupafolin, (󳵳)5𝜇M of eupafolin, (e)10𝜇M of eupafolin, and (I)20𝜇M of eupafolin. Data are presented as mean ± SD of three independent experiments.

700

600

500 1000 400 800 300 600 200 400 (min/O.D.) V

1/ 100 (min/O.D.) 200 V 1/ 0 0 −4 −2 02468−6 −4 −100 0246810 −200 1/[tyrosine] (mM−1) −2 1/[tyrosine] (mM−1) −400 −200

(a) (b)

Figure 2: Lineweaver-Burk plot of tyrosinase inhibition of (a) onopordin (4) and (b) eupafolin (7) with various concentrations of tyrosine. (a) (X) Without onopordin, (◼)10𝜇M of onopordin, (󳵳)60𝜇M of onopordin, and (×)100𝜇M of onopordin. (b) (◼) Without eupafolin, (󳵳) 28 𝜇M of eupafolin, (×)56𝜇M of eupafolin, and (X)112𝜇M of eupafolin. Data are presented as mean ± SD of three independent experiments.

as a mixed-type inhibitor, while eupafolin (7)actedasa However, further investigations are required to determine competitive inhibitor and strongly inhibited the activity of their mechanisms of action. mushroom tyrosinase, with an IC50 value of 56.0 ± 5.0 𝜇M. Tyrosinaseisabletousemono-,di-,andtrihydroxyphenolsas substrates; among these, dihydroxyphenols (catechols) show 3.2. Optimization of HPLC Conditions. To obtain the chem- the maximum activity, indicating that the enzyme is most ical information and valid chromatographic conditions, the active with catechol as a substrate [28, 29]. Also, the catechol columns, mobile phase compositions, detection wavelength, structure has been reported to bind copper ions and thus and gradient elution procedure were investigated in this may compete with tyrosinase for the available copper ions study. [30], leading to tyrosinase dysfunction. This suggests that a Twokindsofreverse-phasecolumns,LiChroCART250- catechol moiety in the B ring attached to an 𝛼,𝛽-unsaturated 4 C18 column (250 mm × 4.6 mm i.d., 5 𝜇m) and Hypersil carbonyl group forms a skeleton similar to that of l-Dopa. ODS-C18 column (250 mm × 4.6 mm i.d., 5 𝜇m), were inves- Therefore, compounds 4 and 7 might bind to the binuclear tigated; the Hypersil ODS-C18 column was found to be more active site of the enzyme and compete with tyrosine and l- suitableandgavegoodpeakseparationandsharppeaks. Dopa as a substrate. The replacement of the substrate and the The effect of mobile phase compositions (acetonitrile inhibition of tyrosinase activity of compounds 4 and 7 might (ACN)/0.1% FA, M/0.1% FA, and M/0.1% acetic acid (AA)) inhibit the melanogenesis. Thus, onopordin4 ( )andeupafolin on chromatographic separation was investigated and it was (7) with good antioxidant and antityrosinase effects can be found that there was no obviously distinction between considered candidates for cosmetic or therapeutic purposes ACN/0.1% FA, M/0.1% FA, and M/0.1% AA. While adding in humans for aging and hyperpigmentation treatments. 0.1% AA, some peaks overlapped and the resolution of 6 The Scientific World Journal

Table 3: Calibration curves, linear ranges, and LODs and LOQs of onopordin (4)andeupafolin(7)byHPLC.

2 Analytes Calibration curve linear range (𝜇g/mL) 𝑅 (𝑛=3)LOD(𝜇g/mL) LOQ (𝜇g/mL) 4 𝑦 = 509933𝑥 − 1863.7 0.50–15.0 0.9999 0.05 0.26 7 𝑦 = 62667𝑥 + 4543.5 1.0–10.0 0.9994 0.01 0.11

14 4 12 10 8 2 6 7 (B) 4 Intensity (mV) Intensity 2 4 2 7 (A) 0 0 10 20 30 40 50 60 70 80 90 100 110 Retention time (min)

Figure 3: (A) The chromatogram of mixture standard compounds: nodifloretin (2), onopordin (4), and eupafolin (7). (B) Chromatographic fingerprint of PNM (2010-06-PNM) by HPLC-UV at 326 nm. separation was slightly decreased. Considering the high- (LOD) and quantification (LOQ) were determined at signal- toxicity and price of ACN and the better separation condition, to-noise ratios (S/N)of3and10,respectively.Theresultsare the binary mixture of M/0.1% FA was chosen for gradient shown in Table 3. elution. Intra- and interday variability were utilized to evaluate The wavelength for the detection of PNM and the precision. The mixed standard solution at low, medium, active constituents in this plant was selected by the UV- and high concentrations in one day (𝑛=3)andon Vis (200–400 nm) detector. Due to a full-scan experiment of three consecutive days was analyzed, respectively. The results PNM, its UV spectrum showed maximum adsorption at the (Table 4) indicated that the mean intraday R. S. D. values wavelengths of 210, 273, and 326 nm. The chromatographs of compounds 4 and 7 were less than 3.97% and the mean monitored at 210 and 273 nm revealed more peaks than at interday R. S. D. values of compounds 4 and 7 were less than 326 nm within 15–20 min. However, due to the fact that some 6.99%. peaks were missing between 40 and 70 min and due to the Repeatability of this method was assessed by analyzing presence of serious baseline noise, some peaks seen at 210 and five independently prepared samples (2010-06-PNM). R. S. 273 nm were not well separated. Meanwhile, signals response D. values of content and retention time of compounds 4 and for flavones and flavonols is commonly referred to as band 7 were all less than 2.2%, which indicated good repeatability. I (usually 300–380 nm) and band II (usually 240–280 nm). The recovery test was evaluated by standard addi- In order to detect more common peaks while achieving tion method. Onopordin (4, 0.20 mg/g) and eupafolin (7, suitable detection of PNM and active constituents, 326 nm 0.15 mg/g) were spiked into the sample (2010-06-PNM) and was selected as the most appropriate detection wavelength. then extracted and analyzed using the proposed procedure. The HPLC chromatograms of standards and PNM are shown As shown in Table 5, the recoveries of compounds 4 and 7 in Figure 3. were in the range of 93.50–97.33% with an R. S. D. value less than 2.40%. Therefore, this HPLC-UV method was considered precise, accurate, and sensitive enough for the 3.3. Method Validation of Quantitative Analysis. Linearity quantitative evaluation of active compounds 4 and 7 in P. was examined with standard solutions. According to the nodiflora. antioxidant and antityrosinase assay (Table 2), nodifloretin (2), onopordin (4), cirsiliol (5), and eupafolin (7)arethe 3.4. Sample Analysis. The established analytical method was major active compounds, but 5 was in a small amount and applied for the quantitative analysis of two active compo- 2 was not well separated from the unknown side-peak in the nents, 4 and 7, in three different batches of the aerial part tested sample; thus, only compounds 4 and 7 were selected of P. no difl ora collected at the same location in different as the representative markers in this study. Each calibration years and seasons (Figure 4). Each sample was analyzed in curve contained five different concentrations and was per- triplicate to determine the mean content, and the peaks formed in triplicate. The linearity for each compound was in chromatograms were identified by comparing the reten- established by plotting the peak area (𝑦) versus concentration tion times and the online UV spectra with those of the (𝑥) by the external standard method. The limits of detection standards. The contents of compounds 4 and 7 were 0.048 The Scientific World Journal 7

(C)

Intensity (mV) Intensity 7 4 7 (B) 4 (A)

0 102030405060708090100110 Retention time (min)

Table 4: Analytical results of intra- and interday variability of onopordin (4)andeupafolin(7).

Precision (𝑛=3) Analytes Concentration Intraday R.S.D. Interday R.S.D. (𝜇g/mL) (%) (%) 0.8 2.34 1.29 4 6 2.58 4.68 12 2.89 1.30 2 2.31 6.99 7 4.5 3.49 1.50 8.5 3.97 2.95

Table 5: Recoveries of onopordin (4)andeupafolin(7) in the aerial part of P. no difl ora (2010-06-PNM) (𝑛=3).

Analytes Original (mg/g) Spiked (mg/g) Found (mg/g) Recovery (%) R.S.D (%) 4 0.545 0.200 0.732 93.50 2.40 7 0.381 0.150 0.527 97.33 0.91

and 0.072 mg/g in 2007-02-PNM, 0.296 and 0.474 mg/g in about the main significant secondary metabolites represented 2009-06-PNM, and 0.545 and 0.381 mg/g in 2010-06-PNM, by onopordin (4)andeupafolin(7). The results indicated that respectively. The quantitative analysis results showed that the the contents of active constituents varied greatly among the crude extract of PNM, even in the different seasons (spring PNM samples collected in different periods, with the total and summer), generally contained the two selected active contents of active constituents being higher in PNM collected constituents. From the results obtained, the contents of active in the summer. This could be helpful for further evaluation of compounds were higher in the summer than in the spring. It the quality of PNM. is known that the different growing place, climate, and harvest processing may affect the level of these compounds. Although PNM in the autumn and in the winter was not collected and Conflict of Interests detected, the results showed PNM in blossom season (May to August, summer), yielding more active compounds, might The authors declare that there is no conflict of interests. serve the good quality and quantity of PNM when used as a cosmetic or herbal medicine additive. Acknowledgments 4. Conclusions The authors are thankful for the financial support provided A HPLC-UV chromatographic fingerprint analysis of P. by the National Science Council of Taiwan (NSC 99-2320-B- nodiflora was established. The antioxidant assay and the 037-021-MY2) and Bioworld Beauty Development Company chromatogram profile of this plant could provide information Limited Research Foundation (S100004). 8 The Scientific World Journal

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Research Article Flavonoids in Juglans regia L. Leaves and Evaluation of In Vitro Antioxidant Activity via Intracellular and Chemical Methods

Ming-Hui Zhao,1 Zi-Tao Jiang,1 Tao Liu,1 andRongLi2

1 Tianjin Key Laboratory of Food Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China 2 College of Science, Tianjin University of Commerce, Tianjin 300134, China

Correspondence should be addressed to Zi-Tao Jiang; [email protected]

Received 13 April 2014; Revised 28 May 2014; Accepted 2 June 2014; Published 15 July 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 Ming-Hui Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Flavonoids are rich in Juglans regia L. leaves. They have potent antioxidant properties, which have been related to regulating immune function and enhancing anticancer activity. Herein, qualitative and quantitative determination of flavonoids from J. regia leaves was carried out using high performance liquid chromatography coupled with tandem mass spectrometry with electrospray ionization and negative ion detection (HPLC-ESI-MS/MS) by comparison of the retention times and mass spectral fragments with standard substances or related literatures. Seventeen compounds were identified and major components are quercetin-3-O-rhamnoside (453.11 𝜇g/g, dry weight), quercetin-3-O-arabinoside (73.91 𝜇g/g), quercetin-3-O-xyloside (70.04 𝜇g/g), kaempferol-O-pentoside derivative (49.04 𝜇g/g), quercetin-3-O-galactoside (48.61 𝜇g/g), and kaempferol-O-pentoside (48.46 𝜇g/g). The in vitro intracellular antioxidation indicated that flavonoids from J. regia leaves could reduce the reactive oxygen species (ROS) level in RAW264.7 cells and showed good radical scavenging activities. These results proved to be more related to the flavonoids that could be considered in the design of new formulations of dietary supplements or functional foods.

1. Introduction flavonoids. Regarding J. regia leaves, there are a few reports on polyphenolic compounds of J. regia leaves from Por- Juglans regia L. leaves are good sources of flavonoids. tuguesecultivars.Inthepreviousstudies[1, 4, 5], four Flavonoids have potent antioxidant properties, which have flavonoids and five phenolic acids were identified includ- been related to regulating immune function and enhancing ing quercetin-3-O-galactoside, auercetin-3-O-arabinoside, anticancer activity. The plant belonging to the genus of Juglans auercetin-3-O-xyloside, auercetin-3-O-rhamnoside, 3- and from Juglandaceae family is a deciduous tree and native to the 5-O-caffeoylquinic acids, 3- and 4-p-coumaroylquinic acids, region stretching from the Balkans eastward to the Himalayas and p-coumaric acid. In addition, two flavonoids were par- and southwest China. Now it is widely distributed in Asia, tially identified such as quercetin-3-O-pentoside derivative southernandeasternEurope,andtheUnitedStates.Itsfruitis and kaempferol-3-O-pentoside derivative. In addition, the avaluableandnutritionalnut,whoseoilisrichinunsaturated extract of Portuguese cultivars can significantly inhibit the fatty acids, tocopherols, and phytosterols [1]. J. regia leaves growth of Gram-positive bacteria, in particular Bacillus are also used as a traditional medicine in China and Europe cereus, and shows some antioxidant activities [5, 6]. However, and have shown various health benefits for the treatment of to the best of our knowledge, there is no report on flavonoids skin inflammations, venous insufficiency, and ulcers. More- of J. regia grown in other countries and regions including over, the researches in pharmacology and therapeutics have China. Due to the influences of geographical environment, shown that J. regia leaves have hypoglycaemic, antioxidative, temperature, exposure time, rainfall, and other factors, the antimicrobial, and antihypertensive effects [2, 3]. same plant grown in different countries will be very different Recently, a large number of researches are mainly focused in the chemical composition. Therefore, it is also necessary to on the extraction/isolation and the antioxidant effect of study the flavonoids of J. regia leaves grown in China. 2 The Scientific World Journal

This study aimed to qualitatively and quantitatively deter- the analysis, it is necessary to purify the extract by use of AB- mine the flavonoids of native J. regia leaves by HPLC-ESI- 8 macroporous adsorption resin. Purification of the extract MS/MS and hoped to provide a reliable basis for use of this will be beneficial for qualitative and quantitative analyses of plant resource. Ten compounds are first found among all low levels of the compounds. The extract was purified by identified flavonoids from J. regia. Further, the antioxidant use of AB-8 macroporous adsorption resin with a column activities of flavonoids of native J. regia leaves were evaluated chromatographic mode, in which the AB-8 resin was used using intracellular and chemical methods. as an adsorbent and packed in a cylindrical glass column. Initially, an appropriate volume of 1.0mg/mL solution (pH 2. Materials and Methods 2.0) of the extract was dropped into the column at a flow rate of 2 bed volume (BV)/h. Then, whole flavonoids were 2.1. Plant Material. The leaves of J. regia, which were iden- first adsorbed onto the AB-8 resin. The adsorbed flavonoids tified by plant taxonomist Dr. Xuchun Wang, were collected were eluted off the AB-8 resins by 70% ethanol at a 2BV/h from Tianjin, China, in August 2012. The leaves were dried at flow.Ethanolintheeluatewasremovedbyarotarythinfilm ∘ 60 C in a vacuum drying oven, ground to 60–80 mesh size, distillation method. The residue was then freeze-dried and and stored at room temperature in a desiccator until use. the purified flavonoid extract powders from J. regia leaves were obtained. 2.2. Chemicals. Epicatechin, quercetin-3-O-galactoside, and quercetin-3-O-rhamnoside standards were purchased 2.5. HPLC-ESI-MS/MS Analysis. All analyses were per- from Shunbo Co. (Shanghai, China). 5-O-Caffeoylquinic formed using a Zorbax SB-C18 column (250 mm × ∘ acid (neochlorogenic acid), quercetin-3-O-arabinoside, and 4.6 mm i.d., 5 𝜇mparticlesize)at30Cwithabinary quercetin-3-O-glucuronide standards were obtained from phase at a flow rate of 0.3 mL/min. The mobile phase was Yifang S & T Co. (Tianjin, China). All standards were at least a mixture of 0.1% (v/v) formic acid in methanol solutions 98% purity. (phase A) and 0.1% (v/v) formic acid in water (phase B). 󸀠 󸀠 2 ,7 -Dichlorofluorescin diacetate (DCFH-DA) was pur- The gradient of phase A was 50%–72% (0–10 min) and then chased from Beyotime Institute of Biotechnology (Shanghai, washeldfor72%phaseAtotheend.Theinjectionvolumes China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), dimethyl of both the solution of purified flavonoid extract and the sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2)-2,5-di- solution of the mixed standards were 5 𝜇L. The concentration phenylterazolium bromide (MTT) were purchased from of mixed standard substances was 8 𝜇g/mL. ESI was operated Sigma-Aldrich (Shanghai, China). Methanol was purchased in negative ion mode. The working conditions for the ionization source were as follows: a capillary voltage of 4 kV, from Merck (Darmstadt, Germany). Formic acid was pur- ∘ chased from Tedia Co. Inc. (Fairfield, OH, USA). All the a source temperature of 350 C, and a nitrogen dry gas of chemicals used were of analytical or HPLC grade. Deionized 10 L/min and a high purity nitrogen (>99%) was used as water was purified by a Milli-Q system (Millipore, Bedford, nebulizing and collision gas. MA, USA) and used throughout. Qualitative analysis of all compounds in the purified RAW264.7 cells (Monocytic leukemia cell mouse macro- flavonoid extract was carried out by using HPLC-ESI- phage) were obtained from American Type Culture Collec- MS/MS. When the authentic standards were available, the compounds were identified by comparing their retention tion (Manassas, VA, USA). AB-8 macroporous adsorption 2 resinwasobtainedfromNankaiHechengS&TCo.(Tianjin, times and MS spectra with those of the reference standards. China). Otherwise, the compounds were proposed mainly based on the combined data of the following parameters including molecular ions, relative positions in the chromatogram, 2.3. Preparation of Extract from Leaves of J. regia. Ethanol 2 solution (60%, v/v) was used for the extraction. Firstly, 5 g fragmentsreleasedinMS experiments, and by comparison of leave samples was placed in a microwave vitreous flask with the related literatures. and extracted with 175 mL of 60% ethanol. The flavonoids Quantitative analysis of the purified flavonoid extract and other polar compounds were extracted by microwave was performed by HPLC-ESI-MS/MS in multiple reactions treatment for 7 min with a microwave power of 400 W monitoring mode and monitoring the characteristic prod- ∘ 2 at 85 C. Then, the extract was filtered and the fat-soluble uct ions selected from MS spectra. The compounds were components in the filtrate were removed using isopyknic quantified using an external standard method when commer- petroleum ether. Finally, the ethanol extract was concentrated cial standards were available. The linear calibration curves in a rotary evaporator to remove ethanol and then freeze- were performed with at least five different concentrations dried. Thereby, the lyophilized extract from J. regia leaves was of the related reference standards. Calibration curves were obtained. established by plotting the peak areas versus the corresponding concentrations of each injection standard substance. 2.4. Purification of Extract from Leaves of J. regia. The Other compounds were quantified using an internal standard obtained extract contains not only flavonoids but also other method. polar compounds as well as other impurities. In addition, the content of some flavonoids is very low. In order to identify 2.6. Validation of Assay. The limit of detection (LOD) and such low levels of the compounds to improve the accuracy of limit of quantification (LOQ) for each reference standard The Scientific World Journal 3

𝜆 were determined at signal to noise (S/N ratio) of 3 and 10, em 525 nm. Intracellular antioxidant activity was expressed respectively. as relative fluorescence units. The precision of the method was evaluated by analyzing the mixed standard solution or real sample in six replicates. 2.9. DPPH Radical Scavenging Activity. The DPPH free The precision was defined as the relative standard deviation radical scavenging test was analyzed according to previous (RSD, %) from six successive injections. method described by Al-Duais et al. [9]withsomemodifica- The accuracy of method was evaluated by recovery test. tions. An aliquot of 0.5 mL sample solution (2.5–20.0 mg/mL) Appropriate amounts of the mixed standard solutions were was mixed with 4.0 mL of 0.2 mM DPPH ethanol solution. added to the definite amounts of the solution of purified The mixture was left in the dark at room temperature for flavonoid extract. Then, the mixture was processed and 30 min and absorbance was measured at 517 nm. In addition, analyzed using the proposed method. The recovery was 0.5 mL of ethanol with 4.0 mL of 0.2 mM DPPH ethanol calculated according to the following equation: solution was used as the control, and 0.5 mL of sample solution with 4.0 mL of ethanol was used as the blank. Recovery (%) The radical-scavenging activity was calculated based on the following equation: total detection amount − original amount =( ) × 100. 𝐴 −𝐴 added amount ( ) = 100 − 𝑖 𝑏 × 100. Radical scavening activity % 𝐴 (1) 𝑐 (2)

2.7. Cell Viability Assay. The cell viability assay using 𝐴𝑖 is absorbance of sample solution; 𝐴𝑏 is absorbance of the RAW264.7 cells was used to evaluate the cytotoxicity and blank; 𝐴𝑐 is the absorbance of the control. sensitivity of the purified flavonoid extract from J. regia L. The results of the DPPH reduction in the purified leaves according to the previous literature [7]withsome flavonoid extract solution were linearized. The half maxi- 5 modifications. RAW264.7 cells (1×10/well) were seeded mum inhibitory concentration of DPPH radicals (IC50)was into a 96-well clear-walled flat bottomed plate (Nunc A/s, calculated according to the standard curve. All the results Denmark) in 100 𝜇Lofgrowthmedium.Afterbeingincu- were measured three times and expressed in microgram per ∘ bated at 37 C under 5% CO2 for 24 h, the growth medium milliliter. wasremovedandthewellswerewashedwith100𝜇LPBS 𝜇 twice. The cells were then treated with 100 Lofthepurified 2.10. Statistical Analysis. Statistical analysis was undertaken flavonoid extract solutions in different concentrations (0.1– using the general linear model procedure from Statistics 𝜇 1000 g/mL) for another 24 h. In order to avoid the effect of Analysis System (SAS) (Version 9.2, SAS Institute Inc., Cary, sample’s color on the final reading, the medium (containing NC, USA). All determinations were based on three replicate sample) was removed and wells were washed with PBS; samples, and the results for content are shown as mean values. then, 5 mg/mL MTT was added. After 4 h of treatment, an Differences between the means of sample were analyzed by appropriate amount of DMSO was added to dissolve the the least significant differences test at a probability level of MTT formation product. After the crystal was completely 0.05. dissolved, the absorbance was measured at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). 3. Results and Discussion 3.1. Qualitative and Quantitative Analysis. The total ion 2.8. Intracellular Antioxidation Assay. The intracellular anti- chromatogram of the purified flavonoid extract from J. oxidation was performed as previously described by Qian regia leaves is presented in Figure 1(a), and the one of the et al. [8] with some modifications. Briefly, RAW264.7 cells reference substances is shown in Figure 1(b).Basedonthe 5 2 at a density of 1×10 /well were seeded into clear bottomed comparison fragmentation, MS and MS spectra with those black 96-well plate (Nunc A/s, Denmark) in 100 𝜇Lofgrowth of standards, and combining mass spectrometric cleav- ∘ medium and incubated at 37 C under 5% CO2.After24h, ageofcompoundsandtherelatedliteratures,theresults the medium was removed and the wells were washed with of qualitative and quantitative analysis of the seventeen 100 𝜇LPBStwice.Triplicatewellswerethentreatedfor compounds are shown in Table 1. In general terms, a coum- 1h with 100𝜇L of the purified flavonoid extract solutions arin compound, two phenolic acids, and fourteen flavonoids with different concentrations (final concentration ranges, 25– were determined including 5-O-caffeoylquinic acid, epicat- 500 𝜇g/mL) plus 10 𝜇M DCFH-DA. After this incubation, all echin, 3-p-coumaroylquinic acid, syringetin-O-hexoside, wellswerewashedwithPBStoremoveanytracesofDCFH- myricetin-3-O-glucoside, myricetin-3-O-pentoside, escu- DA and 100 𝜇Lof300𝜇MH2O2 was applied to the cells. The letin, taxifolin-pentoside, quercetin-3-O-glucuronide, quer- blankwellsweretreatedwithDCFH-DAandgrowthmedium cetin-3-O-galactoside, quercetin-3-O-pentoside derivative, without H2O2, and the control cells were treated with DCFH- quercetin-3-O-xyloside, quercetin-3-O-arabinoside, querce- DA and H2O2. The outer wells were filled with PBS. The tin-3-O-rhamnoside, kaempferol-O-pentoside, kaempferol- fluorescenceineachwellwasmeasuredat5minintervals O-pentoside derivative, and kaempferol-O-rhamnoside. 𝜆 over a 1 h period by a microplate reader with ex 488 nm and As far as we know, epicatechin, syringetin-O-hexoside, 4 The Scientific World Journal

Table 1: The retention time, fragment ions, tentative identification, and quantification results of flavonoids and phenolic acidsin Juglans regia L. Leaves. − [M–H] 2 Content Peak 𝑅𝑡 (min) MS (𝑚/𝑧) Compound Reference (𝑚/𝑧) (𝜇g/g)e 1 8.7 353 191, 179, 135 5-O-Caffeoylquinic acid (neochlorogenic acid) 29.72 ± 1.85 Std. 2 9.0 289 202.2, 123.0 Epicatechina 8.86 ± 1.87 Std. 3 9.5 337 191, 163, 118.9 3-p-Coumaroylquinic acid 6.72 ± 0.13b Ref. [10, 11] 4 11.1 507 327.3 Syringetin-O-hexosidea 5.99 ± 0.47c Ref. [12] 5 11.2 339 158.6 Unknowna 38.63 ± 3.00c 6 12.0 479 316 Myricetin-3-O-glucosidea 21.73 ± 1.69c Ref. [12, 13] 7 13.1 449 316 Myricetin-3-O-pentosidea 8.88 ± 0.69c Ref. [13, 14] 8 13.2 177 131.3, 115.2, 92.3 Esculetina 9.79 ± 0.76c Ref. [15, 16] 9 13.3 435 285, 150.8 Taxifolin-pentosidea 18.23 ± 1.42c Ref. [17, 18] 10 13.5 477 300.9, 150.8 Quercetin-3-O-glucuronidea 4.30 ± 0.23c Std. 11 13.8 463 300.0, 270.8 Quercetin-3-O-galactoside (hyperoside) 48.61 ± 0.11 Std. 12 14.3 433 300, 270.9 Quercetin-3-O-pentoside derivative 16.31 ± 0.24d Ref. [1, 5, 11] 13 14.7 433 299.7, 271.1 Quercetin-3-O-xyloside 70.04 ± 2.77d Ref. [1, 11, 19, 20] 14 14.9 433 301.0, 299.9, 270.8 Quercetin-3-O-arabinoside 73.91 ± 0.32 Std. 15 15.3 447 300.1, 284.2, 254.9 Quercetin-3-O-rhamnoside (huercitrin) 453.11 ± 11.65 Std. 16 16.2 417 285.0, 254.7, 226.9 Kaempferol-O-pentosidea 48.46 ± 3.78c Ref. [21, 22] 17 16.8 417 284.7, 254.7 Kaempferol-O-pentoside derivative 49.04 ± 1.82c Ref. [11, 21, 22] 18 17.4 431 285.2 Kaempferol-O-rhamnosidea 28.96 ± 1.26c Ref. [23, 24] aCompounds identified for the first time in J. regia leaves; bexpressed in equivalents of 5-O-caffeoylquinic acid; cexpressed in equivalents of quercetin-3-O- rhamnoside; dexpressed in equivalents of quercetin-3-O-arabinoside; emean ± RSD; Ref.: literature where the compound has been characterized by MS analysis; Std.: standard.

acids were described to exist in Portuguese cultivars, namely, 11 15 10 14 3-O-caffeoylquinic acids, 4-p-coumaroylquinic acids, and 9 17 1 8 13 p-coumaric acid, but these compounds were not detected in 4 5 16 18 2 6 7 the present study. 3 12 Moreover, according to the results of quantitative analy- Relative intensity Relative sis, the major components in J. regia leaves are quercetin-3- 0 246810121416182022 O-rhamnoside (453.11 𝜇g/g, dry weight), quercetin-3-O-ar- Time (min) abinoside (73.91 𝜇g/g), quercetin-3-O-xyloside (70.04 𝜇g/g), 𝜇 (a) kaempferol-O-pentoside derivative (49.04 g/g), and quercetin-3-O-galactoside (48.61 𝜇g/g), while in Portuguese J. regia

1 14 leaves are quercetin-3-O-galactoside, 3-O-caffeoylquinic 10 acids, quercetin-3-O-xyloside, 5-O-caffeoylquinic acids, and 2 15 11 quercetin-3-O-arabinoside. The obvious differences existed in chemical composition of flavonoids and phenolic acids

Relative intensity Relative between Chinese cultivar and Portuguese cultivars. 0 246810121416182022 Time (min) 3.2. Validation of Assay. The LOD and LOQ of the identified (b) compoundswereintherangeof1.85–9.93ng/mLand6.18– 33.11 ng/mL, respectively. It could be seen that the LOQ Figure 1: (a) Total ion chromatogram of the purified flavonoid values for all compounds were very low, which indicated the extract from Juglans regia L. leaves and (b) total ion chromatogram proposed method was sufficiently sensitive. of the mixture of reference substances. Peak numbers refer to Table 1. The precision of the method expressed in terms of RSD was lower than 5% from six replicate injections. The recoveries of these detected compounds were between 94.34% and myricetin-3-O-glucoside, myricetin-3-O-pentoside, escule- 96.5% with RSD of 0.15–1.87%. The described above results tin, taxifolin-pentoside, quercetin-3-O-glucuronide, kaemp- indicated that the developed method for the quantitative ferol-O-pentoside, and kaempferol-O-rhamnoside are analysis of flavonoids in J. regia leaves was suitable and reported in this species for the first time. Some other phenolic accurate. The Scientific World Journal 5

350 of the absorption at 517 nm. A linearity relationship between DPPH radical scavenging activity and the concentrations of 300 2 the purified flavonoid extract (𝑦 = 2.4453𝑥 − 4.8043; 𝑅 = 0.9994) 250 was obtained. The values of radical scavenging rate of the purified flavonoid extract were in the ranges of 1.06%– 200 56.33%. With the increase of the concentration of the extract, the scavenging activity was strengthened. The IC50 value was 150 22.41 𝜇g/mL.

100 4. Conclusions Relative ﬂuorescence intensity ﬂuorescence Relative 50 The present study suggested that the methods herein 0 employed were useful and accurate for characterization of 0 10 20 30 40 50 60 the flavonoids in J. regia leaves. Seventeen compounds were Treatment time (min) identified, of which nine compounds including epicatechin, Control 250 𝜇g/mL syringetin-O-hexoside, myricetin-3-O-glucoside, myricetin- 25 𝜇g/mL 500 𝜇g/mL 3-O-pentoside, esculetin, taxifolin-pentoside, quercetin-3- 50 𝜇g/mL Blank O-glucuronide, kaempferol-O-pentoside, and kaempferol- 100 𝜇g/mL O-rhamnoside are reported for the first time. Furthermore, the flavonoids in J. regia leaves were not cytotoxic, which Figure 2: Intracellular radical scavenging activities of the purified did not significantly inhibit the growth of RAW264.7 cells. flavonoid extract from leaves of Juglans regia L. in RAW264.7 cells. ± These flavonoids showed strong radical scavenging activities Data are reported as the mean SD of three replicates. both intracellularly and extracellularly. The results provided a theoretical basis for a further study on the natural extracts from J. regia leaves. 3.3. Cell Viability Assay. The reductases in living RAW264.7 cells could convert MTT into purple-colored formazan dye. Basedonthisreason,thenumberoflivingRAW264.7cells Conflict of Interests could be expressed as enzyme activity level. Relative cell The authors declare that there is no conflict of interests viabilities are 95.65% and the standard deviation of three regarding the publication of this paper. replicates is ±4.59.Itisdifficulttofindanobviousdifference between high concentration group (1000 𝜇g/mL) and low concentration group (0.1 𝜇g/mL) of the purified flavonoid Acknowledgments extract. This indicated that the purified flavonoid extract wasalmostnoharmtoRAW264.7cellsandtheintakedose The present research was financially supported by the Key was also independent of the concentrations of the purified Natural Science Foundation of Tianjin (Grant no. 120029) flavonoid extract in the ranges of 0.1 𝜇g/mL–1000 𝜇g/mL. and the Science and Technology Development Foundation of Tianjin Universities (Grant no. 20110608). 3.4. Intracellular Antioxidant Activity. In this assay, DCFH- DA was used as a marker to evaluate the level of intracellular References reactive oxygen species (ROS). Nonfluorescent DCFH-DA dyecouldfreelypenetrateintocellsandwashydrolyzedinto [1] J. S. Amaral, P. Valentao,P.B.Andrade,R.C.Martins,andR.˜ DCFH by intracellular esterase. Then, DCFH was oxidated M.Seabra,“Docultivar,geographicallocationandcropseason influence phenolic profile of walnut leaves?” Molecules,vol.13, to form fluorescent DCF by cellular ROS (generated by no. 6, pp. 1321–1332, 2008. H2O2). So the fluorescence intensity of DCF could reflect the level of cellular ROS. As shown in Figure 2,thescavenging [2]M.Cheniany,H.Ebrahimzadeh,K.Vahdati,J.E.Preece,A. effects of the purified flavonoid extract on cellular ROS Masoudinejad, and M. Mirmasoumi, “Content of different groups of phenolic compounds in microshoots of Juglans regia were compared with H2O2 nonstimulated blank and sample cultivars and studies on antioxidant activity,” Acta Physiologiae nontreated control groups. The treatment of the purified Plantarum,vol.35,no.2,pp.443–450,2013. flavonoid extract could decrease the fluorescence intensity of DCF. This indicated that the purified flavonoid extract could [3] M. Gˆırzu, A. Carnat, A.-M. Privat, J. Fialip, A.-P.Carnat, and J.- L. Lamaison, “Sedative effect of walnut leaf extract and juglone, be absorbed into cells and showed strong scavenging activity an isolated constituent,” Pharmaceutical Biology,vol.36,no.4, to ROS. pp. 280–286, 1998. [4] J. S. Amaral, S. Casal, J. A. Pereira, R. M. Seabra, and B. P. P. 3.5. DPPH Radical Scavenging Activity. The DPPH free radi- Oliveira, “Determination of sterol and fatty acid compositions, calscavengingtestprovidedaneasyandrapidwaytoevaluate oxidative stability, and nutritional value of six walnut (Juglans the antiradical activities of antioxidants. The antioxidants regia L.)cultivarsgrowninPortugal,”JournalofAgriculturaland could scavenge the DPPH radicals, which caused a reduction Food Chemistry,vol.51,no.26,pp.7698–7702,2003. 6 The Scientific World Journal

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Research Article Chemical Characterization of Fruit Wine Made from OblaIinska Sour Cherry

Milica PanteliT,1 Dragana DabiT,2 Saša MatijaševiT,3 Sonja DavidoviT,3 Biljana DojIinoviT,4 Dušanka MilojkoviT-Opsenica,1 Civoslav TešiT,1 and Maja NatiT1

1 Faculty of Chemistry, University of Belgrade, P.O. Box 51, 11158 Belgrade, Serbia 2 Innovation Center, Faculty of Chemistry Ltd, University of Belgrade, 11158 Belgrade, Serbia 3 FacultyofAgriculture,UniversityofBelgrade,Nemanjina6,Zemun,11080Belgrade,Serbia 4 Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, 11000 Belgrade, Serbia

Correspondence should be addressed to Maja Natic;´ [email protected]

Received 31 March 2014; Accepted 2 June 2014; Published 29 June 2014

Academic Editor: Wanchai De-Eknamkul

Copyright © 2014 Milica Pantelic´ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This paper was aimed at characterizing the wine obtained fromcinska, Oblaˇ a native sour cherry cultivar. To the best of our knowledge, this is the first paper with the most comprehensive information on chemical characterization of Oblacinskaˇ sour cherry wine. The chemical composition was characterized by hyphenatedhromatographic c methods and traditional analytical techniques. A total of 24 compounds were quantified using the available standards and another 22 phenolic compounds were identified based on the accurate mass spectrographic search. Values of total phenolics content, total anthocyanin content, and radical scavenging −1 −1 activity for cherry wine sample were 1.938 mg gallic acid eqv L , 0.113 mg cyanidin-3-glucoside L , and 34.56%, respectively. In general, cherry wine polyphenolics in terms of nonanthocyanins and anthocyanins were shown to be distinctive when compared to grape wines. Naringenin and apigenin were characteristic only for cherry wine, and seven anthocyanins were distinctive for cherry wine.

1. Introduction anthocyanins are considered as major group of phenolic compounds [8]. This class of compounds was shown Oblacinskaˇ sour cherry is a native cultivar. High fruit quality, to prevent cardiovascular diseases and to possess strong self-fertility, and high demand in domestic and foreign fruit anti-inflammatory and anticarcinogenic activity [9, 10]. In markets makes the Oblacinskaˇ sour cherry the most planted the literature, several papers reported cyanidin-3-glucoside, cultivar in Serbian commercial orchards [1]. The fruit is of cyanidin-3-rutinoside, cyanidin-3-glucosylrutinoside, cya- “morello” type, small to medium in size, with dark red and nidin-3-soph-oroside, pelargonidin-3-glucoside, peonidin- thin skin. The flesh is red, medium-firm, juicy, quite sour 3-rutinoside and cyanidin-3-arabinosylrutinoside as main aromatic and of high quality [2]. Serbia is one of the leading anthocyanins in sour and sweet cherry fruits [5, 10, 11]. world producers of this commercially very important fruit Sour cherries are characterized with sufficient acid level species [3]. and are preferred for winemaking, using very similar proce- Generally, cherries are important sources of polyphe- dure to the grape wine making process [4]. Continuing our nols such as anthocyanins, flavan-3-ols and flavonols [4]. research in the field of chemical characterization of fruits Also, they are characterized by a high phenolic acid con- and wines from Serbia we found interesting to screen phy- tent, especially hydroxycinamic and hydroxybenzoic acid tochemical composition of wine obtained from Oblacinskaˇ derivates [5, 6]. Wide ranges of phenolic secondary metabo- sourcherrycultivar.Previouslywehavereportedonchemical lites are associated with antioxidizing capacity, nutritional, composition of peach wine produced from Redhaven cultivar and therapeutic value of cherries [7]. In sour cherries belonging to same genus (Prunus)ascherry[12]. Special 2 The Scientific World Journal attention was given to composition of polyphenols, an impor- grape wines (Vranac-East Serbia (W1), Cabernet Sauvignon- tant group of phytochemicals which are reported to be associ- Central Serbia (W2), Cabernet Sauvignon-East Serbia (W3), ated with antioxidant activity and have several health effects. Cabernet Sauvignon-North Serbia (W4) and Frankovka- In this work, two methods based on hyphenated techniques, North Serbia (W5)). which combine chromatographic and spectral methods, were Cherry wine was made using technological procedure for used for target search and establishing polyphenolic profiles. red wine production, which includes sour cherry mushing Ultra High Performance Liquid chromatography (UHPLC) (disintegration), mushed sour cherry maceration, alcoholic coupledwithhybridmassspectrometerwhichcombinesthe fermentation and removal of skins, seeds, and stalks from Linear Trap Quadrupole (LTQ) and OrbiTrap mass analyzer the wine. Harvesting of Oblacinskaˇ sour cherry was done was used to identify phenolics. Quantification of phenolics at its commercial ripening stage, when fruits showed the was done using UHPLC coupled with a diode array detector highest values of soluble solids (which were proved by hand (DAD) and connected to a triple-quadrupole mass spectrom- refractometer) having dry stem scar. Also, fruits showed eter. Also, the overall antioxidant activities, total phenolic uniform color across at least 90% of the fruit skin with content (TPC), total anthocyanin content (TAC), and mineral the characteristic traits of a given cultivar. Harvest was content were determined. In order to gain insight into the followed by mushing, disintegration of the fruit and disposal value of the polyphenolic composition and nutritional quality of husks and stalks in fermentation containers. Temperature- ∘ of cherry wine, red wines produced from three different grape controlled fermentors were used for fermentation (18–20 C) cultivars were also analyzed. The results obtained for cherry with appropriate enzymes facilitating separation of colored wine were compared with the results of selected red wines. and aromatic matters during the maceration.

2.4. Samples Preparation. All wine samples were filtered 2. Materials and Methods through a 0.45 𝜇m PTFE filters before analyzing. For all tests, ∙ 2.1. Materials and Chemicals. 2,2-Diphenyl-1-picrylhydrazyl samples were appropriately diluted. Solid phase extraction ∙ (DPPH ), standards of gentisic acid, (+)-catechin, (+)-gallo- (SPE) was used for separation of anthocyanin and non- catechin, aesculin, (−)-epigallocatechin, p-hydroxybenzoic anthocyanin fraction. First, C18 cartridges were precon- − − ditioned by passing through 10 mL ethyl acetate, 10 mL acid, protocatechuic acid, ( )-epicatechin, ( )-gallocatechin 3 gallate, rutin, ellagic acid, naringin, (−)-epigallocatechin methanol, and 10 mL of 0.1 mol/dm aqueous HCl, sequentially. Then, the amounts of 0.5 mL of extracts were applied. gallate, myrcetin, quercetin, naringenin, luteolin, chrysin, 3 pinocembrin, galangin, trans-resveratrol, apigenin, and hes- Cartridges were washed with 10 mL of 0.1 mol/dm aqueous peretinwerepurchasedfromFlukaAG(Buch,Switzerland). HCl in order to remove sugars, acids, and other water- The other compounds namely cis,trans-abscisic acid, gallic soluble compounds. Cartridges were dried by allowing a acid, chlorogenic acid, caffeic acid, p-coumaric acid, nitric currentofnitrogengastopassthrough,for5minutes,and acid, and hydrogen peroxide were supplied by Sigma Aldrich after that, rinsed with 5 mL ethyl acetate in order to collect (Steinheim, Germany). Methanol (HPLC grade), acetonitrile, non-anthocyanins fraction. The adsorbed anthocyanins were and formic acid (both of them MS grade), sodium car- eluted from the cartridges with 1 mL acidic methanol. bonate, potassium chloride, acetic acid, hydrochloric acid, sodium acetate, ethyl acetate, and Folin-Ciocalteu reagent 2.5. Determination of TPC. The concentration of total soluble were purchased from Merck (Darmstadt, Germany). Ultra- phenolics was determined spectrophotometrically on a UV- pure water (TKA Germany MicroPure water purification Vis spectrophotometer (GBC UV-Visible Cintra 6) and was −1 system, 0.055 𝜇Scm ) was used to prepare standard solu- measured using Folin-Ciocalteu method, with some modi- tions and dilutions. All other reagents were of analytical fications13 [ ]. This reagent oxidizes phenols present in the grade. Syringe filters (13 mm, PTFE membrane 0.45 𝜇m) were solution. An aliquot (0.1 mL) of appropriately diluted samples purchased from Supelco (Bellefonte, PA, USA). The SPE and a standard solution of gallic acid were mixed with 6 mL cartridges Strata C18-E (500 mg/3 mL) were obtained from deionized water and 0.5 mL of Folin-Ciocalteu reagent. After 3 Phenomenex (Torrance, CA, USA). 6min,1.5mLof2mol/dm sodium carbonate was added with ∘ mixing. After incubation for 60 min at 40 C, the absorbance −1 wasmeasuredat765nmanditwasproportionaltothetotal 2.2. Preparation of Standard Solutions. A 1000 mg L stock quantity of phenolic compounds. Gallic acid was used as a solution of a mixture of polyphenolics and cis,trans-abscisic standard. TPC was expressed as the g gallic acid equivalent acid were prepared in methanol. Dilution of the stock (GAE) per L of sample. solution with methanol yielded the working solution at concentrations of 0.025, 0.050, 0.100, 0.250, 0.500, 0.750, and −1 1.000 mg L . Calibration curves were obtained by plotting 2.6. Determination of TAC. In an acid environment there is thepeakareasofthecompoundsidentifiedagainstthe a balance between the two forms of anthocyanins, colored concentration of the standard solution. and the colorless, depending on the pH. Total anthocyanin contents were determined using the pH-differential method, by measuring the absorbance of the sample at pH = 1 (KCl, −1 −1 2.3. Wine Samples. The tests were performed on one sample 0.025 mol L ) and pH = 4.5 (NaOAc/HOAc, 0.4 mol L ) of cherry wine-East Serbia (SCW) and five samples of [14]. Measurements were performed at two wavelengths, at The Scientific World Journal 3

520nm and 700nm, against a blank cell filled with dis- 4000 V; sheet gas pressure 40 arbitrary units, sweep pressure tilled water. Anthocyanin concentrations were calculated and 0 arbitrary units, and auxiliary gas pressure was 8 arbitrary ∘ expressed as g malvidin-3-glucoside (mal-3-glu) equivalents units; capillary temperature at 300 C; skimmer offset was per 1 L sample. The total absorbance of the extract was 0 V. Mass spectrometry data were acquired in a negative determined by the formula: mode. Collision-induced fragmentation experiments were performed using argon as the collision gas, and collision 𝐴 = [(𝐴 −𝐴 ) −(𝐴 −𝐴 ) ]. 520 700 pH1 520 700 pH4,5 (1) energy was set to 30 eV. Selected reaction monitoring (SRM) experiment for quantitative analysis was performed using two 2 The content of total anthocyanins (TAC) was determined MS fragments for each compound, which were previously through the following formula: defined as dominant in PIS (precursor ion scan) experiments. UHPLC ± MS/MS Orbitrap analysis of polypheno- −1 (𝐴⋅MW ⋅ DF ⋅ 1000) ( ) = , lic compounds were performed using a Thermo Scien- TAC mg L (𝜀⋅1) (2) tific liquid chromatography system consisting of a qua- where 𝐴 is absorbance, MW is molecular weight (MW = ternary Accela 600 pump and Accela Autosampler, con- −1 493.2 g mol for malvidin-3-glucoside), DF is dilution factor, nected to a linear ion trap-orbitrap hybrid mass spec- 1 is cuvette path length in cm, 𝜀 is molar absorptivity (𝜀 = trometer with heated-electrospray ionization probe (HESI- −1 −1 II, ThermoFisher Scientific, Bremen, Germany). Phenolics 28000 Lmol cm for malvidin-3-glucoside). were identified and quantified in wine samples according to the corresponding spectral characteristics: mass spectra, 2.7. Determination of the RSA. Radical scavenging activity, exact mass, characteristic fragmentation, and characteristic the ability to scavenge DPPH free radicals, was determined retention time. Xcalibur software (version 2.1) was used spectrophotometrically [15]. Amounts of 0.1 mL of appro- for instrument control, data acquisition and data analysis. priately diluted samples solutions were mixed with 4 mL Internet database of accurate mass spectrometry data, Chem- of methanol DPPH radical solution. The mixtures were Spider (http://www.chemspider.com/),wasusedasarefer- placed in the dark, at room temperature. After an incubation ence library to identify compounds of interest. MS spectra period of 30min, the absorbance was measured at 515nm to ∙ were acquired by full range acquisition covering 100–1000 −1 determine the concentration of remaining DPPH .Relative mz . For fragmentation study, a data dependant scan was antioxidant activity was calculated using the following for- performed by deploying the collision-induced dissociation mula: (CID). The normalized collision energy of the collision- (𝐴 −𝐴 ) induced dissociation (CID) cell was set at 35 eV. ( ) =( DPPH sample ) ⋅ 100, (3) Separation of polyphenolics was performed on a Hyper- RSA % 𝐴 DPPH sil gold C18 (100 × 2.1 mm, 1.9 𝜇m) from Thermo Fisher 𝐴 Scientific. The mobile phase consisted of (A) water +0.1% DPPH is the absorbance of methanol solution of DPPH formic acid and (B) acetonitrile + 0.1% formic acid. A linear 𝐴 −1 radical, sample istheabsorbanceinthepresenceofwine. gradient program at a flow rate of 0.300 mL min was used: Measurements were performed in triplicate and the results 0.0–1.0 min 5% B, 1.0–9.9 min from 5% to 95% (B), 9.9-10 min were expressed as mean values. from95%to5%(B),then5%(B)for3min.Theinjection volume was 5 𝜇L. The mass spectrometer was operated in 2.8. Identification of Polyphenolic Compounds Using LC- negative ionization mode. HESI-source parameters were as MS/MS Analysis. All experiments were performed using a follows: source voltage 3 kV, capillary voltage −20 V, tube ∘ Thermo Fisher Scientific instruments. lens voltage −150 V, capillary temperature 275 C, sheath and Separation, determination and quantification of com- auxiliary gas flow (N2) 30 and 8 (arbitrary units). pounds of interest in each sample were performed using Separation of anthocyanins was performed on a Hypersil Dionex Ultimate 3000 UHPLC system equipped with a diode gold C18 (100 × 2.1 mm, 1.9 𝜇m) from Thermo Fisher Scien- array detector (DAD) and connected to a triple-quadrupole ∘ tific. The mobile phase consisted of (A) water + 1% formic mass spectrometer. Elution was performed at 40 Con acid and (B) acetonitrile. A linear gradient program at a × 𝜇 −1 Syncronis C18 column (100 2.1 mm, 1.7 mparticlesize, flow rate of 0.300 mL min was used: 0.0–2.0 min 5% B, Thermo Fisher Scientific, USA). The mobile phase consisted 2.0–12.0 min from 5% to 95% (B), 12.0–12.2 min from 95% of (A) water + 0.2% formic acid, and (B) acetonitrile, which to 5% (B), then 5% (B) for 3 min. The injection volume were applied in the following gradient elution: 0.0–2.0 min was 5 𝜇L. The mass spectrometer was operated in positive 5% B, 2.0–12.0 min from 5% to 95% (B), 12.0-12.1 min from ionization mode. HESI-source parameters were as follows: 95%to5%(B),then5%(B)for3min.Theflowratewasset source voltage 5 kV, capillary voltage 40 V, tube lens voltage −1 ∘ to 0.4 mL min and the detection wavelength to 280 nm. The 125 V, capillary temperature 275 C, sheath and auxiliary gas 𝜇 injection volume was 5 L. flow (N2) 30 and 8 (arbitrary units). Quantitative analysis of each sample was performed on a TSQ Quantum Access Max triplequadrupole mass 2.9. Determination of Minerals. All wine samples were evap- spectrometer (Thermo Fisher Scientific, Basel, Switzerland), orated down to half their original volume by rotary evapora- ∘ equipped with heated electrospray ionization (HESI) source. tion under reduced pressure at 40 Candthesesolutionswere The ion source settings were set as follows: spray voltage was prepared by microwave digestion using an Ethos 1 microwave 4 The Scientific World Journal

E P 100 R K 80 H 60 M L O S U G I T W 40 F N V B C J Relative Relative 20 A D abundance 0 0123456789101112131415 Time (min)

Figure 1: UV chromatogram of investigated standards at 280 nm: Gallic acid (A); Protocatechuic acid and (+)-Gallocatechin (B); Aesculin (C); (−)-Epigallocatechin (D); p-Hydroxybenzoic and Gentisic acid (E); Chlorogenic acid (F); (+)-Catechin (G); Caffeic acid (H);− ( )-Epicatechin (I); (−)-Gallocatechin gallate (J); Rutin (K); p-Coumaric and Ellagic acid (L); Naringin (M); (−)-Epigallocatechin gallate (N); Myrcetin (O); cis,trans-Abscisic acid (P); Luteolin (R); Quercetin and trans-Resveratrol (S); Naringenin (T); Apigenin (U); Chrysin and Pinocembrin (V); Hesperetin and Galangin (W).

Table 1: Total phenolics contents, total anthocyanin contents, and with the content of total phenolics and anthocyanins. The radical scavenging activity in six wine samplesa. results show satisfactory correlation coefficient for TPC −1 −1 and RSA, indicating a statistically strong linear relationship Sample TPC (g GAE L )TAC(gmal-3-gluL)RSA(%) (𝑟 = 0.990). General conclusion can be made regarding ± ± ± SCW 1.94 0.04 0.12 0.01 34.56 0.18 comparison of TPC, TAC and RSA values: in the case of W1 1.76 ± 0.02 0.08 ± 0.01 32.06 ± 0.09 cherry wine all the values are in the range of the results W2 2.28 ± 0.11 0.17 ± 0.02 46.41 ± 0.20 obtained for all investigated wines. W3 2.50 ± 0.11 0.10 ± 0.01 48.60 ± 0.13 W4 1.19 ± 0.01 0.17 ± 0.02 21.00 ± 0.05 3.2. Quantitative and Qualitative Characterization of Wine W5 1.69 ± 0.02 0.22 ± 0.02 31.21 ± 0.24 Samples. Great help in determining the polyphenolic profiles aThe values shown are mean ± standard deviation of three replications. comes from the hyphenated techniques, which combine chromatographic and spectral methods. Here UHPLC-DAD MS/MS and UHPLC ± MS/MS Orbitrap analysis were uti- system (Advanced Microwave Digestion System, Milestone, lized in order to obtain a comprehensive picture of individ- Italy). About 10 g of evaporated solutions, 5 mL of 65% HNO3 ual flavonoids and phenolic acids, both in qualitative and and 1 mL 30% H2O2 were mixed and transferred by pouring quantitative sense. A total of 24 compounds were quantified into the microwave digestion vessel. After effervescence using the available standards (Table 2). UV chromatogram subsided samples were cooled for five minutes, transferred of investigated standards at 280 nm is shown in Figure 1. into volumetric flasks, and diluted to 25 mL with deionized The limits of detection and quantification, and the recoveries H2O. Blank was prepared in the same way. All analyses of the analytes were determined according to the method described by Gaˇsicetal.[´ 17]. Calibration curves revealed were performed on a Thermo Scientific iCAP 6500 Duo ICP 2 (Thermo Fisher Scientific, Cambridge, UK). good linearity, with 𝑅 values exceeding 0.99 (peak areas vs. concentration). Recoveries determined for phenolic acids were 60% to 80%, while for flavonoids they were 80% to 120%. 3. Results and Discussion Additionally, 22 phenolic compounds were identified based on accurate mass search. In the absence of standards, the 3.1. Determination of TPC, TAC and RSA. Total phenolic identification of the corresponding compound was based on contents, total anthocyanin contents, and antioxidant activity − the search for the [M–H] deprotonated molecule together of six wine samples are listed in Table 1.Basedonthese with the interpretation of its fragmentations. Their mean results,itcanbeseenthatthehighestTPCvalueispresented −1 expected retention times (𝑡𝑅), calculated mass, found mass, in wine Cabernet Sauvignon-East Serbia (2.50 g GAE L mean mass accuracy (ppm), and MS/MS fragments for each wine), followed by the Cabernet Sauvignon-Central Serbia −1 of the identified compound and their distribution in wines (2.28 g GAE L wine). The lowest TPC was recorded in −1 are summarized in Table 3. Cabernet Sauvignon-North Serbia (1.19 g GAE L wine). −1 Characteristic compounds are specified and major dif- TPCvalueforcherrywinewas1.94gGAEL wine, which is ferences between cherry wine and grape wine samples are in agreement with the data found in the literature [16]. The emphasized in the following discussion. Total of 7 phenolic anthocyanin contents in studied wine samples were in the −1 acids were quantified in cherry wine using available standards range from 0.08 g mal-3-glu L wine (in Vranac) to 0.22 g (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, gen- −1 mal-3-glu L wine (Frankovka). All wine samples showed tisic acid, chlorogenic acid, caffeic acid, and p-coumaric acid). distinct radical-scavenging activities. The highest values were Two isomers of caffeoylquinic acids and syringic acid were found for Cabernet Sauvignon-East Serbia (48.60%) and found based on accurate mass search. Here, it is important to Cabernet Sauvignon-Central Serbia (46.41%) and the lowest point out a higher content of protocatechuic acid, chlorogenic for Cabernet Sauvignon-North Serbia (21.00%). The results acid, caffeic acid, and p-coumaricacidinwineproduced obtained for the relative antioxidant activity were compared from sour cherry when compared to grape wine samples (see The Scientific World Journal 5

−1 Table 2: Contents of phenolics and cis,trans-abscisic acid (mg kg ) in wine samples (ND = not detected compound). Results are expressed −1 as mg L .

Compound 𝑡𝑅, min SCW W1 W2 W3 W4 W5 Gallic acid (A)a 1.79 1.10 28.57 20.73 30.39 12.56 20.86 Protocatechuic acid (B) 3.73 23.89 7.93 6.11 4.52 5.38 5.19 (−)-Gallocatechin (B) 3.78 ND ND 4.62 6.02 5.47 2.59 Aesculin (C) 4.78 0.35 0.54 0.37 0.40 0.36 0.45 (−)-Epigallocatechin (D) 4.89 1.01 0.87 0.87 1.07 0.91 0.82 𝑝-Hydroxybenzoic acid (E) 5.10 6.65 4.30 7.45 5.34 0.37 10.45 Gentisic acid (E) 5.12 0.27 0.16 0.42 0.24 1.00 0.14 Chlorogenic acid (F) 5.22 3.57 0.60 0.58 0.58 ND 0.58 (+)-Catechin (G) 5.30 1.31 3.54 5.86 6.13 5.21 14.09 Caffeic acid (H) 5.52 13.88 1.56 2.35 1.25 1.42 1.60 (−)-Epicatechin (I) 5.65 3.92 ND 3.26 3.20 1.86 6.86 (−)-Gallocatechin gallate (J) 5.79NDNDNDNDND2.79 Rutin (K) 6.08 0.23 0.23 0.23 0.24 0.23 ND 𝑝-Coumaric acid (L) 6.20 23.42 3.62 7.77 3.19 5.86 2.98 Ellagic acid (L) 6.24 ND 2.16 2.73 1.99 2.04 1.79 Naringin (M) 6.46 ND 0.31 0.90 0.90 0.79 0.97 (−)-Epigallocatechin gallate (N) 6.79 ND 2.58 0.90 2.36 1.03 ND Myricetin (O) 6.93 ND 0.21 0.22 0.28 0.25 0.29 cis, trans-Abscisic acid (P) 7.43 1.06 0.17 0.30 0.21 0.11 0.10 Quercetin (S) 7.60 ND ND ND 0.03 ND ND Resveratrol (S) 7.65 ND ND ND 8.83 ND ND Naringenin (T) 8.06 0.15 ND ND ND ND ND Apigenin (U) 8.20 0.06 ND ND ND ND ND Hesperetin (W) 9.51 ND 0.27 ND 0.39 0.42 ND aCorresponding to Figure 1.

Table 2) which is in accordance with findings reported for the interpretation of its fragmentations (Table 4). Antho- the sour cherry fruit [18]. Total content of phenolic acids in cyanin profile of cherry wine was shown to be distinctive −1 cherry wine was found to be 72.78 mg L , which is much when compared to grape wines. Differences are obvious −1 higher than in grape wines, where it varies from 28.63 mg L from the visual inspection of the two base peak chro- −1 in Cabernet Sauvignon-North Serbia, to 48.90 mg L in matograms obtained in positive ion mode. Only as an Vranac. example, in Figure 2 two base peak chromatograms, for The following findings further highlight characteristic cherry wine (a) and grape wine Cabernet Sauvignon-Central profile of cherry wine: naringenin and apigenin were found Serbia (b) are presented. Extracted ion chromatograms in −1 −1 only in cherry wine (0.15 mg L and 0.06 mg L ); ellagic positiveionmodeandMS/MSdataofthemostabun- acid, myricetin and naringin were not found in cherry wine, dant anthocyanins found in sour cherry wine are pre- but they were found in all grape wine samples. The gallic sented in Figure 3: (a) cyanidin-3-glucosylrutinoside and acid was the major phenolic compound in grape wines −1 (b) cyanidin-3-rutinoside. The only anthocyanin common (12.56–30.39 mg L ), while its content was up to 30 times to all tested samples was cyanidin-3-glucoside. As it was lower in cherry wine. already stated in the introduction section, published lit- From the qualitative profile obtained from the UHPLC- erature indicates seven anthocyanins to occur in cherries. MS/MS Orbitrap analysis (Table 3)onecanconcludethat Apart from these seven, two more namely delphinidin-3- isomers of caffeoylquinic acids, quercetin-3-O-hexosides, rutinoside and pelargonidin-3-glucosylrutinoside were iden- and caffeoyl-hexosides were found only in cherry wine. tified in this study. Other derivatives of delphinidin, mal- Coumaroyl-hexosides, trans-caftaric acid, dihydroquercetin- vidin, and petunidin were not found in cherry wine, while 3-O-rhamnoside, procyanidin B type isomer 3, myricetin- theirpresencewasconfirmedingrapewines,exceptfor 3-O-hexoside, dihydromyricetin-3-O-rhamnoside, and lute- malvidin-3-caffeoylglucoside which was detected in two olin, were found only in grape wine samples. wines: samples W4 and W5. When comparing anthocyanins A total of 24 anthocyanin derivatives of were identified in all investigated wines, a total of seven compounds + based on the search for the M molecular ion together with were distinctive for cherry wine (delphinidin-3-rutinoside, 6 The Scientific World Journal ), calculated mass, found mass, 𝑅 𝑡 ppm MS/MS fragments SCW W1 W2 W3 W4 W5 − ] M–H [ MS/MS Orbitrap. Target compounds, mean expected retention times ( − Found − ] M–H [ , min Calculated 7.22 253.0506 253.0497 3.6 151, 181 + + + + + ND 7.347.34 255.0663 269.0455 255.0651 269.0443 4.7 4.5 213 183, 197 ND + + + + ND ND + + + + ND 5.55 285.0405 285.0393 4.2 133, 213 ND + + + + + 𝑅 𝑡 -rhamnoside 4.22 465.1023 465.1015 1.7 319 ND + + + + ND -rhamnoside 3.97 449.1074 449.1064 2.2 303 ND + + + + + O O -hexoside 1 2.56 463.0882 463.0859 5.0 179, 301 + ND ND ND ND ND -hexoside 2 2.99 463.0882 463.0860 4.8 179, 301 + ND ND ND ND ND -hexoside 4.14 479.0831 479.0810 4.4 317 ND + + + + + a O O O a a a -Caftaric acid 2.54 311.0407311.0393 4.5 135, 149, 179 ND + Confirmed using available standards. Compound Caffeoylquinic acid isomertrans Quercetin-3- 2.52 1 353.0867 353.08553.4 179, 191 + ND ND ND Galangin Chrysin Pinocembrin Quercetin-3- Procyanidin B type isomer 1Caffeoyl-hexosideCaffeoyl-hexosideCaffeoylquinic acid isomerCoumaroyl-hexoside 1Procyanidin B type 3.14 isomer 2Coumaroyl-hexoside 2Coumaroyl-hexoside 3Dihydroquercetin-3- 3.61 2 3.52 1 2 3.60 577.1351 3.69 3.64 353.0867 3.72 341.0874 3.89 341.0874 577.1351 325.0929 577.1325 325.0929 353.0856 325.0929 341.0869 4.5 341.0874 577.1325 325.0913 1.5 325.0919 0.0 325.0913 425 4.5 4.93.1 179, 3.1 179,135 4.9 179, 135 425 163 + 163 163 + ND + + 191+ ND ++ ND + + +NDND + ND ND + ND + ND + ND + + ND + ND + + + + + + + + + + + + Dihydromyricetin-3- Procyanidin B type isomer 3Myricetin-3- Syringic acidCinnamic acidLuteolin 4.09 577.1351 4.44 4.56 577.1325 197.0455 147.0452 4.5 197.0447 147.0448 425 4.1 2.7 ND + 153 103 + ND + + ND + + + + + + + + + ND + Table 3: Characterization of phenolic compounds in wine samples using UHPLC a mean mass accuracy (ppm), and MS/MS fragments (+ stands for detected and ND for not detected compound). The Scientific World Journal 7 ), calculated mass, found mass, mean 𝑅 𝑡 1.3317ND+++++ 0.2317ND+++++ 0.2331ND+++++ 0.6301ND+++++ 0.6317ND+++++ 0.6317ND+++++ 0.4303ND+++++ − − − − − − − ppm MS/MS fragments SCW W1 W2 W3 W4 W5 + Found M + , min Calculated M 𝑅 𝑡 -coumaroylglucoside 6.68 625.1543 625.1547 -coumaroylglucoside 6.93 639.1700 639.1696 0.6 331 ND + + + + + p -coumaroylglucoside 6.43 625.1543 625.1547 p -coumaroylglucoside 6.68 639.1700 639.1696 0.6 331 ND + + + + + - p - p - . - Compound cis cis trans trans Figure 2 a Peak numbers correspond to 101112131415 Peonidin-3-glucoside Peonidin-3-rutinoside Delphinidin-3-acetylglucoside Malvidin-3-glucoside Cyanidin-3-acetylglucoside Petunidin-3-acetylglucoside 5.58 5.71 5.60 5.93 5.65 5.99 463.1231 507.1123 609.1810 493.1336 491.1174 521.1280 463.1228 507.1125 609.1808 493.1334 0.6 491.1174 0.3 521.1281 0.4 0.0 301, 301 463 331 287 + ND ND ND ND + ND + + + ND ND + + + ND + + + + + + + + Peak number 123456789 Delphinidin-3-rutinoside Delphinidin-3-glucoside Cyanidin-3-glucosylrutinoside Cyanidin-3-sophoroside Cyanidin-3-pentosylrutinoside Pelargonidin-3-glucosylrutinoside 4.77 Cyanidin-3-glucoside 5.15 5.10 5.25 5.12 Cyanidin-3-rutinoside 5.31 611.1598 Petunidin-3-glucoside 465.1023 757.2191 727.2072 611.1598 741.2228 5.30 611.1598 465.1021 757.2170 5.34 727.2069 0.0 611.1597 5.38 741.2225 449.1074 0.4 2.8 0.4 0.2 595.1653 0.4 303, 449.1072 479.1180 449 287, 449, 611 287, 449, 581 303 271, 595.1644 433, 0.4 579 287, 449 479.1186 + + 1.5 + + ND ND ND + + 287 ND 287, ND ND 449 + ND ND ND ND ND ND ND + ND ND ND + ND ND + ND ND ND + ND + ND + ND ND + ND + ND + + ND ND + + 1617 Cyanidin-3-pentoside Peonidin-3-acetylglucoside 6.21 6.20 505.1331 419.0968 505.1334 419.0966 0.5 287 ND + + + + + 18 Malvidin-3-acetylglucoside 6.24 535.1436 535.1437 19202122 Petunidin-3- Pelargonidin-3-glucoside Malvidin-3-caffeoylglucoside Malvidin-3- 6.39 6.31 655.1649 433.1125 655.1643 433.1125 0.00.9 271 331 + ND ND ND ND ND ND ND ND + ND 23 Petunidin-3- 24 Malvidin-3- Table 4: Characterization of anthocyanins in wine samples using UHPLC + MS/MS Orbitrap. Target compounds, mean expected retention times ( a mass accuracy (ppm), and MS/MS fragments (+ stands for detected and ND for not detected compound). 8 The Scientific World Journal

100 100 O 90 OH 90 80 OH O+ 80 Peonidin-3-rutinoside 70 O 70 11 Compound 11 OH OH 60 O + 60 O O Chemical formula: C28H33O15 50 ∗=7+8 OH 50 m/z: 609.1814 40 OH OH OH 40 22 + 23 30 3+4 OH 30 14 20 20 21 # = 16 + 17 + 18 Relative abundance Relative Relative abundance Relative 1 12 24 10 ∗ 19 10 # 0 0 0123456789101112131415 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min) Time (min)

(a) (b)

Figure 2: Base peak chromatograms of (a) cherry wine and (b) Cabernet Sauvignon-Central Serbia in positive ion mode.

5.15 NL: 2.93E6 5.34 NL: 5.37E5 757.2170 m/z = 757.2148–757.2224 595.1644 100 100 m/z = 595.1628–595.1688 F: FTMS + c ESI full ms F: FTMS + c ESI full ms 80 80 [100.00–900.00] MS V1 [100.00–900.00] MS V1 60 60 40 40 Relative Relative 20 Relative abundance 20 abundance 0 0 0123456789101112131415 0 1 2 3 4 5 6 7 8 9 101112131415 Time (min) Time (min)

287 NL: 1.01E5 287 NL: 5.62E4 100 RT: 5.17 AV: 1 F: ITMS 100 RT: 5.33 AV: 1 F: ITMS 80 + c ESI d full ms2 80 + c ESI d full ms2 60 [email protected] 60 [email protected] 40 [195.00–770.00] 40 [150.00–610.00] Relative Relative 20 Relative 449

abundance 20 241.23 320.09 433.04 501.13 611 738.33 abundance 231.16 317.19 407.61 491.57 576.99 0 0 200 250 300 350 400 450 500 550 600 650 700 750 150 200 250 300 350 400 450 500 550 600 m/z m/z

(a) (b)

Figure 3: Extracted ion chromatograms and MS/MS spectra of (a) cyanidin-3-glucosylrutinoside and (b) cyanidin-3-rutinoside.

cyanidin-3-sophoroside, cyanidin-3-pentosylrutinoside, pel- produced from this cultivar. TPC, TAC and RSA values of argonidin-3-glucosylrutinoside, cyanidin-3-rutinoside, pe- cherry wine were in the range of the results determined for onidin-3-rutinoside, and pelargonidin-3-glucoside). red wines. The results suggest that cherry wine contains a high concentration of different polyphenols with high antioxidant 3.3. Determination of Minerals. The content of minerals is potential. Polyphenolic profile of cherry wine varied consid- presented in Table 5. Most common element in all samples erably when compared to grape wines. Caffeic acid, chloro- was potassium. Cherry wine contains up to five times higher genic acid, protocatechuic acid, and p-coumaric acid were the amounts of this mineral than grape wines (content ranging main phenolic acids found in cherry wine, and the contents −1 −1 from 246.200 mg kg , in Vranac, to 1373.100 mg kg in of these acids were much higher in cherry wine than in grape cherry wine). Higher contents of P, Ca and Mg were found wines tested. As for flavonoids, naringenin and apigenin were in cherry wine than in grape wine samples. Toxic elements foundonlyincherrywine.Atotalofsevenanthocyaninswere (As,CdandPb)werefoundinsmallamountsinalltested found in cherry wine only (delphinidin-3-rutinoside, cy- −1 wines (the allowable levels are 0.2 mg kg forPbandAs,and anidin-3-sophoroside, cyanidin-3-pentosylrutinoside, pelar- −1 gonidin-3-glucosylrutinoside, cyanidin-3-rutinoside, peon- 0.01 mg kg for Cd) [19]. idin-3-rutinoside, and pelargonidin-3-glucoside). 4. Conclusions This paper was aimed at characterizing the wine obtained Conflict of Interests from a native cultivar of sour cherry Oblacinska.ˇ The significance of this work is primarily in chemical charac-terization The authors declare that there is no conflict of interests and comprehensive data collection on the sour cherry wine regarding the publication of this paper. The Scientific World Journal 9

Table 5: The amounts of minerals in cherry wine and grape wine [5] L. Gao and G. Mazza, “Characterization, quantitation, and samples. distribution of anthocyanins and colorless phenolics in sweet cherries,” JournalofAgriculturalandFoodChemistry,vol.43, Mineral SCW W1 W2 W3 W4 W5 no. 2, pp. 343–346, 1995. −1 Al (mg kg ) 0.200 0.070 0.071 0.421 0.055 0.090 [6] J. J. Macheix, A. Fleuriet, and J. Billot, “Phenolic acids and −1 As (𝜇gkg ) 0.093 0.060 0.063 0.108 0.042 0.073 coumarins,” in Fruit Phenolics,J.J.MacheixandA.Fleuriet, −1 B(mgkg ) 2.760 1.364 2.101 1.919 1.690 2.082 Eds.,pp.17–39,CRCPress,BocaRaton,Fla,USA,1990. −1 Ca (g kg ) 0.084 0.035 0.023 0.041 0.020 0.022 [7] F. Blando, C. Gerardi, and I. Nicoletti, “Sour cherry (Prunus −1 Cd (𝜇gkg ) 0.093 0.002 0.063 0.051 0.042 0.073 cerasus L) anthocyanins as ingredients for functional foods,” −1 Journal of Biomedicine and Biotechnology,vol.2004,no.5,pp. Co (𝜇gkg ) 0.577 0.755 0.313 0.501 0.143 0.512 −1 253–258, 2004. 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