Nutraceuticals: Recent Advances of Bioactive Food Components

Journal of Chemistry

Guest Editors: Pyo-Jam Park, Thomas Ty Wang, Eun-Kyung Kim, and Qian Zhong-Ji Nutraceuticals: Recent Advances of Bioactive Food Components Journal of Chemistry

Nutraceuticals: Recent Advances of Bioactive Food Components

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

Nutraceuticals: Recent Advances of Bioactive Food Components Pyo-Jam Park, Thomas Ty Wang, Eun-Kyung Kim, and Qian Zhong-Ji Volume 2016, Article ID 5874726, 2 pages Specific Anthocyanin Contents of Whole Blue Maize Second-Generation Snacks: An Evaluation Using Response Surface Methodology and Lime Cooking Extrusion Anayansi Escalante-Aburto, Néstor Ponce-García, Benjamín Ramírez-Wong, Patricia Isabel Torres-Chávez, Juan de Dios Figueroa-Cárdenas, and Roberto Gutiérrez-Dorado Volume 2016, Article ID 5491693, 8 pages Ultrasound-Assisted Extraction of Total Flavonoids from Corn Silk and Their Antioxidant Activity Ling-Li Zheng, Guan Wen, Min-Yong Yuan, and Feng Gao Volume 2016, Article ID 8768130, 5 pages Health, Wellness, and Safety Aspects of the Consumption of Kombucha Mindani I. Watawana, Nilakshi Jayawardena, Chaminie B. Gunawardhana, and Viduranga Y. Waisundara Volume 2015, Article ID 591869, 11 pages Characterization of Calcium Compounds in Opuntia ficus indica as a Source of Calcium for Human Diet Isela Rojas-Molina, Elsa Gutiérrez-Cortez, Moustapha Bah, Alejandra Rojas-Molina, César Ibarra-Alvarado, Eric Rivera-Muñoz, Alicia del Real, and Ma. de los Angeles Aguilera-Barreiro Volume 2015, Article ID 710328, 8 pages New Approach to Enrich Pasta with Polyphenols from Grape Marc V.Marinelli,L.Padalino,D.Nardiello,M.A.DelNobile,andA.Conte Volume 2015, Article ID 734578, 8 pages Evaluation of the Stability of the Total Antioxidant Capacity, Polyphenol Contents, and Starch Hydrolase Inhibitory Activities of Kombucha Teas Using an In Vitro Model of Digestion Mindani I. Watawana, Nilakshi Jayawardena, Shakkya J. Ranasinghe, and Viduranga Y. Waisundara Volume2015,ArticleID684561,9pages Antioxidant Peptide Derived from Spirulina maxima Suppresses HIF1𝛼-Induced Invasive Migration of HT1080 Fibrosarcoma Cells Won Suk Kim, Won Kyo Jung, and Sun Joo Park Volume 2015, Article ID 308602, 7 pages Chemical Composition, Antioxidant Potential, and Antibacterial Activity of Essential Oil Cones of Tunisian Cupressus sempervirens Aicha Ben Nouri, Wissal Dhifi, Sana Bellili, Hanene Ghazghazi, Chedia Aouadhi, Ameur Chérif, Mohamed Hammami, and Wissem Mnif Volume 2015, Article ID 538929, 8 pages Inhibitory Effects of 4-(4-Methylbenzamino)benzoate on Adipocyte Differentiation Jin Taek Hwang, Sanghee Kim, Bo-ra Yoon, Inwook Choi, and Sang Yoon Choi Volume 2015, Article ID 171570, 4 pages First Evaluation of the Biologically Active Substances and Antioxidant Potential of Regrowth Velvet Antler by means of Multiple Biochemical Assays Yujiao Tang, Byong-Tae Jeon, Yanmei Wang, Eun-Ju Choi, Pyo-Jam Park, Hye-Jin Seong, Sang Ho Moon, and Eun-Kyung Kim Volume 2015, Article ID 975292, 7 pages Hindawi Publishing Corporation Journal of Chemistry Volume 2016, Article ID 5874726, 2 pages http://dx.doi.org/10.1155/2016/5874726

Editorial Nutraceuticals: Recent Advances of Bioactive Food Components

Pyo-Jam Park,1 Thomas Ty Wang,2 Eun-Kyung Kim,3 and Qian Zhong-Ji4

1 Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea 2Diet, Genomics and Immunology Lab, Beltsville Human Nutrition Research Center, United States Department of Agriculture, Beltsville, MD 20705, USA 3Division of Food Bio Science, College of Biomedical and Health Sciences, Konkuk University, Chungju 380-701, Republic of Korea 4Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China

Correspondence should be addressed to Pyo-Jam Park; [email protected]

Received 24 January 2016; Accepted 26 January 2016

Copyright © 2016 Pyo-Jam Park 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.

Nutraceuticals are numerous and have been studied intensely Calcium compounds of Opuntia ficus-indica are reported for basic science and applied research. The term is generally by I. Rojas-Molina et al. from Mexico which have a deficiency used to refer to those chemicals that may have biological in calcium intake. They characterize the distribution of significance, for example, antioxidants, disease resistance, calcium compounds in soluble and insoluble dietary fiber and regulating the immune systems, but are not established extracted from Opuntia ficus-indica. as essential nutrients. It utilizes traditional and rapidly Enriching pasta with grape marc is studied by V.Marinelli advancing analytical methods and instrumentation such as et al. from Italy. Grape marc is an interesting source of natural differential solvent extraction, gas or liquid chromatography compounds as polyphenols and fibers. This is the study to coupled with tandem mass spectrometry, nuclear magnetic enrich fresh and dry pasta with grape marc extracts obtained resonance and electron-spin resonance, and gene and protein by means of UAE using only water as solvent extraction. arrays for fractionation, purification, chemical and genetic Antioxidant peptide is obtained from Spirulina maxima fingerprinting, derivatization, modification, synthesis, and by W. S. Kim et al. This paper deals with the antioxidant clinical trials of photochemicals starting from cell-line to peptide derived from Spirulina maxima possessing an effec- animal and patient studies. The goal of this special issue is to tive constituent in antitumor progression products based on provide a platform for scientists all over the world to promote, the mechanism of tumor progression through ROS and the share, and discuss various new issues and developments in the HIF1𝛼 signaling pathway. area of nutraceuticals research. This issue compiles biological activities of bioactive food Tunisian Cupressus sempervirens chemical composition components. and some biological activities are analyzed by A. B. Nouri Kombucha is described in a review and research paper. et al. Some of the studies already focused on chemistry and Kombucha tea is produced by fermenting sugared black tea biological activities of Cupressus sempervirens; however they with a mixed culture of yeast and bacteria. Kombucha tea are originated from different areas in the world. This study is has gained immense popularity in recent times due to many to ascertain the chemical composition of Tunisian Cupressus associated health benefits. The therapeutic effects of this sempervirens to evaluate its antioxidant and antibacterial beverage are thought to be derived from the chemical compo- activities. sition of this beverage, mainly the polyphenols and secondary 4-(4-Methylbenzamino)benzoate is observed to sup- metabolites which are produced during fermentation. The press adipocyte differentiation by J. T. Hwang et al. 4- stability of antioxidant activity and polyphenol contents were (4-Methylbenzamino)benzoate has estimated adipogenesis- also investigated by M. I. Watawana et al. suppressing activity compared to resveratrol or genistein. 2 Journal of Chemistry

The biologically active substances of regrowth velvet antler are described in this issue. In this study, regrowth velvet antler is subjected to extraction by DW to allow determination of its constituent biologically active substances, including uronic acid, sulfated GAGs, sialic acid, uracil, hypoxanthine, and uridine. In addition, the antioxidant activities of RVA are determined by assessing DPPH, H2O2,hydroxyl,andABTS radicalscavengingactivityaswellasFRAPandORAC. We thoroughly reviewed these papers and we believe that they contribute to the applications of bioactive food components. Moreover, we believe that the papers in this special issue will be milestones for the advancement for the next generation of nutraceuticals researchers. Pyo-Jam Park Thomas Ty Wang Eun-Kyung Kim Qian Zhong-Ji Hindawi Publishing Corporation Journal of Chemistry Volume 2016, Article ID 5491693, 8 pages http://dx.doi.org/10.1155/2016/5491693

Research Article Specific Anthocyanin Contents of Whole Blue Maize Second-Generation Snacks: An Evaluation Using Response Surface Methodology and Lime Cooking Extrusion

Anayansi Escalante-Aburto,1,2,3 Néstor Ponce-García,1,4 Benjamín Ramírez-Wong,1 Patricia Isabel Torres-Chávez,1 Juan de Dios Figueroa-Cárdenas,2 and Roberto Gutiérrez-Dorado5 1 DIPA, Universidad de Sonora, Boulevard Luis Encinas y Rosales s/n, 83000 Hermosillo, SON, Mexico 2CINVESTAV-Unidad Queretaro,´ Libramiento Norponiente No. 2000, Fraccionamiento Real de Juriquilla, 76230 Queretaro,´ QRO, Mexico 3Universidad de Monterrey, Avenida Ignacio Morones Prieto No. 4500 Poniente, 66238 San Pedro Garza Garc´ıa, NL, Mexico 4UAEMex, Campus Universitario “El Cerrillo”, El Cerrillo Piedras Blancas s/n, 50200 Toluca, MEX, Mexico 5Programa Regional del Noroeste para el Doctorado en Biotecnolog´ıa, Universidad Autonoma´ de Sinaloa, Avenida de Las Americas´ y Boulevard Universitarios s/n, 80010 Culiacan,SIN,Mexico´

Correspondence should be addressed to Benjam´ın Ram´ırez-Wong; [email protected]

Received 24 September 2015; Accepted 3 January 2016

Academic Editor: Patricia Valentao

Copyright © 2016 Anayansi Escalante-Aburto 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.

Lime cooking extrusion (LCE) is a widely applied technology for producing second-generation snacks, as an alternative to traditional nixtamalization (TN). Pigmented maize has been used to produce snacks with similar organoleptic characteristics to TN products and to obtain a product with additional functional benefits due to the anthocyanic compounds contained in those grains. However, during the process, anthocyanins are degraded, and several chemical modifications occur. Response surface methodology is applied to evaluate extrusion factors and their effects on the response variables of extrudates. The aim of this study was to evaluate the changes in specific anthocyanins after extrusion in second-generation blue maize snacks. Three anthocyanins were identified and quantified by HPLC-UV-DAD: cyanidin 3-glucoside and pelargonidin 3-glucoside, which have been previously reported in blue maize and its products, and cyanidin 3,5-diglucoside. Higher retention values were found in the extrudates making LCE a viable option for producing second-generation blue maize snacks.

1. Introduction positions [1]. Anthocyanidins are not accumulated as such but rather in a glycosylated form, that is, linked to sugars, Anthocyanins represent the major group of water-soluble in which case they are known as anthocyanins. The sugars pigments in the plant kingdom and are widely distributed in attached to anthocyanidins provide high solubility and sta- food crops (e.g., vegetables, roots, tubers, and cereals). Several bility. Generally, the sugar molecule is linked to the phenolic investigations have reported not only nontoxic and nonmu- acidatthe3position,althoughitcanbelinkedatthe5and7 tagenic effects but also therapeutic and antioxidant activities positions as well [2]. Cyanidin 3-glucoside has been reported of anthocyanic compounds. The aglycones (basic forms) of as the most abundant anthocyanin contained in pigmented anthocyanins are termed anthocyanidins. These structures maize (approximate from 48% up to 87%), but pelargonidin are based on the flavylium ion or 2-phenyl benzopyrylium, 3-glucoside, peonidin 3-glucoside, and malvidin 3-glucoside which presents hydroxyl and methoxyl groups in different can also be present [3]. The pigmented maize cultivated in 2 Journal of Chemistry

Table 1: Experimental design of LCE showing different combinations of FM, 𝑇, and LC and experimental values of anthocyanin contents of the extrudates. FM 𝑇 LC C3,5-diG C3G Pel3G ∘ −1 1 % C% mg⋅kg Tx Process factors Response variables

𝑋1 𝑋2 𝑋3 𝑌1 𝑌2 𝑌3 1 19 (0) 130 (0) 0.13 (0) 16.98 28.10 1.63 215(−1.682) 130 (0) 0.13 (0) 16.47 27.22 1.71 3 19 (0) 130 (0) 0.13 (0) 17.09 28.17 1.72 4 19 (0) 130 (0) 0.13 (0) 17.06 27.61 1.66 5 21.38 (1) 141.89 (1) 0.2 (1) 16.97 28.07 1.73 6 19 (0) 130 (0) 0.13 (0) 17.03 28.21 1.76 7 19 (0) 130 (0) 0.25 (1.682) 17.29 28.36 1.75 8 21.38 (1) 118.11 (−1) 0.05 (−1) 16.89 27.41 1.59 9 19(0) 130(0) 0(−1.682) 16.75 27.77 1.78 10 21.38 (1) 141.89 (1) 0.05 (−1) 16.92 27.96 1.72 11 16.62 (−1) 141.89 (1) 0.05 (−1) 16.58 27.26 1.70 12 16.62 (−1) 118.11 (−1) 0.2 (1) 16.48 26.67 1.64 13 23 (1.682) 130 (0) 0.13 (0) 16.84 27.64 1.53 14 21.38 (1) 118.11 (−1) 0.2 (1) 16.89 27.48 1.69 15 19 (0) 110 (−1.682) 0.13 (0) 16.69 27.18 1.61 16 16.62 (−1) 141.89 (1) 0.2 (1) 16.40 26.96 1.70 17 19 (0) 150 (1.682) 0.13 (0) 16.65 27.23 1.67 18 16.62 (−1) 118.11 (−1) 0.05 (−1) 16.59 26.97 1.64 19 19 (0) 130 (0) 0.13 (0) 16.70 27.26 1.53 20 19 (0) 130 (0) 0.13 (0) 16.69 27.26 1.47 1 Numbers in parentheses are the coded levels. Tx: treatment; FM: feed moisture; 𝑇: temperature; LC: lime concentration; C3,5-diG: cyanidin 3,5-diglucoside; C3G: cyanidin 3-glucoside; Pel3G: pelargonidin 3-glucoside.

∘ American and Asiatic countries contains high amounts of at 5 C in black polyethylene bags until use. Commercial lime these compounds; however, during processing, anthocyanins (Calhidra de Sonora, SA de CV, Hermosillo, SON, Mexico) degrade due to high pH, temperature, and light exposure [4]. and distilled water were used. Extrusion is a technology widely used to obtain different maize products, such as expanded snacks. Lime cooking 2.2. Methods extrusion (LCE) is considered a faster and effective alternative method to traditional nixtamalization (TN) processing, the 2.2.1. Lime Cooking Extrusion (LCE). The process was per- latter based on the addition of Ca(OH)2 to maize and water ∘ formed according to the method described by Escalante- at 100 C to form a solution that is cooked for 20–40 min and Aburtoetal.[4].Tenkilogramsofbluemaizewaspassed steeped for at least 16 h [5]. LCE processing involves ther- through a blade mill (Pulvex SA de CV, Model 200, serial mal treatment, low moisture, and high-pressure conditions, among other factors (incorporation of additives and condi- 1030401, Mexico, DF) with a 0.8 mm mesh. Twelve hours tioning of flours). Many studies [6, 7] have been conducted before the extrusion experiment, the ground maize was on pigmented maize using LCE to retain more anthocyanins, divided into 20 batches of 300 g, and commercial lime and limiting the losses due to processing. However, none of these distilled water were added to each batch according to the studies have analyzed the profile of anthocyanins as being experimental design shown in Table 1. affected by multiple process factors using response surface To obtain the extrudates, the experiment was performed methodology (RSM), a statistical method used to investigate in a single screw extruder (Brabender Instruments, Model theeffectsoffactorsincomplexprocesses,andexaminedhow E19/25 D, OHG Duisburg, Germany). The temperatures in these factors influence the different anthocyanins contained the first, second, and third zones were set at 60, 80, and in the extrudates. The aim of this study was to evaluate the ∘ ∘ 110 C,respectively,andthefourthzonevariedfrom110Cto changes in specific anthocyanin contents that occurred in ∘ extruded products elaborated with LCE, applying RSM. 150 C (Table 1). The screw had a diameter of 19 mm, and the nominal compression ratio was 3 : 1. The other extrusion con- 2. Materials and Methods ditions were a screw speed of 120 rpm, feed hopper of 50 rpm, and die opening diameter of 4 mm. The extrudates were 2.1. Raw Material. Blue maize was obtained from a local cooled at room temperature and stored in sealed bags until market in Toluca, Mexico. The grains were cleaned and stored thechemicalanalyseswereperformed. Journal of Chemistry 3

X1 = FM 3 3 2 3 Y1 = C3,5-diG X =T 2 2 y=𝛽k0 + ∑ 𝛽kiXi + ∑ 𝛽kiiX + ∑ ∑ 𝛽kijXiXj +𝜀 Y2 = C3G X = i=1 i=1 i=j j≠+1 3 LC Y3 = Pel3G

Figure 1: Empirical black box modeling of the process variables and the response variables.

2.2.2. Anthocyanin Profile by HPLC Analysis. The extraction Thismathematicalexpressionisusedtomodelthe of anthocyanins was based on the methodology of Abdel- response variables (𝑌1–𝑌3), where the 𝑘 value changes from Aal and Hucl [8]. Extrudate samples were ground (Moulinex, 1to4;𝛽𝑘0 represents a constant, 𝛽𝑘𝑖 the linear coefficient, model 980-18, France) and passed through a number 60 𝛽𝑘𝑖𝑖 the quadratic coefficient, 𝛽𝑘𝑗𝑖 the interaction effect of sieve.Twomgofsamplewasweighedintoa50mLcentrifuge theresponsevariables,and𝜀 the experimental error. The tube (tube 1) with 20 mL of acidified ethanol (ethanol + HCl response variable data were recorded, and an analysis of 1 N, 85 : 15 v/v) and agitated (Wrist Action Shaker, model 75, variance (ANOVA) was performed with a confidence level of Burrell Corp., EUA) for 1 h. Then, the solution was cen- 95%. Moreover, a backward regression analysis was applied, trifuged (Thermo Scientific, model Heraeus Biofuge Primo and nonsignificant factors𝑃 ( > 0.1)wereeliminatedfrom R., Germany) at 27,200 ×g for 15 min, and the supernatant the second-order polynomial equation. Then, a new equation was recovered in a 50 mL tube. Twenty mL of acidified wasrecalculatedtoachievethefinalpredictivemodelfor ethanol was added to the residue contained in tube 1, and each response variable. RSM was used [9] and contour plots the above agitation and centrifugation steps were repeated. for each determination were obtained using Design Expert The supernatants from both steps were combined, giving Software V.7.0.0. an approximate volume of 40 mL, and then concentrated to a final volume of 10mL in a nitrogen evaporator (N- ∘ 3. Results and Discussion EVAP™ 112, Organomation Associates, Inc., EUA) at 40 C. The extracts were passed through a membrane of 0.45 𝜇m ∘ HPLC analyses demonstrated that of the five anthocyanins (Merck,Millipore,Darmstadt,Germany)andstoredat2 Cin analyzed only three were present in raw blue maize and its amber vials until analysis. Three extracts from each treatment extrudates. Cyanidin 3,5-diglucoside, cyanidin 3-glucoside, were obtained. and pelargonidin 3-glucoside were identified. C3,5-diG was An HPLC instrument with UV-vis and a DAD detector the first anthocyanin eluted. The glucose molecules affected (Agilent Technologies, model 1260 Infinity, Germany) was thepolarity,degreeofglycosylation,andnatureofthesugar used.Theseparationwasperformedinareverse-phaseC- moiety, and thus the elution order was galactoside > gluco- 18 column (Waters, model Spherisorb ODS–2, 5 𝜇m, 250 side > arabinoside [10]. Moreover, the presence of multiple × 46 mm, Ireland). The mobile phases were A, methanol, glycosidic substituents affected the retention characteristics, and B, formic acid (5%). The gradient used was as follows: provoking a faster elution of the compounds, as in the case of 0–30 min 25% A, 75% B; 31–60 min 40% A, 60% B; flow ∘ C3,5-diG. rate, 1.5 mL/min; column temperature, 35 C; detection wavelength, 535 nm; injection volume, 20 𝜇L. The analyses were 3.1. Effects of FM, 𝑇, and LC on C3,5-Diglucoside Con- performed in triplicate. centration. The coefficients of the second-order equations, Standards of five specific anthocyanins, cyanidin 3,5- ANOVA, and determination coefficients of the effects of FM, diglucoside (cyanidin chloride), cyanidin 3-glucoside (kuro- 𝑇,andLContheC3,5-diGareshowninTable2. manin chloride), cyanidin 3-galactoside (idaein chloride), The results demonstrated that FM in its linear and pelargonidin 3-glucoside (callistephin chloride), and mal- 2 quadratic (FM) terms exerts highly significant𝑃 ( < 0.0024) vidin 3-glucoside (oenin chloride), were prepared, and cali- and significant (𝑃 < 0.0200) effects, respectively, on the C3,5- bration curves were obtained for concentrations from 0.025 diG concentration. In addition, temperature in its quadratic to 1.0 mg/mL. 2 term (𝑇) presented a very significant effect𝑃 ( < 0.092) ontheanthocyanin.Thepredictionmodelequationwasas 2.3. Experimental Design and Statistical Analysis. Acentral follows: composite experimental design of three factors and five levels 2 2 was chosen (Table 1). The independent variables were feed C3,5-diG = −3.92 + 0.81 (HA) −0.01(HA) − 0.0007 (𝑇) . (1) moisture, FM (𝑋1, 15%–23%); lime concentration, LC (𝑋2, 0%–0.25%); and fourth-zone extruder temperature, 𝑇 (𝑋3, Table 1 displays the average values of the concentration of ∘ −1 −1 110–150 C), coded with levels of −1.682, −1, 0, +1, and +1.682. C3,5-diG, which varied from 16.40 mg⋅kg to 17.29 mg⋅kg . The response variables were cyanidin 3,5-diglucoside (C3,5- −1 −1 C3,5-diG was the second most abundant compound after diG, mg⋅kg ), cyanidin 3-glucoside (C3G, mg⋅kg ), and C3G. The latter situation could occur because diglycosylated −1 pelargonidin 3-glucoside (Pel3G, mg⋅kg ). The empirical anthocyanins have higher stability than monoglycosylated model representing the interaction between the independent ones, which diminishes the susceptibility to degradation by and response variables is presented in Figure 1. temperature and UV radiation [11]. Moreover, when the 4 Journal of Chemistry

Table 2: Coefficients of second-order equations (prediction models) 3.2. Effects of FM, 𝑇, and LC on the Cyanidin 3-Glucoside and analysis of variance, showing the relationship between process (C3G) Concentration. The coefficients of the second-order factors and response variables in the extrudates. equations and ANOVA of the effect of FM, 𝑇,andLCare shown in Table 2. The FM presented a very significant effect in Coefficient C3G C3,5-diG Pel3G the linear and quadratic terms, while 𝑇 in the quadratic term 2 Intercept (𝑇) showed a very significant effect (𝑃 < 0.0195)ontheC3G 𝛽 27.78 16.93 1.63 values. The prediction model equation was as follows: Linear ∗ ∗∗∗ 2 2 𝛽1 0.28 0.16 −0.018 n.s. C3G = −16.56 + 1.25 (FM) − 0.029 (FM) − 0.001 (𝑇) . (2) 𝛽2 0.13 n.s. −0.003 n.s. 0.029 n.s. TheaveragevaluesofC3Gwereintherangeof26.67to 𝛽3 0.042 n.s. 0.50 n.s. −0.003 n.s. ⋅ −1 Quadratic 28.36 mg kg (Table 1). There were slight losses of these ∗∗ anthocyanins, as the extrudates retained between 77.1 and 𝛽11 −0.16 n.s. −0.11 −0.002 n.s. −1 ∗ ∗ 82% of C3G compared with raw blue maize (34.60 mg⋅kg ). 𝛽22 −0.24 −0.10 −0.002 n.s. ∗ Figure 3(a) shows the effects of FM and 𝑇 on the C3G 𝛽33 0.061 n.s. 0.020 n.s. 0.049 ∘ content. Increased FM and 𝑇 of 130 C resulted in the highest Interactions concentrations of these compounds. The interaction FM ∗𝑇 𝛽 − 12 0.070 n.s. 0.026 n.s. 0.005 n.s. did not show a significant effect; nevertheless, there were sig- 𝛽 13 0.098 n.s. 0.042 n.s. 0.013 n.s. nificant effects only of the individual terms, meaning that the 𝛽 − − 23 0.006 n.s. 0.001 n.s. 0.009 n.s. results could be related to the findings reported by Yue C3G, cyanidin 3-glucoside; C3,5-diG, cyanidin 3,5-diglucoside; Pel3G, and Xu [14]. These authors concluded that although antho- ∘ pelargonidin 3-glucoside; 𝛽1, feed moisture; 𝛽2, final extruder temperature; ∗ cyanins degraded at temperatures higher than 100 C, thermal 𝛽3, lime concentration; n.s., not significant (𝑃>0.1); significant𝑃<0.1 ( ); ∗∗ 𝑃 < 0.05 ∗∗∗ 𝑃 < 0.01 stability increased in compounds with glucose or galactose very significant ( ); highly significant ( ). substituents. Moreover, there was a significant production of anthocyanidins such as delphinin and malvidin (present in pigmented maize) when their corresponding anthocyanins ∘ retention of C3,5-diG in the extrudates was evaluated in were submitted to heating at 100 C for 30 minutes in condi- comparison to the values obtained in raw blue maize, it tions of very low moisture. was found that between 91.2 and 96.2% of the anthocyanin Figure 3(b) shows the effects of FM and LC. A significant was retained in the products. The stability of C3,5-diG to effect of FM can be observed; as these process factors the processing conditions applied in this investigation was increased, the concentration of C3G grew higher. It has been demonstrated. Our results differ from the findings of Ziliˇ c´ reported that LC and pH affect the anthocyanin concentra- et al. [12], as the blue maize used in this study contained 10 tion.Inanalkalinemedium,anthocyanincompoundsdimin- −1 times more C3,5-diG (17.98 mg⋅kg )thantheconcentrations ish in content or are completely destroyed. However, there ⋅ −1 were no significant effects of LC. Figure 3(c) shows the effects reported by those authors, which were 0.08 and 1.65 mg kg ∘ ∘ for multicolored and light blue maize kernels, respectively. of temperature and LC. Between 130 Cand140C, the C3G According to Mendez´ et al. [5], the genotype, genetic content in the extrudates was higher, independently of LC. antecedents, physical properties, and relation of pericarp Sanchez-Madrigal´ et al. [15] evaluated the effect of LC addi- and endosperm (structures that concentrate the phenolic tion on the concentration of C3G extracted from blue maize compounds) affected the anthocyanin content of the maize nixtamalized flours obtained by extrusion. Those authors grains. concluded that the concentration of C3G increases without Figure 2(a) shows the effects of FM and 𝑇 on the C3,5- significant effects from the calcium source added. diG concentration of extrudates. It was observed that in ∘ a range of 21–23% FM and at 𝑇 of 120 and 130 Cthe 3.3. Effects of FM, 𝑇, and LC on Pelargonidin 3-Glucoside anthocyanin reached its maximum concentration. When the (Pel3G) Concentration. The coefficients of the second-order HA ∗ LC interaction was analyzed (Figure 2(b)), a directly equations, ANOVA, and determination coefficients of the proportional effect of FM and C3,5-diG was observed: as effects of FM, 𝑇, and LC on Pel3G concentration in the extru- FM increases, the C3,5-diG content of the extrudates also dates are shown in Table 2. The results demonstrated that 2 increases, independently of LC. LC in its quadratic term (LC) presented a significant effect Salinas Moreno et al. [13] reported the presence (𝑃 < 0.0273) on Pel3G concentration. The prediction model of C3G, Pel3G, peonidin 3-glucoside, three acylated 󸀠󸀠 equation was as follows: anthocyanins, cyanidin-3-(6 malonylglucoside), cyanidin- 󸀠󸀠 󸀠󸀠 2 3-(3 ,6 dimalonylglucoside), and four unidentified Pel3G = 1.76 − 2.15 (LC) +8.79(LC) . (3) anthocyanins. This investigation demonstrated that C3,5- diglucoside was present in blue maize and its extrudates. Table 1 displays the average values of Pel3G in the extrudates, −1 Figure 2(c) showed the effect of the 𝑇∗LC interaction which are in a range of 1.47–1.78 mg⋅kg .Lowamounts on C3,5-diG concentration. Only 𝑇 significantly affected this of these compounds were lost in the extrudates compared −1 response variable, and the maximum average values of C3,5- with raw blue maize (2.29 mg⋅kg ), as between 64.1 and ∘ diG were found at 130 C. 77.7% of Pel3G was retained in the products. The Pel3G Journal of Chemistry 5 )

17.10 ) −1 17.01 −1 kg · kg 16.80 · 16.84

16.50 16.68 16.20 -diglucoside (mg -diglucoside 5 -diglucoside (mg -diglucoside , 16.52 5 3 , 15.90 3 16.35 Cyanidin 23.0 Cyanidin 110.00 21.0 23.0 0.00 120.00 21.0 FM (%)19.0 0.06 130.00 ∘ 19.0 0.13 17.0 140.00 C) FM (%) 17.0 0.19 15.0 150.00 LC (%) Temperature ( 15.0 0.25 (a) (b)

) 17.01

−1 kg

· 16.84 (mg 16.68

lucoside 16.52 -dig 16.35

150.00

Cyanidin 3,5 Cyanidin 0.00 140.00 0.06 Temperature130.00 ( 0.13 120.00 0.19 ∘ LC (%) C ) 110.00 0.25 (c)

Figure 2: Effects of the processing factors (a) FM ∗𝑇,(b)FM∗ LC, and (c) 𝑇∗LC on C3,5-diG content in extruded blue maize snacks.

concentration in the extrudates was higher than that reported compounds added, due to the higher stability to thermal by Sanchez-Madrigal´ et al. [15] in blue maize nixtamalized processing. flours obtained by extrusion processing. This result corrob- Figure 4(a) shows the effect of the FM ∗𝑇interaction on orates the different chemical transformations of anthocyanic Pel3G concentration in the extrudates. Maximum concentra- ∘ compounds during processing, in addition to the differences tionsofthesecompoundswerefoundat130C and low levels in the anthocyanin profile for each maize variety, despite of FM (14%), which agreed with the work by Garzon´ and the same grain coloration. Even when Pel3G was present Wrolstad [16] with respect to anthocyanin stability at higher at minimum concentrations, it had a high stable chemical processing temperatures. structure. When the effect of the FM ∗ LC interaction was analyzed GarzonandWrolstad[16]reportedthatPel3Gexerted´ (Figure 4(b)), it was observed that, at minimum and maxi- a very significant effect on the degradation velocity of mum LC (0–0.25%), Pel3G reached its highest concentration anthocyanin contained in certain food products with these independently of FM. Figure 4(c) shows the effect of the 𝑇∗ 6 Journal of Chemistry

28.00 ) 28.00 ) −1 −1 kg ·

27.52 kg · 27.70 27.05 27.40 26.58 -diglucoside (mg -diglucoside 3

-diglucoside (mg -diglucoside 27.10 3 26.10 26.80 Cyanidin Cyanidin 0.25 150.0 23.0 0.19 140.0 21.0 23.0 0.13 Temperature130.0 ( 19.0 21.0 120.0 17.0 19.0 0.06 17.0 ∘ 110.0 15.0 FM (%) FM (% LC (%) C) ) 15.0 0.00 (a) (b)

27.85

)

− kg

· 27.61 (mg 27.37

27.13

26.89 Cyanidin 3-glucoside Cyanidin

150.0 0.25 140.0 0.19 Temperature130.0 ( 0.13 120.0 0.06 ∘ 110.0 0.00 LC (%) C) (c)

Figure 3: Effects of the processing factors (a) FM ∗𝑇,(b)FM∗ LC, and (c) 𝑇∗LC on C3G content in extruded blue maize snacks.

LC interaction, where Pel3G presented higher degradation at In general, the results indicated higher retention per- ∘ LC 0.05–0.16% and 𝑇 110–120 C. centages due to the short time of residence of raw material, Although the interaction of FM and 𝑇 did not show implying minimum conditions of thermal treatment and significant effects and only the individual effects were sig- hence higher retention of pigments. Anthocyanins showed nificant, our results could be related with the findings certain sensitivity to the individual and combined effects of reportedbyYueandXu[14].Theyfoundthatevenwhen processing, such as temperature, pressure, treatment time, anthocyanins were degraded at temperatures higher than and the mechanisms and kinetics of degradation [17]. As we ∘ 100 C, the thermal stability of these compounds with glucose can see in the models, Pel3G was the only anthocyanin that or galactose substituents was better. Moreover, higher pro- showed a significant effect of LC, which was correlated with duction of anthocyanidins such as cyanidin, delphinin, and the pH. Andres-Bello´ et al. [18] reported that, with increasing malvidin (present in pigmented maize) was observed when pH, anthocyanins become paler in color; however, if the food their anthocyanins were subjected to temperatures higher matrix contains substances capable of acting as copigments, ∘ than 100 C and very low moisture conditions. the color may be retained and stabilized to a certain extent. Journal of Chemistry 7

) 1.69

−1 1.78

) · 1

1.66 − (mg

kg 1.74 ·

1.64 (mg

1.71 -glucoside 1.62 1.67

1.59

largonidin 3 largonidin 1.63 Pe

23.0 Pelargonidin 3-glucoside Pelargonidin 21.0 150.0 0.25 23.0 19.0 140.0 0.19 21.0 130.0 17.0 0.13 19.0 Temperature ( 120.0 FM (%) LC (%) 15.0 17.0 ∘ 110.0 0.06 C) 0.00 15.0 FM (%) (a) (b)

1.85

)

−1 kg

· 1.78

mg (

1.72

1.65

1.58

0.25 Pelargonidin 3-glucoside Pelargonidin 0.19 150.0 0.13 140.0 130.0 0.06 Temperature ( 120.0 LC (%) ∘ 110.0 0.00 C) (c)

Figure 4: Effects of the processing factors (a) FM ∗𝑇,(b)FM∗ LC, and (c) 𝑇∗LC on Pel3G content in extruded blue maize snacks.

When anthocyanins are processed in an alkaline medium were exposed to high temperatures. Feed moisture, temper- (pH 7), the pericarp is not completely hydrolyzed by calcium ature, and their interactions showed significant effects on hydroxide (lime), and the release of ferulic acid is lower. It the anthocyanin content. Cyanidin 3-glucoside was the most has been reported that ferulic acid can act as a copigment of abundant anthocyanin in the extruded snacks at the highest values at feed moisture contents (21–23%) and temperatures anthocyanins, increasing their retention during processing. ∘ on the fourth zone of the extruder (130–140 C), with no significant effects of LC. Cyanidin 3,5-diglucoside due to 4. Conclusions its chemical composition showed higher stability and had the highest values at feed moisture contents of 21–23% and ∘ Higher retention percentages of anthocyanins were observed temperature of 130 C, regardless of the LC. On the other in the extruded snacks, even when an alkaline medium was hand, pelargonidin 3-glucoside was significantly affected by ∘ induced with the lime cooking extrusion and the materials LC, showing the highest values at temperature of 150 C, and 8 Journal of Chemistry

lime concentration range of 0–0.25%, regardless of the feed [12] S. Ziliˇ c,´ A. Serpen, G. Akillioglu,ˇ V.Gokmen,¨ and J. Vancetoviˇ c,´ moisture content. These results indicated that it is possible to “Phenolic compounds, carotenoids, anthocyanins, and antiox- produce snacks with beneficial or nutraceutical compounds idant capacity of colored maize (Zea mays L.) kernels,” Journal using the lime cooking extrusion process. of Agricultural and Food Chemistry,vol.60,no.5,pp.1224–1231, 2012. [13]Y.SalinasMoreno,G.SalasSanchez,´ D. Rubio Hernandez,´ and Conflict of Interests N. Ramos Lobato, “Characterization of anthocyanin extracts from maize kernels,” Journal of Chromatographic Science,vol. The authors declare that there is no conflict of interests 43,no.9,pp.483–487,2005. regarding the publication of this paper. [14] X. Yue and Z. Xu, “Changes of anthocyanins, anthocyanidins, and antioxidant activity in bilberry extract during dry heating,” Acknowledgment Journal of Food Science,vol.73,no.6,pp.C494–C499,2008. [15] M. A.´ Sanchez-Madrigal,´ A. Quintero-Ramos, F. Mart´ınez- Escalante-Aburto thanks CONACYT for the postdoctoral Bustos et al., “Effect of different calcium sources on the bioactive scholarship provided. compounds stability of extruded and nixtamalized blue maize flours,” Journal of Food Science and Technology,vol.52,no.5, References pp.2701–2710,2015. [16] G. A. GarzonandR.E.Wrolstad,“Comparisonofthestabilityof´ [1] M. Horbowicz, R. Kosson, A. Grzesiuk, and H. Dębski, “Antho- pelargonidin-based anthocyanins in strawberry juice and con- cyanins of fruits and vegetables, their occurrence, analysis and centrate,” Journal of Food Science,vol.67,no.4,pp.1288–1299, role in human nutrition,” Vegetable Crops Research Bulletin,vol. 2002. 68,no.1,pp.5–22,2008. [17] A. V. Durge, S. Sarkar, and R. S. Singhal, “Stability of antho- [2]M.Y.Salinas,R.J.Soria,andT.E.Espinosa,Aprovechamiento cyanins as pre-extrusion colouring of rice extrudates,” Food yDistribucion´ de Ma´ız Azul en el Estado de Mexico´ ,INIFAP,1st Research International,vol.50,no.2,pp.641–646,2013. edition, 2010. [18] A. Andres-Bello,V.Barreto-Palacios,P.Garc´ ´ıa-Segovia, J. Mir- [3] A. Castaneda-Ovando,˜ M. D. L. Pacheco-Hernandez,´ M. E. Bel, and J. Mart´ınez-Monzo,´ “Effect of pH on color and texture Paez-Hern´ andez,´ J. A. Rodr´ıguez, and C. A. Galan-Vidal,´ of food products,” Food Engineering Reviews,vol.5,no.3,pp. “Chemical studies of anthocyanins: a review,” Food Chemistry, 158–170, 2013. vol.113,no.4,pp.859–871,2009. [4] A. Escalante-Aburto, P.I. Torres-Chavez,B.Ram´ ´ırez-Wong, and N. Ponce-Garc´ıa, “Pigmented maizes: anthocyanin profile and content,” in Handbook of Anthocyanins. Food Sources, Chemical Applications and Health Benefits, Nova Science Publishers, New York,NY,USA,1stedition,2015. [5]L.I.R.Mendez,´ J. D. D. F. Cardenas,´ M. R. Gomez,´ and L. L. M. Lagunas, “Nutraceutical properties of flour and tortillas made with an ecological nixtamalization process,” Journal of Food Science, vol. 78, no. 10, pp. C1529–C1534, 2013. [6] J. Aguayo-Rojas, S. Mora-Roch´ın, E. O. Cuevas-Rodr´ıguez et al., “Phytochemicals and antioxidant capacity of tortillas obtained after lime-cooking extrusion process of whole pigmented mexican maize,” Plant Foods for Human Nutrition,vol.67,no.2,pp. 178–185, 2012. [7] A. Escalante-Aburto, B. Ram´ırez-Wong, P. I. Torres-Chavez´ et al., “Obtaining ready-to-eat blue corn expanded snacks with anthocyanins using an extrusion process and response surface methodology,” Molecules,vol.19,no.12,pp.21066–21084,2014. [8] E.-S. M. Abdel-Aal and P. Hucl, “A rapid method for quanti- fying total anthocyanins in blue aleurone and purple pericarp wheats,” Cereal Chemistry, vol. 76, no. 3, pp. 350–354, 1999. [9]R.H.Myers,D.C.Montgomery,andC.M.Anderson-Cook, “The analysis of second-order response surfaces,” in Response Surface Methodology. Process and Product Optimization Using Designed Experiments, John Wiley & Sons, Hoboken, NJ, USA, 3rd edition, 2009. [10]V.HongandR.E.Wrolstad,“UseofHPLCseparation/photo- diode array detection for characterization of anthocyanins,” Journal of Agricultural and Food Chemistry,vol.38,no.3,pp. 708–715, 1990. [11] M. T. Escribano-Bailon,´ C. Santos-Buelga, and J. C. Rivas- Gonzalo, “Anthocyanins in cereals,” Journal of Chromatography A, vol. 1054, no. 1-2, pp. 129–141, 2004. Hindawi Publishing Corporation Journal of Chemistry Volume 2016, Article ID 8768130, 5 pages http://dx.doi.org/10.1155/2016/8768130

Research Article Ultrasound-Assisted Extraction of Total Flavonoids from Corn Silk and Their Antioxidant Activity

Ling-Li Zheng,1 Guan Wen,2 Min-Yong Yuan,1 and Feng Gao2

1 Department of Pharmacy, The First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China 2Department of Chinese Traditional Herbal, Agronomy College, Sichuan Agriculture University, Chengdu 611130, China

Correspondence should be addressed to Feng Gao; [email protected]

Received 14 September 2015; Accepted 29 December 2015

Academic Editor: Alberto Ritieni

Copyright © 2016 Ling-Li Zheng 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.

Object. Ultrasound-assisted extraction of total flavonoids from corn silk and their antioxidant activities were studied. Methods. Response surface methodology was adopted to optimize the extraction conditions and antioxidant activities of the extracted total flavonoids were detected through ferric reducing antioxidant power (FRAP) assay. Results. Through a three-level, three-variable Box-Behnken design of response surface methodology (RSM) adopting yield as response, the optimal conditions were determined as follows: ultrasonic power 500 W, extraction time 20 min, material solvent ratio 1 : 20, and ethanol concentration 30%. Under the optimum conditions, the extraction yield of total flavonoids was 1.13%. FRAP value of total flavonoids extracted from corn silkwas 467.59 𝜇mol/L. Conclusion. The total flavonoids of corn silk could be developed as food natural antioxidant reagents.

1. Introduction alternative to conventional extraction technique, was adopted in our study. In addition, ultrasound also shows a mechan- Corn silk (Zea mays L.) is one of the Chinese traditional ical effect, allowing greater penetration of solvent into the herbals and contains flavonoids, sterol, alkaloids, carbohy- sample matrix, increasing the contact surface area between drates, inorganic elements, vitamins, and other chemical solid and liquid phases. Meanwhile, Box-Behnken design constituents [1, 2] which show remarkable bioactivities, such of response surface methodology (RSM) was applied in the as diuresis, hypoglycemic, bacteriostatic, antihypertensive, determination of the optimal extraction conditions with the enhancing immune, and anticancer activity [3]. The extracts independent variable of material-solvent ratio, extraction of corn silk are approved by FDA as OTC drug [4]. time, and ethanol concentration. The antioxidant activities of Flavonoids are effective components of many Chinese extracted total flavonoids were also determined using reliable herbal medicine with the function of antihypertensive, reduc- method [10, 11]. ing blood lipid, being antibacterial and antitumor, enhancing immune, antioxidant, and eliminating free radicals [5]. Therefore, it is helpful to look for an effective method to 2. Materials and Methods extract flavonoids from corn silk. However, the literature on efficient extraction of corn 2.1. Instruments. UV-1100 spectrophotometer, ultrasonic ex- silk is limited. Traditional methods (distillation or liquid tractor, rotary evaporator, multiuse recycle water vacuum solvent extraction) for the extraction of flavonoids from pump, grinder, and electronic balance. plant often need long extraction times, use of large amounts of solvent, and low efficiencies. Moreover, flavonoids are 2.2. Reagent. Corn silk was harvested in Sichuan Agricul- thermally unstable and easily degrade during the extraction. tural University green nursery garden, Rutin standard was In order to increase the extraction yield, ultrasound-assisted obtained from Sichuan Institute for Food and Drug (99% extraction [6–9], which is inexpensive, simple, and efficient purity, batch number 130910), ethanol, sodium hydroxide, 2 Journal of Chemistry aluminum nitrate, sodium nitrite, ferrous sulfate, concen- 0.25 trated hydrochloric acid, and sulfuric acid were of analyt- 0.2 ical grade and purchased from Chengdu Kelong Chemical 0.15 Reagent Company. TPTZ was purchased from Beijing Solar- 0.1 bio Science and Technology Company for the preparation of (%) Yield 0.05 0 the FRAP solution. 0 5 101520253035 Extraction time (min) 2.3. Determination of Standard Curve. AtotalofRutinstand- Figure 1: Effects of extraction time on yield of total flavonoids. ard solution was prepared by dissolving Rutin reference material in 70% ethanol; Rutin solution (0.0 mL, 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL) was accurately 0.4 pipetted into 25 mL volumetric flasks, respectively. Then, 0.3 12 mL of the 70% ethanol and 2 mL of the sodium nitrate 0.2 solution were added. The mixture was shaken up and placed Yield (%) Yield 0.1 for 10 min, followed by the addition of 2 mL 10% nitric acid 0 aluminum solution, and was shaken up. After 10 min, 20 mL 1 : 05 1 : 10 1 : 15 1 : 20 1 : 25 sodium hydroxide was added to scale. The mixture was Material/solvent ratio (g/mL) deposited for 5 min, and then the absorbance of the solution was measured at 500 nm with the reagent blank as reference. Figure 2: Effects of material solvent ratio on yield of total flavonoids. Make the standard curve with the concentration of Rutin standard solution as abscissa and absorbency as vertical; the 2 regression equation was 𝑌 = 8.3968𝑥 − 0.0043, 𝑅 = 0.9998. the crude flavonoids were obtained and certain concentration of flavonoids solution was prepared with distilled water. A volume of 0.3 mL sample solution was pipetted, and 2.7 mL 2.4. Single Factor Experiment. Fixed extraction conditions ∘ FRAP solution (preheated to 38 C, prepared by 10 mmol/L are as follows: ultrasonic time 20 min, material solvent ratio ⋅ 1 : 20, ethanol concentration 30%, ultrasonic temperature TPTZ working solution, 20 mmol/L FeCl3 6H2O, 0.3 mol/L ∘ 60 C, ultrasonic power 500 W, and one-time extraction. The Hac buffer at 1 : 1 : 10, pH = 3.6) was added. The mixture supernatant was taken after being centrifuged for 10 min was shaken up and placed for 15 min. We keeping record of the absorbance values at 593 nm. Absolute ethanol was used under 3000 r/min, and then the absorbance was measured 𝜇 according to 2.1. Extraction time (10 min, 15 min, 20 min, as blank control for zeroing. FeSO4 concentration ( mol/L) 25 min, and 30 min), material liquid ratio (1 : 5, 1 : 10, 1 : 15, could be obtained from the standard curve according to the 1 : 20, and 1 : 25), and ethanol concentration (20%, 30%, 40%, absorbance and defined as FRAP value. Sample with higher 50%, and 60%) were selected as the key variables. Their FRAP has higher antioxidant ability. impact on the yield of the total flavonoids was tested separately: 2.6.2. Determination of FeSO4 Standard Curve. Avolumeof 6.08 mg FeSO4 was dissolved with distilled water, 0.25 mL 𝐶 Among them, the flavonoids content (mg/mL) = 18 mol/L H2SO4 was added, and the mixture was diluted 𝑋∗diluted multiples; flavonoids yield (%) = [𝐶 ∗ 𝑉/ withdistilledwatertothescaleof50mL,andthenaniron 1000 W] ∗ 100%; nail was put into it. 5 mL of the solution was pipetted into 𝑋: the concentration of flavonoids, calculated by 50 mL volumetric flask and was diluted with distilled water standard curve (mg/mL); to the scale to make 800 𝜇mol/L FeSO4 standard solution. The 200, 400, and 600 𝜇mol/L standard solutions were prepared 𝑊: sample quality (g); sequentially. Regression equation of the standard curve was 𝑉 2 : the original volume of extracting solution (mL). obtained as 𝑦 = 0.0021𝑥 + 0.3210, 𝑅 = 0.9996.

2.5. The Response Surface Analysis Factor Levels Design [12]. 3. The Results and Analysis According to single factor experiments, coded level of the three factors for Box-Behnken design of RSM were settled 3.1. Single Factor Results as follows: ethanol concentration (20%, 30%, and 40%), extraction time (15 min, 20 min, and 25 min), and material 3.1.1. Effect of Extraction Time. From Figure 1, the extraction liquid ratio (1 : 10, 1 : 15, and 1 : 20). yield of total flavonoids increased with the extension of time and reached maximum value at 20 min, but after 20 min, the 2.6. FRAP Method to Determinate the Antioxidant yield decreased, which might be due to denaturation of the total flavonoids through long period of ultrasonication. Activity of Flavonoids [13, 14] 2.6.1. Antioxidant Activity Determination. The total flavon- 3.1.2. Effect of Solid Liquid Ratio. From Figure 2, as ethanol oids were vacuum-concentrated and dried after being ex- scaled up, the extraction yield increased with the increase tracted under the optimal extraction conditions, and then of the dissolved extracts, which reached top at ratio of Journal of Chemistry 3

1.2 1 1.3 0.8 1.2 0.6 0.4 1.1 Yield (%) Yield 0.2 1 0 0.9 0 10 20 30 40 50 60 70 0.8 Ethanol concentration (%) 0.77 Figure 3: Effects of ethanol concentration on yield of flavonoids. 25.000 40.00 23.00 35.00 21.00 Table 1: Experimental design and data for response surface analysis. 19.00 30.00 17.00 25.00 A B: B 15.00 20.00 A: Factors Flavonoids extraction yield (%) Run 𝐴𝐵𝐶Experimental data Predictive value Figure 4: Response surface for effect of the extracting time and 1 −1 0 −1 1.01 1.08 ethanol concentration on yield. 2 110 0.97 0.93 3 01−1 0.98 1.00 4 000 1.06 1.08 5 1−10 0.91 0.89 6 000 1.08 1.08 1.3 7 000 1.01 1.08 1.2 1.1 8 0−11 0.99 0.97 1 9 000 1.03 1.08 −1 0 1 0.9 10 0.94 0.91 0.8 −1 −1 0 11 0.75 0.79 0.7 12 −1 1 0 1.00 1.00 101 13 1.07 1.09 20.00 40.00 14 10−1 0.82 0.84 18.00 35.00 16.00 30.00 15 011 1.07 1.09 14.00 C 12.00 25.00 16 0−1−1 0.91 0.91 : C 10.00 20.00 A: A 17 000 1.22 1.08 Figure 5: Response surface for effect of the material solvent ratio and ethanol concentration on yield. 1 : 20 (g/mL). Then the extraction yield decreased due toa dilution effect on flavonoids. Variance analysis results suggested that the effect of the 3.1.3. Effect of Ethanol Concentration. From Figure 3, the factors in flavonoids extraction yield were 𝐵, 𝐶,and𝐴 in extraction yield of total flavonoids increased gradually and turn. While material solvent ratio and extracting time had reached top at 30%. Then the yield dropped sharply, caused significant effects on the total flavonoids extraction rate, by enhanced volatilization with high ethanol concentration. ethanol concentration showed little effect. Effect of cross- term 𝐴𝐶 was significant, indicating the significant interaction 3.2. The Response Surface Method to Optimize the Extraction of ethanol concentration and material solvent ratio. From Table 2, the relationship of the regression curve line with 3.2.1. The Response Surface Analysis Factor Levels of Design the actual statistical data was well described and used to Results. Based on single factor experiments, extracting time, determine the optimum conditions. material solvent ratio, and ethanol concentration were selected, to study the effects of different combinations of the three factors by Box-Behnken design of RSM adopting 3.2.3. Response Surface Analysis. Comparison of Figures 4– Design-Expert V8.0.6 (Stat-Ease, Inc.) (Table 1). 6 showed that extracting time played a critical role for achieving higher extraction yields, observed as steep curve 3.2.2. Regression Model of Flavonoids Extraction Yield and the line with optimum value of 21 min. This may due to the Significance Test. The regression equation was obtained as strengthened medium motion caused by ultrasound and increased effect of vibrating homogenization and cavitation, 𝑅1 = 1.08 + 8.750𝑒 − 003 ∗ 𝐴 + 0.058 ∗ 𝐵 +0.044∗𝐶 which accelerated the dissolution of the total flavonoids − 0.048 ∗ 𝐴 ∗ 𝐵 + 0.080 ∗ 𝐴 ∗ 𝐶 +2.500𝐸 and improved the extraction rate. The flat curve line and nonsignificant response value change of the other two factors (1) (ratio of material solvent ratio and ethanol concentration) − 003 ∗ 𝐵 ∗ 𝐶 − 0.01 ∗𝐴2 − 0.073 ∗ 𝐵2 − 0.020 indicated their little influence on the flavonoids yield. Their ∗𝐶2. optimum values were about 1 : 20 and 20 min, respectively. 4 Journal of Chemistry

Table 2: Variance analysis of the regression model.

Sources of variance Sum of squares Degrees of freedom The mean square 𝐹 Pr >𝐹 Significant Model 0.15 9 0.016 3.29 0.0655 ∗ −4 −4 𝐴-Ethanol concentration 6.125 × 10 1 6.125 × 10 0.12 0.7371 𝐵-extracting time 0.026 1 0.026 5.27 0.0553 ∗ 𝐶-material solvent ratio 0.015 1 0.015 3.05 0.1242 −3 −3 𝐴𝐵 9.025 × 10 19.025× 10 1.80 0.2218 𝐴𝐶 0.026 1 0.026 5.10 0.0484 ∗ −5 −5 −3 𝐵𝐶 2.500 × 10 12.500× 10 4.982 × 10 0.9457

𝐴2 0.042 1 0.042 8.39 0.0231 ∗

𝐵2 0.022 1 0.022 4.41 0.0739 ∗ −3 −3 𝐶2 1.684 × 10 11.684× 10 0.34 0.5805 −3 The residual error 0.035 7 5.018 × 10 −3 −4 Loss of quasi item 7.725 × 10 32.575× 10 0.38 0.7762 −3 Pure error 0.027 4 6.850 × 10 Sum 0.18 16 ∗ means the difference is significant at the 0.05 level.

experiment data in good effect. Through response surface 1.3 methodology (RSM) of yield, the optimal conditions were 1.2 determined as follows: extraction time 20 min, solid-liquid 1.1 ratio 1 : 20, and ethanol concentration 30%. Under the opti- 1 mum conditions, the extraction yield of total flavonoids 0.9 was 1.13%, which allowed higher extraction yields with 0.8 0.7 lower temperature and extraction time when compared with conventional solvent extraction methods. With stable results, this method offered a theoretical basis for industrial and 20.00 25.00 18.00 23.00 experimental extraction of total flavonoids from corn silk. 16.00 21.00 14.00 19.00 FRAP value of the total flavonoids extracted from corn silk C 12.00 17.00 B : C 10.00 15.00 B: was determined as 467.59 𝜇mol/L, which indicated good antioxidant activities of the total flavonoids from corn silk. Figure 6: Response surface for effect of the extracting time and ethanol concentration on yield. Conflict of Interests The authors declare that there is no conflict of interests 3.2.4. The Determination of the Optimal Process Conditions regarding the publication of this paper. and Validation. The optimal conditions were determined by Design-Expert as follows: extraction time 21.45 min, material solvent ratio 1 : 20, and ethanol concentration 33.75%. Authors’ Contribution Under the optimum conditions, the extraction yield of total Ling-Li Zheng and Guan Wen contributed equally. flavonoids was 1.129%, which well fits the theoretical value, proving the reliability of extraction parameters obtained from response surface methodology. Acknowledgments This research was supported by Sichuan Sci-Tech Support 3.3. To Generate the Determination Results of Flavonoid Anti- Plan (no. 2015SZ0105) and Department of Sichuan Provincial oxidant Capacity. The FRAP value is 467.59 𝜇mol/L, which Health (no. 130388). indicated that the total flavonoids of corn silk have good antioxidant activity. References

4. Conclusions [1]Q.-L.Hu,L.-J.Zhang,Y.-N.Li,Y.-J.Ding,andF.-L.Li,“Puri- fication and anti-fatigue activity of flavonoids from corn silk,” In this study, the optimization of ultrasound-assisted extrac- International Journal of Physical Sciences,vol.5,no.4,pp.321– tion of total flavonoids from corn silk and evaluation of 326, 2010. their antioxidant activity were conducted. On the basis of [2] S.-C. Ren, Q.-Q. Qiao, and X.-L. Ding, “Antioxidative activity of single-factor test and Box-Behnken experimental design, five flavones glycosides from corn silk (Stigma maydis),” Czech the quadratic regression model was established to fit the Journal of Food Sciences, vol. 31, no. 2, pp. 148–155, 2013. Journal of Chemistry 5

[3] K. Hasanudin, P. Hashim, and S. Mustafa, “Corn silk (Stigma maydis) in healthcare: a phytochemical and pharmacological review,” Molecules,vol.17,no.8,pp.9697–9715,2012. [4] C.-M. Liu, X.-L. Zhao, Z.-Q. Liu, and J.-P. Xing, “Isolation and extraction of total flavonoids from Epimedium Koreanum Nakai by supercritical fluid extraction,” Chemical Research in Chinese Universities,vol.20,no.6,pp.707–710,2004. [5] J. Wang, Y.-M. Zhao, C.-Y. Guo et al., “Ultrasound-assisted extraction of total flavonoids from Inula helenium,” Pharmacog- nosy Magazine,vol.8,no.30,pp.166–170,2012. [6]Y.C.Zhang,C.M.Liu,J.Li,Y.J.Qi,Y.C.Li,andS.N.Li, “Development of ‘ultrasound-assisted dynamic extraction’ and itscombinationwithCCCandCPCforsimultaneousextraction and isolation of phytochemicals,” Ultrasonics Sonochemistry, vol. 26, pp. 111–118, 2015. [7]Y.C.Zhang,C.M.Liu,Y.J.Qi,Y.C.Li,andS.N.Li,“Devel- opment of circulating ultrasound-assisted online extraction coupled to CCC and CPC for simultaneous extraction and isolation of phytochemicals: application to Ligusticum chuanxiong Hort,” Industrial Engineering Chemistry Research,vol.54,no.11, pp. 3009–3017, 2015. [8]E.Garcia-Castello,A.Rodriguez-Lopez,L.Mayor,R.Balles- teros, C. Conidi, and A. Cassano, “Optimization of conventional andultrasoundassistedextractionofflavonoidsfromgrapefruit (Citrus paradisi L.) solid wastes,” LWT—Food Science and Technology,vol.64,no.2,pp.1114–1122,2015. [9] C. Carrera, A. Ruiz-Rodr´ıguez, M. Palma, and C. G. Barroso, “Ultrasound-assisted extraction of amino acids from grapes,” Ultrasonics Sonochemistry,vol.22,pp.499–505,2015. [10] Y. C. Hai, N. M. Hosakatte, H. M. Sang, Y.C. Yong, L. Eun-Jung, and P. Kee-Yoeup, “Comparison of conventional and ultrasound-assisted methods for extraction of nutraceutical compounds from Dendrobium candidum,” CyTA-Journal of Food, vol. 12, no. 4, pp. 355–359, 2014. [11] B. Singh, H. K. Sharma, and B. C. Sarkar, “Optimization of extraction of antioxidants from wheat bran (Triticum spp.) using response surface methodology,” Journal of Food Science and Technology,vol.49,no.3,pp.294–308,2012. [12] W.B. Kong, N. Liu, J. Zhang et al., “Optimization of ultrasound- assisted extraction parameters of chlorophyll from Chlorella vulgaris residue after lipid separation using response surface methodology,” Journal of Food Science and Technology,vol.51, no. 9, pp. 2006–2013, 2012. [13] A. C. Martins, L. Bukman, A. M. M. Vargas et al., “The antioxidant activity of teas measured by the FRAP method adapted to the FIA system: optimising the conditions using the response surface methodology,” Food Chemistry,vol.138,no.1,pp.574– 580, 2013. [14] J. Chanderesh, K. Atul, Z. Robel, A. Anghesom, B. Lwam, and J. K. Jeevan, “FRAP (Ferric reducing ability of plasma) assay and effect of Diplazium esculentum (Retz) Sw. (a green vegetable of North India) on central nervous system,” International Journal of Photoenergy,vol.3,pp.228–231,2012. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 591869, 11 pages http://dx.doi.org/10.1155/2015/591869

Review Article Health, Wellness, and Safety Aspects of the Consumption of Kombucha

Mindani I. Watawana, Nilakshi Jayawardena, Chaminie B. Gunawardhana, and Viduranga Y. Waisundara Functional Food Product Development Project, National Institute of Fundamental Studies, Hantane Road, 20000 Kandy, Sri Lanka

Correspondence should be addressed to Viduranga Y. Waisundara; [email protected]

Received 10 September 2015; Revised 24 November 2015; Accepted 2 December 2015

Academic Editor: Tzortzis Nomikos

Copyright © 2015 Mindani I. Watawana 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.

Functional foods have been identified as whole foods and fortified, enriched, or enhanced products which have a potentially beneficial effect on health when consumed as part of a varied diet on a regular basis, at effective levels. As consumer awareness on functional food escalates, the interest towards conducting scientific studies in this field has also proportionately increased. Many of the traditional food products are known to possess bioactive components, thus qualifying as functional food. Kombucha tea is produced by fermenting sugared black tea with a mixed culture of yeast and bacteria. Kombucha tea has gained immense popularity in recent times due to many associated health benefits. The therapeutic effects of this beverage are thought to be derived from the chemical composition of this beverage, mainly the polyphenols and secondary metabolites which are produced during fermentation. However, the safety aspects of the beverage also need to be taken into account when qualifying the beverage as a functional food. Nevertheless, Kombucha tea could be easily recognized as a beverage which is able to replace the consumption of carbonated beverages due to its possession of health benefits and therapeutic properties.

1. Kombucha: Preparation and to a mild vinegar-like taste, thus increasing the consumer Fermentation Process acceptabilityoftheflavorandothersensoryaspectsofthe beverage [4, 5]. The microbial composition of the Kombucha Kombucha tea is known under different names throughout teacultureisknowntovaryfromoneculturetoanother theworldsuchasredteafungus,Champignondelongue depending on factors such as geographic location, climate, vie, Ling zhi, kocha kinoko, Chainii grib, and Chainii kvass the local species of bacteria and yeast, and the source of [1]. It is traditionally prepared by fermenting sugared black the inoculum [6]. In some studies it has been proven that tea with a symbiotic culture of yeast and bacteria. This the use of different Kombucha starter cultures can cause a beverageisthoughttohaveoriginatedinChinaover2000 development of different antioxidant activity pathways even years ago [2], while there are many historical reports of though the same substrate has been used [1, 7]. this beverage being consumed in countries such as Russia, The yeast and bacteria involved in this microbial fer- Germany, and the Middle East as well [3]. In many countries mentation form a mat-like pellicle known as a “tea fungus.” this beverage is produced in large-scale for commercial use The yeast component of this culture commonly consists as well as in domestic conditions. Despite being a fermented of osmophilic yeast species, while the bacterial component beverage, the flavor of Kombucha tea is considered to be includes acetic acid bacteria. In many studies it has been satisfactory and nonacrimonious, though mildly acidic and found that the dominant acetic acid bacterial species found in mildly alcoholic, similar in taste to apple cider [4]. As the the microbial cultures are Acetobacter xylinum, A. xylinoides, fermentation progresses, the taste of Kombucha tea changes A. aceti, A. pausterianus, and Bacterium gluconicum [5]. from a pleasantly fruity, sour, lightly, and sparkling flavor The dominant yeast strains were found out to be Kloeckera 2 Journal of Chemistry

Approximately 50 g of white sugar is dissolved in 1 liter of boiling concentration of the compounds found in this beverage also water differ with the starter culture, amount of starter culture used, typeoftea,andtypeofsugar[6].Eventhoughtraditionally black tea is being used as the substrate, Kombucha prepared Approximately 5 g of tea leaves are added to the mixture and allowed to infuse for 5 min by other substrates such as green tea and oolong tea are commonly available. Green tea has shown to have a better stimulation effect on the Kombucha fermentation compared The tea leaves and filtered and allowed to cool to room with black tea fermentation and this leads to formation of temperature the product in a shorter period of time [11, 12]. In some rare instances this beverage has been prepared by lemon The cooled tea is added to a sterile glass jar and inoculated balm tea, mulberry tea, jasmine tea, and peppermint tea with the freshly grown Kombucha starter culture [6, 11, 13]. The resulting broth is filtered out and the liquid portion is consumed. The tea fungus which is initially added to the tea is called the “mother tea fungus,” where during The fermentation is allowed to be carried out the fermentation the development of a “daughter tea fungus” takes place. The cellulosic pellet rests on top of the tea broth andproducesafreshthinlayerofthedaughtermatwhich The pellicle which is formed is removed and the liquid portion is strained away for consumption isavailableasanewlayerabovetheoldKombuchaculture mat. This new daughter mat is formed with each successful Figure 1: The typical method of preparation of the Kombucha fermentation step, and it is used to reinoculate a new batch beverages using sugared black tea. of tea. At the beginning of the fermentation, a small portion of previously prepared Kombucha broth may be added to the new tea to decrease the pH in order to stop the growth of undesired microorganisms [8]. As the fermentation pro- spp., Schizosaccharomyces pombe, Saccharomyces ludwigii, S. gresses the appearance of gas bubbles will occur due to cerevisiae, Torulaspora spp., Zygosaccharomyces bailii, and carbonic acid produced during the fermentation. Pichia spp.,[5].Thefungal-likestructureisformedbythe presence of these microbes in a zoogleal mat. This mat is produced due to the formation of a thin layer of floating 2. Composition of Kombucha Tea and How It cellulosetowhichthecellmassofbacteriaandyeastis Differs from Black Tea attached [6]. The cellulose is produced by the bacterial component of the microbial consortium. A. xylinum is primarily The microbes in this culture are able to ferment the sugared knowntoberesponsibleforthecelluloseproductionand black tea and produce a complex cocktail of molecules. At the this cellulose network enhances the association between the end of the fermentation process, the resulting beverage would bacteria and the fungi [8]. It has been reported that the consistofsugars,polyphenolssuchascatechins,organicfood caffeineandrelatedxanthinesfoundinteahavetheability acids, lysine, fiber, ethanol, amino acids, essential elements to stimulate the synthesis of this cellulose production by suchasNa,K,Ca,Cu,Fe,Mn,Ni,andZn,water-solublevita- the bacteria [8]. This powerful symbiotic association and its mins such as vitamin C, vitamin B, and vitamin B2,catalase, byproducts have the ability to inhibit the growth of potential carbon dioxide, substances which act as antibiotic substances, contaminating bacteria [6]. The bacterial cell and yeast cell and some hydrolytic enzymes [1, 14]. The chemical structures 4 6 −1 numbers are generally thought to reach 10 –10 cfu mL in of the commonly produced acids are shown in Figure 2. As aKombuchaculturewhichhasbeenallowedtofermentfor the fermentation progresses, the yeast component of this a span of approximately 10 days [9]. Many scientific studies mixed culture is able to break down sucrose to produce have proven that yeast outnumbers the bacterial count [5, glucose, fructose, and carbon dioxide which gives off the 10]. As the fermentation progresses, the acidity of the broth effervescence and the sparkling appearance. The acetic acid increases due to the production of organic acids. Due to bacteria have the ability to convert glucose to gluconic acid high acidity-induced oxygen starvation, the number of viable andfructoseintoaceticacid.Also,theyeastcomponentis microbial cells present in Kombucha tea decreases as well able to produce ethanol which is then oxidized into acetalde- [10]. The number of yeast and bacterial cells in the broth hyde by the bacterial counterpart of this colony [15]. The yeast hasbeenreportedtobemorethanthecellnumberinthe prefers fructose as the substrate when producing ethanol [8]. cellulosic pellicle [5]. The acetic acid produced by the acetic acid bacteria has the In terms of the traditional preparation, the starter culture ability to stimulate the production of ethanol by yeast, and is added to a sugared black tea infusion and the fermentation ethanol in turn can facilitate the acetic acid bacteria to grow is allowed to happen for a period of 3 to 10 days under and produce acetic acid. The ethanol and acetic acid present ambient temperature [6]. A flow-chart containing the general in the Kombucha broth have been reported to be involved preparation method is shown in Figure 1. Nevertheless, under in the antimicrobial activity of the broth against pathogenic domestic conditions of preparation, the amount of tea used bacteria, thus providing a protection against contamination for the fermentation process and the method of preparation of the tea fungus [8]. It has been reported that even if the differ according to personal preferences. The flavor and byproducts of the fermentation process decrease, the pH Journal of Chemistry 3

OH OH OH O OH O OH O HO OH OH HO HO OH O OH OH O OH Glucuronic acid Gluconic acid Malic acid

O O O OH OH O O CH3 HO HO HO OH O OH OH Oxalic acid L-lactic acid Citric acid Figure 2: The most abundant organic acids found in Kombucha tea as a result of the fermentation process.

value of this beverage has a buffering capacity. This buffering Table 1: Comparison of anionic mineral concentration of Kom- capacity is due to carbon dioxide dissociation, and when this bucha tea and Black tea [7]. process happens, production of amphiprotic hydrocarbonate − Anion Kombucha tea (mg/g) Black tea (mg/g) anion (HCO3 ) occurs. This anion has the ability to react with − + F 3.20 ± 0.16 1.20 ± 0.06 hydrogen ions (H ) from organic acids and inhibit further − + Cl 0.96 ± 0.04 3.12 ± 0.13 change of H concentration in the broth and thus contribute − to formation of buffer in the system [16]. Br 0.04 ± 0.01 0.04 ± 0.01 − ± ± Even though Kombucha is based on the preparation NO3 0.18 0.01 0.34 0.02 2− of black tea, other than the fermentation process, it is HPO4 0.04 ± 0.01 0.08 ± 0.01 2− considered different due to the chemical contents themselves. SO4 1.02 ± 0.04 4.20 ± 0.17 − Tea (Camellia sinensis), in general, is one of the most popular I 1.04 ± 0.08 0.44 ± 0.04 beverages used worldwide and it has been consumed by many, for centuries. There are three major types of teas, namely,black,green,andoolong.Outofthesethreetypes, other [18]. Kombucha tea is known to exhibit an increase black tea is the most popular and it accounts for nearly 80% in the radical scavenging properties during the fermentation of the tea consumed worldwide [15]. This form of tea is possess, showing a higher antioxidant capacity than black tea prepared by the infusion of dried leaves of Camellia sinensis [19]. in hot water. The tea leaves, after picking, are air dried thencrushedtoreleasetheenzymesandthenallowedfor enzymatic fermentation. Then, they are dried again to obtain 3. Health Benefits Associated with the final product. Some of the main components of tea are Consumption of Kombucha purine alkaloids such as caffeine, theaflavins, gallotannins, triterpene, saponins, flavonoids, mineral compounds, car- The American Diabetic Association has defined functional bohydrates, and vitamins [15, 16]. Black tea is known to food to be products including whole foods and fortified, possess a higher radical scavenging property and the major enriched, or enhanced foods, which have a potentially bene- contributor of this property is known to be polyphenols [17]. ficial effect on health when consumed as part of a varied diet Depending on the geographical location, climate, season, soil on a regular basis, at effective levels [20]. Kombucha beverage fertility, and plantation method, a variation can be seen in is known to possess many prophylactic and therapeutic this particular property [17]. Kombucha tea differs from its benefits; it is believed to help in digestion, give relief against parental food by the antioxidant content, starch hydrolase arthritis, act as a laxative, prevent microbial infections, inhibitory activity, anionic mineral composition, and acid combat stress and cancer, provide relief against hemorrhoids, content. Kumar et al. [18] have demonstrated the presence impartapositiveinfluenceonthecholesterollevels,and of the anionic minerals such as fluoride, chloride, bromide, facilitate excretion of toxin as well as blood cleansing [1, iodide, phosphate, sulphate, and nitrate in both black and 6, 8]. This beverage is also associated with influencing the Kombucha tea. Table 1 shows a comparison of anionic min- gastrointestinal microbial flora in humans by acting as a eral concentration of Kombucha tea and Black tea. They have probiotic drink and helping in balancing the intestinal flora, further revealed that the anionic mineral compositions of thus facilitating the normalization of intestinal activities to Kombucha and black tea are significantly different from each a certain extent [1, 21, 22]. It is also known to have the 4 Journal of Chemistry

Table 2: Some constituents of Kombucha related to health claims and their recommended values for consumption.

Amount present in Amount present in LD50 value in rats via Recommended maximum level as per Chemical constituent Kombucha green tea Kombucha black tea oral route (mg/kg of standard guidelines (g/L) (g/L) body weight) 9.51 ± 0.35 (on day 15) 6.17 ± 0.3 (on day 15) Acetic acid 2.1 g per day [62] 3310 [63] [33] [33] 1.57 ± 0.14 (on day 15) 1.5 ± 0.17 (on day 15) Glucuronic acid Not available Not available [33] [33] FDA requirement substance added 0.15 ± 0.02(onday15) 0.33 ± 0.07 (on day 15) directly to the human food affirmed as Lactic acid 3730 [65] [33] [33] generally recognized as safe (GRAS) [64] Citric acid Not available Not available 3000 mg/kg per day [66] 3000 [67] 0.14 mg/kg per day (the scientific basis forthisconclusionisnotavailableand Oxalic acid 0.03 (on day 3) [33] 0.11 (on day 3) [33] 7500 [69] therefore no safe dietary dose for oxalic acid can be established) [68] Acetaldehyde Not available Not available 0–2.5 mg/kg per day [70] 661 [71] abilitytoimprovethehealthofhair,skin,andnails,reduce fermented products such as fermented milk, the amount of stress and nervous disturbances, reduce insomnia, relieve studies done on this beverage is very low. Nevertheless, the headaches, reduce the craving for alcohol of an alcoholic subsequent sections of this review summarize the purported person, and prevent the formation of bladder infections health benefits of Kombucha based on available scientific [6, 8]. Reducing the kidney calcification is also known to evidence and reports. In addition, given the fermentation be a beneficial effect of this beverage [6]. Reduction of process involved in producing the beverage, the review also menstrual disorders and menopausal hot flashes, improving summarizes the safety issues and aspects of caution which eye sight, cellular regeneration, stimulation of glandular need to be borne in mind when consuming the beverage. systemsinthebody,relievingbronchitisandasthmaand the enhancement of general metabolism are a few more of the health benefits which have been claimed to be associated 3.1. Antimicrobial Activity. Kombucha tea is known to show with consumption of the Kombucha broth [6]. The beneficial a remarkable antimicrobial activity against a broad range effects of this beverage are known to be attributed to the of microorganisms. Many scientific studies have been done presence of the metabolic products released into the broth on this subject and the Kombucha broth has demonstrated during the fermentation, although most of the health benefits inhibitory activity against many pathogenic microorganisms arehypothesizedtobeduetoitsradicalscavengingpotential. of both Gram positive and Gram negative origin [8]. Kom- The microbial community has the ability to enhance the bucha tea has demonstrated the ability to inhibit the growth of radical scavenging activity of black tea by the fermentation pathogens such as Helicobacter pylori (the causative organism process[8].Also,thepresenceofglucuronicacidimparts of peptic ulcers), Escherichia coli (the causative organism beneficial properties to this drink [9]. Glucuronic acid is of common diarrhea), Entamoeba cloacae, Pseudomonas normally produced by a healthy liver and is a highly water aeruginosa, Staphylococcus aureus, Staphylococcus epider- soluble carboxylic acid. This acid can be converted into mis, Agrobacterium tumefaciens, Bacillus cereus, Aeromonas glucosamine and chondroitin-sulfate which are associated hydrophila, Salmonella typhimurium, Salmonella enteritidis, with collagen and also the fluid that acts as lubricate in Shigella sonnei, Leuconostoc monocytogenes, Yersinia entero- the joints [23]. Butyric acid produced by the microbial colitica, Campylobacter jejuni, and Candida albicans [8, 24, consortium in the fermentation process is known to protect 25]. This antimicrobial activity of the broth is attributed human cellular membranes. In combination with glucuronic tothelowpHvalueofthisbeverage,especiallyowing acid, this complex has the ability to strengthen the walls of gut tothepresenceofaceticacidinparticularandarange and give protection against parasites [8]. Table 2 shows some of other organic acids and catechins which are shown in of the constituents of Kombucha related to health claims and Figure2aswellasmanylargeproteinswhichareproduced their recommended values for consumption. Although it may during the fermentation [8, 24]. Acetic acid and catechins appear that many reports which are available on Kombucha are specially known to inhibit a range of Gram positive are based on personal experiences and testimonials, in recent and Gram negative microorganisms [6]. It has also been times, scientists have provided scientific evidence elucidating demonstratedthatthebrothmaycontainantibioticsub- the therapeutic effects of the beverage from in vitro as well as stances which give the antimicrobial property [24, 26]. Some in vivo studies. Table 3 shows the bioactivities of Kombucha studies have demonstrated that Kombucha tea shows not and the mode of studies which have been carried out. only antibacterial activity but also antifungal activity [27]. However, in contrast to the number of studies done on other The antifungal activity is attributed to the production and Journal of Chemistry 5

Table 3: Bioactivities of Kombucha and mode of studies which have been carried out. Bioactivities References Antihypercholesterolemic effect In vitro studies on rats [33] Antioxidant activity against chromate In vivo studies on rats [47] Antioxidant activity against lead In vivo studies on rats [3] Antistress activity against cold and hypoxia In vivo studies on rats [46] In vitro studies on human peripheral blood Cytogenetic activity lymphocytes [72] Healing activity against indomethacin-induced acute In vivo studies on mice [50] gastric ulceration Hypoglycaemic effect In vivo studies on mice [6, 73]

Inhibitory activity towards CCl4 induced hepatic injury In vivo studies on rats [45] Longevity In vivo studies on mice [74] Paracetamol induced hepatotoxicity In vivo studies on mice [47] Prevention of weight loss in diabetes In vivo studies on rats [75] Protective effect on chromosomal aberrations induced by In vitro studies on human peripheral lymphocytes [76] gamma radiation in human peripheral lymphocytes

presence of acetic acid in this beverage. In recent times, who work under unhealthy environments, such as workers theimmergenceofresistantstrainsofpathogensassociated in mines and polar expeditors [14]. When the human body with human diseases has been widely seen, and the use is exposed to such conditions for a prolonged period of time, of Kombucha tea as an antimicrobial product can be used the normal microbial consortium of microbes in the intestine to overcome this problem [27]. In this aspect, it has been changes due to the unnatural conditions, psychoemotional demonstrated that the antimicrobial activity of Kombucha discomfort, and drastic change in the diet. This may lead tea prepared form green tea shows a higher activity than to the disappearance of the protective gut microbes and the Kombucha tea prepared traditionally from black tea [27]. immergence of harmful secondary infections by opportunis- tic microbes. This shift in the gut microbiota can lead to many health issues such as allergies, autoimmune diseases, multiple 3.2. Probiotic Effects. Probiotics are known to be living sclerosis, and transplant infectious disease. The change in the microorganisms; when administrated in adequate amounts, gut microbiota can be corrected to some extent with the help they are able to result in health benefits. Most often, the of Kombucha tea. In light of these mentioned possibilities, bacterial component of a probiotic mixture comes from scientists have started to consider this beverage to be used by Lactobacillus or Bifidobacterium or a cocktail of these two astronauts as a supplement to their diet in outer space [14]. strains. In support of these lines, there can be a few common yeast types such as Saccharomyces boulardii and S. cerevisiae in this mixture as well [14]. Probiotic microbes are known to 3.3. Anticancer Properties. Dietary phytochemicals have been play a vital role in the wellness of human health. Probiotic identified as effective anticancer agents. Thus, there is a recent microorganisms provide a balance in intestinal microbiota, trend of consuming food rich in these bioactive compounds. normalizing processes in gut and boosting the immune Scientific studies have claimed that Kombucha has anticancer system. In addition, they help in improving digestion, fighting effects as well [8]. The Central Oncological Research Unit in against harmful bacterial overgrowths, and achieving mental Russia and the Russian Academy of Sciences in Moscow have clarity and mood stability and against psychological con- conducted population studies on this fermented beverage ditions such as anxiety and depression. Many studies have andhavefoundthatthedailyconsumptionofKombucha claimed that this beverage not only is a probiotic but also broth has a correlation with an extremely high resistance acts as a symbiotic, a combination of prebiotics and probiotics to cancer [8]. Scientists have come up with many possible [8,21].Aprebioticselectivelyhelpsthegrowthandactivityof mechanisms for the anticancer ability of this beverage. For the consortium of beneficial microbes present in the human instance, it has been reported that the ability of this fermented gut [21]. The bacteria and yeast present in this beverage act beverage to act as an anticancer agent is due to the presence as probiotics and the microcellulose which is present can of tea polyphenols and the secondary metabolites produced help in the growth of the beneficial microbes present in the during the fermentation process [6, 28]. Many studies have intestine [14]. The popularity of this beverage as a probiotic shown that the ability of the tea polyphenols present in this and a synbiotic has increased in recent times as scientists have fermented beverage to inhibit gene mutations, inhibit the found that this beverage can be used to give the required proliferation of cancer cells, and induce cancer cell apoptosis nutrition and help maintain health and wellness in humans and the ability to terminate metastasis have been highlighted 6 Journal of Chemistry as possible mechanisms for the anticancer properties [29– toxin removal action of Kombucha tea is known to help in 31]. It has also been noted that the consumption of Kom- obtaining relief from gout, rheumatism, arthritis, and kidney bucha tea can help cancer patients to reequilibrate blood stones which are conditions associated with the accumulation pH which usually increases more than 7.56 in the course of toxic substances in the body [8]. of the illness. Additionally, cancer patients lack L-lactic acid in their connective tissues; this can also be corrected by the consumption of Kombucha, where the fermentation 3.5. Antioxidant Activity. The popular definition of an antiox- process produces lactic acid as a byproduct [8]. Many of the idant is any substance, when present at low concentrations compounds which were identified to be present in Kombucha compared with that of an oxidizable substrate, that signifi- tea such as polyphenols, gluconic acid, glucuronic acid, lactic cantly delays or inhibits oxidation of the substrate [36]. This acid, and vitamin C are known to have the ability to reduce bioactivity could be broadly presented in the form of (1) the occurrence of stomach cancer. It has also been found out scavenging properties of molecules, (2) binding of prooxidant that Kombucha contains D-saccharic acid-1,4-lactone (DSL) metals, and (3) inhibition of prooxidant enzymes. Many which is known to inhibit the activity of glucuronidase, an studies have proven the effect of these antioxidant properties enzyme which is thought to be indirectly related to cancer on many human diseases such as cancer and diabetes [7]. The [26]. Glucuronidase has the ability to hydrolyze glucuronide primary mechanism of the action of antioxidants in these and produce cancer-causing aglycones [32]. It was reported disease conditions is to remove free radical intermediates, that the polyphenols present in the Kombucha tea pos- and these free radicals are generated in oxidation reactions sess antitumor properties, thus acting as a cancer-blocking which happen across the human body. Free radicals have the agent [26]. Another study reports presence of dimethyl2- ability to start multiple chain reactions which will eventually (2-hydroxy-2-methoxypropylidene) malonate and vitexin in lead to cell damage or the death of the affected cell [36]. the ethyl acetate fraction of Kombucha tea which has shown When an antioxidant comes in contact with free radicals, cytotoxic effects at a concentration of 100 𝜇g/mL [28]. they have the ability to oxidize themselves and inhibit other oxidation reactions which lead to harmful chain reactions. Theoxidativestresscausedbyfreeradicalsplaysanimportant 3.4. Detoxification. Detoxification is the complex process role in many of the commonly prevalent human diseases such of removal of toxic substances from the body of a living as Parkinson’s disease, coronary heart disease, and cancer, the organism. This process can be physiological or medicinal. In reasons being the lack of appropriate nutrition and exercise, the human body, this process is carried out mainly by the air pollution in the environment, and smoking [2]. In order to liver. Detoxification helps in the maintenance of a healthy counterbalance this oxidative stress caused by free radicals, it liverandisalsoknowntoplayapartincancerprevention.The is important to incorporate antioxidant containing food stuff enzymes, bacterial acids, and other secondary metabolites to the daily diet [36]. produced by the microbes during the fermentation process During the Kombucha fermentation, many compounds done in the preparation of Kombucha tea have displayed with radical scavenging properties are released from the the ability to detoxify body [8]. In addition, most of the tea leaves themselves [1]. Polyphenols and catechins are the enzymes and bacterial acids found in Kombucha tea are main group of compounds which are found in tea belonging very similar to the chemicals produced by the body for the to flavanol group [1, 27, 37]. Polyphenols are considered purpose of the detoxifying process. Thus, incorporation of as having high levels of broad antioxidant properties since Kombucha tea into one’s diet may result in the reduction of they have the ability to scavenge free radicals and reactive the detoxification load pressured on the liver. Many scientific oxygen species (ROS) [2]. Polyphenols are about 30% of studieshavereportedthatthisabilityismainlyduetothe the total dry weight of fresh tea leaves and epigallocatechin, capacity of glucuronic acid to bind with toxic molecules epigallocatechin-3-gallate, epicatechin-3-gallate, and epicat- which enter the body and also the ability to increase excretion echin are the most prominent types of polyphenols found of these molecules from the physiology by the help of kidneys in tea leaves [19]. Kombucha tea when prepared using green and intestines [33]. The process of strong binding between tea, black tea, and tea waste material has been shown to have the glucuronic acid molecules and the toxin is known as a high radical scavenging activity [19]. When the complex glucuronidation [34]. Glucuronic acid is produced due to phenolic compounds are present in an acidic environment or the oxidation process of glucose during the fermentation when enzymes liberated by bacteria and yeasts in tea fungus process [34]. This acid is the most significant detoxifier are present, degradation of complex molecules to small in human body; thus, it has the ability to bind to the molecules happens and this causes an increase in the total toxins in the liver and encourage them to flush out of the phenolic compounds available in the Kombucha tea broth body [35]. Similar to glucuronic acid, malic acid is also a [2]. Therefore, when the fermentation happens, the total byproduct of the fermentation which helps in detoxifying phenolic content increases [2]. The production of compounds the liver [2]. Other than supporting the detoxification of the possessing radical scavenging properties depends on the liver, consumption of Kombucha tea is also known to help culture period and starter origins where they decide which excrete heavy metal substances and environmental pollutants metabolites are to be produced [7]. However, prolonged from the human body through the kidneys [9]. It is also fermentation is not suitable as accumulation of organic acids beneficial in the biotransformation of indigenous metabolites can cause a harmful effect when Kombucha tea is directly such as bilirubin and excess of steroid hormones [2]. The consumed [2]. Journal of Chemistry 7

3.6. Hepatoprotective Effects. Hepatoprotection is the ability during the Kombucha tea fermentation. It is able to enhance to prevent the damage occurring to the liver by toxic the blood circulation and helps prevent constipation [8]. substances [22, 38]. Many studies carried out on cell lines Oxalic acid, which is also produced as a byproduct, can be andanimalmodelshaveshownthatKombuchabrothshows useful in the production of adenosine triphosphate (ATP) hepatoprotective activity against various environmental pol- [6]. Bacteria found in the Kombucha mat produce gluconic lutants [6]. Many of the environmental pollutants have the acid by breakdown of caprylic acid which can prevent certain ability to induce hepatotoxicity and damage the liver. Many types of yeast-based infections and candidiasis [33]. Butyric scientific studies were carried out to assess the ability of this acid is a production of yeast available in the Kombucha mat tea broth to effectively attenuate the physiological changes and helps in protecting the human cellular membrane, where which are caused by many of the hepatotoxicity-causing it combines with gluconic acid and strengthens the gut walls agents such as aflatoxin B1 [39], cadmium chloride [40], in conditions such as candidiasis [6]. Moreover, healing of tert-butyl hydroxyperoxide [41], and acetaminophen [42, 43]. gastric ulcers in rats due to Kombucha tea has been evident Carbon tetrachloride (CCl4) is a xenobiotic that induces the as per histopathological and biochemical studies [50]. The − lipid peroxidation and it forms a free radical CCl3 ,and usage of this beverage to eliminate the growth of gray hair this involves accumulating lipid derived oxidants which leads andtheusageintheimprovementofeyesightareafewof to liver injury [44]. Kombucha tea consumption has been the health benefits which have been claimed as well [8]. demonstratedtoinhibittheactivityofCCl4 and prevent Oral supply of Kombucha tea at a dose of 5 mg/kg of body liver injury in rats [45]. In vivo studies have suggested weight in alloxan-induced diabetes rats has depicted better that Kombucha tea is capable of preventing paracetamol inhibition on 𝛼-amylaseandlipaseenzymeactivityinthe induced hepatotoxicity [46]. Studies have been carried out plasma and pancreas and also better suppression of increased to investigate how Kombucha tea can induce oxidative stress blood glucose levels [48]. Thus, Kombucha tea has potential in Albino rats by chromate (VI) [47]. Studies have also hypoglycemic and antilipidemic activity as well. been carried out to find protective effects of Kombucha tea against thioacetamide-induced hepatotoxicity and the results have shown that the antioxidant activity of polyphenol 4. Safety Issues and Controlling Potential substances of Kombucha tea is responsible for this function Hazards of Kombucha [22]. These studies have further explained that Kombucha tea prevents the apoptotic cell death of the hepatocytes SinceKombuchaisacomplexmixtureofmicroorganisms, which is triggered from the exposure of the liver to the it is essential to discuss the safety of Kombucha tea for environmental toxins [22]. Histological analyses of alloxan- consumption. As mentioned previously, bacteria and fungi induced diabetes rats given a diet containing Kombucha tea in the Kombucha zoogleal mat are capable of forming a have revealed protective liver-kidney functions [48]. This powerful symbiosis which can inhibit the growth of contam- is supported by the reduction in the activity of aspartate inating bacteria [8, 51]. Nevertheless, pathogenic microor- transaminase, alanine transaminase, and gamma-glutamyl ganisms can contaminate the Kombucha tea throughout the transpeptidase in the plasma, as well as in the creatinine urea preparation. Due to fermentation, the pH reaches ≤ 4.2. concentrations [48]. Pathophysiological evidences are also However,untilthisisachieved,thereisahighpossibility available for hepatoprotective effects of Kombucha tea in rat for contaminations to occur [52]. Mold contamination can models [41, 43]. occur on Kombucha cultures, especially with Penicillium and Aspergillus, when the Kombucha tea is home-made. Aspergillus species are known to cause carcinogenic and 3.7. Other Therapeutic Effects. There are many allegations of toxigenic effects [53]. Therefore, it is important to be cau- health benefits and uses related to this beverage, some of tious when administering infected beverages by immune- which are not discussed in this review in further detail. For compromised individuals. It is essential to concern that that instance, the microbial mat produced in the fermentation the efficacy of Kombucha tea as a therapeutic beverage is had been used to produce artificial skin in Nossa Senhora hardly proven by clinical trials involving human subjects [54]. da Conceic¸ao˜ Hospital from Lagarto, SE, Brazil [49]. Some Beside, even though consumption of Kombucha claims to researchers have used this skin to accelerate the healing havenoadversesideeffects,therehavebeenafewexceptions process and also as an antiseptic by adhering it to open over past years. Few case reports and case series question injuries, the so-called Bioskin [49]. The bacterial cellulose the safety of Kombucha tea with suspected liver damage, which is produced during the Kombucha tea fermentation metabolic acidosis, and cutaneous anthrax infections [54]. process has many potential applications in fields such as food Allergic reactions and an uncomfortable stomach are a result and biopharmaceuticals. The ability and trend to use bacterial of consuming Kombucha tea by people with acid sensitivities cellulose in these fields are due to the high purity and the and renal insufficiencies [53]. Several studies have confirmed unique physicochemical properties which are present in the that Kombucha can cause nausea, shortness of breath, throat fermented beverage. In addition, this bacterial cellulose is tightness, headache, dizziness, and jaundice [34, 53]. One case preferred in instances where plant based cellulose cannot be revealed that two persons had developed allergic reactions, used [5]. In the food industry bacteria-based cellulose is used a third person developed jaundice, and another developed as food matrices, thickeners, dietary fibers, stabilizes, and nausea,vomiting,andheadandneckpain.Thecasereports binders [5]. Lactic acid is one of the organic acids produced an etiological association of all four patients since all of them 8 Journal of Chemistry had consumed Kombucha tea in proximity to the onset of form a substantial conclusion about the safety aspects in the symptoms and their subsequent disappearance after cessation consumption of Kombucha tea. of drinking the beverage [55]. Some individuals have reported Using clean and sanitized utensils during preparation of that they felt dizziness and nausea after consumption of Kombucha tea helps in preventing any contamination. It is Kombucha tea [56]. However, it was not explained whether better to keep the preparation and fermentation areas clean these symptoms are a result of unusual toxins developed in a to control growth of any microorganisms since tea must be ∘ particular batch of Kombucha tea. cooled to about 20 C within two hours prior to adding the Overfermentation can increase the availability of high Kombucha culture into the tea. It is also important to control acetic acid concentrations, and this might lead to the leeching the pH during the fermentation process since overproduction of some chemical contaminants from the fermentation vessel of acetic acid can be hazardous. According to the British or packaging materials. There is evidence that severe lead Columbia Center for disease control, the fermentation should poisoning can be caused by regular use of Kombucha tea be terminated at the pH 4.2 [56]. Overproduction of alcohols which was brewed in a ceramic pot [57, 58]. This might andcarbondioxidecanalsobepreventedbypasteurizing have occurred due to Kombucha tea being acidic and the the finished product [57]. Adding 0.1% sodium benzoate and resulting reactions caused by some ceramics. Most of the 0.1% potassium sorbate to the finished product, followed ceramics contain very low levels of lead which would not be by refrigeration, can also be carried out for safety purposes of any danger when brewing Kombucha tea. Nevertheless, [61]. Refrigeration of the final product is the most common if the Kombucha is steeped in them for a long time, then method followed by the commercial producers of Kombucha high amounts of lead can dissolve in the tea [6, 57]. It tea [13]. is important to use glass containers for the preparation and storage of Kombucha tea to prevent leaching of toxic elements such as lead into the beverage. In addition to acetic 5. Conclusions acid, Kombucha tea contains several organic acids [6, 33]. The chemical composition of the Kombucha tea depends on Some of these metabolites have the potential to damage type of tea leaf variety, amount of sugar, and fermentation liver and kidney at high concentrations, as evident by few and composition of tea fungus. However, there are a very case reports [56, 59, 60]. Kombucha tea is contraindicated few studies focused on the safety of consuming Kombucha in pregnant and lactating women [53]. Studies have also tea. Most of the health properties of Kombucha are related reported the presence of Bacillus anthrax in Kombucha tea to the acidic composition of the beverage and acetic acid, fermented under unhygienic conditions [61]. The source of which is the major acidic component to inhibit the fungal B. anthrax was found to be cows and anthrax was passed growth.Overproductionofacidscanbecontrolledduring to an individual who rubbed it on his skin to alleviate the the fermentation duration as well as the packaging of the pain. final product. Yet, the importance of studying the safety of Due to the detoxification effects of Kombucha tea, toxic Kombucha tea consumption is important as there are only a materials are forced to be excreted from the body. However, very few such studies been carried out throughout the years. if the kidneys are not working properly, these individuals may Since this a popular beverage around the world, investigating not be able to successfully discharge the toxic materials. Thus, the advantages and disadvantages of consuming this beverage it is recommended to drink plenty of water to facilitate the in similar capacities can be extremely meaningful. According elimination of toxins to overcome this problem [53]. From to literature, there is no evidence about systematic human this aspect, it is essential to give attention to the abnormal trials being done using Kombucha tea. This could be an area odour or colour development in Kombucha tea to overcome in which future research could be focused in establishing this any adverse side effects. Domestic cultivation of Kombucha is beverage as a functional food. Since studies carried out to one of the most possible ways of contamination by pathogenic assess qualitative and quantitative properties of constituents bacteriaandyeasts.Sincethisteafungusisbeinggrown of Kombucha are scattered, scientific research should be under aseptic conditions and it is propagated from one carried out to clarify the health beneficial claims and safety house to another, the transfer of contaminations is high aspects, which might help in promoting this beverage among [13, 53]. A study reported that consumption of Kombucha consumers. Despite the scattered safety issues, production mightalsobeahealthriskforHIV-positivepatients[6]. and consumption of this beverage in a safe manner can It was discovered that acute renal failure may occur with be used to substantiate the stand of this beverage as a lactic acidosis and hyperthermia due to consumption of this replacement for carbonated beverages. Attention of relevant beverage. The possibility of getting infected by toxins is higher authorities should be focused on establishing national and when Kombucha tea is consumed in large quantities. Another international policies and regulations regarding the safety report states that one individual died due to perforations of Kombucha consumption and mass production of this of the intestinal tract and severe acidosis, who consumed beverage as a readily available commercial beverage. home-made Kombucha tea 4 oz per day for two months [52]. Another individual who consumed Kombucha tea coming from the same initial zoogleal mat had suffered from cardiac Conflict of Interests arrest and severe acidosis [52]. Despite these reports, most of these studies are limited to a very small number of The authors declare that there is no conflict of interests individuals. Hence, further studies should be carried out to regarding the publication of this paper. Journal of Chemistry 9

Acknowledgments [14] N.O.Kozyrovska,O.M.Reva,V.B.Goginyan,andJ.-P.DeVera, “Kombucha microbiome as a probiotic: a view from the per- The authors wish to acknowledge the financial support spective of post-genomics and synthetic ecology,” Biopolymers rendered by the National Institute of Fundamental Studies, and Cell,vol.28,no.2,pp.103–113,2012. Hantane Road, Kandy, Sri Lanka. Funding provided for Mrs. [15] A. Morshedi and M. H. Dashti-Rahmatabadi, “Chronic con- Chaminie B. Gunawardhana as a research student through sumption of Kombucha and black tea prevents weight loss in National Research Council of Sri Lanka, Grant no. 14-13, is diabetic rats,” Iranian Journal of Diabetes and Obesity,vol.2,no. also acknowledged. 2, pp. 23–26, 2010. [16] D. Kaczmarczyk and S. Lochynski,´ “Products of biotransformation of tea infusion—properties and application,” Polish Journal References of Natural Sciences,vol.29,no.4,pp.381–392,2004. [1] R. V. Malbaˇsa,E.S.Loncar,ˇ J. S. Vitas, and J. M. Canadanoviˇ c-´ [17]S.Jayasekera,A.L.Molan,M.Garg,andP.J.Moughan, Brunet, “Influence of starter cultures on the antioxidant activity “Variation in antioxidant potential and total polyphenol content of kombucha beverage,” Food Chemistry,vol.127,no.4,pp.1727– of fresh and fully-fermented Sri Lankan tea,” Food Chemistry, 1731, 2011. vol. 125, no. 2, pp. 536–541, 2011. [2] T. Srihari and U. Satyanarayana, “Changes in free radical [18] S. D. Kumar, G. Narayan, and S. Hassarajani, “Determination scavenging activity of Kombucha during fermentation,” Journal of anionic minerals in black and Kombucha tea using ion of Pharmaceutical Sciences and Research,vol.4,no.11,pp.1978– chromatography,” Food Chemistry, vol. 111, no. 3, pp. 784–788, 1981, 2012. 2008. [3] P. Dipti, B. Yogesh, A. K. Kain et al., “Lead induced oxidative [19] R. Jayabalan, P. Subathradevi, S. Marimuthu, M. Sathishkumar, stress: beneficial effects of Kombucha tea,” Biomedical and and K. Swaminathan, “Changes in free-radical scavenging Environmental Sciences,vol.16,no.3,pp.276–282,2003. ability of kombucha tea during fermentation,” Food Chemistry, vol.109,no.1,pp.227–234,2008. [4]A.J.Marsh,O.O’Sullivan,C.Hill,R.P.Ross,andP.D. [20]C.M.Hasler,A.S.Bloch,andC.A.Thomson,“Positionofthe Cotter, “Sequence-based analysis of the bacterial and fungal American Dietetic Association: functional foods,” Journal of the compositions of multiple kombucha (tea fungus) samples,” Food American Dietetic Association,vol.104,no.5,pp.814–826,2004. Microbiology,vol.38,pp.171–178,2014. [21] R. J. S. Junior,´ R. A. Batista, S. A. Rodrigues, L. X. Filho, [5] W. N. Goh, A. Rosma, B. Kaur, A. Fazilah, A. A. Karim, and R. and A.´ Silva Lima, “Antimicrobial activity of broth fermented Bhat, “Fermentation of black tea broth (Kombucha): I. Effects with Kombucha colonies,” Journal of Microbial & Biochemical of sucrose concentration and fermentation time on the yield of Technology,vol.1,no.1,pp.72–78,2009. microbial cellulose,” International Food Research Journal,vol.19, no. 1, pp. 109–117, 2012. [22]N.Kabiri,M.Setorki,andM.A.Darabi,“Protectiveeffects of Kombucha tea and silimarin against thioacetamide induced [6]R.Jayabalan,R.V.Malbaˇsa,E.S.Loncar,ˇ J. S. Vitas, and hepatic injuries in wistar rats,” World Applied Sciences Journal, M. Sathishkumar, “A review on kombucha tea—microbiology, vol.27,no.4,pp.524–532,2013. composition, fermentation, beneficial effects, toxicity, and tea fungus,” Comprehensive Reviews in Food Science and Food [23] N. Yavari, M. M. Assadi, M. B. Moghadam, and K. Larijani, Safety,vol.13,no.4,pp.538–550,2014. “Optimizing Glucuronic acid production using tea fungus on grape juice by response surface methodology,” Australian [7] S.-C. Chu and C. Chen, “Effects of origins and fermentation Journal of Basic and Applied Sciences,vol.5,no.11,pp.1788– time on the antioxidant activities of Kombucha,” Food Chem- 1794, 2011. istry,vol.98,no.3,pp.502–507,2006. [24]G.Sreeramulu,Y.Zhu,andW.Knol,“Kombuchafermentation [8] C. Dufresne and E. Farnworth, “Tea, Kombucha, and health: a and its antimicrobial activity,” JournalofAgriculturalandFood review,” Food Research International,vol.33,no.6,pp.409–421, Chemistry,vol.48,no.6,pp.2589–2594,2000. 2000. [25] G. Sreeramulu, Y. Zhu, and W. Knol, “Characterization [9]A.L.Teoh,G.Heard,andJ.Cox,“YeastecologyofKombucha of antimicrobial activity in Kombucha fermentation,” Acta fermentation,” International Journal of Food Microbiology,vol. Biotechnologica,vol.21,no.1,pp.49–56,2001. 95,no.2,pp.119–126,2004. [26] M. Deghrigue, J. Chriaa, H. Battikh, K. Abid, and A. Bakhrouf, [10]C.ChenandB.Y.Liu,“Changesinmajorcomponentsoftea “Antiproliferative and antimicrobial activities of Kombucha fungus metabolites during prolonged fermentation,” Journal of tea,” African Journal of Microbiology Research,vol.7,pp.3466– Applied Microbiology,vol.89,no.5,pp.834–839,2000. 3470, 2013. [11] A. S. Velicanski,D.D.Cvetkovi´ c,´ S. L. Markov, V. T. Tumbas, [27] H. Battikh, K. Chaieb, A. Bakhrouf, and E. Ammar, “Antibacte- and S. M. Savatovic,´ “Antimicrobial and antioxidant activity of rial and antifungal activities of black and green kombucha teas,” lemonbalmKombucha,”Acta Periodica Technologica,vol.38, Journal of Food Biochemistry,vol.37,no.2,pp.231–236,2013. pp.165–172,2007. [28] R. Jayabalan, P.-N. Chen, Y.-S. Hsieh et al., “Effect of sol- [12] C. J. Greenwalt, R. A. Ledford, and K. H. Steinkraus, “Determi- vent fractions of kombucha tea on viability and invasiveness nation and characterization of the antimicrobial activity of the of cancer cells-characterization of dimethyl 2-(2-hydroxy-2- fermented tea Kombucha,” LWT—Food Science and Technology, methoxypropylidine) malonate and vitexin,” Indian Journal of vol.31,no.3,pp.291–296,1998. Biotechnology,vol.10,no.1,pp.75–82,2011. [13] A. Velicanski,´ D. Cvetkovic,´ and S. Markov, “Characteristics of [29]A.H.Conney,Y.P.Lu,Y.R.Lou,andM.T.Huang,“Inhibitory kombucha fermentation on medicinal herbs from lamiaceae effects of tea and caffeine on UV-induced carcinogenesis: family,” Romanian Biotechnological Letters,vol.18,no.1,pp. relationship to enhanced apoptosis and decreased tissue fat,” 8034–8042, 2013. European Journal of Cancer Prevention,vol.2,pp.28–36,2002. 10 Journal of Chemistry

[30] C. Ioannides and V. Yoxall, “Antimutagenic activity of tea: [46] T. Pauline, P.Dipti, B. Anju et al., “Studies on toxicity, anti-stress role of polyphenols,” Current Opinion in Clinical Nutrition and and hepato-protective properties of Kombucha tea,” Biomedical Metabolic Care,vol.6,no.6,pp.649–656,2003. and Environmental Sciences,vol.14,no.3,pp.207–213,2001. [31] A. M. Bode and Z. Dong, “Signal transduction pathways: targets [47]M.SaiRam,B.Anju,T.Paulineetal.,“EffectofKombucha for green and black tea polyphenols,” Journal of Biochemistry tea on chromate(VI)-induced oxidative stress in albino rats,” and Molecular Biology,vol.36,no.1,pp.66–77,2003. Journal of Ethnopharmacology,vol.71,no.1-2,pp.235–240, [32] K. S. Kumar, N. Sastry, H. Polaki, and V. Mishra, “Colon cancer 2000. prevention through probiotics: an over view,” Journal of Cancer [48] A. Aloulou, K. Hamden, D. Elloumi et al., “Hypoglycemic and Science and Therapy,vol.7,pp.081–092,2015. antilipidemic properties of Kombucha tea in alloxan-induced [33] R. Jayabalan, S. Marimuthu, and K. Swaminathan, “Changes in diabetic rats,” BMC Complementary and Alternative Medicine, content of organic acids and tea polyphenols during kombucha vol. 12, no. 1, article 63, 2012. tea fermentation,” Food Chemistry,vol.102,no.1,pp.392–398, [49] C. Vicente, B. Sebastian,´ B. Fontaniella, A. Marquez,L.X.´ 2007. Filho, and M.-E. Legaz, “Bioskin as an affinity matrix for the [34]R.Vijayaraghavan,M.Singh,P.V.L.Raoetal.,“Sub-acute(90 separation of glycoproteins,” Journal of Chromatography A,vol. days) oral toxicity studies of Kombucha tea,” Biomedical and 917, no. 1-2, pp. 55–61, 2001. Environmental Sciences, vol. 13, no. 4, pp. 293–299, 2000. [50]D.Banerjee,S.A.Hassarajani,B.Maity,G.Narayan,S.K. [35]N.K.Nguyen,N.T.N.Dong,P.H.Le,andH.T.Nguyen, Bandyopadhyay, and S. Chattopadhyay, “Comparative healing “Evaluation of the glucuronic acid production and other bio- property of kombucha tea and black tea against indomethacin- logical activities of fermented sweeten- black tea by Kombucha induced gastric ulceration in mice: possible mechanism of layer and the co-culture with different Lactobacillus Sp. strains,” action,” Food and Function,vol.1,no.3,pp.284–293,2010. International Journal of Modern Engineering Research,vol.4,no. [51] N. Khan and H. Mukhtar, “Tea and health: studies in humans,” 5, pp. 12–17, 2014. Current Pharmaceutical Design,vol.19,no.34,pp.6141–6147, [36] Y.Shebis,D.Iluz,Y.Kinel-Tahan,Z.Dubinsky,andY.Yehoshua, 2013. “Natural antioxidants: function and sources,” Food and Nutri- [52] Food safety assessment of Kombucha tea recipe and food safety tion Sciences,vol.4,no.6,pp.643–649,2013. plan.FoodIssue,NotesfromtheField,BCCentreforDisease [37] M. W. L. Koo and C. H. Cho, “Pharmacological effects of Control, An Agency of the Provincial Health Services Authority, green tea on the gastrointestinal system,” European Journal of http://www.bccdc.ca/resource-gallery/Documents/Educational Pharmacology,vol.500,no.1–3,pp.177–185,2004. %20Materials/EH/FPS/Food/kombucha1.pdf. [38] E. A. Adewusi and A. J. Afolayan, “Areview of natural products [53] A. S. Kole, H. D. Jones, R. Christensen, and J. Gladstein, “Acase with hepatoprotective activity,” Journal of Medicinal Plants of Kombucha tea toxicity,” Journal of Intensive Care Medicine, Research,vol.4,no.13,pp.1318–1334,2010. vol.24,no.3,pp.205–207,2009. [39] R. Jayabalan, S. Baskaran, S. Marimuthu, K. Swaminathan, and [54] E. Ernst, “A systemic review of the clinical evidence,” Research in Complementary medicine,vol.10,no.2,pp.85–87,2003. S. E. Yun, “Effect of kombucha tea on aflatoxin B1 induced acute hepatotoxicity in albino rats-prophylactic and curative studies,” [55] R. Srinivasan, S. Smolinske, and D. Greenbaum, “Probable Journal of Applied Biological Chemistry,vol.53,no.4,pp.407– gastrointestinal toxicity of Kombucha tea: is this beverage 416, 2010. healthy or harmful?” Journal of General Internal Medicine,vol. [40] N. K. Ibrahim, “Possible protective effect of Kombucha Tea Fer- 12, no. 10, pp. 643–644, 1997. ment on cadmium chloride induced liver and kidney damage [56] Z. Kovacevic, G. Davidovic, J. Vuckovic-Filipovic, M. A. in irradiated rats,” World Academy of Science, Engineering and Janicijevic-Petrovic, K. Janicijevic, and A. Popovic, “A toxic Technology,vol.55,pp.1097–1102,2011. hepatitis caused the kombucha tea—case report,” Macedonian [41] S. Bhattacharya, R. Gachhui, and P. C. Sil, “Hepatoprotective Journal of Medical Sciences,vol.7,no.1,pp.128–131,2014. properties of kombucha tea against TBHP-induced oxidative [57] F. Bolle, W. Brian, D. Petit, K. Boutakhrit, G. Feraille, and stress via suppression of mitochondria dependent apoptosis,” J. Van Loco, “Tea brewed in traditional metallic teapots as a Pathophysiology,vol.18,no.3,pp.221–234,2011. significant source of lead, nickel and other chemical elements,” [42]J.Abshenas,A.Derakhshanfar,M.H.Ferdosi,andS. Food Additives and Contaminants Part A,vol.28,no.9,pp.1287– Hasanzadeh, “Protective effect of kombucha tea against 1293, 2011. acetaminophen-induced hepatotoxicity in mice: a biochemical [58]T.G.Phan,J.Estell,G.Duggin,I.Beer,D.Smith,andM.J. and histopathological study,” Comparative Clinical Pathology, Ferson, “Lead poisoning from drinking Kombucha tea brewed vol.21,no.6,pp.1243–1248,2012. in a ceramic pot,” Medical Journal of Australia,vol.169,no.11-12, [43] Y. Wang, B. Ji, W. Wu et al., “Hepatoprotective effects of kom- pp.644–646,1998. bucha tea: identification of functional strains and quantification [59] F. A. Durazo, C. Lassman, S. H. B. Han et al., “Fulminant liver of functional components,” Journal of the Science of Food and failure due to usnic acid for weight loss,” The American Journal Agriculture, vol. 94, no. 2, pp. 265–272, 2014. of Gastroenterology,vol.99,no.5,pp.950–952,2004. [44]T.I.Jeon,S.G.Hwang,N.G.Parketal.,“Antioxidativeeffect [60] L. K. Massey, H. Roman-Smith, and R. A. L. Sutton, “Effect of chitosan on chronic carbon tetrachloride induced hepatic of dietary oxalate and calcium on urinary oxalate and risk injury in rats,” Toxicology,vol.187,no.1,pp.67–73,2003. of formation of calcium oxalate kidney stones,” Journal of the [45] G. S. Murugesan, M. Sathishkumar, R. Jayabalan, A. R. Bin- American Dietetic Association,vol.93,no.8,pp.901–906,1993. upriya, K. Swaminathan, and S. E. Yun, “Hepatoprotective [61] S. C. Smolinske, “Herbal product contamination and toxicity,” and curative properties of Kombucha tea against Carbon Journal of Pharmacy Practice, vol. 18, no. 3, pp. 188–208, 2005. tetrachloride-induced toxicity,” Journal of Microbiology and [62] P. C. Sherertz, Acetic Acid,DivisionofHealthHazardsControl, Biotechnology,vol.19,no.4,pp.397–402,2009. Virginia Department of Health, 2015, http://www.vdh.virginia Journal of Chemistry 11

.gov/epidemiology/DEE/PublicHealthToxicology/documents/ pdf/aceticacid.PDF. [63] FAO nutrition meetings, report series no. 40 A, B, C WHO/ Food add./67.29, Joint FAO/WHO Expert Committee on Food Additives which met at Rome, 13–20 December, 1965 Geneva, 11–18 October, 1966, http://www.inchem.org/documents/jecfa/ jecmono/40abcj37.htm. [64]FRCcodeofFederalRegulations,Title21,Vol.3,https://www .accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? fr=184.1061. [65] Material Safety Data Sheet, 2009, http://www.dogee.org/lab/ chemicals/915.pdf. [66] International Food Standards, World Health Organization, and Food and Agriculture Organization of the United Nations, “Cordex Alimentarius, Cordex Stan 192-1995,” 2015. [67] Material Safety Data Sheet, 2013, http://www.sciencelab.com/ msds.php?msdsId=9923494. [68] Oxalic Acid, Summary Report, EMEA/MRL/891/03-FINAL, Committee for Veterinary Medicinal Products, The European Agency for the Evaluation of Medicinal Products, Veterinary Medicines and Inspections, 2003. [69] Dietary Reference Intakes (DRIs): Estimated Average Require- ments, United States National Academy of Sciences, Washing- ton, DC, USA, 2015. [70]J.Doull,C.D.Klaassen,andM.D.Amdur,Casarett and Doull’s Toxicology, Macmillan Publishers, New York, NY, USA, 2nd edition, 1980. [71] Science Lab, Material Safety Data Sheet, Science Lab, 2013, http://www.sciencelab.com/msds.php?msdsId=9922768. [72] J. Mrdanovic,´ G. Bognadovic,´ D. Cvetkovic,´ A. Velicanski,´ and D. Cetojeviˇ c-Simin,´ “The frequency of sister chromatid exchange and micronuclei in evaluation of cytogenetic activity of Kombucha on human peripheral blood lymphocytes,” Archive of Oncology,vol.15,no.3-4,pp.85–88,2007. [73] C. Shenoy, “Hypoglycemic activity of bio-tea in mice,” Indian JournalofExperimentalBiology,vol.38,no.3,pp.278–279, 2000. [74]A.M.Hartmann,L.E.Burleson,A.K.Holmes,andC.R. Geist, “Effects of chronic kombucha ingestion on open-field behaviors, longevity, appetitive behaviors, and organs in c57- bl/6 mice: a pilot study,” Nutrition,vol.16,no.9,pp.755–761, 2000. [75]A.Morshedi,M.H.Dashti,M.H.Mosaddegh,A.Rafati,andA. S. Salami, “The chronic effect of Kombucha tea consumption on weight loss in diabetic rats,” Journal of Medicinal Plants,vol.5, no. 2, pp. 17–22, 2006. [76] K. Cavusoglu and P. Guler, “Protective effect of Kombucha mushroom (KM) tea on chromosomal aberrations induced by gamma radiation in human peripheral lymphocytes in vitro,” Journal of Environmental Biology,vol.31,no.5,pp.851–856, 2010. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 710328, 8 pages http://dx.doi.org/10.1155/2015/710328

Research Article Characterization of Calcium Compounds in Opuntia ficus indica as a Source of Calcium for Human Diet

Isela Rojas-Molina,1 Elsa Gutiérrez-Cortez,2,3 Moustapha Bah,1 Alejandra Rojas-Molina,1 César Ibarra-Alvarado,1 Eric Rivera-Muñoz,4 Alicia del Real,5 and Ma. de los Angeles Aguilera-Barreiro6

1 Laboratorio de Investigacion´ Qu´ımica y Farmacologica´ de Productos Naturales, Facultad de Qu´ımica, Universidad Autonoma´ de Queretaro,´ Cerro de las Campanas S/N, 76010 Queretaro,´ QRO, Mexico 2Facultad de Ingenier´ıa, Universidad Autonoma´ de Queretaro,´ Carretera a Chichimequillas s/n, 76140 Queretaro,´ QRO, Mexico 3FES-Cuautitlan,´ Universidad Nacional Autonoma´ de Mexico,´ Laboratorio de Procesos de Transformacion´ y Tecnolog´ıas Emergentes en Alimentos, Km 2.5 Carretera Cuautitlan-Teoloyucan,´ San Sebastian´ Xhala, 54714 Cuautitlan´ Izcalli, MEX, Mexico 4Departamento de Nanotecnolog´ıa, Centro de F´ısica Aplicada y Tecnolog´ıa Avanzada, Universidad Nacional Autonoma´ de Mexico,´ Campus Juriquilla, A.P. 1-1010, 7600 Queretaro,´ QRO, Mexico 5Departamento de Ingenier´ıa Molecular de Materiales, Centro de F´ısica Aplicada y Tecnolog´ıa Avanzada, Universidad Nacional Autonoma´ de Mexico,´ Campus Juriquilla, A.P. 1-1010, 7600 Queretaro,´ QRO, Mexico 6Facultad de Ciencias Naturales, Universidad Autonoma´ de Queretaro,´ Avenida de las Ciencias S/N, 76230 Queretaro,´ QRO, Mexico

Correspondence should be addressed to Isela Rojas-Molina; [email protected]

Received 24 September 2015; Accepted 6 December 2015

Academic Editor: Nick Kalogeropoulos

Copyright © 2015 Isela Rojas-Molina 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.

Analyses of calcium compounds in cladodes, soluble dietary fiber (SDF), and insoluble dietary fiber (IDF) of Opuntia ficus indica are reported. The characterization of calcium compounds was performed by using Scanning Electron Microscopy, Energy Dispersive Spectrometry, X-ray diffraction, and infrared spectroscopy. Atomic Absorption Spectroscopy and titrimetric methods were used for quantification of total calcium and calcium compounds. Whewellite (CaC2O4⋅H2O), weddellite (CaC2O4⋅(H2O)2.375), and calcite (CaCO3) were identified in all samples. Significant differences𝑃 ( ≤ 0.05) in the total calcium contents were detected between samples. CaC2O4⋅H2O content in cladodes and IDF was significantly higher𝑃 ( ≤ 0.05)incomparisontothatobservedinSDF, whereas minimum concentration of CaCO3 was detected in IDF with regard to CaCO3 contents observed in cladodes and SDF. 2+ Additionally, molar ratio oxalate : Ca in all samples changed in a range from 0.03 to 0.23. These results support that calcium bioavailability in O. ficus indica modifies according to calcium compounds distribution.

1. Introduction interest in monitoring and increasing consumption of dietary calcium. Moreover, recently, health professionals have been In Mexico, 21% of people over 20 years have a deficiency in taking action on the matter [3]. calcium intake. Calcium content in the Mexican diet covers In addition, it has been demonstrated that a high bioavail- only 50% of the recommended daily intake (1200 mg/day) ability of calcium from the diet improves bone health [4], [1]. Calcium deficiency causes skeletal system diseases, that althoughcalciumisdistributedindifferentfoodstuffssuchas is, osteoporosis, which is a public health problem, due milk and dairy foods that provide more than 80% of calcium to the fact that this disease is among the eight leading to the human diet. Furthermore, the calcium bioavailability in causes of hospital morbidity with a prevalence of 8% in the milk and dairy foods is significant, and, due to this, mineral Mexican population [2]. For this reason, there is an amplified absorption is associated with absorption promoters such as 2 Journal of Chemistry lactose among other factors [5]. In developing countries, cal- ultrapure, and distilled water were obtained from J. T. Baker cium intake from dairy products is limited by the high costs (DF, Mexico). The Total Fiber Dietary Kit (TDF-1100 A) was of these foodstuffs, as well as the problems associated with purchased from Sigma (St. Louis, MO, USA) as also were lactose intolerance [6]. These facts restrict the consumption oxalic acid and potassium bromide standards. of animal products causing a reduction of calcium in the diet. Consequently, Mexico and Central American countries 2.3. Extraction of Soluble and Insoluble Dietary Fiber from depend on nixtamalized products as their primary source O. ficus indica. The dried material was mixed with distilled of calcium in their diet [7]. Thus, it is necessary to propose water (4% w/w). This suspension was homogenized using alternative sources of calcium to improve daily intake of this a blender (IKA-WERKE, Mod. Eurostar BSC.S1) (450 rpm mineral. Opuntia ficus indica cladodes (pads) represent a for 20 min). Subsequently, the suspension was left to stand potential source of calcium in human diet, due to the fact forfourhourstoensurehydrationofthesolids.Then,the that calcium content in pads increases with the growing stage suspension was placed in the feed tank of a disk centrifuge [8, 9]. (DIDACTA Italia, Mod. TAG1/d), which was operated at McConnandNakata[10]observedareductionincalcium 450 rpm. The speed of centrifuge was increased gradually availability in prickly pear cactus by using an in vitro assay; until it reached 7000 rpm. Next, the feed valve of the this was attributed in part to the presence of calcium oxalate centrifuge was opened to allow the flow of soluble solids crystals. In addition, Contreras-Padilla et al. [11] reported that throughthegravityringsandtheupperhopperofequipment, the concentration of oxalate in O. ficus indica in different while the insoluble solids (insoluble fiber) were retained in phases of maturity appears to have a cyclic tendency that the bowl of the centrifuge. The insoluble dietary fiber was ∘ could be determined by the presence of calcium content in dehydrated in Teflon pans at 80 kPa and 40 Cinavacuum thesoil,theplant’sneedsduringactivegrowth,andseasonal oven (Barnstead International, Mod. 3618) for 35 min, until and environmental conditions. a humidity content of 4% (w/w). Soluble solids recovered On the other hand, several researches have shown the were mixed with ethyl alcohol at 95% (v/v) in a 1 : 2 v/v ratio. presence of calcium compounds in mucilage cell and cell This suspension was subjected to vacuum filtration at 4 kPa to walls of Opuntia ficus indica [12, 13]. However, there is no remove excess water and alcohol in order to obtain the soluble previous report about the distribution of these compounds dietary fiber. Finally, this precipitate was dehydrated at the extracted from the matrix of O. ficus indica with the purpose same conditions as before. of increasing the bioavailability of calcium from this Cac- taceae. 2.4. Separation of Oxalate Crystals. Suspensions of dried The goal of the present research was to characterize the material (cladodes, soluble dietary fiber, and insoluble dietary distribution of calcium compounds in soluble and insoluble fiber) and distilled water (4% w/w) were prepared. These dietary fiber extracted from O. ficus indica cladodes at a late suspensions were processed as was reported by Malainine et maturation stage (400 g of weight), in order to underlie the al. [14]. potentialofthiscactusasasourceofcalciumtohelpthe formation of bone mass. These findings will promote the 2.5. Chemical Characterization utilization of powder of O. ficus indica,withnocommercial value, due to the fact that this cactus is not consumed as a 2.5.1. Total, Soluble, and Insoluble Dietary Fiber Content in vegetable, which can be used as a dietary supplement with Dehydrated Cladodes of O. ficus indica. Total dietary fiber, an affordable price to increase calcium intake in the Mexican soluble dietary fiber (SDF), and insoluble dietary fiber (IDF) population. in samples were analyzed according to methods 991.42 and 993.19 [15], respectively, by using a dietary fiber kit. 2. Materials and Methods 2.5.2. Characterization of Calcium Compounds in Cladodes, 2.1. Samples. Opuntia ficus indica cladodes were cultivated Soluble, and Insoluble Dietary Fiber of O. ficus indica by Scan- in an experimental field in Silao, Guanajuato (Rancho Los ning Electron Microscopy (SEM) and Energy Dispersive Spec- Lorenz), Mexico, with organic fertilizer and harvested during trometry (EDS). The morphology of calcium compounds the spring of 2014. The O. ficus indica cladodes of 400 g was analyzed in a Scanning Electron Microscopy (Jeol JSM (100 days of maturation stage) were washed with distilled 6060LV, Japan). Prior to the analysis, the samples were water and the thorns were manually removed. Then, O. ficus × fixed on an aluminum specimen holder with carbon tape indica cladodes were dehydrated by drying cladode slices (2 and dried under critical point conditions in a Cryo-SEM 2 cm) in a force air oven (BG model E102). The dehydration ∘ preparation system (Quorum Technologies, Mod. PP30105, process was carried out at 50 C during 70 minutes, each pan UK) operated with liquid CO2. Subsequently, the mounted containing 5 kg of O. ficus indica slices. The dry material samples were then sputter coated with gold. The micro- was milled using a hammer mill (PULVEX 200, DF, Mexico) compositional analysis of the samples was carried out using equipped with a 0.8 mm screen. an Energy Dispersive Spectrometer (INCA x-sight) provided with software (Oxford Instrument, UK). Each sample was 2.2. Chemicals and Reagents. Ethyl alcohol (95% v/v) reac- turned to move the focus position of the microscope. Further, tive grade, hydrochloric acid analytical grade, nitric acid surface views of isolated samples were taken to obtain Journal of Chemistry 3 themicrographs.Theconditionsoftheanalysiswerehigh 3. Results and Discussion vacuum, 20 KV electron acceleration voltage, and secondary electron mode. Additionally, standards of pure compounds 3.1. Soluble and Insoluble Dietary Fiber Content. The SDF 2.53 ± 0.90 43.44 ± were observed with comparative purposes. and IDF contents in samples were and 1.69%, respectively. These results differ from those reported by Hernandez-Urbiola´ et al. [9]. These authors found that, 2.5.3. Characterization of Calcium Compounds in Cladodes, in nopal pads with 100 days of age (400 g of weight approxi- Soluble, and Insoluble Dietary Fiber of O. ficus indica by mately),theSDFandIDFcontentswere8and52%,respec- X-Ray Diffraction. Before analysis, samples were calcinated ∘ tively. Nutrient profile of cladodes from different harvests in a furnace (Nabertherm, Mod. L-P 330, GER) at 168 C and regions varies due to the fact that this profile depends in order to decompose organic matter and to prevent the on environmental factors, that is, edaphic factors at the formation of new mineral compounds or to avoid decay of cultivation site, the season, and the age of the plant [8, 20]. A calcium compounds commonly present in the Opuntioideae higher content of IDF with respect to SDF is attributed to the subfamily as was previously reported [14, 16–18]. The samples 𝜇 process of lignification, where polyphenolic polymer lignin were ground to a fine powder and passed through a 150 m is formed due to maturation of cladodes. On the contrary, screen. The powder samples were then densely packed into young Opuntia cladodes lack lignin [21]. The increase of fibers an aluminum sample holder. The X-ray diffraction patterns in cladodes involves mainly cellulose and hemicelluloses [22]; of the samples were recorded on a diffractometer (Rigaku these compounds in conjunction with lignin constitute IDF Miniflex) operating at 35 kV and 15 mA, with a CuK𝛼 radi- [23]. Total dietary fiber (TDF) content in O. ficus indica in the ation wavelength of 𝜆 = 1.5406 A.˚ The measurements were ∘ ∘ present study is higher than TDF values in cactus pear (fruit) obtained from 10 to 70 on a 2𝜃 scale with a step size of 0.05 . reported by Jimenez-Aguilar´ et al. [24]. Spectrum analysis software (Materials Data Inc. Jade V 5.0) was used for the samples analysis. 3.2. SEM and EDS Analysis. Figure 1 shows representative images of microscopic examinations of samples. Presence 2.5.4. Characterization of Calcium Compounds in Cladodes, of calcium oxalate crystals in O. ficus indica cladodes is Soluble, and Insoluble Dietary Fiber of O. ficus indica by evident in Figure 1(a) (see arrows) in accordance with pre- Infrared (IR) Analysis. The IR spectra of dehydrated samples vious reports [10, 12, 13, 25]. Biomineralized calcium oxalate of O. ficus indica cladodes, SDF, and IDF were recorded on a crystallites in Cactaceae species were identified either as IR-Bruker Vector 33 spectrophotometer in the spectral range CaC2O4⋅2H2O (weddellite) or as CaC2O4⋅H2O(whewellite). −1 between 4000 and 400 cm ,usingtheKBrpellettechnique Whewellite druses differ from weddellite druses principally (4 mg of the powdered sample dispersed in 100 mg of KBr). by their stellate shapes, with individual crystallites having acute sharp points emerging from the center of the druse. 2.5.5. Total Calcium and Oxalate Content in Cladodes, Soluble, On the other hand, weddellite druses are usually made up and Insoluble Dietary Fiber of O. ficus indica Cladodes. Total of individual tetragonal crystals [26]. In this study, also calcium and oxalate content was determined according to weddellite crystals were detected (see Figure 1(b)) according AOAC Official Method 983.27 and 974.24, respectively [15]. to Malainine et al. [14] and Saenz et al. [25]. Figure 1(c) The oxalate concentration was measured with a double beam shows crystalline calcium oxalate in IDF extracted from O. atomic absorption (Analyst 300 Perkin Elmer), equipped ficus indica. As it can be seen, the quantity of crystals in with a deuterium lamp, background corrector, and a hollow IDF is more than that detected in cladodes (see arrows). The size of druses ranged from 150 to 250 𝜇m; these crystals are cathode lamp. The operating parameters for calcium were a larger than druses observed by Rodr´ıguez-Garc´ıa et al. [8] hollowcathodelampwithawavelengthof422.7nm,70psi and Saenz et al. [25]. These authors reported that the crystal of acetylene, nitrous oxide as an oxidant, and slit aperture of size of whewellite of O. ficus indica cladodes (from 60 to 0.7 mm and for oxalates 12 psi of dry air at 70 psi of acetylene 200 g of weight) ranged from 30 to 70 𝜇m. At this point, it is withawavelengthof422.7nm,10mAlampcurrent,anda important to mention that, in the present study, the weight of 0.7nmslitwidth. cladodes was 400 g (100 days of age) and the oxalate crystal size increases as a function of maturation [26]. Figure 1(d) 2.5.6. Calcium Carbonate Content in Cladodes, Soluble, and shows a detail of a vessel from the xylem of O. ficus indica with InsolubleDietaryFiberofO.ficusindica. Calcium carbonate a calcium oxalate crystal adhered to the vascular tissue with a content in samples was analyzed by volumetric analysis high content of lignin. Calcium oxalate crystals are present in according to AOAC [19]. all tissues of O. ficus indica cladodes [13]; nevertheless, SEM analysis shows that the presence of calcium oxalate crystals 2.6. Statistical Analysis. Three samples were used for each in IDF is very noticeable. This result is in agreement with preparation and all the assays were carried out in triplicate. those of Ginestra et al. [13]; indeed, these authors found The results are expressed as mean values and standard that calcium oxalate crystals are strongly associated with an deviation (SD). The results were analyzed using one-way alcohol-insoluble residue (constituted by vascular bundles, analysis of variance (ANOVA) followed by Tukey’s test with clumps of parenchyma, and skin) obtained from a cell-wall 𝛼 = 0.05 and using the Statgraphics procedure (Graphics fractionation of powdered lyophilized cladodes. Figure 2(a) Software System, Manugistics, Inc., USA). shows a prismatic crystal in SDF extracted from O. ficus 4 Journal of Chemistry

(a) (b)

Figure 1: SEM images of cladodes and insoluble dietary fiber of O. ficus indica, (a) calcium oxalate crystals (whewellite) in O. ficus indica cladodes, (b) calcium oxalate crystals (weddellite) in O. ficus indica cladodes, (c) calcium oxalate crystals in insoluble dietary fiber extracted from O. ficus indica, and (d) vessel from the xylem of O. ficus indica with a calcium oxalate crystal (whewellite).

Ca Au Spectrum 1 O

K Ca C

Mg K Au

1 23456789 (keV) Full scale 528cts cursor:0.000keV (a) (b)

Figure 2: SEM images and EDS analysis of soluble dietary fiber extracted from O. ficus indica, (a) prismatic crystal in soluble dietary fiber with elemental analysis by EDS and (b) prismatic crystal emerging from a globular structure located in soluble dietary fiber.

indica. A qualitative EDS analysis of this material revealed calcium carbonate or calcium-magnesium carbonate. Glob- thepresenceofcalcium,oxygen,carbon,potassium,and ular structures were detected by microscopic observation of magnesium (inserted in Figure 2(a)). Contreras-Padilla et the SDF (Figure 2(b)). Furthermore, a quadratic crystal is al.[18]havereportedsimilarcrystallinestructures.These emerging from a globular structure (see white arrow). In authors associate this composition with the presence of this regard, Contreras-Padilla et al. [18] found that calcium Journal of Chemistry 5

Ww Ww Ww 47-1743> Calcite-CaCO3 Ww that peaks of crystalline compounds different to calcite, Ww Ww Ww Ww Ww whewellite, and weddellite are very weak and the strongest calcite and whewellite reflection peaks are superimposed with Cladodes other compound peaks or possibly they are amorphous.

3.4. Infrared Analysis. Figure 4 shows the infrared emission Wd Wd Insoluble dietary fiber spectra of SDF and IDF isolated from O. ficus indica and cladodes. The infrared spectra confirm the existence of Intensity (a.u.) Intensity calciumcarbonateinSDF.Thisissupportedbythepresence C G −1 2− −1 2− G SS of bands near 1420 cm (]as CO3 )and875cm (CO3 CC C C Soluble dietary fiber out-of-plane bending vibration). The intensity of these bands C C C C indicates a large amount of calcite. Nevertheless, this spectrum shows no evident absorption bands for oxalate, whereas infrared spectra of cladodes and IDF reveal the presence of 10 20 30 40 50 60 70 whewellite and calcite. It is important to denote that calcite 2𝜃 (deg.) is in trace amounts in IDF. Calcium oxalate is responsible −1 −1 for bands at 1625 cm (]as OCO), 1312 cm (]s OCO), and Figure 3: X-ray diffraction patterns of O. ficus indica samples −1 (cladodes, insoluble, and soluble dietary fiber). Ww: whewellite; Wd: 750 cm (OCO deformations). These findings are consistent weddellite; C: calcite; G: glauberite; S: spurrite. with the X-ray diffraction results. Finally, the presence of −1 anintensebandcenteredat1070–1080cm indicates a high content of silicon oxide (SiO2)inallsamplesasitwas carbonate crystals grow into prismatic forms comparable to observed in the X-ray diffraction results. Biomineralized the crystal observed in SDF extracted from experimental silicon in plants has been related with a structural role in the samples. These authors suggest that the growth of these cell wall and defense as a mineral barrier to both the invasion crystal structures can be correlated with the age of the plant. of pathogen and insect attacks, as well as the translocation of water and salts [27]. 3.3. X-Ray Diffraction Analysis. Figure 3 corresponds to the X-rays diffraction patterns of O. ficus indica samples 3.5. Total Calcium, Calcium Oxalate, and Calcium Carbonate (cladodes, SDF, and IDF). These diffractograms revealed the in O. ficus indica. The average total calcium, oxalate, and presence of calcium oxalate monohydrate (whewellite) in calcium carbonate contents in O. ficus indica samples are showninTable1.Itisevidentthattotalcalciumcontentin cladodes and IDF, which fit with the PDF # 20–0231 of 𝑃 ≤ 0.05 ICDD-JCPDS database. Monje and Baran [17] and Contreras- cladodes is significantly higher ( )incomparison Padilla et al. [18] reported two characteristic peaks in X- tocalciumcontentinSDFandIDF.Nevertheless,itisworth rays diffraction patterns for oxalate monohydrate at 14- noting that calcium content in SDF is higher than calcium ∘ 15 and 24-25 diffraction angle 2𝜃 in different Cactaceae content in IDF. This means that calcium content in SDF is on average 18.24% higher than calcium content in IDF. Calcium species belonging to the Opuntioideae subfamily, including 𝑃 ≤ 0.05 O. ficus indica; both peaks are evident in cladodes and oxalate content in SDF significantly decreases ( )with IDF samples. In contrast, these peaks were not detected respect to that in cladodes and IDF. In addition, the highest in SDF samples. Furthermore, characteristic peaks in X- concentration of calcium carbonate was detected in cladodes, rays diffraction patterns for calcium carbonate at 29-30, whilethecontentofthiscompoundisapproximately50% ∘ 39-40, and 45–50 diffraction angle 2𝜃 observed by the higherinSDFincomparisonwiththatobservedinIDF.These same authors were identified in SDF only. This indicates the results may be explained as follows: for salts (ionic solids) that dissociate into ions in water, such as the compounds presence of CaCO3 (PDF # 47-1743) in these samples. These 𝐾 findings are in agreement with the distribution of crystalline contained in O. ficus indica, a solubility product ( sp)is 𝐾 ∘ compounds observed by the SEM and EDS analyses. Two typically given. In the case of CaCO3, sp (25 C) = 3.36 ∘ −9 ∘ −9 small Bragg reflections located on 14.36 and 32.2 in the IDF × 10 ,while,forCaC2O4, 𝐾sp (25 C) = 2.32 × 10 [28]. sample are indicative of the presence of weddellite crystals The smaller the solubility product of a substance, the lower (CaC2O4⋅(H2O)2.375) (PDF # 75-1314); nevertheless they do its solubility. This means that CaCO3 is more soluble in notappearintheSDFsample.Thisresultisinagreementwith water than CaC2O4; consequently, this fact justifies a major Malainine et al. [14]; these authors also reported the presence concentration of oxalate in IDF compared with that in SDF of weddellite crystals in Opuntia ficus indica cladodes. The and a higher content of calcium carbonate in SDF with other two crystalline compounds containing calcium were respect to IDF. detected in SDF sample: spurrite (Ca5(SiO4)2CO3)(PDF# Calcium carbonate in plants has been related to a mech- 2+ 13-0496) and glauberite (Na2Ca(SO4)2) (PDF # 74-2340). anism to control soluble Ca levels within plant tissues [29]. These two compounds reveal the presence of other elements Similarly, calcium oxalate has a role as a calcium regulator and suchassodium,silicon,andsulfur.Theanalysisperformed other functions, that is, mechanical support, intracellular pH with the MDI Jade software showed no presence of other regulation, ion balance detoxification, and gravity perception crystalline compounds. This could be attributed to the fact between others [30]. 6 Journal of Chemistry

Cladodes 0.9

0.7

0.5 ox Transmittance (a.u.) Transmittance 0.2 ox SiO2 0.8 Insoluble dietary fiber

0.7

0.6 ox ox 0.5 ox 0.4 2− Soluble dietary fiber CO3

0.3

0.2

0.1 Transmittance (a.u.)Transmittance (a.u.) Transmittance

0.0 4000 3000 2000 1000 0 −1 Wavenumber (cm )

Figure 4: Infrared spectra of Opuntia ficus indica samples (cladodes, insoluble, and soluble dietary fiber). ox: CaC2O4⋅H2O(whewellite).

Table 1: Total calcium, oxalate, calcium carbonate content, and molar ratio oxalate : calcium in Opuntia ficus indica samples (cladodes, soluble, ∗ and insoluble dietary fiber) .

Calcium Calcium Total calcium content Oxalate content oxalate content carbonate content Molar ratio Samples (mg/g) (mg/g) (mg/g) (mg/g) oxalate : calcium dry matter dry matter dry matter dry matter a a a a a Cladodes 32.33 ± 2.90 6.71 ± 0.80 8.31 ± 0.80 70.81 ± 3.40 0.12 b b b b b Soluble dietary fiber 18.99 ± 1.30 0.27 ± 0.20 1.25 ± 0.20 49.67 ± 1.87 0.03 c a a c c Insoluble dietary fiber 13.08 ± 1.10 5.39 ± 0.17 6.61 ± 0.17 21.95 ± 2.17 0.23 ∗ Values ± SD followed by the same letter are not significantly different (𝑃 < 0.05).

In a food evaluation, the chelating agent to mineral and calcium in O. ficus indica pads at different maturity stages chelated molar ratio is an important factor for determining was lower than 1, suggesting that the bioavailability of calcium potency of mineral bioavailability. The World Health Organi- is not compromised. zation considers this value as a good index as a preliminary criterion for mineral bioavailability [31]. 4. Conclusions In order to predict the bioavailability of calcium in 2+ samples [oxalate]/[Ca ],ratioswerecalculated(Table1). Calcium carbonates and calcium oxalates were detected in 2+ The values obtained from this molar ratio (oxalate : Ca ) cladodes, IDF, and SDF of O. ficus indica. Nevertheless, are below the critical level of 1, known to impair calcium significant differences in total calcium, calcium carbonate, 2+ 2+ bioavailability. This means that molar oxalate : Ca ratios ≥ 1 calcium oxalate contents, and molar oxalate : Ca ratio were are indicative of calcium unavailability [32]. These results are observed in all samples. This means that calcium bioavailabil- in agreement with those reported by Contreras-Padilla et al. ity in O. ficus indica varies according to calcium compounds [11].Theseauthorsfoundthatthemolarratiobetweenoxalate distribution. Journal of Chemistry 7

Conflict of Interests [12] M. E. Malainine, A. Dufresne, D. Dupeyre, M. Mahrouz, R. Vuong, and M. R. Vignon, “Structure and morphology of clado- The authors declare that they have no conflict of interests. des and spines of Opuntia ficus-indica. Cellulose extraction and characterisation,” Carbohydrate Polymers,vol.51,no.1,pp.77– Acknowledgments 83, 2003. [13] G. Ginestra, M. L. Parker, R. N. Bennett et al., “Anatomical, This work was supported by CONACYT (Basic Science chemical and biochemical characterization of cladodes from Projects, Convocation 2011-02, Project no. 167769) and Fondo prickly pear [Opuntia ficus-indica (L.) Mill.],” Journal of Agricul- para el Fortalecimiento de la Investigacion´ UAQ-2012 (FOFI- tural and Food Chemistry,vol.57,no.21,pp.10323–10330,2009. UAQ-2012, Project no. FCQ-2012-20), Mexico. The authors [14] M. E. Malainine, A. Dufresne, D. Dupeyre, M. R. Vignon, and thank Q. Flora Lazaro´ Torres (FES-Cuautitlan-UNAM)´ for M. Mahrouz, “First evidence for the presence of weddellite crystallites in Opuntia ficus indica parenchyma,” Zeitschriftur f¨ her technical support. Naturforschung,vol.58,no.11-12,pp.812–816,2003. [15] AOAC, OfficialMethodsofAnalysisoftheAssociationofOffi- References cial Analytical Chemists, Methods 991.41 and 993.19, AOAC, Gaithersburg, Md, USA, 17th edition, 2000, edited by: W. [1] G. Olaiz-Fernandez,´ J. Rivera-Dommarco, T. Shamah-Levy et Horwitz. al., “Nutricion,”´ in Encuesta Nacional de Salud y Nutricion´ , [16] I. Hoffman, M. Schnitzer, and J. R. Wright, “Application of pp. 87–97, Instituto Nacional de Salud Publica,´ Cuernavaca, thermogravimetry to the analysis of carbonates occurring in Mexico,´ 1st edition, 2006. soils. II.—Analysis of carbonates in soils,” Journal of the Science [2]P.Clark,F.Carlos,andJ.L.Vazquez´ Mart´ınez, “Epidemiology, of Food and Agriculture, vol. 11, no. 3, pp. 167–172, 1960. costs and burden of osteoporosis in Mexico,” Archives of [17] P.V.Monje and E. J. Baran, “Complex biomineralization pattern Osteoporosis,vol.5,no.1-2,pp.9–17,2010. in cactaceae,” Journal of Plant Physiology,vol.161,no.1,pp.121– [3] M. A. Aguilera-Barreiro, J. A. Rivera-Marquez,´ H. M. Trijillo- 123, 2004. Arriaga, J. A. Tamayo y Orozco, E. Barreira-Mercado, and M. E. [18] M. Contreras-Padilla, E. M. Rivera-Munoz,˜ E. Gutierrez-´ Rodr´ıquez-Garc´ıa, “Intake of dehydrated nopal (Opuntia ficus Cortez,A.R.DelLopez,´ and M. E. Rodr´ıguez-Garc´ıa, “Char- indica) improves bone mineral density and calciuria in adult acterization of crystalline structures in Opuntia ficus-indica,” Mexican women,” Food and Nutrition Research,vol.57,pp.1–9, JournalofBiologicalPhysics,vol.41,no.1,pp.99–112,2015. 2013. [19] AOAC, OfficialMethodsofAnalysisoftheAssociationofOfficial [4] R. P. Heaney, “Bone health,” American Journal of Clinical Analytical Chemists,W.Horwitz,Ed.,AOAC,Washington,DC, Nutrition,vol.85,pp.300S–303S,2007. USA, 16th edition, 1980. [5] K. D. Cashman, “Milk minerals (including trace elements) and [20] F. C. Stintzing and R. Carle, “Cactus stems (Opuntia spp.): a bone health,” International Dairy Journal,vol.16,no.11,pp. review on their chemistry, technology, and uses,” Molecular 1389–1398, 2006. Nutrition and Food Research,vol.49,no.2,pp.175–194,2005. [6]J.L.Rosado,C.Gonzalez,M.E.Valenciaetal.,“Lactose [21] B. C. Pena-Valdivia˜ and B. A. Sanchez-Urdaneta,´ “Nopalito maldigestion and milk intolerance: a study in rural and urban and cactus pear (Opuntia spp.) polysaccharides: mucilage and Mexico using physiological doses of milk,” Journal of Nutrition, pectin,” Acta Horticulturae,vol.728,pp.241–248,2006. vol.124,no.7,pp.1052–1059,1994. [22] H. M. Ram´ırez-Tob´ıas, C. Lopez-Palacios,´ J. R. Aguirre-Rivera, [7] I. Rojas-Molina, E. Gutierrez,´ A. Rojas et al., “Effect of andJ.A.Reyes-Aguero,¨ “Hydroponic cactus pear produc- temperature and steeping time on calcium and phosphorus tion, productivity and quality of nopalito and fodder,” in content in nixtamalized corn flours obtained by traditional Hydroponics—A Standard Methodology for Plant Biological nixtamalization process,” Cereal Chemistry,vol.86,no.5,pp. Researches, T. Asao, Ed., chapter 10, pp. 199–224, InTech, Rijeka, 516–521, 2009. Croatia, 2012. [8]M.E.Rodr´ıguez-Garc´ıa, C. de Lira, E. Hernandez-Becerra´ et [23] J. L. Slavin, “Position of the American Dietetic Association: al., “Physicochemical characterization of nopal pads (Opuntia health implications of dietary fiber,” Journal of the American ficus indica) and dry vacuum nopal powders as a function of Dietetic Association,vol.108,no.10,pp.1716–1731,2008. the maturation,” Plant Foods for Human Nutrition,vol.62,no. [24] D. M. Jimenez-Aguilar,´ J. M. Lopez-Mart´ ´ınez, C. Hernandez-´ 3, pp. 107–112, 2007. Brenes et al., “Dietary fiber, phytochemical composition and [9]M.I.Hernandez-Urbiola,´ M. Contreras-Padilla, E. Perez-´ antioxidant activity of Mexican commercial varieties of cactus Torrero et al., “Study of nutritional composition of nopal pear,” Journal of Food Composition and Analysis,vol.41,pp.66– (Opuntia ficus indica cv. Redonda) at different maturity stages,” 73, 2015. The Open Nutrition Journal,vol.4,pp.1–6,2010. [25] C. Saenz, M. Yoong, F. Figuerola, I. Chiffelle, and A. M. [10] M. M. McConn and P. A. Nakata, “Oxalate reduces calcium Estevez, “Cactus pear cladodes powders as a source of dietary availability in the pads of the prickly pear cactus through fibre: purification and properties,” International Journal of Food formation of calcium oxalate crystals,” Journal of Agricultural Sciences and Nutrition,vol.63,no.3,pp.283–289,2012. and Food Chemistry,vol.52,no.5,pp.1371–1374,2004. [26] P. V. Monje and E. J. Baran, “Characterization of calcium [11] M. Contreras-Padilla, E. Perez-Torrero,´ M. I. Hernandez-´ oxalates generated as biominerals in cacti,” Plant Physiology,vol. Urbiola et al., “Evaluation of oxalates and calcium in nopal pads 128, no. 2, pp. 707–713, 2002. (Opuntia ficus-indica var. redonda) at different maturity stages,” [27] C. Exley, “Silicon in life: a bioinorganic solution to bioorganic Journal of Food Composition and Analysis,vol.24,no.1,pp.38– essentiality,” JournalofInorganicBiochemistry,vol.69,no.3,pp. 43, 2011. 139–144, 1998. 8 Journal of Chemistry

[28] D. D. Ebbing and S. D. Gammon, “Solubility and complex-ion equilibria,” in General Chemistry, L. Lockwood and A. White, Eds., pp. 710–715, Brooks/Cole Publishers, Belmont, Calif, USA, 10th edition, 2013. [29]Y.Sugimura,T.Mori,I.Nittaetal.,“Calciumdepositionin idioblasts of mulberry leaves,” Annals of Botany,vol.83,no.5, pp. 543–550, 1999. [30] P. V. Monje and E. J. Baran, “Characterization of calcium oxalate biominerals in Pereskia species (Cactaceae),” Zeitschrift fur¨ Naturforschung C,vol.64,pp.763–766,2009. [31] B.P.Gargari,S.Mahboob,andS.V.Razavieh,“Contentofphytic acid and its mole ratio to zinc in flour and breads consumed in Tabriz, Iran,” Food Chemistry, vol. 100, no. 3, pp. 1115–1119, 2007. [32] M. R. Bhandari and J. Kawabata, “Assessment of antinutritional factors and bioavailability of calcium and zinc in wild yam (Dioscorea spp.) tubers of Nepal,” Food Chemistry,vol.85,no. 2, pp. 281–287, 2004. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 734578, 8 pages http://dx.doi.org/10.1155/2015/734578

Research Article New Approach to Enrich Pasta with Polyphenols from Grape Marc

V. Marinelli, L. Padalino, D. Nardiello, M. A. Del Nobile, and A. Conte

Services Center of Applied Research, University of Foggia, Via Napoli 25, 71122 Foggia, Italy

Correspondence should be addressed to A. Conte; [email protected]

Received 7 September 2015; Revised 27 October 2015; Accepted 28 October 2015

Academic Editor: Somdet Srichairatanakool

Copyright © 2015 V. Marinelli 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.

Food industry produces significant amount of waste that represents a problem for the sector. However, by-products are also promising sources of compounds which may be reused for their nutritional properties. The aim of this work is to exploit wine- making by-products, obtaining an extract by ultrasound-assisted extraction only using water as solvent. The characteristics of spaghetti enriched with grape marc were assessed and compared to control samples. In particular, total phenolic and flavonoids contents, the antioxidant activity, the cooking quality, and the sensory acceptability were evaluated at various steps of pasta production. The enriched spaghetti showed higher total phenolic and flavonoids contents and higher antioxidant activity than the control pasta. In addition, low cooking losses were found. In terms of sensory properties fortified pasta is acceptable as the traditional product, thus demonstrating that it is possible to exploit food waste to better satisfy consumer demand for healthy food products in a more sustainable perspective.

1. Introduction due to the environmental consequences of by-products, food industry points towards formulation of products enriched Food industry and in particular fruit and vegetable pro- with bioactive compounds that can be also extracted from cessing generally produce significant wastes that represent by-products [2]. In the scientific literature several extraction the major disposal problem for food sector, from both the environmental and the economic point of view. However, techniques have been proposed. Some of them are traditional it is worth noting that by-products can be also promising systems based on the extraction power of solvents or heat sources of compounds, being rich in carotenoids, polyphe- application and other ones are unconventional techniques of nols, tocopherols, vitamins, and other substances [1]. In new generation as the ultrasound-assisted extraction (UAE). general, agroindustrial wastes are disposed, utilized as animal This extraction technique is preferred compared to the feed or as fertilizer, but their use as sources of natural food classical extraction methods because it allows to shorten the ingredients recently recorded considerable attention from extraction time, reduces the organic solvent waste, increases food research. The interest for by-products is due to the fact the extraction yield, and enhances the quality of extracts that they represent raw materials at low cost and are widely and it is possible to use water as solvent [3, 4]. The UAE is available. In addition, the natural compounds contained based on sound mechanical waves that go beyond human in by-products could allow satisfying consumer demand hearing, whose frequencies are superior to 20 kHz. This type for food products with new functional properties, typical of extraction requires a liquid medium previously selected of compounds from plant world, which are natural and that allows ultrasonic wave to propagate up to the product, health-promoting. It is widely recognized that an adequate also preserving the integrity of the molecules that can be consumption of fruit and vegetables plays an important role thermolabile, thermostable, hydrosoluble, and liposoluble. in the prevention of diseases, for example, reduced risk of This is possible due to the cavitation forces resulting from the heart disease and stroke, as well as certain types of cancer ultrasound application. This phenomenon could be explained [1]. Due to this consumer demand for healthier foods and as the formation and the final collapse of microbubbles 2 Journal of Chemistry into the liquid medium that may cause different mechanical Sigma-Aldrich (Milan, Italy). All reagents were of analytical effects, such as turbulent streaming, particle collisions, and grade. cell wall disruption. These mechanical effects could cause a greater penetration of the solvent into the food matrix 2.3. Extraction Process. Grape marc extract (GME) was and, consequently, an increase of the mass transfer rates obtained by means of UAE (USR-1500-50WL, Weal s.r.l., of the bioactive compounds from the food matrix into the Milan, Italy) using only water as solvent. In the reactor grape extraction solvent [3]. Under these conditions the transfer of marc was suspended in water at a ratio of 1 : 10 (w/v) and compounds to be extracted is facilitated and the extraction ultrasonically treated for 60 minutes at acoustic frequency time is reduced. of 25 kHz and with ultrasonic power density of 50 W/L. The According to the literature consulted, most of the studies obtained extract was centrifuged at 10000 rpm for 10 minutes on by-products are focused on optimization conditions for at room temperature and then it was filtered by means of the extraction to obtain potential bioactive compounds, but 0.45 𝜇m PTFE filters (Teknokroma, Sant Cugat del Valles,´ ∘ the applications of the extracted substances to foods are very Barcelona, Spain) and stored at −20 Cuntiltheanalytical scarce. Calvo et al. [5] have designed sausages formulation determinations. enriched with lycopene by means of direct addition of tomato skins, previously dried and ground. The same group of 2.4. Total Phenols, Flavonoids, and Antioxidant Activity researchersalsostudiedtheinfluenceonthephysicochemical Determination. To determine total phenols, flavonoids, and sensorial properties of raw and cooked hamburgers and antioxidant activity, the extraction was performed as enriched with dry tomato peels, demonstrating that the described by Biney and Beta [8] from all the various samples: addition of tomato peel to meat products results in healthier dough, extruded fresh spaghetti, pasteurized fresh spaghetti, product due to both lycopene and fiber content present in dry spaghetti, and cooked fresh and cooked dry spaghetti. tomato by-product [6]. Ozvural¨ and Vural [7] incorporated ∘ The samples were dried at 30 C, grounded, and sieved grape seed flour, rich in polyphenols obtained from wine by- through an 800 𝜇m sieve. For total phenols and antioxidant products, into frankfurters. activity determination, 2 g of powdered sample was mixed In this context, grape marc is taken into account, being with 20 mL of acidified methanol (HCl/H2O:MeOH, an interesting source of natural compounds as polyphenols 20:80),while,inthecaseofflavonoids,thesamequantityof (anthocyanins, catechins, flavonols, and phenolic acids) and sample was combined with 10 mL of acidified methanol. The fibers [3]. The aim of this work was to enrich fresh and dry mixtures were included in 50 mL centrifuge tubes and shaken pasta with grape marc extracts obtained by means of UAE at room temperature in darkness for 2 h at 300 rpm using using only water as solvent extraction, thus demonstrating orbitalshaker(HS260BASIC,IKA,Staufen,Germany). that it is possible to increase polyphenols amount and antiox- ∘ Next, the samples were centrifuged at 5 Cfor15minutesat idant activity of pasta without compromising the sensory 10000 rpm (5804R, Eppendorf, Milan, Italy) and supernatant characteristics and valorize wine-making by-products that was collected and filtered (PTFE, 0.45 𝜇m) prior to the are generally discarded. analytical determinations.

2. Material and Methods 2.5. Spaghetti Preparation. Commercial durum wheat semolina was purchased from Agostini Mill (Montefiore 2.1. Raw Materials. Grape marc made up of skins, seeds, and dell’Aso, Italy). Semolina was mixed with proper amount of stalks was provided by a local company of Foggia (Southern water or grape marc extract (GME) in the rotary shaft mixer Italy), during the 2014 harvest. The samples were dried at 30– ∘ ∘ (Namad, Rome, Italy) at 25 Cfor20mintodistributeliquid 35 C in a dryer (SG600, Namad, Rome, Italy) for 48 hours. uniformly throughout the semolina particles. The dough The dried grape marc was reduced in a fine powder bya was extruded with a 60VR extruder (Namad) as described hammer mill (16/BV-Beccaria s.r.l., Cuneo, Italy) and then by Padalino et al. [9]. After extrusion, pasta was pasteurized ∘ ∘ stored at 4 C until further utilization. with steam for 3 min at 90 C (Namad, Rome, Italy). The pasteurization system uses air at room temperature to 2.2. Chemicals. Folin-Ciocalteu reagent, gallic acid monohy- cool down the pasta after the thermal treatment. Then, the drate, methanol, hydrochloric acid, ABTS (2,2-azino-bis(3- extruded pasta was dried in a dryer (SG600; Namad). The ethylbenzothiazoline-6-sulfonic acid diammonium salt), drying process conditions applied were in accordance with potassium persulfate (K2S2O8), Trolox (6-hydroxy-2,5,7,8- Padalino et al. [9]. tetramethylchroman-2-carboxylic acid), aluminium chloride (AlCl3), sodium nitrite (NaNO2), sodium hydroxide solution 2.6. Sensory Analysis. Fresh-extruded and dry spaghetti sam- (NaOH), and quercetin were supplied from Sigma-Aldrich ples were submitted to a panel of 15 trained tasters (seven men (Milan, Italy). Anhydrous sodium carbonate (Na2CO3)was and eight women, aged between 28 and 45 years) in order to supplied from Carlo Erba (Milan, Italy). For the preparation evaluate the sensory attributes. The panelists were selected of the phosphate buffered saline (PBS), the following on the basis of their sensory skills (ability to accurately salts were used: sodium phosphate dibasic heptahydrate determine and communicate the sensory attributes as appear- (HNa2O4P⋅7H2O) and sodium phosphate monobasic mono- ance, odor, flavor, and texture of a product). The panelists hydrate (H2NaO4P⋅H2O). These were purchased from were also trained in sensory vocabulary and identification of Journal of Chemistry 3 particular attributes by evaluating durum wheat commercial potassium persulfate were utilized. The ABTS radical cation ∙+ spaghetti [10]. They were asked to indicate color, homogene- (ABTS ) was obtained by reacting ABTS stock solution ity,andresistancetobreakingoffreshanddryuncooked with 2.45 mM potassium persulfate (final concentration) and spaghetti. In addition, to color and homogeneity, odor was allowingthemixturetostandinthedarkatroomtemperature ∙+ also evaluated for fresh-extruded and pasteurized spaghetti. for 12–16 h. The ABTS solution was diluted with 5 mM Elasticity, firmness, bulkiness, adhesiveness, color, odor, and phosphate buffered saline, pH 7.4(PBS), and absorbance 0.70 taste were evaluated on fresh and dry cooked spaghetti. To the ± 0,02 at 734 nm. Then, 200 𝜇L of sample extract was added aim, a nine-point scale, where 1 corresponded to extremely ∙+ to 2 mL of ABTS diluted solution and after 3 minutes at unpleasant, 9 to extremely pleasant, and 5 to the threshold ∘ 30 C the mixture was measured through a spectrophotometer acceptability, was used to quantify each attribute. On the basis (UV1800, Shimadzu Italia s.r.l., Milan, Italy) at 734 nm. A cal- of the abovementioned attributes, panelists were also asked ibration curve was previously built using 6-hydroxy-2,5,7,8- to score the overall quality of both cooked and uncooked tetramethylchroman-2-carboxylic acid (Trolox) as standard, products using the same nine-point scale [9]. 2 at concentrations between 0.98 and 250 𝜇M(𝑅 = 0,9997) and the antioxidant activity was expressed as 𝜇moli Trolox 2.7. Cooking Quality. The optimal cooking time (OCT) was equivalents for gram of dry weight (dw). All analyses were evaluated according to the AACC [11] approved method 66– carried out in triplicate. 50. The cooking loss, that is, the amount of solid substance lost into the cooking water, was determined according to the AACC [11] approved method 66–50. The swelling index 2.8.3. Determination of Total Flavonoids. Total flavonoids and the water absorption of cooked pasta (grams of water content both in GME and in all the extracts was determined per gram of dry pasta) were determined according to the by aluminum chloride colorimetric method, according to procedure described by Padalino et al. [12]. Moreover, cooked Huang and Ho [15] with modifications, using quercetin as standard. Extracts (0.5 mL), prepared as previously spaghetti samples were submitted to hardness and adhesive- 𝜇 ness analysis by means of a Zwick/Roell model Z010 Texture described, were mixed with 2 mL of distilled water and 150 L of a 5% sodium nitrite (NaNO2) solution. After 6 minutes, Analyzer (Zwick Roell Italia s.r.l., Genoa, Italy) equipped with 𝜇 a stainless steel cylinder probe (2 cm diameter). The hardness 150 Lofa10%aluminumchloride(AlCl3)solutionwas (mean maximum force, N) and adhesiveness (mean negative added and the mixture was allowed to stand for 6 minutes. area, Nmm) were measured according to the procedure Finally, 1 mL of 1 M sodium hydroxide (NaOH) was added described by Padalino et al. [12]. Six measurements for each andtotalvolumewasmadeupto5mLwithdistilledwater. spaghettisamplewereperformed. Then, the solutions were mixed and for each sample the absorbance was read in triplicate against blank at 415 nm. The standard curve was prepared using quercetin as standard 2 2.8. Chemical Analysis in the range 3,13–500 mg/L (𝑅 = 0,9981) and total amount of flavonoids was expressed in mg of quercetin/100 g of dry 2.8.1. Determination of Total Phenolic Compounds. Total phe- weight (dw). nolic compounds were determined by UV-vis spectropho- tometry according to Folin-Ciocalteu method [13]. In particular, GME was 1 : 10 diluted with water before analysis, while 2.9. Statistical Analysis. Experimental data were compared by extracts obtained from samples relative to each production a one-way analysis of variance (ANOVA). Duncan’s multiple step of spaghetti, previously described, were analyzed without range test, with the option of homogeneous groups (𝑃< any dilution. Specifically, 0.5 mL of grape marc or pasta 0.05), was carried out to determine significant differences extract was mixed with 2.5 mL of Folin-Ciocalteu reagent between spaghetti samples. STATISTICA 7.1 for Windows (diluted 1 : 10 with water) and, after 5 minutes, 2 mL of (StatSoft, Inc., Tulsa, OK, USA) was .used Na2CO3 (75 g/L) was added. The sample was kept in darkness at room temperature for 2 hours. Distilled water was used as 3. Results and Discussion control sample. The absorbance was measured at 765 nm by an UV-vis spectrophotometer (UV1800, Shimadzu Italia s.r.l., In this study the total phenolic and flavonoids content Milan, Italy). Total phenolic compounds were quantified by a together with the antioxidant activity were evaluated on both 2 calibration curve previously built (3–200 mg/L; 𝑅 = 0,9989) control and enriched samples: dough, extruded fresh pasta, using standard solution of gallic acid, and the total phenolic and fresh-pasteurized/dry pasta (uncooked and cooked). content was expressed as mg gallic acid/100 g of dry weight Details on chemical characterization, cooking quality (only (dw). All tests were carried out in triplicate. for dry pasta), and sensory properties are reported below separately. 2.8.2. Determination of Antioxidant Activity. The antioxidant activity was assessed using ABTS test, which is based on the 󸀠 3.1. Chemical Quality. Total phenolic (mg gallic acid/ ability of antioxidants to interact with the radical cation 2,2 - 100 g dw), flavonoids (mg quercetin/100 g dw), and antioxi- ∙+ azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ) dant activity (𝜇moli Trolox/g dw), measured by ABTS assay, inhibiting its absorption at 734 nm, according to the method of GME were shown in Table 1. The obtained results indicate of Re et al. [14]. 7 mM ABTS stock solution and 140 mM that the extract has a high content of polyphenols, equal 4 Journal of Chemistry

Table 1: Total phenols, total flavonoids, and antioxidant activity of the grape marc extract.

Sample Total phenols Total flavonoids Antioxidant activity (mg gallic acid/100 g dw) ± SD (mg quercetin/100 g dw) ± SD (𝜇moli Trolox/g dw) ± SD GME 443 ± 1.7 405 ± 12 518 ± 7. 4 GME: grape marc extract.

900 9

g) bb 800 bb b 8 b b 700 b a a 7 100 a 600 b 6 b 500 5 a a aa a 400 4 a 300 b b a Trolox/g) moli 3 𝜇

200 ( 2 a (mg gallic acid/ (mg 100 1 0 0 Dough Dough spaghetti spaghetti Cooked dry Cooked Cooked dry Cooked spaghetti spaghetti spaghetti spaghetti Dry spaghetti Dry spaghetti Extruded fresh Extruded fresh Extruded Pasteurized fresh Pasteurized fresh spaghetti Pasteurized fresh Pasteurized fresh spaghetti Cooked pasteurized Cooked Cooked pasteurized Cooked CNT CNT ACTIVE ACTIVE Figure 1: Total phenolic content of spaghetti samples in each step Figure 2: Antioxidant activity of spaghetti samples in each step of of the production process. Results are expressed as means ± SD for the production process. Results are expressed as means ± SD for a,b a,b 𝑛=3. Data in columns with different superscripts are significantly 𝑛=3. Data in columns with different superscripts are significantly different (𝑃 < 0.05). CNT: samples without the addition of grape different (𝑃 < 0.05). CNT: samples without the addition of grape marc extract; ACTIVE: samples with the addition of grape marc marc extract; ACTIVE: samples with the addition of grape marc extract. extract.

to 443 mg acid gallic/100 g dw, greater than that reported led to a significant increase in antioxidant capacity. As in the by Gonzalez-Centeno´ et al. [3], who studied the effect of case of total phenols, also in this case, the ACTIVE samples acoustic frequency, ultrasonic power density, and extraction proved an antioxidant activity greater than the corresponding time of grape pomace by UAE. Moreover, the extract was CNT sample. In fact, according to Alonso et al. [17] there characterized by a high flavonoid content and antioxidant is a positive correlation between the antioxidant activity activity, 405 mg quercetin/100 g dw, and 518 𝜇moli Trolox/g of and the total polyphenolic content of samples. In every step dw, respectively. of production process a statistically significant difference The phenolic compounds of the experimental samples between CNT and ACTIVE samples was detected. According were expressed as mg gallic acid for 100 g dw and shown in to our findings, the entire pasta process did not cause any Figure 1. In every step of the spaghetti production process, significant change in antioxidant activity; in fact, after extru- the phenolic content of the ACTIVE samples was higher sion an increase of antioxidant activity was observed, which than spaghetti without any extract. In particular, among remains constant in the subsequent steps. Only after cooking the experimental samples, the extruded fresh pasta showed adecreaseofantioxidantactivitywasfound,probablybecause the highest quantity of bioactive compounds (737 mg gallic heat degraded phenols, thus decreasing their concentration. acid/100 g dw). As it can be inferred by the figure, the phenolic The flavonoid content of both uncooked and cooked content decreased as temperature increased; in fact the spaghetti is presented in Figure 3. The addition of grape cooked dry pasta revealed the lowest phenolic value, probably marc extract to spaghetti significantly increased the flavonoid due to the combination of drying and cooking procedure. content in every phase of the production process. The highest The same trend was observed by Pasqualone et al. [2], who flavonoids content was recorded for the extruded fresh pasta, studied biscuits enriched with grape marc extract. According which presented 58 mg quercetin/100 g dw, greater than the to Abdel-Aal and Rabalski [16] the effect of cooking on dough sample enriched with the extract. According to the phenols is not always the same but depends on the type of obtained results, it is possible to infer that the extrusion bioactivecompoundandtypeofproduct. phase increased the flavonoids amount, as observed also in ABTS assay was used to evaluate the antioxidant activity. the case of total phenols and antioxidant activity, probably The results are shown in Figure 2 and expressed as 𝜇moli because this step frees the bounded compounds. The pas- Trolox per gram of dry weight. According to obtained data, teurization and the drying process exerted little effect on the fortification of spaghetti with aqueous grape marc extract flavonoids content; in fact, these phases recorded 56 and Journal of Chemistry 5

Table2:Cookingqualityofdryspaghettisamples.

Sample OCT (min) Cooking loss (%) Swelling index Water Hardness (N) Adhesiveness (Nmm) absorption (%) CNT 10.00 6.47 ± 0.04a 2.06 ± 0.08a 165 ± 0.51a 7. 32 ± 0.52a 1.04 ± 0.08a ACTIVE 10.00 4.53 ± 0.21b 1.73 ± 0.013b 135 ± 0.51b 7. 2 5 ± 0.56a 0.84 ± 0.05b CNT: samples without the addition of grape marc extract. ACTIVE: samples with the addition of grape marc extract. a,bData in columns with different superscripts are significantly different𝑃 ( < 0.05).

80 with carotenoids from tomato peels had similar swelling

g) b 70 b b index value of control sample (100% wheat flour). 100 60 b 50 b As compared to the CNT sample, ACTIVE sample a a a 40 a b recorded lower adhesiveness value. This could be due to the 30 a fact that the antioxidant compounds generally form with the 20 a 10 gluten proteins a stronger gluten network that entraps the (mg quercetin/ (mg 0 starch granules, slowing down the amylose release during cooking. On the contrary, the hardness value of ACTIVE

Dough sample was similar to that of the CNT sample. spaghetti Cooked dry Cooked spaghetti spaghetti Dry spaghetti

Extruded fresh Extruded 3.3. Sensory Quality. The sensory properties of the investi- Pasteurized fresh Pasteurized fresh spaghetti gated samples were evaluated by means of a group of trained Cooked pasteurized Cooked CNT panelistsandtheresultsarelistedinTables3and4forfresh- ACTIVE extruded/pasteurized and dry spaghetti samples (uncooked and cooked), respectively. Sensory data of uncooked fresh- Figure 3: Total flavonoids of spaghetti samples in each step of the extruded/pasteurized spaghetti samples (E-ACTIVE and P- production process. Results are expressed as means ± SD for 𝑛= a,b 3. Data in columns with different superscripts are significantly ACTIVE) showed that addition of grape marc extract deter- different (𝑃 < 0.05). CNT: samples without the addition of grape mined a slight decrease in overall quality as compared to the marc extract; ACTIVE: samples with the addition of grape marc CNT, even though no statistically significant differences were extract. observed among samples. Specifically, samples E-ACTIVE and P-ACTIVE recorded the smallest color scores with respect to the CNT samples. In fact, the spaghetti samples 53 mg quercetin/100 g dw, respectively. Only after cooking a containing GME showed a light brown color in comparison significant decrease was observed, especially in the cooked to the bright yellow color of the CNT sample. Pasta color dry spaghetti. is essential for assessing pasta quality. Generally, consumers prefer pasta with a bright yellow color [20]. Moreover, the 3.2. Cooking Quality. Cooking quality is an important lowest color score has been observed for both pasteurized parameter for dry pasta evaluation. The cooking perfor- samples P-CNT and P-ACTIVE with respect to extruded mances of the investigated spaghetti samples in terms of opti- samples. Most probably, the high temperature during the mum cooking time, cooking loss, water absorption, swelling pasteurization temperature promoted development of Mail- index, hardness, and adhesiveness are shown in Table 2. As lard reaction, giving to pasta a brownish color that slightly canbeobservedfromTable2therewasnodifferencein affects panelist judgment. Regarding the extruded and the optimum cooking time. In fact, for both samples studied, the pasteurized cooked spaghetti samples, the incorporation of optimum cooking time was around 10 min. GME caused a little rise of overall quality, even though The spaghetti sample with grape marc extract showed no significant difference was observed between the studied alowercookinglossthantheCNTsample.Onepossible samples. In particular, E-ACTIVE and P-ACTIVE spaghetti explanation could be due to the capacity of the antioxidant samples recorded a decline in adhesiveness and bulkiness compounds from grape marc extract present in the form of (high score) in comparison with the E-CNT and P-CNT. complex with proteins around the starch granules, encapsu- Besides, the addition of GME did not determined significant lating them during cooking and restricting excessive swelling differences in the other sensorial attributes. Concerning dry and diffusion of the amylose content [18]. Also Rizk et al. [18] spaghetti (uncooked and cooked) the addition of the GME foundareductionofcookinglossinsampleenrichedwith did not cause any significant differences in overall quality antioxidant compounds from tomato peels (carotenoids) with respect to the CNT sample (Table 4). with respect to the control sample (100% wheat flour). Regarding the water absorption and swelling index, 4. Conclusions ACTIVE sample recorded the lowest values with respect to CNT sample (Table 2). These results are in agreement with The reutilization of wine-making by-products was proposed RizkandTolba[19]whoalsoobservedthatpastaenriched with success to enrich fresh or dry pasta from durum wheat. 6 Journal of Chemistry a a a a 0.31 0.31 0.30 0.30 ± ± ± ± 7. 02 7. 0 8 7. 2 3 7. 24 a a a a 0.20 0.20 0.24 0.24 ± ± ± ± 7. 2 1 7. 2 1 7. 2 0 7. 2 0 a a a a 0.37 0.37 0.27 0.27 ± ± ± ± 7. 2 1 7. 2 1 7. 0 4 7. 0 4 a a a a 0.31 0.31 0.27 0.27 ± ± ± ± 7. 32 7. 2 0 7. 3 5 7. 2 5 b a a,b a,b 0.24 0.28 0.24 0.28 ± ± ± ± 7. 5 0 6.75 7. 2 5 7. 0 0 b b a a,b ess Adhesiveness Color Odor Taste Overall quality 0.23 0.25 0.27 0.27 ± ± ± ± 7. 5 0 6.95 6.98 7. 3 8 a a a a 0.34 0.34 0.41 0.41 ± ± ± ± 7. 2 1 7. 2 1 7.07 7.07 a a a a ). 0.27 0.27 0.24 0.24 ± ± ± ± 𝑃 < 0.05 7. 4 5 7. 4 5 7. 5 0 7. 5 0 a a a a 0.17 0.17 0.33 0.33 ± ± ± ± 7. 4 5 7. 8 0 7. 5 0 7. 2 0 a a a a 0.18 0.18 0.36 0.36 ± ± ± ± Table 3: Sensory characteristics of fresh-extruded (E) and fresh-pasteurized (P) uncooked and cooked spaghetti samples. 7. 9 2 7. 9 2 7. 8 0 7. 8 0 Uncooked spaghetti Cooked spaghetti b a a,b a,b 0.18 0.23 0.18 0.23 ± ± ± ± Color Odor Overall quality Elasticity Firmness Bulkin Data in columns with different superscripts are significantly different ( Sample E-CNTE-ACTIVE 7.25 P-CNT 7.52 P-ACTIVE 7.08 7.30 a,b E-CNT: fresh-extruded spaghetti samples without theE-ACTIVE: addition fresh-extruded of spaghetti grape samples marc with extract. theP-CNT: addition fresh-pasteurized of spaghetti grape samples marc without extract. theP-ACTIVE: addition fresh-pasteurized of spaghetti grape samples marc with extract. the addition of grape marc extract. Journal of Chemistry 7 a a 0.26 0.27 ± ± 7. 31 7. 3 7 a a 0.38 0.36 ± ± 7. 5 3 7. 6 4 a a 0.42 0.29 ± ± 7. 31 7. 2 5 a a 0.25 0.42 ± ± 7. 2 5 7. 2 0 a a 0.33 0.23 ± ± 7. 2 0 7. 3 8 a a 0.32 0.26 ± ± 7. 2 1 7. 4 5 a a s Bulkiness Adhesiveness Color Odor Taste Overall quality 0.33 0.24 ± ± 7. 2 5 7. 2 0 a a 0.30 0.34 ). ± ± 7. 2 5 7. 3 0 𝑃 < 0.05 a a 0.21 0.24 ± ± Table 4: Sensory characteristics of dry uncooked and cooked spaghetti samples. 7. 2 1 7. 4 7 a a 0.23 0.28 ± ± 7.41 7. 3 0 Uncooked spaghetti Cooked spaghetti a a 0.27 0.32 ± ± Color Resistance to break Overall quality Elasticity Firmnes Data in columns with different superscripts are significantly different ( Sample CNTACTIVE 7.40 7.61 a,b CNT: samples without the addition ofACTIVE: grape samples marc with extract. the addition of grape marc extract. 8 Journal of Chemistry

Theobtainedresultsshowthatitispossibletousegrape [7] E. B. Ozvural¨ and H. Vural, “Grape seed flour is a viable marc aqueous extract, obtained from ultrasound extraction, ingredient to improve the nutritional profile and reduce lipid instead of simple water to produce pasta, without altering oxidation of frankfurters,” Meat Science, vol. 88, no. 1, pp. 179– its sensory characteristics. In fact, according to sensory 183, 2011. analysis no significant differences have been found among [8] K. Biney and T. Beta, “Phenolic profile and carbohydrate the experimental samples. Moreover, enriched spaghetti was digestibility of durum spaghetti enriched with buckwheat flour characterized by a higher content of phenolic compounds, and bran,” LWT—Food Science and Technology,vol.57,no.2,pp. flavonoids, and consequently antioxidant activity compared 569–579, 2014. to the control sample. Therefore, data demonstrated with a [9] L.Padalino,M.Mastromatteo,L.Lecce,S.Spinelli,F.Conto,` and concrete example that it is possible to reuse an agroindustrial M. A. Del Nobile, “Chemical composition, sensory and cooking waste such as grape marc to design new foods with healthful quality evaluation of durum wheat spaghetti enriched with pea flour,” International Journal of Food Science and Technology,vol. properties. In this way, it is possible to face environmental 49,no.6,pp.1544–1556,2014. problemsandsatisfyatthesametimeconsumerdemandfor [10] ISO Standards, “Alimentary pasta produced from durum wheat food products with a recognized premium quality. semolina—estimation of cooking quality by sensory analysis— part 2: routine method,” ISO 11036/7304-2:2008, 2008. Abbreviations [11] AACC. American Association of Cereal Chemists, “Approved methods of the American Association of Cereal Chemistry,” ABTS: 2,2-Azino-bis(3-ethylbenzothiazoline)-6- Method 66-50, AACC, Saint Paul, Minn, USA, 2000. sulfonic acid diammonium salt [12] L.Padalino,M.Mastromatteo,L.Lecce,S.Spinelli,F.Conto,` and Trolox: 6-Hydroxy-2,5,7,8-tetramethylchroman-2- M. A. Del Nobile, “Chemical composition, sensory and cooking carboxylic acid quality evaluation of durum wheat spaghetti enriched with pea PBS: Phosphate buffered saline flour,” International Journal of Food Science & Technology,vol. GME: Grape marc extract 49,no.6,pp.1544–1556,2014. CNT: Samples without the addition of grape [13] S. Spinelli, A. Conte, L. Lecce, A. L. Incoronato, and M. marc extract A. Del Nobile, “Microencapsulated propolis to enhance the ACTIVE: Samples with the addition of grape marc antioxidant properties of fresh fish burgers,” Journal of Food extract. Process Engineering,vol.38,no.6,pp.527–535,2015. [14] R.Re,N.Pellegrini,A.Proteggente,A.Pannala,M.Yang,andC. Rice-Evans, “Antioxidant activity applying an improved ABTS Conflict of Interests radical cation decolorization assay,” Free Radical Biology and Medicine, vol. 26, no. 9-10, pp. 1231–1237, 1999. The authors declare that there is no conflict of interests regarding the publication of this paper. [15] Y.-S. Huang and S.-C. Ho, “Polymethoxy flavones are responsible for the anti-inflammatory activity of citrus fruit peel,” Food Chemistry,vol.119,no.3,pp.868–873,2010. References [16] E.-S. M. Abdel-Aal and I. Rabalski, “Effect of baking on free and bound phenolic acids in wholegrain bakery products,” Journal [1] A. Schieber, F. C. Stintzing, and R. Carle, “By-products of plant of Cereal Science,vol.57,no.3,pp.312–318,2013. food processing as a source of functional compounds—recent [17] A.M.Alonso,D.A.Guill´ en,C.G.Barroso,B.Puertas,andA.´ developments,” Trends in Food Science and Technology,vol.12, Garc´ıa, “Determination of antioxidant activity of wine byprod- no. 11, pp. 401–413, 2001. ucts and its correlation with polyphenolic content,” Journal of [2] A. Pasqualone, A. M. Bianco, V. M. Paradiso, C. Summo, G. Agricultural and Food Chemistry,vol.50,no.21,pp.5832–5836, Gambacorta, and F. Caponio, “Physico-chemical, sensory and 2002. volatile profiles of biscuits enriched with grape marc extract,” [18]E.M.Rizk,S.H.Bedier,andM.A.Elgendy,“Utilizationof Food Research International,vol.65,pp.385–393,2014. carotenoid pigments extracted from tomato peel as natural [3] M. R. Gonzalez-Centeno,´ K. Knoerzer, H. Sabarez, S. Simal, antioxidants and colorants in sunflower oil and spaghetti,” C. Rossello,´ and A. Femenia, “Effect of acoustic frequency and Egyptian Journal of Agricultural Research,vol.92,no.1,pp.309– power density on the aqueous ultrasonic-assisted extraction of 321, 2014. grape pomace (Vitis vinifera L.)—a response surface approach,” [19] E. M. Rizk and K. H. Tolba, “Isolation and stabilization of Ultrasonics Sonochemistry,vol.21,no.6,pp.2176–2184,2014. carotenoids from sweet red pepper and utilization as food [4] Y. Tao, Z. Zhang, and D.-W. Sun, “Kinetic modeling of colorants,” Arab Universities Journal of Agricultural Sciences,vol. ultrasound-assisted extraction of phenolic compounds from 10, pp. 221–234, 2002. grape marc: influence of acoustic energy density and temper- [20]A.Debbouz,W.J.Pitz,W.R.Moore,andB.L.Dappolonia, ature,” Ultrasonics Sonochemistry,vol.21,no.4,pp.1461–1469, “Effect of bleaching on durum-wheat and spaghetti quality,” 2014. Cereal Chemistry,vol.72,no.1,pp.128–131,1995. [5]M.M.Calvo,M.L.Garc´ıa, and M. D. Selgas, “Dry fermented sausages enriched with lycopene from tomato peel,” Meat Science,vol.80,no.2,pp.167–172,2008. [6]M.L.Garc´ıa,M.M.Calvo,andM.D.Selgas,“Beefhamburgers enriched in lycopene using dry tomato peel as an ingredient,” Meat Science,vol.83,no.1,pp.45–49,2009. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 684561, 9 pages http://dx.doi.org/10.1155/2015/684561

Research Article Evaluation of the Stability of the Total Antioxidant Capacity, Polyphenol Contents, and Starch Hydrolase Inhibitory Activities of Kombucha Teas Using an In Vitro Model of Digestion

Mindani I. Watawana, Nilakshi Jayawardena, Shakkya J. Ranasinghe, and Viduranga Y. Waisundara Functional Food Product Development Project, National Institute of Fundamental Studies, Hantane Road, 20000 Kandy, Sri Lanka

Correspondence should be addressed to Viduranga Y. Waisundara; [email protected]

Received 7 September 2015; Revised 9 October 2015; Accepted 12 October 2015

Academic Editor: Dimitris P. Makris

The objective of this study was to evaluate and compare antioxidant and starch hydrolase inhibitory activity of three different types of Kombucha beverages prepared by three pellicles with different microbial compositions. The fermentation process was carried out for 7 days and the assessments of antioxidant and starch hydrolase inhibitory activities as well as tea phenolic compounds were carried out. These parameters were also evaluated after subjecting the final fermented samples to gastric and duodenal digestion in an in vitro digestion model. The pH had a statistically significant decrease during the period of fermentation. The total phenolics content and antioxidant activities had increased during the fermentation process as well as when subjected to digestion. The starch hydrolase inhibitory activities also increased in a similar manner during the different phases. The 𝛼-amylase and 𝛼-glucosidase inhibitory activities showed statistically significant increases (𝑃 < 0.05) as the fermentation progressed, while an increase was observed after being subjected to pancreatic and duodenal digestion as well. All three types of tea showed a higher 𝛼-amylase inhibitory activity than 𝛼-glucosidase inhibitory activity.

1. Introduction and symbiotic beverage [2–4]. This beverage is traditionally prepared by fermenting sugared black tea with a symbiotic The interest towards conducting systematic studies on tradi- mixed culture of yeast and bacteria [5]. It is believed that tionally consumed food items for the purpose of maintaining this beverage originated in China and it is popular for its good health and well-being has increased during recent high antioxidant activity and starch hydrolase inhibitory times. Many of the traditional food products are known activities which have been systematically demonstrated by to possess bioactive components which enable them to be many recent studies [5–8]. The period of fermentation is considered as functional foods. Essentially, this category of typically known to be a minimum of 3 days with a maximum food products includes whole foods and fortified, enriched, of 60 days depending on the native practices, whereby the or enhanced foods, which have a potentially beneficial effect brothisfilteredandconsumedaftertheprocess[5].The on health when consumed as part of a varied diet on a flavor of this tea varies widely from sparkling flavor to regularbasis,ateffectivelevels[1].Kombucha,afermented a mild vinegar-like taste [9]. The studies focusing on the tea beverage, has gained immense popularity throughout antioxidant activity of the beverage have also demonstrated the world in recent times as a functional food, especially the possession of bioactive ingredients of therapeutic interest, in Western and Mediterranean regions, primarily due to mainlythepolyphenolsandsecondarymetabolitesdeveloped numerous associated health benefits such as anticancer, anti- during the fermentation process itself. diabetic, anti-inflammatory, hepatoprotective, and detoxifi- The bacterial component of the Kombucha culture con- cation as well as due to the ability to act as a probiotic sists of strains such as Acetobacter xylinum, A. xylinoides, 2 Journal of Chemistry

A. aceti, A. pausterianus, and Bacterium gluconicum [9]. andinfusedfor5minafterwhichtheywerefilteredthrough The dominant yeast strains are Zygosaccharomyces bailii, a sterile sieve. Sucrose (10%) was dissolved in each beverage Schizosaccharomyces pombe, Saccharomyces ludwigii, S. cere- and the preparation was left to cool to room temperature at ∘ visiae, Kloeckera spp., Torulaspora spp., and Pichia species [9, 24 ± 3 C. The cooled tea was aseptically inoculated with each 10]. Many scientific studies have proven that yeast outnumber of the freshly grown tea fungi for 7 days. The fermentation ∘ the bacterial count during the fermentation and it has also was carried out at 24 ± 3 C. Sampling was carried out only been discovered that the number of yeast and bacterial cells once per day in order to avoid contamination. The fermented in the broth is more than the number of cells in the cellulosic teas were centrifuged at 7240 ×g for 10 min prior to the assays pellicle [8, 10, 11]. The organic acids produced during the and analyses. The pH values were measured with a hand-held fermentation decrease the pH in the broth, which leads to pH meter (Testo 206 PH1, Keison, London, UK), while the acidity-induced oxygen starvation. Due to this process, the TA was measured according to the method by Chen and Liu number of viable pathogenic microbial cells, if present at [10]. all, decreases as well, resulting in a beverage which is safe for consumption despite being of microbial origin [11]. The 2.2.EnumerationofBacteriaandFungiPopulationinthe acetic acid bacteria present in this consortium have the ability Broth and Pellicle. Enumerations of the overall population of to produce cellulose which forms a zoogleal mat, where bacteriaandyeastinthebrothandpellicleofthefermented this network enhances the association between bacteria and beverages were determined according to the method by Chen yeast [12]. Caffeine and related xanthines found in tea have and Liu [10]. Both figures were expressed as colony-forming the ability to stimulate the synthesis of this cellulose by the units per mL (cfu/mL). bacteria [7, 12]. Many factors such as climate, geographic location, local species of bacteria and yeast, and source of 2.3. Determination of the Total Phenolics Content and Antiox- inoculum have been known to play a significant role in idant Activity. The method by Huang et al. [14] was used deciding the microbial composition of the Kombucha culture for determining the total phenolics content and the Oxygen [12]. RadicalAbsorbanceCapacity(ORAC)assaywascarried One objective of this study was to evaluate the enhance- out according to Prior et al. [15]. Results for the total ment of the antioxidant activity, the changes in polyphenolic phenolics content were expressed as milligrams of gallic acid contents, and the starch hydrolase inhibitory activity in equivalents (GAE) per milliliters (mg GAE/mL), while the sugared black tea fermented with three Kombucha cultures ORAC values were expressed as micromoles of trolox equiv- of differed microbial composition. The obtained results were alents (TE) per milliliters (𝜇mol TE/mL). The di(phenyl)- compared with the unfermented values of tea prior to (2,4,6-trinitrophenyl)iminoazanium (DPPH) and superoxide the fermentation process. Additionally it was necessary to radical scavenging activity assays were carried out according quantify and study the bioaccessible antioxidant capacity to the method described by Lee et al. [5]. All assays were and starch hydrolase inhibitory activity. This is essentially carried out using the Synergy HTX multimode microplate the antioxidant and starch hydrolase inhibitory properties reader and Gen5 software (Biotek, Winooski, VT, USA). which are released from the fermented beverage and subsequently released for absorption during the intestinal and 𝛼 𝛼 duodenal digestion processes [13]. An in vitro digestion 2.4. Determination of the -Amylase and -Glucosidase 𝛼 model was utilized for this purpose, where the fermented Inhibitory Activities. The -amylase inhibitory activity was tea samples were subjected to gastric and duodenal digestion evaluated according to the method by Liu et al. [16] while the 𝛼 phases. Measurement of the total antioxidant capacity (TAC), -glucosidase inhibitory activity was carried out according total polyphenolics content, and starch hydrolase inhibitory tothemethodbyLeeetal.[17].Acarbosewasusedasthe activity before and after the in vitro digestion phases were positive control for both assays and the data were expressed quantified of all three different Kombucha teas prepared from as IC50 (mg/mL). the varied strains. 2.5. In Vitro Digestion Process. The in vitro digestion model 2. Materials and Methods was adapted from Ryan et al. [18]. In brief, the fermented tea samples at day 7 of the process were transferred to clean The bacterial and fungi strains pellicular mats used for amber bottles and mixed with saline (balanced salt solution) the study were verified and authenticated according to the to create a final volume of 20 mL. The samples were acidified to pH 2.0 with 1 mL of a porcine pepsin preparation (0.04 g method by Marsh et al., [8] using DNA amplification and ∘ high-throughput sequencing. The results are reported in pepsin in 1 mL 0.1 M HCl) and incubated at 37 Cinashaking water bath at 3000 ×g for 1 h. After gastric digestion, 500 𝜇Lof Table 1. Black tea dust was obtained from Watawala Plan- ∘ tations, Sri Lanka. All other reagents, chemicals, and HPLC each sample was extracted and stored at −20 C. The pH was standards used for the study were purchased from Sigma then increased to 5.3 with 0.9 M sodium bicarbonate followed Chemicals (St. Louis, MO, USA). by the addition of 200 𝜇Lofbilesaltsglycodeoxycholate (0.04gin1mLsaline),taurodeoxycholate(0.025g in1mL 2.1. Preparation of Kombucha Teas and Determination of saline), taurocholate (0.04 g in 1 mL saline), and 100 𝜇Lof the pH and Titratable Acidity (TA). One gram of black tea pancreatin (0.04 g in 500 𝜇L saline). The pH of each sample (Camellia sinensis) dust was added to 100 mL of boiling water wasincreasedto7.4with1MNaOH.Sampleswereincubated Journal of Chemistry 3 85.3 5.77 5.89 Pichia Dekkera Zygosaccharomyces 1.12 Other 3.04 0.55 2.26 6.59 84.3 0.92 Acetobacter Lactococcus Leuconostoc Lactobacillus Bifidobacterium Gluconacetobacter 5.21 83.2 5.22 Pichia Dekkera Zygosaccharomyces 1.58 1.65 Other 6.37 1.54 82.3 3.59 3.49 Acetobacter Lactococcus Leuconostoc Lactobacillus Bifidobacterium Gluconacetobacter 84.1 5.24 6.28 Pichia Table 1: Relative abundances of the major bacterial and fungi genera in the tea fungus pellicles used for the study. Dekkera Zygosaccharomyces K1 K2 K3 0.81 Other 4.38 2.85 2.92 85.6 0.78 0.69 Bacteria Fungi Bacteria Fungi Bacteria Fungi Leuconostoc Lactococcus Bifidobacterium Gluconacetobacter Other 6.35 Other 5.85 Other 4.26 Lactobacillus Acetobacter 4 Journal of Chemistry

∘ inashakingwaterbathat95rpmat37C for 2 h to complete tea is the key determinant. Gluconacetobacter was generally the intestinal phase of the in vitro digestion process. After observed to be the common and most populated bacterial the intestinal phase, 500 𝜇Lofeachsamplewasextractedand entity in all three types of Kombucha pellicles. In previous ∘ stored at −20 C. studies, it has been identified as the key bacterial strain which enhances the bioaccessibility of polyphenols and antioxidant 2.6. High Performance Liquid Chromatography (HPLC) Deter- activities in Kombucha beverage [11]. According to Table 2, mination of the Phenolic Compounds. A Shimadzu LC2010 the cell concentrations of bacteria and yeast in the broth HPLC system (Kyoto, Japan) equipped with an SPD-M10AVP were higher than the cell concentrations in the cellulose diode array detector (Kyoto, Japan) and a phenomenex pellicle. The Lactobacillus spp. present in all three pellicles Luna C-18(2) column (4.6 mm i.d. × 25 cm, 5 𝜇m) was may possess potential therapeutic properties including acting used for the quantification of (−)-epicatechin (EC), (−)- as a probiotic organism. Such microorganisms are known epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), to provide a balance in intestinal microbiota, normalizing and (−)-epigallocatechin-3-gallate (EGCG), theaflavin (TF), processes in gut and boosting the immune system [21]. The and gallic acid (GA). A gradient profile using two solvents presence of both Gluconacetobacter and Lactobacillus spp. was applied following the method by Jayabalan et al. [19] with mayalsocontributetothefactthattheantioxidantactivity a few modifications for the quantification of EC, ECG, EGC, of the beverages was primarily imparted due to the metabolic EGCG, and GA. The solvents used were as follows: solvent activities of these two microbial genera as shown by previous A: 8% aqueous formic acid; solvent B: acetonitrile/methanol studies [12, 21, 22]. (10 : 90, v/v). A flow rate of 0.9 mL/min was maintained. The gradient was as follows: 0 min—20% B; 7 min—35% B; 14 min—45% B; 21 min—65% B; 25 min—85% B; 32 min— 3.2. pH and TA. Figure 1 shows the pH values of the samples 95% B. The TF content was determined according to the on days 0, 1, and 7 as well as the results when the fermented method by Thanaraj and Seshadri [20] using the same HPLC samples are subjected to pancreatic and duodenal digestion. 𝑃 < 0.05 setup which was used to quantify the epicatechin isomers. A statistically significant decrease ( )wasobservedin The wavelengths of the diode array detector were set at 260, the pH of all three types of Kombucha teas during the period 280, and 320 nm for monitoring of the phenolic compounds. of fermentation starting from day 1 onwards, as compared The concentrations of all the compounds in the extracts with their unfermented counterparts on day 0. Compared were quantified using standard curves and expressed as with the sample values at day 7, a statistically significant 𝑃 < 0.05 micrograms per mL (𝜇g/mL). ( ) change in the pH was observed when the teas were subjected to the gastric and duodenal digestion phases. K1 displays a statistically significantly lower pH (𝑃 < 0.05) 2.7. Statistical Analysis. IBM SPSS Statistics version 21.0 than its nondigested counterpart, whereas K2 and K3 display released in 2012 (IBM Corp., Armonk, NY, USA) for Win- statisticallysignificantlyhigher(𝑃 < 0.05)values.Priortothe dows was used for the statistical analyses. Results were fermentation, the pH of all three teas remained between 6.4 calculated and expressed as mean ± standard error mean and6.5.Byday7ofthefermentationperiod,thepHofthe (SEM) of ≥3 independent analyses. 𝑃 values of <0.05 were teas varied between 4.8 and 4.5. K1 was observed to be the considered to be significant. leastacidicofthethreeteasattheendofthefermentation process with a pH value of 4.8 while K2 and K3 had a value 3. Results and Discussion of 4.5. The decrease in pH during the fermentation process would be due to the production of organic acids by the yeast The initial values of all the analytical parameters prior to the andbacteriapresentintheteafungus.ThedecreaseinpH fermentation process are indicated as day 0 in Tables 1–3 and can be beneficial in terms of maintaining the bioactivity and Figures 1–3. Sample results at day 7 are considered as the preventing the chemical degradation of phenolic compounds. values prior to the digestion process, which are subsequently Previous studies have proven that the organic acids produced subjectedtothepancreaticandduodenaldigestionphases. during the fermentation are gluconic acid, glucuronic acid, L-lactic acid, malic acid, tartaric acid, malonic acid, citric 3.1. Changes in Population of Viable Bacteria and Fungi. acid, and oxalic acid [22, 23]. The TA of the unfermented Table 2 shows the changes of the composition of the overall teas ranged between 0.14 and 0.15, and at the end of the population of bacteria and yeast present in the pellicle prior fermentation process, the TA values ranged between 2.5 and tofermentation,onday1andday7,aswellasinthe 2.7 which are statistically significant increase𝑃 ( < 0.05) brothondays1and7.InallthreeKombuchateatypes,an compared with the unfermented samples. After pancreatic increase in the overall bacteria and yeast population was digestion the TA had statistically significant increase (𝑃< observed.Theincreaseinthebacteriaandyeastpopulation 0.05) in all 3 samples as compared with the day 7 fermented canbecorrelatedtothefactthatthesemicrobeshavethe sample. Following duodenal digestion, the TA values have abilitytoutilizethesubstrateandsuccessfullyandthrive shown a statistically significant decrease (𝑃 < 0.05)compared in the given environment. In addition, bacterial and fungi with the fermented sample at day 7. According to Reiss [24], proportions of the fermented teas matched those of the the optimum consumable acidity level of a beverage is 4.0– corresponding pellicle, thereby suggesting that the fungal 4.5 g/L; thus all three tea types are within the range of the composition of the cellulosic pellicle used to inoculate the acceptable TA level and can be acceptable for consumption. Journal of Chemistry 5

Table 2: Changes to the composition of the overall population of bacteria and fungi present in the broth and the pellicle prior to fermentation (day 0) and on day 1 and day 7.

Days (population of microorganisms) Sample Microorganisms 0 (cfu/mL) 1 (cfu/mL) 7 (cfu/mL) 7 8 Bacteria — 7.1 ± 0.2 × 10 8.4 ± 0.2 × 10 K1 8 9 Fungi — 6.6 ± 0.1 × 10 7. 2 ± 0.2 × 10 6 9 Bacteria — 9.6 ± 0.1 × 10 4.9 ± 0.1 × 10 Broth K2 6 9 Fungi — 7.2 ± 0.1 × 10 5.6 ± 0.2 × 10 6 9 Bacteria — 4.9 ± 0.5 × 10 7. 9 ± 0.2 × 10 K3 6 9 Fungi — 9.9 ± 0.2 × 10 8.2 ± 0.3 × 10 5 6 8 Bacteria 4.7 ± 0.1 × 10 2.9 ± 0.1 × 10 8.5 ± 0.1 × 10 K1 5 5 7 Fungi 4.8 ± 0.2 × 10 6.5 ± 0.1 × 10 7. 6 ± 0.1 × 10 5 5 7 Bacteria 3.8 ± 0.1 × 10 6.9 ± 0.1 × 10 9.5 ± 0.2 × 10 Pellicle K2 5 6 7 Fungi 3.9 ± 0.1 × 10 6.8 ± 0.1 × 10 9.6 ± 0.3 × 10 5 5 7 Bacteria 1.9 ± 0.2 × 10 7. 5 ± 0.5 × 10 9.6 ± 0.1 × 10 K3 5 6 7 Fungi 2.2 ± 0.1 × 10 7. 2 ± 0.2 × 10 8.2 ± 0.1 × 10

Table 3: Changes in the polyphenol contents in the three Kombucha cultures throughout the 7-day period of analysis expressed as mean ± ∗ † SEM. 𝑃 < 0.05 versus the value of each tea at day 0. 𝑃 < 0.05 versus the value of each tea at day 7.

Days/phase of digestion Kombucha Polyphenola 0(𝜇g/mL) 1 (𝜇g/mL) 7 (𝜇g/mL) Pancreatic (𝜇g/mL) Duodenal (𝜇g/mL) ∗ ∗ ∗† EGCG 26.5 ± 1.3 27.5 ± 1.1 36.9 ± 1.5 37.5 ± 1.6 41.5 ± 2.1 ∗ ∗ ∗† EGC 27.8 ± 1.2 27.9 ± 1.1 31.2 ± 1.1 32.5 ± 1.3 34.1 ± 1.1 ∗ ∗ ∗ ECG 10.8 ± 1.1 11.2 ± 1.2 13.6 ± 1.5 13.9 ± 1.6 14.8 ± 1.2 1 ∗ ∗ ∗† EC 9.8 ± 1.4 10.5 ± 1.2 12.9 ± 1.3 13.9 ± 1.1 14.8 ± 1.0 ∗ ∗ ∗† TF 12.6 ± 1.2 13.9 ± 1.1 15.8 ± 1.0 16.7 ± 1.1 17.9 ± 2.5 ∗ ∗ ∗ ∗† GA 31.5 ± 1.9 36.8 ± 1.1 39.7 ± 1.5 40.1 ± 1.9 46.7 ± 1.7 ∗ ∗ ∗† EGCG 26.5 ± 1.3 28.4 ± 1.2 35.9 ± 1.6 36.8 ± 1.4 40.2 ± 1.9 ∗ ∗ ∗† EGC 27.8 ± 1.2 28.7 ± 1.2 33.2 ± 1.2 34.5 ± 1.6 36.2 ± 1.4 ∗ ∗ ∗† ECG 10.8 ± 1.1 12.5 ± 1.1 14.9 ± 1.3 14.9 ± 1.2 15.9 ± 1.1 2 ∗ ∗ ∗† EC 9.8 ± 1.4 14.8 ± 1.4 15.4 ± 1.2 15.9 ± 1.2 16.8 ± 1.0 ∗ ∗ ∗† TF 12.6 ± 1.2 14.8 ± 1.0 16.9 ± 1.3 17.1 ± 1.3 18.5 ± 2.0 ∗ ∗ ∗† GA 31.5 ± 1.9 37.2 ± 1.2 41.6 ± 1.6 42.9 ± 1.7 47.6 ± 1.2 ∗ ∗ ∗† EGCG 26.5 ± 1.3 27.6 ± 1.5 36.1 ± 1.5 37.9 ± 1.6 41.1 ± 1.1 ∗ ∗ ∗† EGC 27.8 ± 1.2 28.1 ± 1.5 34.9 ± 1.2 35.9 ± 1.3 42.9 ± 1.2 ∗ ∗ ∗† ECG 10.8 ± 1.1 13.9 ± 1.0 15.2 ± 1.1 15.4 ± 1.0 17.4 ± 1.1 3 ∗ ∗ ∗† EC 9.8 ± 1.4 15.1 ± 1.2 15.9 ± 1.3 16.1 ± 1.0 16.7 ± 1.1 ∗ ∗ ∗† TF 12.6 ± 1.2 15.1 ± 1.1 17.9 ± 1.2 18.0 ± 1.2 18.9 ± 1.1 ∗ ∗ ∗† GA 31.5 ± 1.9 38.5 ± 1.5 42.1 ± 1.6 43.6 ± 1.2 48.3 ± 1.2 aThe denoted abbreviations are as follows: (−)-epicatechin (EC), (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin-3- gallate (EGCG), theaflavin (TF), and gallic acid (GA).

3.3. Total Phenolics Content. The total phenolics contents 7ofthefermentationaswell.Thereasonfortheincreasein are shown in Figure 2(a). The values of the unfermented total phenolics content during the fermentation process has teas varied between 470 and 476 mg GAE/mL. Following been explained by Blanc [25], where it was demonstrated that the initiation of the fermentation process, the total phenolic the bacteria and yeast present in the Kombucha microbial content shows a statistically significant increase (𝑃 < 0.05) consortium have the ability to liberate enzymes, such as in all 3 fermented samples compared with their unfermented phytase which has the capability of breaking down the counterparts. When the teas were subjected to the in vitro cellulosic backbone of Camellia sinensis to release polyphenol digestion, statistically significant increases (𝑃 < 0.05)were compounds. Partial oxidation of polyphenols can be observed observed in all 3 samples after intestinal digestion. In addi- when the tea is fermented for a longer period of 1-2 months, tion, statistically significant increases𝑃 ( < 0.05)were resulting in a decrease in the total phenolics content and observed in the total phenolics contents following the duode- the antioxidant potential [26]. Thus, extended periods of nal digestion as compared with the undigested sample at day fermentation can lead to the accumulation of unwanted 6 Journal of Chemistry

∗ 7 3 ∗ ∗ ∗ ∗ ∗

* ∗ # ∗ # 6 ∗ ∗# ∗ ∗# # ∗# ∗ 2 ∗

5 ∗ ∗ ∗# ∗ ∗ ∗ (g/mL) 1 4

3 0 K1 K2 K3 K1 K2 K3

Day 0 Pancreatic Day 0 Pancreatic Day 1 Duodenal Day 1 Duodenal Day 7 Day 7 (a) (b)

∗ Figure 1: Changes in the (a) pH and (b) titratable acidity values of the three Kombucha beverages. Error bars represent the SEM. 𝑃 < 0.05 # versus the value at day 0. 𝑃 < 0.05 versus the value at day 7 (prior to being subjected to the digestion phases). metabolites, and, therefore, the beverage may be deemed as be due to the fact that the phenolic compounds present in all therapeutically inadequate for imparting the required health three types of tea samples may possess a better scavenging benefits. activity of peroxide radicals which are generated during the ORAC assay.

3.4. ORAC, DPPH EC50, and Superoxide Scavenging Activities. The ORAC, DPPH EC50, and superoxide scavenging activity 3.5. Starch Hydrolase Inhibitory Activity. The results obtained results are shown in Figures 2(b)–2(d). In observing the for starch hydrolase inhibitory activity are shown in Figure 3. ORAC results, statistically significant increases (𝑃 < 0.05) In all three tea samples statistically significant increases were observed in all 3 samples as the fermentation progresses, (𝑃 < 0.05)inthe𝛼-amylase inhibitory activity and 𝛼- in comparison to the unfermented tea samples. At the glucosidaseactivitywereobservedfollowingthecompletion unfermented stage all three tea samples had ORAC values in of the fermentation process. Prior to fermentation the 𝛼- ranging from 1840 to 1880 𝜇mol TE/mL. As the fermentation amylase inhibitory activity of all three Kombucha samples progressed, the ORAC values had increased. By day 7 of remained in the range of 55-56 𝜇g/mL in terms of the IC50 thefermentationprocess,theORACvalueforallteaswas value. As the fermentation progressed, the inhibitory activity within a range of 2460–2640 𝜇mol TE/mL. In comparing the has increased in a statistically significant𝑃 ( < 0.05)man- values during the digestion processes, statistically significant ner. When subjected to pancreatic digestion, the 𝛼-amylase increases (𝑃 < 0.05) were observed at both phases. It was inhibitory activity remains more so the same compared notedthatK3hadthehighestORACvalueamongall3 with the undigested counterpart. However, after duodenal fermented samples by the end of the fermentation process. digestion the 𝛼-amylase inhibitory activity displayed statis- An increase in the ORAC value represents the maintenance tically significant increases𝑃 ( < 0.05)comparedwiththe of the antioxidant potential throughout the fermentation undigested sample at day 7 of the fermentation process. The period and this could be of therapeutic importance. It is also 𝛼-glucosidase inhibitory activity in the unfermented teas known that the oxidation of the polyphenols can result in ranged between 69 and 70 𝜇g/mL in terms of IC50 value. the formation of stable intermediates which demonstrates As the fermentation progressed, the inhibitory activity has strong antioxidant activity [27]. However, in this sample, the shown statistically significant increases𝑃 ( < 0.05). When increase in the ORAC value was less than K1 and K2 in the the fully fermented samples were subjected to pancreatic duodenal digestion phases as compared with the undigested digestion, the 𝛼-glucosidase inhibitory activity was observed counterparts. Nevertheless, the final ORAC values of all three to be similar to the undigested counterpart. When it was sub- typesofteaswerewithinacomparablerangeattheendofthe jected to duodenal digestion, statistically significant decrease digestion phases. The DPPH EC50 values of the 3 fermented (𝑃 < 0.05)intheIC50 value was observed, as compared Kombucha teas remained within the range of 56–59 mg/kg with the unfermented sample. When considering the results by day 7. As for the DPPH EC50 values and the superoxide obtained for the three Kombucha strains used in this study, scavenging activities, the trend was not as clear as in the it can be noted that the 𝛼-glucosidase inhibitory activity of instances of the ORAC assay. A better correlation between K3 after duodenal digestion was higher than the other two thetotalphenolicscontentandtheORACvaluewasobserved strains. In comparing 𝛼-amylase inhibitory activity and 𝛼- in comparison to the correlation between DPPH EC50 and glucosidase inhibitory activities in all three tea samples it can superoxide scavenging values. This better correlation could be noted that the increase in 𝛼-amylase inhibitory activity Journal of Chemistry 7

# 700 ∗ ∗ 3200 # ∗ ∗ ∗# # ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 600 ∗ ∗ ∗ ∗ ∗ 2400 500

400 TE/mL) mol 𝜇 (mg GAE/mL) (mg ( 1600

300

200 800 K1 K2 K3 K1 K2 K3

Day 0 Pancreatic Day 0 Pancreatic Day 1 Duodenal Day 1 Duodenal Day 7 Day 7 (a) (b) 70 28

55 21 (mg/kg) 50

EC 40 14

25 (%) activity radical scavenging Superoxide 7 K1 K2 K3 K1 K2 K3

Day 0 Pancreatic Day 0 Pancreatic Day 1 Duodenal Day 1 Duodenal Day 7 Day 7 (c) (d)

Figure 2: Changes in the (a) total phenolics content, (b) ORAC, (c) DPPH EC50, and (d) superoxide scavenging activities of the three ∗ # Kombucha beverages. Error bars represent the SEM. 𝑃 < 0.05 versus the value at day 0. 𝑃 < 0.05 versus the value at day 7 (prior to being subjected to the digestion phases).

is higher than the 𝛼-glucosidase inhibitory activity. Thus, 3.6. Changes in the Tea Polyphenol Quantities. The changes it may be concluded that the tea fungus has the ability to in the tea polyphenol contents are shown in Table 3. It can be enhance the 𝛼-amylase inhibitory activity better than the observed that all EGCG, EGC, ECG, EC, TF, and GA showed 𝛼-glucosidase inhibitory activity. 𝛼-amylase is required for a statistically significant increase𝑃 ( < 0.05)byday7com- the subsequent reactions of 𝛼-glucosidase. Therefore, the pared with day 0 or the unfermented counterpart, in all three conclusion above may be considered as an important finding types of Kombucha teas. There was also a statistically signif- in terms of therapeutic effects of the Kombucha beverages. icant increase (𝑃 < 0.05) in the polyphenol contents after The observations also throw light on the therapeutically subjecting the teas to pancreatic and duodenal digestions. beneficial effects of these teas, since the enhancement of Samples subjected to duodenal digestion showed the highest the inhibitors has the ability to retard glucose absorption by value of polyphenol content for all three tea types. Quantities inhibition the enzymes 𝛼-amylase and 𝛼-glucosidase, which of these phenolic compounds were reflective of the total are found in the intestinal brush border [16, 17]. Thus, it has an phenolics contents displayed in Figure 2(a). It was heartening enhanced ability to prevent the breakdown of starch and the to observe an increase in the tea catechins themselves as a subsequent curbing of glucose release into the physiological result of the fermentation process as well as the pancreatic and system of the body. duodenal digestion, given the reported health benefits of this 8 Journal of Chemistry

60 80

55 70 50 ∗ ∗ 60 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 45 ∗ ∗ g/mL) g/mL) ∗ ∗ 𝜇 𝜇 𝜇 50 ( ( 40 # 50 50 ∗ ∗# # IC IC ( g/mL) IC ∗ 40 35

30 30

25 20 K1 K2K3 K1 K2 K3

Day 0 Pancreatic Day 0 Pancreatic Day 1 Duodenal Day 1 Duodenal Day 7 Day 7 (a) (b)

Figure 3: Changes in the (a) 𝛼-amylase and (b) 𝛼-glucosidase inhibitory activities of the three Kombucha beverages. Error bars represent the # SEM. 𝑃 < 0.05 versus the value at day 7 (prior to being subjected to the digestion phases). group of compounds, especially catechin and epicatechin. enhancing the health and wellness; thus this beverage can The structural criteria for compounds to be considered as be considered as a functional food with notable therapeutic potent free-radical scavengers are that these should possess effects. The tea fermented with K3 pellicle was discovered either a 3-hydroxy group on unsaturated C ring, or a 2,3- to be the better Kombucha beverage in terms of having the double bond with the 3-OH group and 4-one in the C ring, highest antioxidant and starch hydrolase inhibitory activity or an ortho-OH substitution pattern in the B ring where following fermentation. Since K3 Kombucha sample contains the OH groups are not glycated [28–30]. Being the major Lactobacillus spp., this beverage can be used as a potentially polyphenolic components, catechin and epicatechin fulfill the good probiotic supplement. Finally, the study can be used first and third structural criteria for effective antioxidants. as a platform to carry out further studies based on different In addition to antioxidant and starch hydrolase inhibitory microbial compositions and their effect on changing the properties, tea polyphenols have displayed antimicrobial and chemical composition of Kombucha beverage. antimycotic activities in vitro [31, 32]. Thus, in addition to enhancing the human microbiome with beneficial microbes, Conflict of Interests the organic compounds produced during the Kombucha fermentation further expand the list of therapeutic effects The authors report no conflict of interests, financial or and benefits gained from the consumption of the fermented otherwise. beverage. Acknowledgment 4. Conclusions The financial and analytical support provided by the National This study was able to demonstrate the ability of the tea Institute of Fundamental Studies, Hantane Road, Kandy, Sri fungus to increase the total phenolics, antioxidant activity, Lanka, is acknowledged. and starch hydrolase inhibitory activities, due to the increase in phenolic compounds. Three types of Kombucha pellicles were studied individually as well as in comparison with each References other. Also, this study was able to demonstrate the statistically [1]C.M.Hasler,A.S.Bloch,C.A.Thomson,E.Enrione,and significant increase𝑃 ( < 0.05) in total phenolics, antioxi- C. Manning, “Position of the American Dietetic Association: dant activity, and starch hydrolase inhibitory activities after functional foods,” Journal of American Diabetic Association,vol. subjecting the fermented beverage to an in vitro digestion 104, pp. 814–826, 2004. 𝛼 model. It was also observed that the -amylase inhibitory [2] N. Yavari, M. M. Assadi, M. B. Moghadam, and K. Larijani, activity in particular was increased after the fermentation “Optimizing glucuronic acid production using tea fungus on process, whereby this increase could be associated with the grape juice by response surface methodology,” Australian Jour- increase in the total phenolics content as well as the increased nal of Basic and Applied Sciences,vol.5,no.11,pp.1788–1794, presence of compounds which have the ability to inhibit 2011. 𝛼-amylase produced during the fermentation process. The [3] T. Srihari and U. Satyanarayana, “Changes in free radical scav- increase in starch hydrolase inhibitory activity contributes in enging activity of Kombucha during fermentation,” Journal of Journal of Chemistry 9

Pharmaceutical Sciences and Research,vol.4,no.11,pp.1978– by mammalian glycosidases,” Journal of Agricultural and Food 1981, 2012. Chemistry,vol.58,no.1,pp.148–154,2010. [4] N. Kabiri, M. Setorki, and M. A. Darabi, “Protective effects [18]L.Ryan,O.O’Connell,L.O’Sullivan,S.A.Aherne,andN.M. of kombucha tea and silimarin against thioacetamide induced O’Brien, “Micellarisation of carotenoids from raw and cooked hepatic injuries in wistar rats,” World Applied Sciences Journal, vegetables,” Plant Foods for Human Nutrition,vol.63,no.3,pp. vol.27,no.4,pp.524–532,2013. 127–133, 2008. [5] Y. H. Lee, C. Choo, M. I. Watawana, N. Jayawardena, and V. Y. [19] R. Jayabalan, S. Marimuthu, P.Thangaraj et al., “Preservation of Waisundara, “Kombucha ‘tea fungus’ enhances the tea polyphe- kombucha tea—effect of temperature on tea components and nol contents, antioxidant activity and alpha-amylase inhibitory free radical scavenging properties,” JournalofAgriculturaland activity of five commonly consumed teas,” Journal of Functional Food Chemistry,vol.56,no.19,pp.9064–9071,2008. Foods,2014. [20] S. N. Thanaraj and R. Seshadri, “Influence of polyphenol oxidase [6] M. I. Watawana, N. Jayawardena, and V. Y. Waisundara, activity and polyphenol content of tea shoot on quality of black “Enhancement of the functional properties of coffee through tea,” Journal of the Science of Food and Agriculture,vol.51,no.1, fermentation by ‘tea fungus’ (Kombucha),” Journal of Food pp. 57–69, 1990. Processing and Preservation,2015. [21] N. O. Kozyrovska, O. M. Reva, V.B. Goginyan, and J.-P.de Vera, [7] M. I. Watawana, N. Jayawardena, C. Choo, and V. Y. Waisun- “Kombucha microbiome as a probiotic: a view from the per- dara, “Application of the Kombucha ‘tea fungus’ for the spective of post-genomics and synthetic ecology,” Biopolymers enhancement of antioxidant and starch hydrolase inhibitory and Cell,vol.28,no.2,pp.103–113,2012. properties of ten herbal teas,” Food Chemistry,vol.194,pp.304– [22]S.L.Markov,R.V.Malbaˇsa,M.J.Hauk,andD.D.Cvetkovic,´ 311, 2016. “Investigation of tea fungus associations. I. The yeasts,” Acta [8]A.J.Marsh,O.O’Sullivan,C.Hill,R.P.Ross,andP.D. Periodica Technologica,vol.32,pp.133–138,2001. Cotter, “Sequence-based analysis of the bacterial and fungal [23] R. V. Malbaˇsa,E.S.Loncar,ˇ J. S. Vitas, and J. M. Canadanoviˇ c-´ compositions of multiple kombucha (tea fungus) samples,” Food Brunet, “Influence of starter cultures on the antioxidant activity Microbiology,vol.38,pp.171–178,2014. of kombucha beverage,” Food Chemistry,vol.127,no.4,pp.1727– [9] W. N. Goh, A. Rosma, B. Kaur, A. Fazilah, A. A. Karim, and 1731, 2011. R. Bhat, “Fermentation of black tea broth (kombucha): effects [24] J. Reiss, “Influence of different sugars on the metabolism of of sucrose concentration and fermentation time on the yield of the tea fungus,” Zeitschriftur f¨ Lebensmittel-Untersuchung und microbial cellulose,” International Food Research Journal,vol.19, -Forschung, vol. 198, no. 3, pp. 258–261, 1994. no. 1, pp. 109–117, 2012. [25] P. J. Blanc, “Characterization of the tea fungus metabolites,” [10]C.ChenandB.Y.Liu,“Changesinmajorcomponentsoftea Biotechnology Letters,vol.18,no.2,pp.139–142,1996. fungus metabolites during prolonged fermentation,” Journal of Applied Microbiology,vol.89,no.5,pp.834–839,2000. [26] R. Jayabalan, P. Subathradevi, S. Marimuthu, M. Sathishkumar, and K. Swaminathan, “Changes in free-radical scavenging [11] C. Dufresne and E. Farnworth, “Tea, Kombucha, and health: a ability of kombucha tea during fermentation,” Food Chemistry, review,” Food Research International,vol.33,no.6,pp.409–421, vol.109,no.1,pp.227–234,2008. 2000. [12] R. Jayabalan, R. V. Malbaˇsa,E.S.Loncar,ˇ J. S. Vitas, and [27] L. R. Fukumoto and G. Mazza, “Assessing antioxidant and M. Sathishkumar, “A review on kombucha tea—microbiology, prooxidant activities of phenolic compounds,” Journal of Agri- composition, fermentation, beneficial effects, toxicity, and tea cultural and Food Chemistry,vol.48,no.8,pp.3597–3604,2000. fungus,” Comprehensive Reviews in Food Science and Food [28] S.-C. Chu and C. H. Chen, “Effects of origins and fermentation Safety,vol.13,no.4,pp.538–550,2014. time on the antioxidant activities of kombucha,” Food Chem- [13] N. Jayawardena, M. I. Watawana, and V. Y. Waisundara, “Evalu- istry,vol.98,no.3,pp.502–507,2006. ation of the total antioxidant capacity, polyphenol contents and [29]D.A.Balentine,S.A.Wiseman,andL.C.M.Bouwens,“The starch hydrolase inhibitory activity of ten edible plants in an in chemistry of tea flavonoids,” Critical Reviews in Food Science vitro model of digestion,” Plant Foods for Human Nutrition,vol. and Nutrition,vol.37,no.8,pp.693–704,1997. 70,no.1,pp.71–76,2015. [30] C. J. Greenwalt, K. H. Steinkraus, and R. A. Ledford, “Kom- [14]D.Huang,B.X.Ou,M.Hampsch-Woodill,J.A.Flanagan, bucha, the fermented tea: Microbiology, composition, and and E. K. Deemer, “Development and validation of oxygen claimed health effects,” JournalofFoodProtection,vol.63,no. radical absorbance capacity assay for lipophilic antioxidants 7,pp.976–981,2000. using randomly methylated 𝛽-cyclodextrin as the solubility [31] C. Pasha and G. Reddy, “Nutritional and medicinal improve- enhancer,” JournalofAgriculturalandFoodChemistry,vol.50, ment of black tea by yeast fermentation,” Food Chemistry,vol. no.7,pp.1815–1821,2002. 89,no.3,pp.449–453,2005. [15]R.L.Prior,H.Hoang,L.Guetal.,“Assaysforhydrophilic [32] H.-L. Alakomi, R. Puupponen-Pimia,¨ A.-M. Aura et al., “Weak- and lipophilic antioxidant capacity (oxygen radical absorbance ening of Salmonella with selected microbial metabolites of capacity (ORACFL)) of plasma and other biological and food berry-derived phenolic compounds and organic acids,” Journal samples,” Journal of Agricultural and Food Chemistry,vol.51,no. of Agricultural and Food Chemistry,vol.55,no.10,pp.3905– 11, pp. 3273–3279, 2003. 3912, 2007. [16] T. Liu, L. Song, H. Wang, and D. Huang, “A high-throughput assay for quantification of starch hydrolase inhibition based on turbidity measurement,” Journal of Agricultural and Food Chemistry, vol. 59, no. 18, pp. 9756–9762, 2011. [17]W.K.Lee,L.L.Wong,Y.Y.Loo,S.Kasapis,andD.J. Huang, “Evaluation of different teas against starch digestibility Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 308602, 7 pages http://dx.doi.org/10.1155/2015/308602

Research Article Antioxidant Peptide Derived from Spirulina maxima Suppresses HIF1𝛼-Induced Invasive Migration of HT1080 Fibrosarcoma Cells

Won Suk Kim,1 Won Kyo Jung,2 and Sun Joo Park3

1 Department of Pharmaceutical Engineering, Silla University, Busan 9491, Republic of Korea 2Department of Biomedical Engineering, Pukyong National University, Busan 8160, Republic of Korea 3Department of Chemistry, Pukyong National University, Busan 8160, Republic of Korea

Correspondence should be addressed to Sun Joo Park; [email protected]

Received 28 July 2015; Revised 7 October 2015; Accepted 8 October 2015

Academic Editor: Eun-Kyung Kim

Copyright © 2015 Won Suk Kim 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.

Hypoxia causes the malignant progression of tumor cells; hence, it has been considered a central issue that must be addressed for effective cancer therapy. The initiation of tumor metastasis requires invasive cell migration. Here, we show that an antioxidant peptide derived from Spirulina maxima suppresses hypoxia-induced invasive migration of HT1080 human fibrosarcoma cells. HT1080 cells treated with a hypoxia-inducing agent, CoCl2, exhibited an increase in invasive migration and intracellular reactive oxygen species (ROS), which is associated with an increase in the expression of hypoxia-induced factor 1𝛼 (HIF1𝛼) accompanied by the activation of PI3K/Akt and ERK1/2. The inhibition of PI3K/Akt and ERK1/2 with specific inhibitors diminished the2 CoCl - induced increase in HIF1𝛼 expression and invasive cell migration. Moreover, CoCl2-induced HIF1𝛼 expression was associated with an increase in the expression of molecules downstream of 𝛽-integrin, such as N-cadherin, vimentin, and 𝛽-catenin. Therefore, the S. maxima peptide effectively attenuated the CoCl2-induced ROS generation and downregulated the HIF1𝛼 signaling pathway involving PI3K/Akt, ERK1/2, and 𝛽-integrin in cells. These results suggest that the S. maxima antioxidant peptide downregulates the HIF1𝛼 signaling pathway necessary for hypoxia-induced invasive migration of HT1080 cells by attenuating intracellular ROS. S. maxima peptide may be an effective constituent in antitumor progression products.

1. Introduction in tumor cell adaptation to hypoxic environments. In particular, HIF1𝛼 regulates the expression of a number of genes The rapid growth and proliferation of tumor cells result in affecting the metastatic progression of tumor cells [4–6]. a dramatic surge in oxygen demand. Tumor hypoxia caused Tumor metastasis is a complex multistep process by which by inadequate oxygen supply is strongly associated with tumor cells disseminate from the primary site, penetrate into tumor propagation, malignant progression, and resistance lymphaticandbloodvessels,andspreadtoothersitesinthe to therapy [1, 2]. Therefore, tumor hypoxia is an important body. HIF1𝛼 expression has been implicated in the increased factor in tumor biochemistry and an important challenge invasive migration of tumor cells during the initiation of in tumor treatment. Accumulating evidence indicates that metastasis [2, 7]. the effect of hypoxia on the malignant progression of tumor Reactive oxygen species (ROS) have been recently iden- cellsismediatedbyaseriesofhypoxia-inducedcellular tified as key mediators involved in tumor propagation and changes that activate anaerobic metabolism, angiogenesis, malignant progression. ROS activate downstream PI3K/Akt, and metastasis and enable tumor cells to survive or escape ERK1/2, and 𝛽-integrin pathways that regulate HIF1𝛼 expres- their oxygen-deficient environment [1, 3]. The transcription sion [8–10]. ROS-induced upregulation of HIF1𝛼 also causes factor hypoxia-inducible factor 1 (HIF1), containing HIF1𝛼 the invasive migration of tumor cells through the regulation and HIF1𝛽 subunits, has been known to be a key regulator of target genes such as N-cadherin, vimentin, and 𝛽-catenin 2 Journal of Chemistry

[2, 7]. Therefore, there has been a marked increase in the fibronectin (25 𝜇g/mL) at room temperature for 1 h and study of the role of ROS and antioxidants in the prevention washed 3 times in DMEM-BSA. DMEM-BSA was added to of tumor progression. the lower compartment of the chamber. Cells were starved Peptides purified from protein hydrolysates have received in DMEM-BSA overnight and treated with 100 𝜇MCoCl2 much attention because of their anticancer, anti-inflamma- or 100 𝜇M peptide as described above, trypsinized, and tion, and antioxidant activities [11, 12]. We recently reported collected. Thereafter, 200 𝜇Lofeachcellsuspension(2× 5 that an antioxidant peptide identified from the enzymatic 10 cells/well in DMEM-BSA) was added to the upper com- ∘ hydrolysates of Spirulina maxima is effective against Fc𝜀RI- partmentofthechamberandincubatedat37Cinahumid- mediated allergic reactions in mast cells [13]. In the present ified atmosphere with 5%2 CO for 24 h. Cells on the upper study, we examined the protective effects of this peptide surface of the membrane were removed, and cells that had against the invasive migration of tumor cells in vitro. migratedtothelowersurfaceofthemembranewerefixed with 3.7% formaldehyde in phosphate buffered saline (PBS), 2. Materials and Methods stained with crystal violet (0.4% dissolved in 10% ethanol) for15min,washedtwicewithPBS,andcountedundera 2.1. Cell Culture and Reagents. HT1080 human fibrosarcoma phase-contrast microscope with a 10x objective lens. Cells cells, obtained from the American Type Culture Collection in 9 randomly selected fields from triplicate chambers were (ATCC), were grown in Dulbecco’s modified Eagle’s medium counted in each experiment. (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 mg/ ∘ 2.5. Measurement of ROS. Dichlorofluorescein diacetate mL streptomycin, in 5% CO2 at 37 C. For the cell-culture (DCF-DA) was used to evaluate the generation of ROS by 4 experiments, cells were passaged at least 3 times and detached oxidative stress. HT1080 cells (4 × 10 cells/well) in 24-well with trypsin-EDTA. Matrigel Invasion Chambers were pur- plates were first incubated with 100 𝜇MCoCl2 or 100 𝜇Mpep- chased from BD Biosciences (USA). Antibodies against tide for 24 h. The cells were then washed with PBS and incu- 𝛼 𝛽 HIF1 , Akt, ERK1/2, N-cadherin, vimentin, -catenin, and bated with 10 𝜇M DCFH-DA for 30 min at room temperature. actin were obtained from BD Biosciences (USA), Santa Cruz Fluorescence was measured by using a fluorescence plate Biotechnology (USA), Cell Signaling Technology (USA), reader. Furthermore, cellular ROS levels were determined and Sigma-Aldrich (USA), respectively. DCFH-DA was pur- by dihydroethidium (DHE) (Sigma-Aldrich) staining and by chased from Molecular Probes. Chemicals and reagents were using the Muse Oxidative Stress Kit (Millipore) according to purchasedformSigma-Aldrich,unlessstatedotherwise. the manufacturer’s instructions.

2.2. Preparation of the Spirulina maxima Peptide. The S. 2.6. Western Blotting. After proper treatment, cells were maxima peptide was prepared as reported by Vo et al. [13]. washed 2 times with PBS, harvested, and solubilized in 2x > The purity of the peptide was 98% according to RP-HPLC sodium dodecyl sulfate (SDS) protein sample buffer contain- assessment and N-terminal sequence analysis. The amino ing 100 mM Tris-HCl (pH 6.8), 200 mM DTT, 4% SDS, 0.4% acid sequence of the final purified peptide was determined bromophenol blue, and 20% glycerol. Equal amounts of pro- to be LDAVNR by electrospray ionization mass spectrometry tein samples were separated by SDS-polyacrylamide gel elec- (ESI/MS). trophoresis (PAGE). The resolved proteins were transferred to polyvinyl difluoride (PVDF) membranes (Millipore Corp.). 2.3. Cell Viability (MTT) Assay. The cytotoxicity of CoCl2 The membranes were blocked by incubation with 1% bovine and/or peptide was determined by the MTT [3-(4,5-dim- serum albumin (BSA) in TBS-T (10 mM Tris-HCl, 150 mM ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] forma- NaCl [pH 7.5], with 0.1% Tween-20) at room temperature for zan assay. HT1080 cells were seeded in 96-well plates at a 1 h and further incubated with the specific primary antibody 3 density of 1 × 10 cells/well in DMEM containing 10% fetal for 1 h. The membranes were washed 3 times with TBS-T bovine serum. Twenty-four hours after seeding, the medium and incubated for 30 min with the appropriate secondary was changed to DMEM containing 0.1% bovine serum antibody conjugated to alkaline phosphatase (AP). Proteins albumin (DMEM-BSA) and the cells were incubated with were detected by colorimetric reactions using the respective 100 𝜇MCoCl2 with or without 100 𝜇M peptide for 24 h. BCIP/NBT substrates. Thereafter, the medium was carefully removed and160 𝜇Lof MTT (0.5 mg/mL final concentration) solution was added to ∘ 3. Results each well prior to incubation for an additional 4 h at 37 Cin 5% CO2. The medium was aspirated without the formazan 3.1. S. maxima Peptide Decreases CoCl2-Induced Invasive crystals and 1 mL of DMSO was added to each well. The Migration of HT1080 Cells. We first examined whether the absorbance was measured on a microplate reader (iMark, peptide purified from S. maxima affects the invasive migra- Bio-Rad) at 540 nm. tion of highly metastatic HT1080 fibrosarcoma cells. HT1080 cells were precultured in the presence or absence of S. 2.4. Invasive Cell Migration Assay. For the invasion assay, maxima peptidefor24h,transferredontoMatrigel-coated thelowersurfaceoftheporousmembranesintheMatrigel transwell membranes, and further incubated for 16 h under Invasion Chambers (BD Biosciences, USA) was coated with preculture conditions. Cells that had migrated to the lower Journal of Chemistry 3

120 120 100 ∗ ∗ 100 ∗ 80 80 † 60 60 †

40 40 Invasive migration (%) migration Invasive 20 20 Invasive migration (%) migration Invasive

0 0 −−−− 100 𝜇 CoCl CoCl2 ( M) −++++ 2 𝜇 0 0 25 50 100 Peptide (𝜇M) 0 25 50 100 Peptide ( M) (a) (b)

Figure 1: S. maxima peptide decreases CoCl2-induced invasive migration of HT1080 cells. HT1080 cells were serum starved in DMEM containing 0.1% bovine serum albumin (DMEM-BSA) for 24 h and precultured in the presence or absence of 100 𝜇MCoCl2 and/or various concentrations of S. maxima peptide for 24 h, transferred onto Matrigel-coated transwell membranes, and further incubated for 16 h under preculture conditions. Migrated cells on the lower surface of the membrane were fixed, stained, and counted. Cells in 9 randomly selected fields were counted in each experiment. Results of at least 3ndent indepe experiments were averaged. All data are presented as mean ± S.D. ∗ † 𝑃 < 0.05 compared with blank; 𝑃 < 0.05 compared with CoCl2 control.

surface of the membrane were fixed, stained, and counted 120 under a phase-contrast microscope. The results demonstrated 100 that treatment with the S. maxima peptide inhibited the invasive migration of HT1080 cells (Figure 1(a)). After treat- 80 ment with the peptide, invasive migration was decreased to 60 97.4%, 84.7%, and 78.3% (𝑃 < 0.05)at25,50,and100𝜇M of peptide, respectively. Interestingly, the inhibitory effect of 40 the S. maxima peptide on the invasive migration of HT1080 (%) Cell viability 20 cells was more dramatic under CoCl2-induced hypoxic con- 𝜇 dition (Figure 1(b)). HT1080 cells incubated with 100 M 0 100 𝜇 CoCl2 demonstrated an approximately 1.7-fold increase in CoCl2 ( M) − ++++ invasive migration compared to that of blank which were Peptide (𝜇M) 002550 100 not incubated with CoCl2. Additional treatment with the Figure 2: Effect of the S. maxima peptide and CoCl2 on the viability S. maxima peptide significantly attenuated CoCl2-induced of HT1080 cells. HT1080 cells were incubated with 100 𝜇MCoCl2 increase in the invasive migration of cells. Compared to and various concentrations of S. maxima peptide for 48 h and control cells without peptide treatment, treatment with 25, assessed using the MTT assay. Results of 3 independent experiments 50, and 100 𝜇M of the peptide reduced the invasive migration were averaged. All data are presented as mean ± S.D. of the cells to 85.2%, 66.2%, and 54.7%, respectively. Since treatment with <100 𝜇MoftheS. maxima peptide did not affect cell viability (Figure 2), these results suggest that the S. peptide effectively attenuated the CoCl2-induced increase in maxima peptide regulates intracellular signaling involved in the ROS levels. Treatment with 100 𝜇MCoCl2 increased the the hypoxic invasive migration of HT1080 cells. intracellular ROS level to approximately 2.57-fold compared to that in blank without CoCl2 stimulation. In contrast, the 𝜇 3.2. S. maxima Peptide Effectively Decreases CoCl2-Induced addition of 100 M S. maxima peptideattenuatedtheCoCl2- ROS Generation in HT1080 Cells. Since many studies have inducedincreaseinROSlevelstosimilarleveltothatin shown that elevated ROS generation under hypoxic condi- control cells without CoCl2 stimulation (Figure 3(a)). Sim- tions is associated with tumor progression and metastasis, ilarly, microscopic fluorescence image and flow cytometry and that the S. maxima peptide decreases ROS production assays showed that the S. maxima peptideiseffectiveinthe in Fc𝜀RI-mediated mast cell activation [13], we attempted to suppression of CoCl2-induced ROS generation (Figures 3(b) examine whether the S. maxima peptide regulates CoCl2- and 3(c)). Figure 3(c) shows that the addition of 100 𝜇M S. induced ROS generation in HT1080 cells. The fluorescent maxima peptide decreased CoCl2-induced ROS generation probeDCFH-DAwasusedtomeasuretheeffectoftheS. (67.42%) in HT1080 cells to approximately 37.14%. Therefore, maxima peptideontheintracellularROSlevelinHT1080 these data suggest that the S. maxima peptide may regulate cells. As shown in Figure 3(a), CoCl2 treatment increased the invasive migration of HT1080 cells by attenuating the the ROS level in HT1080 cells, whereas the S. maxima hypoxia-induced increase in intracellular ROS levels. 4 Journal of Chemistry

100 𝜇 Control CoCl2 ( M)

3.5 ∗ 3

2.5 100 𝜇 + CoCl2 ( M) 2 Peptide (100 𝜇M) peptide (100 𝜇M) † 1.5 † Relative ROS Relative 1

0.5

0 100 𝜇 CoCl2 ( M) −−+ + Peptide (100 𝜇M) −−+ + (a) (b) 100 𝜇 Control CoCl2 ( M) 45 45

34 24.24% 34 67.42%

23 23 Count Count

11 11

00 100 101 102 103 104 100 101 102 103 104

100 𝜇 100 𝜇 + 100 𝜇 Peptide ( M) CoCl2 ( M) peptide ( M) 45 45

34 27.72% 34 37.14%

23 23 Count Count

11 11

00 100 101 102 103 104 100 101 102 103 104 (c)

4 Figure 3: S. maxima peptide attenuates CoCl2-induced ROS level in HT1080 cells. (a) HT1080 cells (4 × 10 cells/well) were incubated in the presence or absence of 100 𝜇MCoCl2 and 100 𝜇M peptide for 24 h. Cellular ROS levels were assessed by DCFH-DA. All data are presented ∗ † as mean ± S.D. 𝑃 < 0.05 compared with blank; 𝑃 < 0.05 compared with CoCl2 control. (b) Representative images of HT1080 cells stained with DCF-DA. Intracellular ROS is observed with fluorescence microscopy (ZEISS, MIC00266). Bar, 10 𝜇m. (c) Cellular ROS levels were determined by dihydroethidium (DHE) staining and flow cytometry assay. Representatives of at least 3 independent experiments are shown in the panel. Journal of Chemistry 5

PD98059 LY294002 100 𝜇 − + − + − + CoCl2 ( M) + − + − + − 120 Peptide (100 𝜇M) −−+ + −−+ + −−+ + ∗ 100 HIF1𝛼 80 p-Akt † † 60 Akt p-ERK1/2 40

ERK1/2 (%) migration Invasive 20

Actin 0 100 𝜇 −++ −+− CoCl2 ( M) PD98059 LY294002 (a) (b) 100 𝜇 −−++ CoCl2 ( M) Peptide (100 𝜇M) −−+ + 𝛽-integrin

N-cadherin

Vimentin

𝛽-catenin Actin

(c)

Figure 4: S. maxima peptide suppresses the HIF1𝛼 signaling pathway necessary for CoCl2-induced invasive migration in HT1080 cells. (a) HT1080 cells were incubated with 100 𝜇MCoCl2 and/or 100 𝜇M peptide for 24 h in the presence or absence of PI3K inhibitor, 10 𝜇MLY294002 or ERK inhibitor, and 10 𝜇M PD98059. Cell lysates were prepared, and the expression level of HIF1𝛼 and phosphorylation levels of Akt and ERK1/2 were examined by western blot analysis. Representatives of 3 independent experiments are shown. (b) Cells were prepared as in (a). ∗ Invasive migration of HT1080 cells was examined. All data are presented as mean ± S.D. 𝑃 < 0.05 compared with blank without CoCl2 † stimulation; 𝑃 < 0.05 compared with CoCl2 control. (c) HT1080 cells were incubated with or without 100 𝜇MCoCl2 and 100 𝜇M peptide for 24 h. Cell lysates were analyzed for HIF1𝛼, 𝛽-integrin, N-cadherin, vimentin, 𝛽-catenin, and 𝛽-actin.

3.3. S. maxima Peptide Downregulates the HIF1𝛼 Signaling these inhibitors (Figure 4(b)), indicating that PI3K/Akt and 𝛼 Pathway Necessary for CoCl2-Induced Invasive Migration of ERK1/2, which are upstream of HIF1 ,areassociatedwith HT1080 Cells. Hypoxia-induced cellular ROS induces the CoCl2-induced invasive migration of HT1080 cells. expression and activation of transcription factor hypoxia- We also examined whether the S. maxima peptide regu- inducible factor 1a (HIF1𝛼), which leads to aggressive cel- lates the PI3K/Akt and ERK1/2 signaling necessary for CoCl2- lularchangesthatareassociatedwithtumorcellinvasion induced invasive migration of HT1080 cells (Figure 4(a)). through the regulation of target genes [2, 7]. Since PI3K/Akt HT1080 cells were incubated with 100 𝜇MCoCl2 for 24 h in and ERK1/2, upstream molecular regulators of HIF1𝛼,have thepresenceorabsenceof100𝜇M S. maxima peptide, and the been reported to be responsible for invasive migration in expression and phosphorylation levels of the related proteins response to oxidative stress during tumorigenesis [14, 15], we were examined. Treatment with the S. maxima peptide atten- first examined whether these proteins are further involved uated the CoCl2-induced increase in the phosphorylation in the CoCl2-induced hypoxic condition of HT1080 cells. levels of Akt and ERK1/2 as well as expression of HIF1𝛼.Fur- HT1080 cells were incubated with 100 𝜇MCoCl2 for 24 h thermore, CoCl2 treatment was found to increase the expres- in the presence or absence of PI3K inhibitor, LY294002 or sion of 𝛽-integrin, another regulator of HIF1𝛼,andthe ERK inhibitor, and PD98059. Thereafter, the expression level S. maxima peptide conversely attenuated CoCl2-induced of HIF1𝛼, phosphorylation levels of Akt and ERK1/2, and increases in the expression of 𝛽-integrin (Figure 4(c)). Since invasive migration of HT1080 cells were examined (Figures many studies have previously indicated that the ROS/HIF1𝛼- 4(a) and 4(b)). CoCl2 treatment increased the phospho- induced activation of 𝛽-integrin is associated with the activa- rylation levels of Akt and ERK1/2 as well as expression tion of N-cadherin, vimentin, and 𝛽-catenin, the underlying level of HIF1𝛼 in HT1080 cells. However, LY294002 or mechanisms were further investigated (Figure 4(c)). The PD98059 treatment reduced the CoCl2-induced increase treatment of HT1080 cells with CoCl2 increased the expres- in the expression of HIF1𝛼 and phosphorylation levels of sion levels of N-cadherin, vimentin, and 𝛽-catenin, whereas Akt and ERK1/2 (Figure 4(a)). The CoCl2-induced invasive the S. maxima peptide treatment conversely attenuated the migration of HT1080 cells was decreased by treatment of CoCl2-induced increase in the expression levels of these 6 Journal of Chemistry proteins. Taken together, we confirmed that the antioxidant S. results suggest that the antioxidant peptide derived from S. maxima peptide downregulates the HIF1𝛼 signaling pathway maxima maybeaneffectiveconstituentinantitumorprogres- necessary for hypoxia-induced invasive migration of HT1080 sionproductsbasedonthemechanismoftumorprogression cells by attenuating intracellular ROS. through ROS and the HIF1𝛼 signaling pathway.

4. Discussion 5. Conclusion

Tumor hypoxia has been regarded as a potential therapeutic The S. maxima peptide attenuates the CoCl2-induced intra- problemsinceitcancauseamoreaggressivemalignantpro- cellular ROS generation and downregulates the HIF1𝛼 sig- gression of tumor cells. Sustained tumor hypoxia increases naling pathway involving PI3K/Akt, ERK1/2, and 𝛽-integrin the potential for invasive growth and metastatic migration in HT1080 cells, which results in a decrease in the CoCl2- and enhances the intrinsic resistance of tumors to cancer induced invasive migration of HT1080 fibrosarcoma cells. treatments [1, 2]. Therefore, an understanding of the effects of hypoxia on tumor physiology is required to counteract tumor progression. Recently, it has been accepted that the ROS- Conflict of Interests induced upregulation of HIF1𝛼 causes the invasive migration of tumor cells through the regulation of genes such as The authors declare no conflict of interests. PI3K/Akt, ERK1/2, and 𝛽-integrin/catenin [8–10]. Therefore, there has been a marked increase in the study of ROS and Acknowledgment antioxidants for the prevention of tumor progression through the HIF1𝛼 signaling pathway. In this study, we showed that This work was supported by a Research Grant of Pukyong an antioxidant peptide derived from S. maxima attenuates National University (year of 2014). CoCl2-induced intracellular ROS generation and downreg- 𝛼 ulates the HIF1 signaling pathway, leading to a decrease in References the invasive migration of HT1080 fibrosarcoma cells. Previous studies have reported that hypoxia-induced [1] D. A. Chan and A. J. Giaccia, “Hypoxia, gene expression, and ROS activate downstream PI3K/Akt and ERK1/2 pathways metastasis,” Cancer and Metastasis Reviews,vol.26,no.2,pp. that regulate HIF1𝛼.TheinhibitionofROSproduction 333–339, 2007. reduces not only the phosphorylation of Akt and ERK1/2 [2] K. Lundgren, B. Nordenskjold,¨ and G. Landberg, “Hypoxia, but also the expression of HIF1𝛼,whichisassociatedwith snail and incomplete epithelial-mesenchymal transition in a decrease in cell migration and invasion [8–10]. The sup- breast cancer,” British Journal of Cancer,vol.101,no.10,pp.1769– pression of PI3K/Akt or ERK1/2 inhibits the migration of 1781, 2009. tumor cells in response to the extracellular matrix and growth [3] M. J. Calzada and L. del Peso, “Hypoxia-inducible factors and factors under hypoxic conditions [16–18]. Therefore, these cancer,” Clinical and Translational Oncology,vol.9,no.5,pp. results suggest a dependency of ROS on the PI3K/Akt and 278–289, 2007. 𝛼 ERK1/2 signaling pathway and HIF1 expression in invasive [4] X. Wang, S. Peralta, and C. T. Moraes, “Mitochondrial alter- 𝛼 tumor cell migration. Our results indicate that these HIF1 ations during carcinogenesis: a review of metabolic transforma- upstream molecules are further involved in CoCl2-induced tion and targets for anticancer treatments,” Advances in Cancer invasive migration of HT1080 cells, and that the antioxidant Research,vol.119,pp.127–160,2013. S. maxima peptide has antitumor effects based on this molec- [5] S. Jing, J. Wang, Q. Liu et al., “Relationship between hypoxia 𝛽 ular mechanism. In addition, the -integrin family, which inducible factor-1𝛼 and esophageal squamous cell carcinoma: a is heterodimeric receptors involved in cell-cell and cell- meta analysis,” Zhonghua Bing Li Xue Za Zhi,vol.43,no.9,pp. extracellular matrix interactions [19], has been reported to 593–599, 2014. promote invasive cell migration [20]. A relationship between [6] C.-M. Tang and J. Yu, “Hypoxia-inducible factor-1 as a thera- the metastatic potential of tumor cells during invasion peutic target in cancer,” Journal of Gastroenterology and Hepa- and quantitative changes in 𝛽-integrin expression has been tology,vol.28,no.3,pp.401–405,2013. reported in several hypoxic cancers [21, 22]. The upregulation 𝛽 [7] S. Cannito, E. Novo, A. Compagnone et al., “Redox mechanisms of -integrin expression is associated with the upregulation switch on hypoxia-dependent epithelial-mesenchymal transi- of epithelial-mesenchymal transition marker proteins such tion in cancer cells,” Carcinogenesis,vol.29,no.12,pp.2267– as N-cadherin and vimentin, and the downregulation of E- 2278, 2008. cadherin which is a cell-cell adhesion molecular marker in [8] C. Shi, X. Zhao, X. Wang, and R. Andersson, “Role of nuclear cancer cells [14, 21]. Therefore, it has been suggested that the factor-𝜅B, reactive oxygen species and cellular signaling in regulation of molecules downstream of 𝛽-integrin such as N/ the early phase of acute pancreatitis,” Scandinavian Journal of E-cadherin, vimentin, and 𝛽-catenin is also important for Gastroenterology,vol.40,no.1,pp.103–108,2005. hypoxia-induced invasive migration of tumor cells. The [9]Y.Liu,Y.Cui,M.Shi,Q.Zhang,Q.Wang,andX.Chen, present results clearly support this hypothesis by demonstrat- “Deferoxamine promotes MDA-MB-231 cell migration and ing the relationship between the expression of HIF1𝛼 and invasion through increased ros-dependent HIF-1𝛼 accumula- N-cadherin, vimentin, and 𝛽-integrin/𝛽-catenin in hypoxia- tion,” Cellular Physiology and Biochemistry,vol.33,no.4,pp. induced invasive tumor cell migration. Taken together, our 1036–1046, 2014. Journal of Chemistry 7

[10] S. H. Lee, Y. J. Lee, and H. J. Han, “Role of hypoxia-induced fibronectin-integrin 𝛽1 expression in embryonic stem cell proliferation and migration: involvement of PI3K/Akt and FAK,” Journal of Cellular Physiology,vol.226,no.2,pp.484–493,2011. [11] D.-H. Ngo, T.-S. Vo, D.-N. Ngo, I. Wijesekara, and S.-K. Kim, “Biological activities and potential health benefits of bioactive peptides derived from marine organisms,” International Journal of Biological Macromolecules,vol.51,no.4,pp.378–383,2012. [12] E. Schlimme and H. Meisel, “Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects,” Die Nahrung,vol.39,no.1,pp.1–20,1995. [13] T.-S. Vo, D.-H. Ngo, K.-H. Kang, S.-J. Park, and S.-K. Kim, “The role of peptides derived from Spirulina maxima in downregulation of Fc𝜀RI-mediated allergic responses,” Molecular Nutrition and Food Research, vol. 58, no. 11, pp. 2226–2234, 2014. [14] W.Yan, Y. Fu, D. Tian et al., “PI3 kinase/Akt signaling mediates epithelial-mesenchymal transition in hypoxic hepatocellular carcinoma cells,” Biochemical and Biophysical Research Commu- nications,vol.382,no.3,pp.631–636,2009. [15] E. J. Gweon and S. J. Kim, “Resveratrol induces MMP-9 and cell migration via the p38 kinase and PI-3K pathways in HT1080 human fibrosarcoma cells,” Oncology Reports,vol.29,no.2,pp. 826–834, 2013. [16]D.J.Webb,D.H.D.Nguyen,andS.L.Gonias,“Extracellular signal-regulated kinase functions in the urokinase receptor- dependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell migration and matrigel invasion,” Journal of Cell Science,vol. 113, no. 1, pp. 123–134, 2000. [17] D. A. Cheresh, J. Leng, and R. L. Klemke, “Regulation of cell contraction and membrane ruffling by distinct signals in migra- tory cells,” TheJournalofCellBiology,vol.146,no.5,pp.1107– 1116, 1999. [18] S. Y. Cho and R. L. Klemke, “Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix,” Journal of Cell Biology,vol.149,no.1,pp.223–236,2000. [19] S. M. Albelda and C. A. Buck, “Integrins and other cell adhesion molecules,” The FASEB Journal,vol.4,no.11,pp.2868–2880, 1990. [20] S. J. Park and Y. J. Jeon, “Dieckol from Ecklonia cava suppresses the migration and invasion of HT1080 cells by inhibiting the focal adhesion kinase pathway downstream of Rac1-ROS signaling,” Molecules and Cells,vol.33,no.2,pp.141–149,2012. [21] C. Sun, Z. Wang, Q. Zheng, and H. Zhang, “Salidroside inhibits migration and invasion of human fibrosarcoma HT1080 cells,” Phytomedicine,vol.19,no.3-4,pp.355–363,2012. [22] S. Prifti, Y. Zourab, A. Koumouridis, M. Bohlmann, T. Strow- itzki, and T. Rabe, “Role of integrins in invasion of endometrial cancer cell lines,” Gynecologic Oncology,vol.84,no.1,pp.12–20, 2002. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 538929, 8 pages http://dx.doi.org/10.1155/2015/538929

Research Article Chemical Composition, Antioxidant Potential, and Antibacterial Activity of Essential Oil Cones of Tunisian Cupressus sempervirens

Aicha Ben Nouri,1 Wissal Dhifi,2 Sana Bellili,1 Hanene Ghazghazi,3 Chedia Aouadhi,4 Ameur Chérif,1 Mohamed Hammami,5,6 and Wissem Mnif1,7

1 LR11-ES31 Biotechnologie et Valorisation des Bio-Geo´ Ressources, Institut Superieur´ de Biotechnologie de Sidi Thabet, Universite´ de la Manouba, BiotechPole de Sidi Thabet, 2020 Sidi Thabet, Tunisia 2UR Ecophysiologie Environnementale et Proced´ es´ Agroalimentaires, Universite´ de la Manouba, BiotechPole de Sidi Thabet, 2020 Sidi Thabet, Tunisia 3Laboratory of Management and Valorization of Forest Resources, National Research Institute of Rural Engineering, Water and Forestry (INRGREF), 1002 Tunis, Tunisia 4Laboratoire d’Epidemiologie´ et Microbiologie Vet´ erinaire,´ Groupes de Bacteriologie´ et Developpement´ Biotechnologique, Institut Pasteur de Tunis, Universite´ El Manar, 1002 Tunis, Tunisia 5USCR de SpectrometriedeMasse,LaboratoiredeBiochimie,Facult´ edeM´ edecine,´ 5019 Monastir, Tunisia 6Institut National de Recherche et d’Analyses Physicochimique (INRAP), Universite´ de la Manouba, BiotechPole de Sidi Thabet, 2020 Sidi Thabet, Tunisia 7Faculty of Sciences and Arts in Balgarn, Bisha University, P.O. Box 60, Balgarn, Sabt Al Alaya 61985, Saudi Arabia

Correspondence should be addressed to Wissem Mnif; w [email protected]

Received 8 May 2015; Revised 4 July 2015; Accepted 7 July 2015

Academic Editor: Patricia Valentao

Copyright © 2015 Aicha Ben Nouri 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 extraction yield of the essential oil (EO) extracted by hydrodistillation from the cones of Tunisian Cupressus sempervirens L. was of 0.518%. The chemical composition was analyzed by GC-MS.esults R showed that this essential oil was mainly composed of monoterpene hydrocarbons (65%) with 𝛼-pinene as the major constituent (47.51%). Its antioxidant activity was ascertained by evaluating the total antioxidant capacity and also by evaluating its inhibitory effect against DPPH and ABTS radicals. In addition, it showed a strong antioxidant power against the DPPH (IC50 =151𝜇g/mL) and ABTS (IC50 = 176.454 𝜇g/mL) radicals scavenging. Moreover, its antibacterial activity was tested against different species of pathogenic bacteria (three Gram-positive and eight Gram- negative bacteria). The bacterial strains susceptible to the evaluated oil were Bacillus subtilis, Escherichia coli, Klebsiella oxytoca, Morganella morganii, Shigella,andVibrio cholerae.

1. Introduction Cupressus sempervirens L. is a medicinal plant. The dried leaves of this plant are used as an emmenagogue and for stom- Cupressus (Cupressaceae),comprisingtwelvespecies,isdis- ach pain [5] as well as for diabetes [6]. Its dried fruit plant is tributed in North America, the Mediterranean region, and subtropical Asia at high altitudes [1]. The geographic area of used for inflammation treatment7 [ ], toothache, and laryngi- Cupressus genus is limited to the northern hemisphere and tis [8] and also as a contraceptive [9], astringent, and antiphra- many species have been studied [2, 3]. In Tunisia, only one stic drug [10]. The dried seed of this tree has been used for species of the genus Cupressus, Cupressus sempervirens L. [4], wounds, ulcers, bruises, sores, pimples, pustules, skin erup- was native. tions, and erysipelas [11]. Cupressus sempervirens essential oil 2 Journal of Chemistry

∘ is used externally for headache, colds, cough, and bronchitis subsequently held isothermal for 4 min; injector port: 250 C; ∘ [12]. detector: 280 C; split ratio: 1 : 50; volume injected: 0.1 mL of Studies on phytochemical compounds of Cupressus sem- 1% solution (diluted in hexane); mass spectrometer: HP5972 pervirens L. revealed that it contains active constituents recording at 70 ev scan time: 1.5 s; mass range: 40–300 amu. such as flavonoids (cupressuflavone, amentoflavone, rutin, Software adopted to handle mass spectra and chromatograms quercitrin, quercetin, and myricitrin), phenolic compounds was ChemStation. The identification of the compounds was (anthocyanidin, catechins flavones, flavonols and isoflavones, based on mass spectra (compared with Wiley 275.L, 6th edi- tannins, and catechol), and essential oils (EO) [13, 14]. It has tion mass spectral library). Further confirmation was done been demonstrated that principals active from Cupressus sem- from Retention Index data generated from a series of alkanes pervirens L. display antiseptic, aromatherapeutic, astringent, retention indices (relative to C9–C28 on the HP-5 and HP- balsamic, and anti-inflammatory activities [15]. Cupressus 20M columns) [18]. sempervirens L. antimicrobial activity has been reported in several studies [14, 16]. 2.4. Antioxidant Activities Even studies have focused on chemistry and biological activities of Cupressus sempervirens L. leaves originating from 2.4.1. Evaluation of Total Antioxidant Capacity. The total anti- different areas in the world [14, 16, 17]; there is no report oxidant capacity is based on the reduction of Mo (VI) to Mo concerning this species (cones) in Tunisia. The aim of this (V) by the oil and the formation of a phosphate subsequent study is to ascertain the chemical composition of the EO of green/Mo (V) complex at acid pH [19]. In a reaction volume Tunisian Cupressus sempervirens L. cones and to evaluate its of 1 mL was added to different concentrations of tested oil and antioxidant and antibacterial activities. standard sulfuric acid (0.6 M), sodium phosphate (28 mM), and ammonium molybdate (4 mM). The solutions were then ∘ 2. Materials and Methods incubated in a water bath at 95 Cfor1hour.Aftercoolingto room temperature, the optical density is measured at 695 nm. 2.1. Plant Material. Cones(theaerialparts)ofCupressus sem- Eachfractionwasanalyzedintriplicate. pervirens L. were collected from Sidi Thabet (North of Tunisia) in March 2014. The botanical identification was achi- 2.4.2. DPPH Radical-Scavenging Activity. The ability of our eved by Pr. Mohammed Chaeib from the Faculty of Sciences essential oil to reduce the DPPH was measured according of Sfax-Tunisia.Female cones were dried at room temperature to the method described by Tuberoso [20]. For each con- for 7 days and used for analyses. Voucher specimens of the centration, one milliliter was added to 0.25 mL of ethanolic plants were deposited in the Herbarium of this laboratory. solution of DPPH. The mixture was stirred vigorously and then incubated at room temperature for 30 min in the dark. 2.2. Extraction of the Essential Oil. Cupressus sempervirens The absorbance illustrating the power of the extract to reduce cones were dried at room temperature. After that, 100 g of dry the free radical DPPH to the yellow-colored diphenylpicryl- matter was used for essential oil extraction by hydrodistilla- hydrazine was measured at 520 nm. So, antiradical activity −1 tion in a Clevenger apparatus for four hours. The resulting is expressed as IC50 (𝜇gmL ), the extract dose required essential oil recovered is dried by anhydrous sodium sulphate to induce a 50% inhibition. The ability to scavenge the ∘ andthenstoredat4C for further analysis. DPPH radical was calculated using the following equation: DPPH scavenging effect = [(𝐴0 −𝐴1)/𝐴0] × 100,where𝐴0 𝐴 2.3. Analysis of the Essential Oil. Cupressus sempervirens L. and 1 were the absorbance of the control and the sample essential oil composition was investigated by GC and GC/MS. after 30 min, respectively. Each experiment was analyzed in The analytical GC was carried out on an HP5890-series II triplicate. gas chromatograph (Agilent Technologies, California, USA) equipped with Flame Ionization Detectors (FID) under the 2.4.3. ABTS Assay. ForABTSassay,weusedthemethod described by Hayouni et al. [21]. The stock solutions included following conditions: the fused silica capillary column, apolar ∙+ HP-5, and polar HP Innowax (30 m × 0.25 mm ID, film thick- 7mMABTS and 2.45 mM potassium persulfate solution. ∘ nessof0.25mm).Theoventemperaturewasheldat50Cfor The working solution was then prepared by mixing the two ∘ ∘ 1min,thenprogrammedatrateof5 C/minto240 C, and held stock solutions in equal quantities and allowing them to react isothermal for 4 min. The carrier gas was nitrogen at a flow for12hatroomtemperatureinthedark.Thesolutionwas ∘ ∙+ rate of 1.2 mL/min; injector temperature: 250 C; detector: then diluted by mixing 1 mL ABTS solution with 50 mL ∘ 280 C;thevolumeinjected:0.1mLof1%solution(dilutedin methanol to obtain an absorbance of 0.7 ± 0.02 units at 734 nm using the spectrophotometer 6305 UV-VIS. Fresh hexane). The percentages of the constituents were calculated ∙+ by electronic integration of FID peak areas without the use ABTS solution was prepared for each assay. 50 𝜇L of each concentration of the essential oil is added with 950 𝜇Lof of response factor correction. GC/MS was performed in a ∙+ Hewlett-Packard 5972 MSD System. An HP-5 MS capillary ABTS solution, allowed to react in the dark and then we fol- column (30 m × 0.25 mm ID, film thickness of 0.25 mm) was low the kinetics of this mixture every 5 min for 30 min. Then directly coupled to the mass spectrometry. The carrier gas was the absorbance was measured at 734 nm. Antiradical activity −1 helium, with a flow rate of 1.2 mL/min. Oven temperature was is expressed as IC50 (𝜇gmL ), the extract dose required to ∘ ∘ ∘ programmed (50 Cfor1min,then50–240 Cat5 C/min) and induce a 50% inhibition. A low IC50 value corresponds to Journal of Chemistry 3

a high antioxidant activity of plant extract. Results are expre- ene -Par -Pinene 70000 -Pinene 3 - 𝛼 𝛽

𝜇 𝛿

ssed in g Trolox equivalents (TE)/mg dry mass. The percen- Limonene -Caryophylene -Cedrol 60000 𝛽 𝛼

tage inhibition of the ABTS cation radical by the samples was -Myrcene -Cymene 𝛽 -Terpinyl acetate -Terpinyl 𝛼 Bornyl acetate Bornyl ortho calculated according to the following formula: Inhibition 50000 Sabinene -Copaene 𝛼 -ol =(𝐴 −𝐴)/(𝐴 × 100) 4 percentage (% inhibition) 0 𝑡 0 ,where 40000

𝐴 𝑡=0 𝐴 Counts

0 is absorbance of control sample ( h) and 𝑡 is Terpinen- -Cubebene -Bisabolene -Cymene 𝛼 Germacrene D

30000 𝛽 -Cubebene para

absorbance of a tested sample in 5 or 30 min. 𝛽 Terpinolene -Humelene

20000 𝛼 -Muurolol Sabinene Terpinyl acetate-4 Terpinyl 𝛼 -Ocimene -Cadinol -Elemene 𝛼 𝛽 Manool -Dimethystyrene -Acorenol 𝛿 -Calamenene Borneol Totarol -Terpineol P 𝛼 , Ferruginol -Muurolene Sabinol 𝛼 -Phellandrene 𝛼 (Z)- Spathulenol Longifolene cis 𝛼 Kaurene 10000 𝛽 Isopimaradiene Cadalene Methyl carvacrol 2.5. Antimicrobial Activities oxide Manoyl 0 5 10 15 20 25 2.5.1. Microorganisms. The tested microorganisms include (min) Gram-positive bacteria, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633,andBacillus cereus ATCC 11778, Figure 1: Essential oil chromatogram from cones of Cupressus sem- and Gram-negative bacteria: Escherichia coli ATCC 8739, pervirens. KlebsiellaoxytocaCECT8207, Salmonella salamae ATCC 43972, Salmonella typhi ATCC 25241, Morganella morganii ATCC 25830, Salmonella anatum ATCC 9270, Vibrio cholerae that obtained for Algerian C. sempervirens (0.26%) [14]and CECT 8265,andShigella ATCC 29930. comparable to that of the Cameroon species (1%) [24]. The major components of Tunisian Cupressus sempervirens L. as reported by Riahi et al. (2012) [23]were𝛼-terpino- 2.5.2. Disk-Diffusion Assay. Antibacterial activity was eval- lene (24.44%), 3-carene (18.60%), 𝛼-limonene (11.61%), ter- uated using the method described by Choi et al. (2006) [22]. pinen-4-ol (10.56%), 𝛽-myrcene (4.89%), camphor (4.62%), The principle of this method is to use Whatman paper discs of and 𝛽-linalool (4.23%). For essential oils of most Cupressus 6 mm in diameter. The discs were impregnated with essential species, 𝛼-pinene and 3-carene were cited as the major com- oil diluted in hexane. A disc soaked in hexane was used as pounds [25]. However in our sample 𝛼-pinene component is negative control. These discs are then deposited on the surface absent. Results by Tapondjou et al. (2005) revealed that the oil ofamiddleswabwithabacterialsuspensiontoanoptical from Cupressus sempervirens L. in Cameroon mainly consists density of 0.5 McFarlend standard. We used the bacterial of 𝛼-pinene (9.9%), terpinen-4-ol (11.2%), and sabinene strains for the culture medium Muller-Hinton. At the end of ∘ (14.8%). the incubation, 24 hours at 37 C, the diameters of the zone of Moreover, 𝛼-pinene and 𝛾-terpinene accounted, respec- inhibition were measured. tively,for39.5and11.56%ofthewholeessentialoilofCupres- sus sempervirens L. cones originating from Greece [26]. The 2.5.3. Minimum Inhibitory Concentration. Abrothmicrodi- cone essential oil of Egyptian Cupressus sempervirens L. lution was used to determine the minimum inhibitory con- showed antibacterial activity [27]. It is in contradiction with centration (MIC). The tests were performed in Muller- the results reported by Cheraif´ et al. (2005) [28]. Indeed, 𝛼- Hinton Broth. The essential oil was dissolved in Tween 80. A pinene is present in the essential oil of Cupressus sempervirens serial doubling of tested oil was prepared in a 96-well micro- L. leaves at a low rate (20%) compared with that of cone 𝜇 titer plate over the range 78 g/mL–1 mg/mL. In each well essential oil. In addition, the proportion of 𝛿-3-carene is 𝜇 containing a concentration was added 100 Lofbacterialsus- important (22.9%) in the essential oil branches of Cupressus pensionof106(0.5MacFarlenstandard).Weincubatedplates ∘ sempervirens L. by comparison with that of cones (7.40%). overnight at 37 C and then measured the optical density at Limonene is of the order of 5.1% in the essential oils branches 620 nm. of Cupressus sempervirens L.Itsrateislowerinconesessential oil(1.75%).Thesameresultshavebeenreportedfor𝛼-terpinyl 3. Results and Discussion acetate whose rates were of 7.5% and 4.11%, respectively, in branches and cones. Furthermore, 𝛽-caryophllene was detec- 3.1. Chemical Composition. The extraction yield of the EO ted as trace in twigs whereas its rate was of 4.53% in cones. from the cones of Tunisian Cupressus sempervirens L. was of Among volatiles, 𝛼-terpinolene was present in the branches 0.518%. The coupling analysis of GC/MS and GC-FID/KI essential oil with an amount of 9.4%. However, it was absent revealed 67 compounds (Figure 1). The major compound was in cones essential oil. 𝛼-pinene (47.51%). It was followed by 𝛿-3-carene, 𝛼-terpinyl Loukis et al. (1991) [26]reportedthat𝛼-pinene and 𝛾- acetate, 𝛽-caryophyllene, and 𝛼-cedrol whose proportions terpinene accounted, respectively, for 39.5 and 11.56% of the were 7.40, 4.11, 4.53, and 4.99%, respectively (Table 1). These whole essential oil of Cupressus sempervirens L. cones origi- results are in accordance with those reported by Boukhris nating from Greece. et al. (2012) [17]forCupressus sempervirens L. collected from Emami et al. (2004) [29] detected 42 compounds in the the random gardens in Sfax, Tunisia, and characterized by essential oil of cones Cupressus sempervirens L. originating 𝛼-pinene (37.14%), 𝛿-3-carene (19.67%), limonene (5.43%), from Egypt. They reported a yield of 0.26% for leaves essential and 𝛼-terpinolene (4.69%) as the most abundant volatiles. oil. Tognolini et al. (2006) [30]reportedthatthe𝛼-pinene is Furthermore,Riahietal.(2012)[23]reportedayieldof themajorcompound.Thisisthesecondmonoterpenehydro- 0.92% for Tunisian Cupressus sempervirens L. It is higher than carbon predominant compound according to the results of 4 Journal of Chemistry

Table 1: Essential oil composition from cones of Cupressus sempervirens.

Number Volatile compound RIa RIb Amount (% of whole EO) Methods of identification 1 Tricyclene 924 1015 0.14 MS, RI 2 𝛼-Pinene 939 1032 47.51 MS, RI, Co-GLC 3 𝛼-Fenchene 953 1044 0.74 MS, RI 4 Camphene 954 1076 0.15 MS, RI, Co-GLC 5 Sabinene 976 1132 0.6 MS, RI, Co-GLC 6 𝛽-Pinene 980 1118 1.53 MS, RI, Co-GLC 7 𝛽-Myrcene 994 1174 1.28 MS, RI, Co-GLC 8 𝛿-3-Carene 1011 1159 7.4 MS, RI, Co-GLC 9 𝛼-Terpinene 1018 1188 0.11 MS, RI, Co-GLC 10 p-Cymene 1026 1280 0.2 MS, RI, Co-GLC 11 m-Cymene 1023 1278 1.12 MS, RI, Co-GLC 12 Limonene 1030 1203 1.75 MS, RI, Co-GLC 13 𝛽-Phellandrene 1006 1176 0.13 MS, RI 14 (Z)-b-Ocimene 1040 1246 0.14 MS, RI 15 (E)-b-Ocimene 1050 1266 0.12 MS, RI 16 g-Terpinene 1062 1266 0.12 MS, RI, Co-GLC 17 𝛼,p-Dimethylstyrene 1080 1452 0.15 MS, RI 18 Fenchone 1087 1406 0.13 MS, RI 19 Terpinolene 1088 1290 0.6 MS, RI, Co-GLC 20 Sabinol 1210 1800 0.24 MS, RI 21 Borneol 1165 1719 0.31 MS, RI 22 Terpinen-4-ol 1018 1188 0.97 MS, RI, Co-GLC 23 𝛼-Terpineol 1189 1706 0.17 MS, RI, Co-GLC 24 Carvacrol, methyl ether 1244 1586 0.07 MS, RI, Co-GLC 25 Bornyl acetate 1295 1597 2.75 MS, RI 26 𝛼-Terpinyl acetate 1350 1677 4.11 MS, RI 27 Terpinyl-4 acetate 1333 1709 0.39 MS, RI 28 𝛿-Elemene 1337 1479 0.24 MS, RI 29 𝛼-Cubebene 1352 1468 0.93 MS, RI 30 𝛼-Ylangene 1372 1493 0.9 MS, RI 31 𝛼-Copaene 1376 1497 1.72 MS, RI, Co-GLC 32 𝛽-Bourbonene 1385 1535 0.15 MS, RI 33 𝛽-Elemene 1391 1600 0.12 MS, RI 34 Longifolene 1413 1575 0.2 MS, RI 35 𝛽-Cubebene 1348 1456 0.69 MS, RI 36 𝛼-Gurjunene 1408 1529 0.37 MS, RI 37 𝛽-Caryophyllene 1415 1612 4.53 MS, RI 38 𝛽-Gurjunene 1432 1612 0.2 MS, RI 39 𝛼-Cedrene 1411 1568 0.14 MS, RI 40 Aromadendrene 1439 1628 0.29 MS, RI 41 𝛼-Humulene 1454 1687 0.64 MS, RI, Co-GLC 42 Alloaromadendrene 1474 1661 0.54 MS, RI 43 Germacrene D 1480 1696 1.14 MS, RI, Co-GLC 44 𝛼-Muurolene 1477 1704 0.15 MS, RI 45 𝛿-Cadinene 1517 1773 0.11 MS, RI 46 cis-Calamenene 1543 1837 0.16 MS, RI 47 Cadina-1,4-diene 1552 1768 0.12 MS, RI 48 𝛽-Bisabolene 1508 1740 1.73 MS, RI 49 Spathulenol 1576 2144 0.21 MS, RI 50 𝛼-Cedrol 1597 2021 4.99 MS, RI, Co-GLC Journal of Chemistry 5

Table 1: Continued. Number Volatile compound RIa RIb Amount (% of whole EO) Methods of identification 51 T-Cadinol 1642 2187 0.25 MS, RI 52 𝛼-Acorenol 1630 2163 0.34 MS, RI 53 𝛽-Acorenol 1637 2212 0.31 MS, RI 54 epi-𝛼-Cadinol 1640 2170 0.11 MS, RI 55 𝛼-Muurolol 1642 2209 0.73 MS, RI 56 𝛽-Eudesmol 1650 2257 0.68 MS, RI 57 𝛼-Eudesmol 1641 2216 0.17 MS, RI 58 𝛼-Cadinol 1649 2255 0.55 MS, RI 59 Cadalene 1674 2200 0.09 MS, RI 60 Sandaracopimara-8(14),15-diene 1960 2255 0.04 MS, RI 61 Manoyl oxide 2010 2396 0.07 MS, RI 62 Isopimara-9-(11),15-diene 1906 2175 0.05 MS, RI 63 Abietadiene 2054 2530 0.05 MS, RI 64 Manool 1989 2376 0.38 MS, RI 65 Kaurene 2048 2399 0.13 MS, RI 66 Totarol 2280 2314 0.31 MS, RI 67 Ferruginol 2295 2330 0.23 MS, RI Total (%)97.69 RI:RetentionIndex;MS:massspectrometry;Co-GLC:coinjection. %: percentage calculated by GC-FID on nonpolar HP-5 capillary column. aApolar HP-5 MS column. bPolar HP Innowax column.

Sacchettietal.(2005)[31]. Furthermore, the most abundant 1.2 volatile compounds in the essential oil of Turkish Cupressus sempervirens L. are 𝛼-pinene and Δ-3-carene. Obviously, the 1 nm composition of essential oils is significantly influenced by the 0.8 organ. It is also influenced by many other factors including 695 environmental factors such as rainfall, sunlight, soil, and 0.6 climatic conditions and agronomic factors such as the date 0.4 of harvest and the density of the culture.

Absorbance at Absorbance 0.2

3.2. Antioxidant Activities. Considering the many aspects of 0 antioxidants and their reactivity, several tests are applied as 0 200 400 600 800 1000 1200 antioxidants. Among them, the total antioxidant capacity, Concentration (𝜇g/mL) DPPH, and ABTS radicals scavenging tests are used to deter- Trolox mine the antioxidant power of essential oil. HE The chemical complexity of essential oils, often a mixture of dozens of compounds with different functional groups, Figure 2: Total antioxidant capacity from cones essential oil of polarity, and antioxidant activity, may lead to scattered Cupressus sempervirens. results, depending on the test employed [31].

values, respectively, in the order of 2.89 mg ET/g DW and 3.2.1. Total Antioxidant Capacity. The total antioxidant activ- 3.39 mg ET/g DW. ity of Cupressus sempervirens L. is expressed as Trolox equiv- alent. The phosphomolybdenum method is based on the reduction of Mo (VI) with Mo (V) by the antioxidant compounds 3.2.2. DPPH Radical-Scavenging Activity. Free radical-scav- and the formation of a green phosphate/Mo complex (V). enging activities of the tested oil and positive control (Trolox) Our results showed that the antioxidant activity is propor- are presented in Figure 3.Infact,Cupressus sempervirens L. tional to the extract concentration (0.0434 𝜇gET/gDW) essential oil remarkably reduced the concentration of DPPH (Figure 2). Baykan Erel et al. (2012) [32] studied the antiox- free radical and transformed its stable, purple color into the idant activity of essential oils of six species of Artemisia. It yellow-colored DPPH-H with an efficiency50 IC =151𝜇g/mL. was noted that only essential oil of Artemisia absinthium and The effect of antioxidant on DPPH radical scavenging was Artemisia arborescens has a total antioxidant activity with thought to be due to its hydrogen-donating ability. DPPH 6 Journal of Chemistry

Table 2: Antibacterial activity from essential oil cones of Cupressus sempervirens.

Concentration (mg/mL) Microorganisms Negative control 1 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 Bacillus subtilis ATCC 6633 −− − ++ + + + + Escherichia coli ATCC 8739 −− − − ++++ + Klebsiella oxytoca CECT 8207 −− − − ++++ + Morganella morganii ATCC 25830 −− ++ + + + + + Shigella ATCC 29930 −− − − ++++ + Vibrio cholerae CECT 8265 −− − − − +++ +

120 45 40 100 35 nm 80 30 534 25 60 20 40 Inhibition (%) Inhibition 15 Absorbance at Absorbance 20 10 5 0 0 0.5 1 1.5 2 2.5 3 0 0 100 200 300 400 500 600 Concentration (𝜇g/mL) Concentration (𝜇g/mL) PI HE (%) 5 min 15 min PI Trolox (%) 10 min 20 min Figure 3: Scavenging of DPPH radical from cones essential oil of Figure 4: Scavenging of ABTS radical from cones essential oil of Cupressus sempervirens. Cupressus sempervirens. radical is a stable free radical and accepts an electron or hyd- that the essential oil of Cupressus sempervirens L. has a strong rogen radical to become a stable diamagnetic molecule [33]. antioxidant against ABTS cation. The antioxidant activity of In two antioxidants screening studies Cupressus semper- essential oil could be assigned to the synergistic effects of two virens L. was reported to display moderate radical-scavenging or more of its components. In this context, Lu and Foo (2001) effect against DPPH [31, 34]. The leaf methanolic extract of [38]reportedthatmostnaturalantioxidantcompoundsoften Egyptian Cupressus sempervirens L. had strong DPPH radi- work synergistically to produce a broad spectrum of anti- cal-scavenging activity as reported by Ibrahim et al. (2009) oxidant properties that create an effective defense system [35]. against free radicals. Cupressus sempervirens L. essential oil Needless to say, potency of antioxidant activity of a sub- consists of a very complex mixture of various chemical classes stance is correlated with the applied method. In a study on (Table 1), which may produce either synergistic or anta- some Iranian conifers the leaf and fruit MeOH extracts of gonistic effects on the process of lipid oxidation [39]. Cupressus sempervirens L. were highly effective in ferric thio- cyanate (FTC) and thiobarbituric acid (TBA) methods [36]. 3.3. Antibacterial Activity. The antibacterial test allowed us to Nevertheless, activity of these extracts was also changeable determine the sensitive strains to the essential oil of Cupressus according to the method. sempervirens L. The screening of this activity was firstly determined by the disk-diffusion method on agar for predicting 3.2.3. ABTS Assay. Essential oil of Cupressus sempervirens the inhibitory activity of the oil on the growth of a bacte- L. had an antioxidant according to the DPPH test. The rial culture. Our results showed that the evaluated oil had results relative to ABTS radical cation scavenging confirmed significant antibacterial effect. In fact, among twelve tested the previous result. In fact, IC50 obtained at the end of strains, six were sensitive to this oil (Table 2). Gram-negative this test is 176.454 𝜇g/mL (Figure 4). Ghazghazi et al. (2010) bacteria (Klebsiella oxytoca, Vibrio cholerae, Shigella,andE. [37] obtained, by studying the essential oil samples of R. coli) were the most sensitive strains with the MIC values canina ofFeijaandAinDraham(NorthofTunisia)dur- that do not exceed 125 𝜇g/mL of essential oil (Table 2). This ing the test of the ABTS, IC50 values of 159.0 𝜇g/mL and finding was in agreement with other findings. For example, 201.8 𝜇g/mL, respectively. From these results we can conclude the cone EO of Egyptian Cupressus sempervirens L. showed Journal of Chemistry 7 antibacterial activity [27]. Cheraif´ et al. (2005) [28]testedthe Acknowledgment antibacterial activity of essential oil Cupressus sempervirens L. on Gram-positive and Gram-negative bacteria and found This study was funded by the Tunisian Ministry of Higher that this essential oil has a moderate activity against tested Education and Scientific Research. bacteria. It is important to mention that the activity was more pronounced for Gram-positive than Gram-negative bacteria. References Similarly, Mazari et al. (2010) [14], when studying the biological activities of essential oils of Cupressus sempervirens L. and [1] I. Rawat, R. Ranjith Kumar, T. Mutanda, and F. Bux, “Dual Juniperus phoenicea,reportedthatbothoilsconstitutesources role of microalgae: phycoremediation of domestic wastewater of antimicrobial agents. There are often large variations in and biomass production for sustainable biofuels production,” the intensity of the antimicrobial activities against Gram- Applied Energy, vol. 88, no. 10, pp. 3411–3424, 2011. negative and Gram-positive bacteria. [2] E.Zavarin,F.W.CobbJr.,J.Bergot,andH.W.Barber,“Variation of the Pinus ponderosa needleoilwithseasonandneedleage,” The EO of Cupressus sempervirens L. presented antibacte- Phytochemistry,vol.10,no.12,pp.3107–3114,1971. rial activity against Gram-positive and Gram-negative bacte- [3] C. Pierre-Leandri, X. Fernandez, L. Lizzani-Cuvelier et al., ria, showing the biggest inhibition with B. subtilis and E. coli. “Chemical composition of cypress essential oils: volatile con- Additionally, the cypress essential oil was found to have mod- stituentsofleafoilsfromsevencultivatedCupressus species,” erate antimicrobial activity when compared to vancomycin JournalofEssentialOilResearch,vol.15,no.4,pp.242–247,2003. (30 mcg) and erythromycin (15 mcg) as antibiotics [40]. [4] A. Cuenod, FloreanalytiqueetsynoptiquedelaTunisie: The antibacterial activity could be affected by the solubil- Cryptogames vasculaires, Gymnospermes et monocotyledones´ , ity of the oil, the diffusion range in the agar and the evapo- Imprimerie S.E.F.A.N, Tunis, Tunisia, 1954. ration [41, 42]. In addition, the antibacterial activities of the [5]V.R.Osorio´ e Castro, “Chromium in a series of Portuguese essential oils suggest their usefulness in the treatment of vari- plants used in the herbal treatment of diabetes,” Biological Trace ous infectious diseases caused by the tested bacteria. Element Research, vol. 62, no. 1-2, pp. 101–106, 1998. [6] M. Assadi, “Cupressaceae,” in Flora of Iran,M.Assadi,M. It is well known that Gram-negative bacteria are more Khatamsaz, A. A. Maassoumi, and V. Mozaffarian, Eds., vol. 21, resistant to essential oil compound antibacterial properties pp. 8–11, Research Institute of Forests and Rangelands, Tehran, than Gram-positive bacteria because of hydrophobic lipo- Iran, 1998. polysaccharide in the outer membrane which provides pro- [7] N. Mascolo, G. Autore, F. Capasso, A. Menghini, and M. P. tection against different agents43 [ ]; however, the obtained Fasulo, “Biological screening of Italian medicinal plants for anti- results indicated that the evaluated oil possesses selective inflammatory activity,” Phytotherapy Research,vol.1,no.1,pp. antibacterial activity and its effect was pronounced against 28–31, 1987. Gram-negative bacteria compared to Gram-positive ones. [8] V. Darias, L. Bravo, R. Rabanal, C. S. Mateo, R. M. G. Luis, and A. M. H. Perez,´ “New contribution to the ethnopharmacological study of the Canary islands,” Journal of Ethnopharmacology,vol. 4. Conclusion 25, no. 1, pp. 77–92, 1989. [9] W. Jochle, “Biology and biochemistry of reproduction and con- traception,” Angewandte Chemie,vol.1,pp.537–549,1962. According to our results, the essential oil of cypress cones is very rich in 𝛼-pinene which is its major component. This [10] M. Ponce-Macotela, I. Navarro-Alegria, M. N. Martinez- Δ 𝛼 𝛽 Gordillo, and R. Alvarez-Chacon, “In vitro antigiardiasic activ- compound is followed by -3-carene, -terpinyl acetate, - ity of plant extracts,” Revista de Investigacion Clinica,vol.46,no. caryophyllene, and cedrol. This composition is different from 5, pp. 343–347, 1994. those found in other studies, and this difference may be due [11] A. Caceres,´ L. M. Giron,S.R.Alvarado,andM.F.Torres,´ to climatic factors. “Screening of antimicrobial activity of plants popularly used in Furthermore, the evaluation of the antioxidant activity by guatemala for the treatment of dermatomucosal diseases,” chemical tests (total antioxidant capacity, DPPH, and ABTS) Journal of Ethnopharmacology,vol.20,no.3,pp.223–237,1987. revealed a variable behavior of the essential oil against the [12] N. J. Montvale, Anonymous. PDR for Herbal Medicines,Thom- used radicals. Moreover, IC50 values calculated are very low son PDR, 2004. such that this reflects the high antioxidant power: they are of [13] K. M. M. Koriem, “Lead toxicity and the protective role of 176.45 and 151 𝜇g/mL for ABTS and DPPH tests, respectively. Cupressus sempervirens,” Revista Latinoamericana de Qu´ımica, The evaluation of the antibacterial activity of the tested essen- vol.37,no.3,pp.230–242,2009. tialoilshowedthatitpossessesasignificantactivity.These [14] K. Mazari, N. Bendimerad, C. Bekhechi, and X. Fernandez, promising results allow us to expand our studies to ascertain “Chemical composition and antimicrobial activity of essential other activities of this essential oil such as antiparasitic, anti- oils isolated from Algerian Juniperus phoenicea L. and Cupressus sempervirens L.,” Journal of Medicinal Plants Research,vol.4,no. fungal, and anticholinesterase activities. 10, pp. 959–964, 2010. [15] J. Bellakhder, La Pharmacopee´ Marocaine Traditionnelle, Edi- tion Ibis Press, Paris, France, 1997. Conflict of Interests [16] J. Zhang, A. A. Rahman, S. Jain et al., “Antimicrobial and antiparasitic abietane diterpenoids from Cupressus sempervirens,” The authors declare that there is no conflict of interests Research and Reports in Medicinal Chemistry,vol.2012,no.2, regarding the publication of this paper. pp. 1–6, 2012. 8 Journal of Chemistry

[17] M. Boukhris, G. Regane, T. Yangui, S. Sayadi, and M. Bouaziz, antioxidants, antiradicals and antimicrobials in foods,” Food “Chemical composition and biological potential of essential Oil Chemistry,vol.91,no.4,pp.621–632,2005. from Tunisian Cupressus sempervirens L.,” Journal of Arid Land [32] S¸. Baykan Erel, G. Reznicek, S. G. S¸enol, N. U.¨ Karabay Studies,vol.22,no.1,pp.329–332,2012. Yavas¸ogulu,˘ S. Konyalioglu,˘ and A. U. Zeybek, “Antimicrobial [18] R. P. Adams, Identification of Essential Oil Components by Gas and antioxidant properties of Artemisia L. species from western Chromatography/Mass Spectrometry, Allured Publishing, Carol anatolia,” Turkish Journal of Biology,vol.36,no.1,pp.75–84, Stream,Ill,USA,1995. 2012. [19] P. Prieto, M. Pineda, and M. Aguilar, “Spectrophotometric [33] J. R. Soares, T. C. P. Dinis, A. P. Cunha, and L. M. Almeida, quantitation of antioxidant capacity through the formation of “Antioxidant activities of some extracts of Thymus zygis,” Free a phosphomolybdenum complex: specific application to the Radical Research, vol. 26, no. 5, pp. 469–478, 1997. determination of vitamin E,” Analytical Biochemistry,vol.269, [34] R. Mothana, R. Gruenert, P. J. Bednarski, and U. Lindequist, no. 2, pp. 337–341, 1999. “Evaluation of the in vitro anticancer, antimicrobial and antiox- [20] C. I. G. Tuberoso, A. Kowalczyk, E. Sarritzu, and P. Cabras, idant activities of some Yemeni plants used in folk medicine,” “Determination of antioxidant compounds and antioxidant Pharmazie,vol.64,no.4,pp.260–268,2009. activity in commercial oil seeds for food use,” Food Chemistry, [35] N. A. Ibrahim, H. R. El-Seedi, and M. M. D. Mohammed, “Con- vol.103,no.4,pp.1494–1501,2007. stituents and biological activity of the chloroform extract and [21] E. A. Hayouni, M. Abedrabba, M. Bouix, and M. Hamdi, essential oil of Cupressus sempervirens,” Chemistry of Natural “The effects of solvents and extraction method on the phenolic Compounds,vol.45,no.3,pp.309–313,2009. contents and biological activities in vitro of Tunisian Quercus [36] S. A. Emami, J. Asili, Z. Mohagheghi, and M. K. Hassanzadeh, coccifera L. and Juniperus phoenicea L. fruit extracts,” Food “Antioxidant activity of leaves and fruits of Iranian conifers,” Chemistry, vol. 105, no. 3, pp. 1126–1134, 2007. Evidence-Based Complementary and Alternative Medicine,vol. [22]Y.M.Choi,D.O.Noh,S.Y.Cho,H.J.Suh,K.M.Kim,andJ.M. 4, no. 3, pp. 313–319, 2007. Kim, “Antioxidant and antimicrobial activities of propolis from [37] H. Ghazghazi, M. G. Miguel, B. Hasnaoui et al., “Phenols, several regions of Korea,” LWT—Food Science and Technology, essential oils and carotenoids of Rosa canina from Tunisia and vol.39,no.7,pp.756–761,2006. their antioxidant activities,” African Journal of Biotechnology, [23] L. Riahi, H. Chograni, S. Ziadi, N. Zoghlami, and A. Mliki, vol. 9, no. 18, pp. 2709–2716, 2010. “Essential oils of inus brutia and Cupressus sempervirens from [38] Y. Lu and L. Y. Foo, “Antioxidant activities of polyphenols from Tunisia: chemical composition and antioxidant activity,” Revue sage (Salvia officinalis),” Food Chemistry,vol.75,no.2,pp.197– de la Societ´ e´ des Sciences Naturelles de Tunisie,vol.38,pp.55–60, 202, 2001. 2012. [39] S. Singh, N. A. Anjum, N. A. Khan, and R. Nazar, “Metal- [24] A. L. Tapondjou, C. Adler, D. A. Fontem, H. Bouda, and C. binding peptides and antioxidant defense system in plants: Reichmuth, “Bioactivities of cymol and essential oils of Cupres- significance in cadmium tolerance,” in Abiotic Stress and Plant sus sempervirens and Eucalyptus saligna against Sitophilus zea- Responses,N.A.KhanandS.Singh,Eds.,pp.159–189,IKInter- mais Motschulsky and Tribolium confusum du Val,” Journal of national, New Delhi, India, 2008. Stored Products Research,vol.41,no.1,pp.91–102,2005. [40] S. Toroglu, “In vitro antimicrobial activity and antagonistic [25] G. Pauly, A. Yani, L. Piovetti, and C. Bernard-Dagan, “Volatile effect of essential oils from plant species,” Journal of Environ- constituents of the leaves of Cupressus dupreziana and Cupressus mental Biology,vol.28,no.3,pp.551–559,2007. sempervirens,” Phytochemistry,vol.22,no.4,pp.957–959,1983. [41] J. Kim, M. R. Marshall, and C.-I. Wei, “Antibacterial activity of [26] A. Loukis, E. Tsitsa-Tzardi, M. Kouladi, and M. Yuemei, “Com- some essential oil components against five foodborne patho- position of the essential oil of Cupressus sempervirens L. cones gens,” JournalofAgriculturalandFoodChemistry,vol.43,no. from Greece,” JournalofEssentialOilResearch,vol.3,pp.363– 11, pp. 2839–2845, 1995. 364, 1991. [42] K. Cimanga, K. Kambu, L. Tona et al., “Correlation between [27] F. F. Kassem, F. M. Harraz, N. A. El-Sebakhy, H. L. De Pooter, chemical composition and antibacterial activity of essential oils andN.M.H.SchampAbou-Schleib,“Compositionofthe of some aromatic medicinal plants growing in the Democratic essential oil of Egyptian Cupressus sempervirens L. cones,” Republic of Congo,” JournalofEthnopharmacology,vol.79,no. Flavour and Fragrance Journal,vol.6,no.3,pp.205–207,1991. 2,pp.213–220,2002. [28] I. Cheraif,´ H. Ben Jannet, M. Hammami, and Z. Mighri, “Con- [43] M. Vaara, “Agents that increase the permeability of the outer tribution al’` etude´ de la composition chimique de l’huile essen- membrane,” Microbiological Reviews,vol.56,no.3,pp.395–411, tielle des rameaux de Cupressus sempervirens L. poussant en 1992. Tunisie,” Journal de la Societ´ eChimiquedeTunisie´ ,vol.7,pp. 75–82, 2005. [29] S. A. Emami, M. H. Khayyat, M. Rahimizadeh, B. S. Fazly- Bazzaz, and J. Assili, “Chemical constituents of Cupressus sempervirens L. cv. cereiformis rehd. essential oils,” Iranian JournalofPharmaceuticalSciences,vol.1,no.1,pp.39–42,2004. [30] M. Tognolini, E. Barocelli, V. Ballabeni et al., “Comparative screening of plant essential oils: phenylpropanoid moiety as basiccoreforantiplateletactivity,”Life Sciences,vol.78,no.13, pp. 1419–1432, 2006. [31] G. Sacchetti, S. Maietti, M. Muzzoli et al., “Comparative evaluation of 11 essential oils of different origin as functional Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 171570, 4 pages http://dx.doi.org/10.1155/2015/171570

Research Article Inhibitory Effects of 4-(4-Methylbenzamino)benzoate on Adipocyte Differentiation

Jin Taek Hwang,1,2 Sanghee Kim,3 Bo-ra Yoon,1 Inwook Choi,1 and Sang Yoon Choi1,2

1 Korea Food Research Institute, Seongnam 463-746, Republic of Korea 2Korea University of Science and Technology, Daejeon 305-350, Republic of Korea 3College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

Correspondence should be addressed to Sang Yoon Choi; [email protected]

Received 12 June 2015; Revised 23 July 2015; Accepted 29 July 2015

Academic Editor: Bartolo Gabriele

Copyright © 2015 Jin Taek Hwang 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 potent suppression of adipocyte differentiation by 4-(4-methylbenzamino)benzoate was discovered during the search fornew antiobesity compounds. 4-(4-methylbenzamino)benzoate was observed to suppress adipocyte differentiation in 3T3-L1 cells by 96.8% at 50 𝜇M without cytotoxicity. In addition, 4-(4-methylbenzamino)benzoate reduced the cellular expression of fatty acid synthase in a concentration-dependent manner, as well as suppressing PPAR-gamma activity, which controls fatty acid storage and glucose metabolism. Based on these results, 4-(4-methylbenzamino)benzoate shows potential as an antiobesity material.

1. Introduction resveratrol or genistein. Therefore, MBAB show great potential as a new antiobesity substance. Obesity, a major factor in the development of heart disease, cancer, hypertension, diabetes, and degenerative arthritis, 2. Materials and Methods is induced by adipocyte differentiation due to hormonal changes and imbalances in energy metabolism caused by 2.1. Materials. MBAB was synthesized using a previously excessive fat intake [1–5]. Adipogenesis in the body is the reported method [13]. Resveratrol and genistein, which were process of cell differentiation by which preadipocytes become used as positive controls, were purchased from Sigma- adipocytes during fat accumulation. Mature adipocytes are Aldrich (St. Louis, MO, USA). The 3T3-L1 cells were pur- differentiated from immature adipocytes such as fibroblasts chased from American Type Culture Collection (Manassas, and form lipid droplets inside cells [6, 7]. VA, USA) and the FAS antibodies from Cell Signaling Studies have shown that natural compounds, resveratrol Technology (Danvers, MA, USA). and genistein, have antiobesity effects [8–11]. Resveratrol and genisteinarecontainedingrapesandbeans,respectively. 2.2. Cell Culture and Differentiation. 3T3-L1 cells were cul- ∘ Although some antiobesity drugs are currently available, such tured in DMEM culture medium with 10% FBS, at 37 C, as orlistat, sibutramine, or sertraline, several side effects have and 5% CO2 conditions. For the differentiation, 3T3-L1 been reported [12]. Thus, studies are being conducted on cells were grown in a 48-well plate until confluence, and compounds with antiobesity effects to replace these drugs. differentiation was induced by incubation with a hormonal In this study, the potent adipogenesis-suppressing activ- cocktail containing 10 𝜇g/mL insulin, 0.5 𝜇M dexamethasone, ity of 4-(4-methylbenzamino)benzoate (MBAB, Figure 1) and 0.5 𝜇MIBMXfor24h.Thecellswerethenincubated wasobservedin3T3-L1cellswithoutcytotoxicity,during with a normal medium containing 10 𝜇g/mL insulin in the the search for new antiobesity substances. MBAB exhib- presence or absence of MBAB, resveratrol, and genistein for ited higher adipogenesis-suppressing activity compared to 8days. 2 Journal of Chemistry

OH O OH OH O O OCH3 OH N H HO HO O

(a) (b) (c)

Figure 1: Chemical structure of MBAB (a), resveratrol (b), and genistein (c).

2.3. Cell Viability. After differentiation was completed, 120 0.5 mg/mL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphen- ∘ 100 yltetrazolium bromide] was added and incubated at 37 C for 4 hours. After eliminating the MTT solution, 200 𝜇Lof 80 DMSO was added and absorbance at 540 nm was measured 60 to determine cell viability. 40 Cell viability (%) Cell viability 2.4. Oil Red O Staining and TG Assay. When differentiation 20 was complete, the cells were washed with PBS twice and 0 fixed with 3.7% formaldehyde. After incubating the cells for MBAB Resveratrol Genistein 1 hour using Oil Red O dye, isopropanol was added, and the 12.5 𝜇M absorbance was measured at 510 nm to determine the amount 25 𝜇M of triglycerides. 50 𝜇M

2.5. Fatty Acid Synthase (FAS) Expression. The 3T3-L1 cells Figure 2: Effect on cell viability in 3T3-L1 cells. The cell viability of ± were washed twice with ice-cold PBS and then lysed with a the vehicle was set at 100%. The data represent the mean SD of the lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 0.5% sodium triplicate experiments. deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 0.2% protease 0.16 inhibitor cocktail, pH 7.2). The collected protein was cen- trifugedat14,000rpmfor5minutes,andthesupernatantwas 0.14 𝜇 collected for protein quantification. Then, 30 gofprotein 0.12 ∗∗ ∗∗ ∗∗

was loaded in 10% SDS-PAGE for electrophoresis, after which nm) it was transferred to a nitrocellulose membrane and reacted 0.1 510 ∗∗ with FAS antibodies (1 : 1000), anti-mouse antibody (1 : 1000), 0.08 and detected by ECL. OD ( 0.06 ∗∗∗ 𝛾 𝛾 2.6. Peroxisome Proliferator-Activated Receptor (PPAR- ) 0.04 𝛾 M M M M M M M M Transcription Activity. The effect on PPAR- activity in 3T3- M 𝜇 𝜇 𝜇 𝜇 𝜇 𝜇 𝜇 𝜇 𝛾 𝜇 L1 cells was measured using a PPAR- transcription factor None assay kit (Cayman Chemical). The 3T3-L1 cells were treated Induction Res 25 Res 50 Gen 50 Gen 25 Res 12.5

with rosiglitazone and each concentration of MBAB, and then Gen 12.5 MBAB 50 MBAB MBAB 25 MBAB the cell extract was added to a dsDNA sequence-coated plate. 12.5 MBAB 𝛾 The PPAR- antibodies and the secondary antibodies were Figure 3: Inhibitory effects on 3T3-L1 adipocyte differentiation. reacted in order, and then a detection reagent was added and Induction: hormone mixture, MBAB: hormone mixture + MBAB, absorbance was measured at 450 nm. Res: hormone mixture + resveratrol, and Gen: hormone mixture + genistein. Three independent experiments were conducted. The 2.7. Statistical Analysis. All data were presented as mean ± results are expressed as the mean ± SD of absorbance at 510 nm. ∗∗ < 0.01 ∗∗∗ < 0.001 standard deviation (SD). The significance of the differences 𝑃 and 𝑃 represent significant differences when between groups was tested using one-way analysis of variance comparing the hormone treatment group (induction group). (ANOVA).

3. Results and Discussion of less than 50 𝜇M, whereas resveratrol and genistein reduced the cell viability in a concentration-dependent manner 3.1. Effects on Cell Viability. The MTT assay showed that (Figure 2). Therefore, our results indicate that MBAB has MBAB did not cause significant cell death at a concentration lower cytotoxicity compared to resveratrol and genistein. Journal of Chemistry 3

(a) (b) (c)

(d) (e) (f)

Figure 4: Photographs of 3T3-L1 adipocytes after Oil Red O staining. (a) Normal (control); (b) induction (hormone mixture); (c) induction (hormone mixture) + 12.5 𝜇M of MBAB; (d) induction (hormone mixture) + 25 𝜇M of MBAB; (e) induction (hormone mixture) + 50 𝜇Mof MBAB; (f) induction (hormone mixture) + 50 𝜇Mofresveratrol.Scalebar=50𝜇m.

3.2. Inhibitory Effects on Adipocyte Differentiation. After 2.0 confirming the absence of cytotoxicity in 3T3-L1 cells treated 𝜇 with MBAB at concentrations of less than 50 M, the effect 1.5 ∗∗ on adipocyte differentiation was measured. As shown in Figure 3, MBAB suppressed the adipocyte differentiation of 3T3-L1 cells in a concentration-dependent manner after 1.0 they were induced to differentiate after being treated with actin (ratio) 𝛽 a hormone mixture. MBAB strongly suppressed adipocyte differentiation at a concentration of 50 𝜇M, which was similar FAS/ 0.5 to the control group. In particular, MBAB exhibited a higher adipocyte differentiation suppressing activity at all concen- 0.0 trations compared to the same concentrations of resveratrol N IN 12.5 25 50 or genistein. Figure 4 shows photographs of 3T3-L1 cells MBAB (𝜇M) treated with MBAB and stained with Oil Red O. Figure 5: Effect on intracellular FAS expression. N: normal; IN: hormone mixture; MBAB: hormone mixture + MBAB. 3.3. Inhibitory Effects on FAS Expression. Fatty acid is synthe- sizedbytheactionoffattyacidsynthase(FAS),anenzyme with a molecular weight of 250 kDa, using malonyl-CoA as substrate [14–17]. FAS is known as important factor in manner. Therefore, it is considered that MBAB impairs lipid the regulation of fat biosynthesis and obesity. Thus, FAS production by suppressing fatty acid biosynthesis through inhibitors can effectively reduce fat production and suppress FAS reduction. obesity. The effect of MBAB on the intracellular expression of 3.4. Inhibitory Effects on PPAR-𝛾 Activity. PPAR-𝛾 regulates FAS was measured as shown in Figure 5. MBAB inhibited fatty acid storage and glucose metabolism [18]. Since the theincreaseinFASproductionin3T3-L1cellsthatwere PPAR-𝛾 signaling pathway is known to be a major target for induced by a hormone mixture in a concentration-dependent the development of antiobesity drugs, the effect of MBAB 4 Journal of Chemistry

70 [8]M.Zhang,K.Ikeda,J.-W.Xu,Y.Yamori,X.-M.Gao,andB.-L. ∗∗ 60 ∗∗ Zhang, “Genistein suppresses adipogenesis of 3T3-L1 cells via multiple signal pathways,” Phytotherapy Research,vol.23,no.5, 50 ∗ pp.713–718,2009. 40 ∗ [9] K. Szkudelska and T. Szkudelski, “Resveratrol, obesity and diabetes,” European Journal of Pharmacology,vol.635,no.1–3, 30 pp. 1–8, 2010.

Inhibition (%) Inhibition 20 [10] C. A. Baile, J.-Y. Yang, S. Rayalam et al., “Effect of resveratrol on fat mobilization,” Annals of the New York Academy of Sciences, 10 vol. 1215, no. 1, pp. 40–47, 2011. 0 [11] N. Behloul and G. Wu, “Genistein: a promising therapeutic M M M M agent for obesity and diabetes treatment,” European Journal of 50 𝜇 25 𝜇 50 𝜇 Pharmacology,vol.698,no.1–3,pp.31–38,2013. 12.5 𝜇 [12] M. Li and B. M. Cheung, “Rise and fall of anti-obesity drugs,” Res World Journal of Diabetes,vol.2,no.2,pp.19–23,2011. MBAB MBAB MBAB [13]S.Hwang,S.Y.Choi,J.H.Leeetal.,“Identificationofapotent Figure 6: Inhibitory effect of MBAB on rosiglitazone induced and noncytotoxic inhibitor of melanin production,” Bioorganic PPAR-𝛾 transcriptional activity. Res: resveratrol. The data are the &MedicinalChemistry,vol.18,no.15,pp.5602–5609,2010. ∗ ∗∗ mean ± SD values of the three experiments. 𝑃 < 0.05, 𝑃 < 0.01 [14] S. Kreuz, C. Schoelch, L. Thomas, W. Rist, J. F. Rippmann, compared with the induction group (control). and H. Neubauer, “Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetes,” Diabetes/Metabolism Research and Reviews,vol.25,no.6,pp.577–586,2009. on PPAR-𝛾 transcriptional activity was measured. The results [15] S. J. Wakil and L. A. Abu-Elheiga, “Fatty acid metabolism: target show that MBAB suppressed rosiglitazone induced PPAR-𝛾 for metabolic syndrome,” JournalofLipidResearch,vol.50,pp. transcriptional activity in a concentration-dependent man- 138–143, 2009. ner, and the degree of inhibition at 50 𝜇Mwas55%compared [16] G. V. Ronnett, E.-K. Kim, L. E. Landree, and Y. Tu, “Fatty acid to control (Figure 6). metabolismasatargetforobesitytreatment,”Physiology & Behavior,vol.85,no.1,pp.25–35,2005. Conflict of Interests [17] M. J. Wolfgang and M. D. Lane, “The role of hypothalamic malonyl-CoA in energy homeostasis,” The Journal of Biological The authors declare that there is no conflict of interests Chemistry, vol. 281, no. 49, pp. 37265–37269, 2006. regarding the publication of this paper. [18] S. I. Anghel, E. Bedu, C. D. Vivier, P. Descombes, B. Desvergne, and W.Wahli, “Adipose tissue integrity as a prerequisite for systemic energy balance: a critical role for peroxisome proliferator- References activated receptor 𝛾,” The Journal of Biological Chemistry,vol. 282,no.41,pp.29946–29957,2007. [1] K. F. Adams, A. Schatzkin, T. B. Harris et al., “Overweight, obesity, and mortality in a large prospective cohort of persons 50 to 71 years old,” The New England Journal of Medicine,vol. 355, no. 8, pp. 763–778, 2006. [2]T.B.HorwichandG.C.Fonarow,“Glucose,obesity,metabolic syndrome, and diabetes relevance to incidence of heart failure,” JournaloftheAmericanCollegeofCardiology,vol.55,no.4,pp. 283–293, 2010. [3] P. Strazzullo, L. D’Elia, G. Cairella, F. Garbagnati, F. P. Cap- puccio, and L. Scalfi, “Excess body weight and incidence of stroke: meta-analysis of prospective studies with 2 million participants,” Stroke,vol.41,no.5,pp.e418–e426,2010. [4]B.C.T.Field,O.B.Chaudhri,andS.R.Bloom,“Obesity treatment: novel peripheral targets,” British Journal of Clinical Pharmacology,vol.68,no.6,pp.830–843,2009. [5] J. Roth, A. L. Szulc, and A. Danoff, “Energy, evolution, and human diseases: an overview,” The American Journal of Clinical Nutrition, vol. 93, no. 4, pp. 875–883, 2011. [6]E.D.RosenandB.M.Spiegelman,“Molecularregulation of adipogenesis,” Annual Review of Cell and Developmental Biology,vol.16,pp.145–171,2000. [7]W.Kiess,S.Petzold,M.Topfer¨ et al., “Adipocytes and adipose tissue,” Best Practice & Research: Clinical Endocrinology & Metabolism,vol.22,no.1,pp.135–153,2008. Hindawi Publishing Corporation Journal of Chemistry Volume 2015, Article ID 975292, 7 pages http://dx.doi.org/10.1155/2015/975292

Research Article First Evaluation of the Biologically Active Substances and Antioxidant Potential of Regrowth Velvet Antler by means of Multiple Biochemical Assays

Yujiao Tang,1,2,3 Byong-Tae Jeon,1,2 Yanmei Wang,3 Eun-Ju Choi,4 Pyo-Jam Park,2,5 Hye-Jin Seong,1,2 Sang Ho Moon,1,2 and Eun-Kyung Kim1,2

1 Division of Food Bio Science, College of Biomedical and Health Sciences, Konkuk University, Chungju 380-701, Republic of Korea 2Korea Nokyong Research Center, Konkuk University, Chungju 380-701, Republic of Korea 3Jilin Sino-ROK Institute of Animal Science, Changchun 130-600, China 4Division of Sport Science, College of Biomedical and Health Sciences, Konkuk University, Chungju 380-701, Republic of Korea 5Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea

Correspondence should be addressed to Sang Ho Moon; [email protected] and Eun-Kyung Kim; [email protected]

Received 31 May 2015; Accepted 2 July 2015

Academic Editor: Patricia Valentao

Copyright © 2015 Yujiao Tang 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.

We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition of biologically active substances and antioxidant potential of different antler segments. RVA was subjected to extraction using DW (distilled water). RVA was divided into 3 segments: T-RVA (top RVA), M-RVA (middle RVA), and B-RVA (base RVA). The T-RVA section possessed the greatest amounts of uronic acid (36.251 mg/g), sulfated GAGs (sulfated glycosaminoglycans) (555.76 mg/g), sialic acid (111.276 mg/g), uridine (0.957 mg/g), uracil (1.084 mg/g), and hypoxanthine (1.2631 mg/g). In addition, the T-RVAsection 󸀠 possessed the strongest antioxidant capacity as determined by DPPH, H2O2 (hydrogen peroxide), hydroxyl, and ABTS (2,2 - azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity as well as FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorbance capacity). The values of those were 53.44, 23.09, 34.12, 60.31, and 35.81 TE/𝜇Mat1mg/mL and 113.57 TE/𝜇Mat20𝜇g/mL. These results indicate that the T-RVA section possesses the greatest amount of biologically active substances and highest antioxidant potential. This is the first report on the biologically active substances and antioxidant potential of RVA.

1. Introduction studies have been conducted on the chemical composition of RVA [3, 4], there have been no comprehensive reports on the Velvet antler consists of the cartilaginous, prequalified antlers composition of biologically active substances and antioxidant of moose, elk, and sika deer, which regrow yearly. The growth potential of RVA. of deer antlers is one of the fastest types of tissue growth in Numerous studies have demonstrated that free radicals mammals. Growing antlers contain nerves and blood vessels are generated by oxidative damage to biomolecules such and are covered with a hairy, skin covering tissue commonly as lipids, nucleic acids, proteins, and carbohydrates [5– known as “velvet” [1]. Velvet antler is a widely used traditional 7]. Overproduction of free radicals and reactive oxygen Asian medicine that has been used clinically in East Asia for species is believed to be associated with cellular and tissue millennia to treat various diseases and as a tonic [2]. Velvet pathogenesis, which leads to several chronic diseases such antler is generally harvested twice per year. The first velvet as cancer, diabetes mellitus, and neurodegenerative and antler harvest occurs after 40–45 days of growth, while a inflammatory diseases8 [ ]. Many medical reports and clinical second harvest occurs after 50–55 days of regrowth, at which observations convincingly show that disease-resistance can point the harvested velvet antler is known as RVA. Although be conferred by enhancing antioxidative processes [9–14]. 2 Journal of Chemistry

Therefore, antioxidant supplementation could prevent or solution (10 mM, 20 𝜇L) was added to 200 𝜇Lofaglycoconju- inhibit oxidative stress induced by ROS. Antioxidants ter- gate sample in a 15 mL polypropylene test tube. The solution minate free radical chain reactions by removing free radical was chilled in an ice bath for 45 min. The reaction was termi- intermediates while inhibiting other oxidation reactions. nated by the addition of 100 𝜇Lof50mMsodiumthiosulfate Because of the clinical potential of antioxidants, significant solution. Next, 500 𝜇L of 4.0 M ammonium acetate (pH 7.5) interest has been focusing on the development of natural and 400 𝜇L of ethanolic solution of 100 mM acetoacetanilide antioxidants that are safe and effective. were added to the solution, which was left standing for In this study, RVA was subjected to extraction by DW 10 min at room temperature. The fluorescence intensity of to allow determination of its constituent biologically active the solution was measured at 471 nm with an excitation substances, including uronic acid, sulfated GAGs, sialic acid, wavelength of 388 nm. uracil, hypoxanthine, and uridine. In addition, the antioxidant activities of RVA were determined by assessing DPPH, 2.3.4. Uracil, Hypoxanthine, and Uridine. Uracil, hypoxan- H2O2, hydroxyl, and ABTS radical scavenging activity as well thine, and uridine were determined as described previously as FRAP and ORAC. [18]. 1 mg of the DW extract was dissolved in 1 mL of 3% methanol solution, after which 1 mL of the resulting 2. Materials and Methods solution was filtered for HPLC analysis. The analysis was performedonanHPLCsystemequippedwithanisocratic 2.1. Materials. Seven specimens of sika deer (Cervus nip- pump (Kyoto, Japan) and RI (refractive index) detector (Lab pon) RVA were collected at the same farm (Fanrong farm, Alliance,model500).Theseparationwasconductedona China). Carbazole, sodium tetraborate, dimethylmethylene ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 5 𝜇m, blue, glycine, sodium thiosulfate, acetoacetanilide, uracil, Agilent Technologies, USA). The mobile phase was 0.07% hypoxanthine, uridine, DPPH, ABTS, potassium persulfate, acetic acid methanol water (3 : 97, v/v; pH 3.5) at a flow rate 󸀠 TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), FL, and AAPH (2,2 - of 1.0 mL/min. A series of standards of uracil, hypoxanthine, azobis(2-amidinopropane) dihydrochloride) were purchased and uridine in the range of 0.625–40.00 ppm were prepared from Sigma-Aldrich (St. Louis, MO, USA). inthemobilephase.Quantificationwascarriedoutbyinte- gration of the peak areas using external standard calibration. 2.2. Preparation of Samples. The RVA specimens were A linear response with a correlation coefficient of 0.999 divided into 3 sections, T-RVA, M-RVA, and B-RVA, lyophil- (𝑛=6) was obtained for the standards. For all experiments, ized and homogenized with a grinder. Next, 10 g of each the extracts and standards were filtered through a 0.45 𝜇m segment was added to 100 mL of DW and subjected to cellulose ester membrane before injection into the HPLC extraction in boiling DW for 1 h. The RVA extracts were system. Detection was performed at a wavelength of 254 nm. filtered (0.25 𝜇m pore size) and lyophilized (yields: T-RVA, 3.87%; M-RVA, 3.61%; B-RVA, 2.66%) in a freeze dryer for 5 2.4. Antioxidant Activity days. 2.4.1. DPPH Radical Scavenging Activity. The DPPH scaveng- 2.3. Analysis of Bioactive Compounds ing activity of each antler extract was measured according to a slightly modified version of the method of Blois [19]. −4 2.3.1. Uronic Acid. Uronic acid content was determined by DPPH solutions (1.5 × 10 M, 100 𝜇L) were mixed with and the carbazole reaction [15]. Briefly, a 50 𝜇Lserialdilution without each extract (100 𝜇L), after which the mixtures were of the standards or samples was placed in a 96-well plate, incubated at room temperature for 30 min. After standing after which 200 𝜇L of 25 mM sodium tetraborate in sulfuric for30min,absorbancewasrecordedat540nmusinga acid was added to each well. The plate was heated for 10 min microplate reader. The scavenging activity was calculated as a ∘ at 100 C in an oven. After cooling at room temperature for percentage using the following equation: 15 min, 50 𝜇Lof0.125%carbazoleinabsoluteethanolwas ∘ (𝐴 −𝐴 ) carefully added. After heating at 100 Cfor10mininanoven control sample ( ) = × 100, (1) andcoolingatroomtemperaturefor15min,theplatewas Inhibition % 𝐴 control read in a microplate reader at a wavelength of 550 nm. 𝐴 where control was the absorbance of the reaction mixture 𝐴 2.3.2. Sulfated GAGs. GAGs content was determined by the without an RVA sample and sample was the absorbance of DMB (dimethylmethylene blue) dye binding method [16]. the reaction mixture with an RVA sample. Briefly, the color reagent was prepared by dissolving 0.008 g of DMB in a solution containing 1.185 g NaCl, 1.520 g glycine, 2.4.2. Hydrogen Peroxide Radical Scavenging Activity. Hydro- 0.47 mL HCl (12 M), and 500 mL DW.Each sample was mixed gen peroxide scavenging activity was determined according into 1 mL of color reagent and the absorbance was read to the method of Muller [20]. A 100 𝜇Lof0.1Mphosphate immediately at 525 nm. buffer (pH 5.0) was mixed with each extract in a 96-microwell plate. A 20 𝜇Lofhydrogenperoxidewasaddedtothe ∘ 2.3.3. Sialic Acid. Sialic acid content was determined based mixture and then incubated at 37 Cfor5min.Afterthe on the procedures described by Matsuno and Suzuki [17]. All incubation, 30 𝜇Lof1.25mMABTSand30𝜇Lofperoxidase solutions were precooled in an ice bath. Sodium periodate (1 unit/mL) were added to the mixture and then incubated Journal of Chemistry 3

∘ 𝐶 𝜇 𝑘 at 37 C for 10 min. The absorbance was recorded at 405 nm by where Trolox is the concentration of Trolox (20 M), is microplate reader and the percentage of scavenging activity thesampledilutionfactor,andAUCistheareabelowthe was calculated using (1). fluorescencedecaycurveofthesample,blank,andTrolox, respectively, calculated by applying the following formula in a 2.4.3. Hydroxyl Radical Scavenging Activity. The hydroxyl Microsoft Excel spreadsheet (Microsoft, Washington, USA): radical scavenging activity of each antler extract was deter- 𝑓5 𝑓10 5 mined according to the method of Chung et al. [21]. Hydroxyl AUC = (0.5 + + +⋅⋅⋅+𝑓𝑛 + )×5, (3) radicals were generated by the Fenton reaction in the pres- 𝑓0 𝑓0 𝑓0 ence of FeSO4. A reaction mixture containing 0.1 mL of where 𝑓0 istheinitialfluorescenceand𝑓𝑛 is the fluorescence 10 mM FeSO4,10mMEDTA,and10mM2-deoxyribosewas 𝑛 mixed with 0.1 mL of the extract solution, after which 0.1 M at time . phosphate buffer (pH 7.4) was added to the reaction mixture to reach a total volume of 0.9 mL. Subsequently, 0.1 mL of 2.5. Statistical Analysis. The results shown are summaries of 10 mM H2O2 was added to the reaction mixture, which was the data from at least 3 experiments. All data are presented as ∘ incubated at 37 Cfor4h.Afterincubation,0.5mLof2.8% mean ± SEM (standard error of the mean). Statistical analyses TCA and 1.0% TBA were added to each mixture, after which were performed using SAS statistical software (SAS Institute, each mixture was placed in a boiling water bath for 10 min. Inc., Cary, NC, USA). Treatment effects were analyzed using Absorbance was measured at 532 nm. Hydroxyl radical scav- one-way ANOVA followed by Dunnett’s multiple range test. enging activity was calculated as a percentage using (1). Results of 𝑃 < 0.05 indicated statistical significance.

2.4.4. ABTS Radical Scavenging Activity. The ABTS scaveng- 3. Results and Discussion ing activity of each antler extract was assessed following the ∙+ method of Arnao et al. [22]. Stock solutions included ABTS 3.1. Bioactive Composition. The biologically active substances solution and potassium persulfate solutions. A working contained in the 3 RVA segments, including uronic acid, solution was prepared by mixing the 2 stock solutions in equal sulfated GAGs, sialic acid, uridine, uracil, and hypoxanthine, quantities and allowing them to react for 12 h. The working arelistedinTables1 and 2. ∙+ solution was diluted with fresh ABTS solution and mixed The uronic acid content, sulfated GAGs content, and with or without each extract. After incubation for 2 h, the sialic acid content of the T-RVA and M-RVA sections were absorbance of each solution was recorded at 735 nm. The significantlygreaterthanthoseoftheB-RVAsection(𝑃< scavenging activity was calculated as a percentage using (1). 0.05). The DW extract of the T-RVA section contained 36.25 mg/g uronic acid, 555.76 mg/g sulfated GAGs, and 2.4.5. FRAP Assay. The FRAP assay was performed accord- 111.28 mg/g sialic acid (Table 1). The DW extract of the ing to the method of Benzie and Strain [23]. Fresh work- RVA contained 0.957 mg/g uridine, 1.084 mg/g uracil, and ing solution was prepared by mixing acetate buffer, TPTZ 1.263 mg/g hypoxanthine (Table 2). ∘ solution, and FeCl3⋅6H2Osolutionandwarmedat37C Uronic acid has been reported to improve circulation and before use. Each extract was allowed to react with the FRAP decrease stroke risk [25]; therefore, our chemical analyses solution in a dark room at room temperature for 30 min. The indicate that the DW extract of T-RVA might possess sim- absorbance of the colored product was measured at 595 nm. ilar activities. Sulfated GAGs, particularly CS (chondroitin Scavenging activity was calculated as a percentage using (1). sulfate), are of particular interest to physicians and phar- macists. Sulfated GAGs are composed of units of amino 2.4.6. ORAC Assay. For ORAC assay, the method of Ou et al. sugar, including D-glucosamine and D-galactosamine, and wasusedwithsomeslightlymodification[24]. The working bond with core proteins to form proteoglycans. Cartilage solution of FL and AAPH radical was prepared daily. Sample, proteoglycans regulate water retention and are integral to the blank, or standard was placed in 96-microwell plate, and the differentiation and proliferation of chondrocytes. The most ∘ plate was heated to 37 C for 15 min prior to the addition prominent sulfated GAG in velvet antler tissue is chondroitin of AAPH. The fluorescence was measured immediately after sulfate [26]. Sialic acid is a water soluble component that the AAPH addition and measurements with fluorescence was efficiently extracted by DW and showed significant filters for an excitation wavelength of 485 nm and an emission accumulation in the T-RVA section. Our findings are similar wavelength of 535 nm were taken every 5 min until the to those of a previous report [27], which showed that the T- relative fluorescence intensity was less than 5% of the value RVA, or “wax piece,” contains sialic acid levels higher than of the initial reading. those of other antler regions. Uracil is a primary mediator The ORAC values, expressed as 𝜇MTroloxequivalents of MAO (monoamine oxidase) inhibition by velvet antler (𝜇MTE/mg)werecalculatedbyapplyingthefollowingfor- extract [28]. Our data indicate that the T-RVA section may mula: contribute the majority of the inhibitory effect on MAO activity produced by velvet antler. In a report by Wang et al. ORAC (𝜇MTE) [29] aimed at identifying the active compound in velvet (𝐶 ×(AUC − AUC )×𝑘) (2) antler responsible for inhibiting MAO-B activity, the author = Trolox sample blank , suggested that the main antiaging compound in velvet antler ( − ) AUCsample AUCblank is hypoxanthine. Zhou et al. [28]showedthaturidinewas 4 Journal of Chemistry

Table 1: Sulfated GAGs, sialic acid, and uronic acid contents of 60 a regrowth velvet antler extracts. 50 T-RVA M-RVA B-RVA c b mg/g mg/g mg/g 40 d c e Sulfated GAGs 555.76 ± 12.48 369.30 ± 19.81 228.98 ± 24.42 30 f e ± ± ± g

Sialic acid 111.28 4.27 79.50 5.20 72.96 1.50 activity scavenging l h

± ± ± M TE/mg) h Uronic acid 36.25 2.96 25.90 2.29 23.11 2.40 ica 20 𝜇 d ( i Table 2: Hypoxanthine, uridine, and uracil contents of regrowth 10 velvet antler extracts. DPPH ra 0 T-RVA M-RVA B-RVA T-RVA M-RVA B-RVA mg/g mg/g mg/g Figure 1: The effect of RVA on DPPH radical scavenging activity. a–i Hypoxanthine 1.08 ± 0.03 1.00 ± 0.01 0.92 ± 0.01 Values not sharing a common letter are significantly different at 𝑃 < 0.05 Uridine 1.26 ± 0.03 1.14 ± 0.04 1.06 ± 0.03 by Dunnett’s multiple range tests. Light gray square, 125 𝜇g/mL; gray square, 250 𝜇g/mL; dark grey square, 500 𝜇g/mL; Uracil 0.96 ± 0.08 0.092 ± 0.03 0.845 ± 0.04 black square, 1,000 𝜇g/mL.

2+ responsible for 34.75% of the Fe -chelating activity of velvet antler. Therefore, the DW extract of T-RVA is expected to 30 show strong antioxidant activity due to its abundance of uridine. Zhou and Li [18] investigated the amounts of uridine, 25 a uracil, and hypoxanthine from ethanol extracted velvet antler, b and the values were 3.7, 3.6 and 3.9 mg/g, respectively. Their 20 d d values were higher than ours. This may be attributed to d extraction method. 15 e scavenging activity scavenging l e M TE/mg) g g ica 𝜇 ( 3.2. Antioxidant Activity. The antioxidant activities of RVA d 10 h ra i may not be attributed to a single mechanism. Therefore, 2 j O 2 5 6 methods were used to evaluate different aspects of the H antioxidant activities of RVA. The antioxidant activities of the DW extracts of RVAwere 0 T-RVA M-RVA B-RVA evaluated by assessing DPPH, H2O2,ABTS,andhydroxyl radical scavenging activity. In addition, FRAP and ORAC Figure 2: The effect of RVA on H2O2 radical scavenging activity. a–i were estimated. Values not sharing a common letter are significantly different at 𝑃 < 0.05 by Dunnett’s multiple range tests. Light gray square, The antioxidant activity of the DW extract of T-RVA was 𝜇 𝜇 𝜇 significantly better than those of the M-RVA and B-RVA 125 g/mL; gray square, 250 g/mL; dark grey square, 500 g/mL; black square, 1,000 𝜇g/mL. sections (𝑃 < 0.05) and appeared to be dose-dependent. The DPPH radical scavenging activity was highest for the T-RVA section (53.44 𝜇MTE/mg, IC50 0.853 mg/mL) and lowest for the B-RVA section (Figure 1). H2O2 (32.20 𝜇MTE/mg; 40 Figure 2)andABTS(60.31𝜇MTE/mg; Figure 3)radical a scavenging activities were also highest for the T-RVA sec- 35 b tion. The hydroxyl radical scavenging activity was high- 30 c c estfortheT-RVAsection(23.09𝜇M TE/mg), whereas the d 25 d activities of M-RVA and B-RVA were similar (Figure 4). e scavenging activity activity scavenging The T-RVA section was the most effective section in l 20 f M TE/mg) g ica g 𝜇

𝜇 d the FRAP assay (35.81 M TE/mg), whereas the activities ( 15 g g ra of B-RVA and M-RVA were similar (Figure 5). In the l 10

ORAC assay, 1,000 mg/mL T-RVA showed excellent activity roxy (121.58 𝜇MTE/mg)(Figure 6). d 5 Hy DPPH radical scavenging activity is often used as a 0 method of evaluating antioxidant activity. DPPH is a stable T-RVA M-RVA B-RVA radical that accepts an electron and/or hydrogen radical from Figure 3: The effect of RVA on hydroxyl radical scavenging activity. donor molecules to form a stable diamagnetic molecule. a–gValues not sharing a common letter are significantly different Therefore, the extracts of velvet antler may have provided an at 𝑃 < 0.05 by Dunnett’s multiple range tests. Light gray square, electron and/or hydrogen radical to neutralize DPPH [30]. In 125 𝜇g/mL; gray square, 250 𝜇g/mL; dark grey square, 500 𝜇g/mL; areportbyLeeandChung[31], the DPPH radical scavenging black square, 1,000 𝜇g/mL. Journal of Chemistry 5

70 activity of velvet antler extract obtained from the upper a section was reported to be 67.1% at an extract concentration 60 b c of 100 mg/mL, which was lower than the activity measured d in our analysis. H2O2 isareactivenonradicalandaclinically 50 d d e important compound due to its ability to penetrate biological 40 e membranes. H2O2 canbeconvertedintomorereactive f

scavenging activity scavenging species, such as singlet oxygen and hydroxyl radicals, thereby

l f M TE/mg)

𝜇 30 g (

ica causing lipid peroxidation or toxicity to cells. Therefore, d h scavenging of hydrogen peroxide can decrease prooxidants’ 20 levels. Our analysis of H2O2 scavenging by velvet antler

ABTS ra 10 produced results similar to those reported by Je et al. [30]. Hydroxyl radicals are extremely reactive and easily react 0 with amino acids, DNA, and membrane components. In this T-RVA M-RVA B-RVA study, the hydroxyl radical scavenging activity of RVA was Figure 4: The effect of RVA on ABYS radical scavenging activity. higher than that of velvet antler as reported by Je et al. a–gValues not sharing a common letter are significantly different [32]. In addition, our analysis of ABTS radical scavenging at 𝑃 < 0.05 by Dunnett’s multiple range tests. Light gray square, activity by RVA identified activity higher than that reported 125 𝜇g/mL; gray square, 250 𝜇g/mL; dark grey square, 500 𝜇g/mL; by Zhao et al. [33]. The FRAP assay treats the antioxidants black square, 1,000 𝜇g/mL. contained in the samples as reductants in a redox-linked colorimetric reaction, allowing assessment of the reducing power of antioxidants [34]. Zhao et al. [33]reportedactivity of 85.8 ± 0.02% by 5 mg/mL velvet antler extract in the FARP 40 assay, which was lower than the activity measured in our a 35 analysis. The ORAC assay has been applied extensively to b b evaluate the antioxidant activities of fruits, vegetables, leaves, 30

) d stems, herbs, and spices. As a result, the ORAC assay is d 25 e commonly mentioned in scientific publications and health e food publications [35]. However, the antioxidant activity of 20 f M TE/mg RVAhas not been evaluated using the ORAC assay. Therefore, 𝜇 g

( g 15 g this is the first report of an assessment of the antioxidant h activity of RVA using the ORAC assay. ORAC value of gallic

FRAP 10 acid was shown 161 ± 4.8 by Zulueta et al. [36], which was 5 higher than the activity of RVA found in our study. 0 T-RVA M-RVA B-RVA 4. Conclusions Figure 5: The effect of RVA on FRAP assay. a–gValues not sharing a common letter are significantly different at 𝑃 < 0.05 by Dunnett’s In the present study, we provided the first comprehensive multiplerangetests.Lightgraysquare,125𝜇g/mL; gray square, evaluation of the biologically active substances of RVA and 250 𝜇g/mL; dark grey square, 500 𝜇g/mL; black square, 1,000 𝜇g/mL. the antioxidant potential of different RVA segments. Future studies are required to further elucidate the other biological activities of the T-RVA, M-RVA, and B-RVA sections and the biological mechanisms underlying their effects. 140 a 120 Conflict of Interests ) 100 The authors declare that there is no conflict of interests b b regarding the publication of this paper. 80 M TE/mg c c 𝜇 ( 60 Acknowledgment d ORAC 40 ThispaperwassupportedbyKonkukUniversityin2015. e e f 20 e f g References 0 T-RVA M-RVA B-RVA [1] C. Li, “Development of deer antler model for biomedical Figure 6: The effect of RVA on ORAC. a–gValues not sharing a research,” Recent Advances & Research Updates,vol.4,no.2,pp. common letter are significantly different at 𝑃 < 0.05 by Dunnett’s 255–274, 2003. multiplerangetests.Lightgraysquare,125𝜇g/mL; gray square, [2]Z.Q.Zhang,Y.Zhang,B.X.Wang,H.O.Zhou,Y.Wang, 250 𝜇g/mL; dark grey square, 500 𝜇g/mL; black square, 1,000 𝜇g/mL. and H. Zhang, “Purification and partial characterization of 6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nippon [17] K. Matsuno and S. Suzuki, “Simple fluorimetric method for Temminck,” Acta Pharmaceutica Sinica,vol.27,no.5,pp.321– quantification of sialic acids in glycoproteins,” Analytical Bio- 324, 1992. chemistry,vol.375,no.1,pp.53–59,2008. [3]Z.F.Ma,D.S.Zhao,Q.D.Zhou,andS.Sun,“Studyon [18] R. Zhou and S. F. Li, “In vitro antioxidant analysis and raising yield of reproductive pilose antler of sika deer,” Journal characterisation of antler velvet extract,” Food Chemistry,vol. of Northeast Forestry University,vol.22,no.6,pp.41–47,1994. 114, no. 4, pp. 1321–1327, 2009. [4] J. F. He, Y. Z. Liu, S. B. Zhu, and L. Shang, “Study on increasing [19] M. S. Blois, “Antioxidant determinations by the use of a stable quality and quantity of reborn antlers of cervus elaphuslinnaeus free radical,” Nature,vol.181,no.4617,pp.1199–1200,1958. and cervus Nippon hortulorun, swinchone,” Natural Science [20] H. E. Muller, “Detection of hydrogen peroxide produced by JournalofHarbinNormalUniversity,vol.20,no.4,pp.91–94, microorganism on ABTS-peroxidase medium,” Zentralblatt fur¨ 2004. Bakteriologie, Mikrobiologie und Hygiene,vol.259,no.2,pp.151– [5]D.A.Butterfield,A.Castegna,C.M.Lauderback,andJ.Drake, 158, 1985. “Evidence that amyloid beta-peptide-induced lipid peroxida- [21] S.-K. Chung, T. Osawa, and S. Kawakishi, “Hydroxyl radical- tion and its sequelae in Alzheimer’s disease brain contribute to scavenging effects of spices and scavengers from brown mustard neuronal death,” Neurobiology of Aging,vol.23,no.5,pp.655– (Brassica nigra),” Bioscience, Biotechnology and Biochemistry, 664, 2002. vol.61,no.1,pp.118–123,1997. [6] W. A. Pryor and N. Y. Ann, “Free radical biology: xenobiotics, [22] M. B. Arnao, A. Cano, and M. Acosta, “The hydrophilic cancer, and aging,” Annals of the New York Academy of Sciences, and lipophilic contribution to total antioxidant activity,” Food vol. 393, no. 1, pp. 1–22, 1982. Chemistry,vol.73,no.2,pp.239–244,2001. [7] E.-K. Kim, S.-J. Lee, J.-W. Hwang et al., “In vitro investigation [23] I. F. F. Benzie and J. J. Strain, “The ferric reducing ability of on antioxidative effect of Inonotus obliquus extracts against plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP oxidative stress on PC12 cells,” Journal of the Korean Society for assay,” Analytical Biochemistry,vol.239,no.1,pp.70–76,1996. Applied Biological Chemistry, vol. 54, no. 1, pp. 112–117, 2011. [24] B. Ou, M. Hampsch-Woodill, and R. L. Prior, “Development and [8] J.-W.Hwang, E.-K. Kim, S.-J. Lee et al., “Antioxidant activity and validation of an improved oxygen radical absorbance capacity protective effect of anthocyanin oligomers on2 H O2-triggered assay using fluorescein as the fluorescent probe,” Journal of G2/M arrest in retinal cells,” Journal of Agricultural and Food Agricultural and Food Chemistry,vol.49,no.10,pp.4619–4626, Chemistry, vol. 60, no. 17, pp. 4282–4288, 2012. 2001. [9] B. R. Bhavnani, “Pharmacology of hormonal therapeutic [25] M. A. Moskowitz, E. H. Lo, and C. Iadecola, “The science of agents,” in The Menopause Comprehensive Management,B.A. stroke: mechanisms in search of treatments,” Neuron,vol.67,no. Eskin, Ed., pp. 229–256, Parthenon Press, New York, NY, USA, 2, pp. 181–198, 2010. 2000. [26] Y. W. Ha, B. T. Jeon, S. H. Moon et al., “Characterization of [10]S.S.Ditchkoff,L.J.Spicer,R.E.Masters,andR.L.Lochmiller, heparan sulfate from the unossified antler of Cervus elaphus,” “Concentrations of insulin-like growth factor-I in adult male Carbohydrate Research,vol.340,no.3,pp.411–416,2005. white-tailed deer (Odocoileus virginianus): associations with [27] B. Jeon, S. Kim, S. Lee et al., “Effect of antler growth period on serum testosterone, morphometrics and age during and after the chemical composition of velvet antler in sika deer (Cervus the breeding season,” Comparative Biochemistry and Physiology nippon),” Mammalian Biology,vol.74,no.5,pp.374–380,2009. A: Physiology,vol.129,no.4,pp.887–895,2001. [28] R. Zhou, J. Wang, S. Li, and Y.Liu, “Supercritical fluid extraction [11] J. A.´ Gomez,´ A. J. Garc´ıa, T. Landete-Castillejos, and L. Gallego, of monoamine oxidase inhibitor from antler velvet,” Separation “Effect of advancing births on testosterone until 2.5 years of age and Purification Technology,vol.65,no.3,pp.275–281,2009. and puberty in Iberian red deer (Cervus elaphus hispanicus),” [29] B. X. Wang, X. H. Zhao, X. W. Yang et al., “Inhibition of Animal Reproduction Science, vol. 96, no. 1-2, pp. 79–88, 2006. lipid peroxidation of deer antler (Rokujo) extract in vivo and [12]C.Li,Z.Jiang,G.Jiang,andJ.Fang,“Seasonalchangesof in vitro,” JournalofMedicalandPharmaceuticalSocietyfor reproductive behavior and fecal steroid concentrations in Pere` WAKAN-YAKU, vol. 5, pp. 123–128, 1988. David’s deer,” Hormones and Behavior,vol.40,no.4,pp.518– [30] J.Y.Je,P.J.Park,D.H.Lim,B.T.Jeon,K.H.Kho,andC.B.Ahn, 525, 2001. “Antioxidant, anti-acetylcholinesterase and composition of bio- [13] Y.-J. Li, T.-H. Kim, H. B. Kwak, Z. H. Lee, S.-Y. Lee, and G.- chemical components of Russian deer velvet antler extracts,” J. Jhon, “Chloroform extract of deer antler inhibits osteoclast Korean Journal for Food Science of Animal Resources,vol.31,no. differentiation and bone resorption,” Journal of Ethnopharma- 3,pp.349–355,2011. cology,vol.113,no.2,pp.191–198,2007. [31] K. A. Lee and H. Y.Chung, “The biological activity of deer antler [14] H. H. Sunwoo, T. Nakano, and J. S. Sim, “Effect of water-soluble extract in vitro,” The Korean Journal of Food and Nutrition,vol. extract from antler of wapiti (Cervus elaphus)onthegrowthof 20,no.2,pp.114–119,2007. fibroblasts,” Canadian Journal of Animal Science,vol.77,no.2, [32] J.-Y. Je, P.-J. Park, E.-K. Kim et al., “Composition of biologically pp. 343–345, 1997. active substances and antioxidant activity of New Zealand deer [15] M.Cesaretti,E.Luppi,F.Maccari,andN.Volpi,“A96-wellassay velvet antler extracts,” Korean Journal for Food Science of Animal for uronic acid carbazole reaction,” Carbohydrate Polymers,vol. Resources,vol.30,no.1,pp.20–27,2010. 54,no.1,pp.59–61,2003. [33] L. Zhao, Y.-C. Luo, C.-T. Wang, and B.-P. Ji, “Antioxidant [16]R.W.Farndale,D.J.Buttle,andA.J.Barrett,“Improvedquanti- activity of protein hydrolysates from aqueous extract of velvet tation and discrimination of sulphated glycosaminoglycans by antler (Cervus elaphus) as influenced by molecular weight and use of dimethylmethylene blue,” Biochimica Biophysica Acta, enzymes,” Natural Product Communications,vol.6,no.11,pp. vol. 883, no. 2, pp. 173–177, 1986. 1683–1688, 2011. Journal of Chemistry 7

[34] S.-Q. Huang, S. D. Ding, and L. P. Fan, “Antioxidant activities of five polysaccharides from Inonotus obliquus,” International Journal of Biological Macromolecules,vol.50,no.5,pp.1183–1187, 2012. [35] K. Thaipong, U. Boonprakob, K. Crosby, L. Cisneros-Zevallos, andD.H.Byrne,“ComparisonofABTS,DPPH,FRAP,and ORAC assays for estimating antioxidant activity from guava fruit extracts,” JournalofFoodCompositionandAnalysis,vol. 19, no. 6-7, pp. 669–675, 2006. [36] A. Zulueta, M. J. Esteve, and A. Fr´ıgola, “ORAC and TEAC assays comparison to measure the antioxidant capacity of food products,” Food Chemistry,vol.114,no.1,pp.310–316,2009.