I a f I E A C a b c d e B f a A R R A A K S J P P A 1 b i o c a ( ( d h 0 Industrial Crops and Products 89 (2016) 225–230 Contents lists available at ScienceDirect Industrial Crops and Products jo ur nal home p age: www.elsev ier .com/ locate / indcrop nfluence of pasteurization on antioxidant and in vitro nti-proliferative effects of jambolan (Syzygium cumini (L.) Skeels) ruit pulp vanise Guilherme Brancoa,∗, Izabel Cristina Freitas Moraesb, liana Janet Sanjinez Argandoñac, Grasiele Scaramal Madronad, Catarina dos Santosa, na Lúcia Tasca Góis Ruize, João Ernesto de Carvalhoe, harles Windson Isidoro Haminiukf Departamento de Ciências Biológicas, Universidade Estadual Paulista (UNESP), 19806-900 Assis, SP, Brazil Departamento de Engenharia de Alimentos, Universidade de São Paulo (USP), 13636-000 Pirassununga, SP, Brazil Faculdade de Engenharia, Universidade Federal da Grande Dourados (UFGD), 79804-070 Dourados, MS, Brazil Departamento de Engenharia de Alimentos, Universidade Estadual de Maringá (UEM), 87020-900 Maringá, PR, Brazil Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA), Universidade Estadual de Campinas (UNICAMP),13081-970 Campinas, SP, razil Departamento de Engenharia de Alimentos, Universidade Tecnológica Federal do Paraná (UTFPR), 873001-006 Campo Mourão, PR, Brazil r t i c l e i n f o rticle history: eceived 25 November 2015 eceived in revised form 19 January 2016 ccepted 24 January 2016 vailable online 21 May 2016 eywords: yzygium cumini (L.) skeels a b s t r a c t Here we analyze jambolan pulp phenolic compounds in order to establish a correlation with antioxi- dant and in vitro anti-proliferative effects, both before and after pasteurization. Total levels of phenolic compounds, flavonoids and anthocyanins were quantified using UV–vis techniques. Major phenolic com- pounds were identified by standard compound co-injection in HPLC-DAD/UV–vis. Antioxidant activity was measured by radical scavenging ability, as determined by DPPH assay. In vitro anti-proliferative activity was determined against nine human tumour cell lines using the methodology described by the Developmental Therapeutics Program at NCI/NIH. Pasteurization led to an increase in the levels of total ambolan asteurization henolic compounds ntioxidant and anti-tumour effects soluble solids (6.7%), phenolic compounds (7.2%) and flavonoids (16.4%). Anthocyanin content was largely preserved (91%) when compared with pulp without treatment. S. cumini preserved 56% of its original antioxidant activity after pasteurization, while thermal treatment revealed cytostatic activity in kidney (786-0) and ovary (OVCAR-3) lineages. Therefore, pasteurization can be applied successfully to S. cumini pulp. © 2016 Published by Elsevier B.V. . Introduction Jambolan (Syzygium cumini (L.) Skeels), also known as jambolão, lack plum, jamun or java plum, belongs to the Myrtaceae fam- ly. This plant possesses fruits that are oblong berries, deep purple r bluish in colour with a pinkish pulp. These fruits are widely onsumed and also used for the treatment of various diseases as n astringent, antiscorbutic, diuretic, antidiabetic, anti-Leishmania Rodrigues et al., 2015), and a treatment for chronic diarrhoea Ayyanar and Subash-Babu, 2012). In addition to all of the above- escribed properties, the fruit from the Jambolan (Syzygium cumini ∗ Corresponding author. E-mail address: igbranco@assis.unesp.br (I.G. Branco). ttp://dx.doi.org/10.1016/j.indcrop.2016.04.055 926-6690/© 2016 Published by Elsevier B.V. (L.) Skeels) plant has also been reported to have strong antioxidant and anti-genotoxic potential (Baliga et al., 2011). S. cumini fruit pulp is a source of phenolic compounds such as flavonoids and phenolic acids (Reynertson et al., 2008; Faria et al., 2011; Tavares et al., 2016). It also contains hydrolysable tannins, which may be the main phenolic compounds responsible for the astringency of the edible parts of fruit (Tavares et al., 2016), and anthocyanins: 3.5-diglucosides of delphinidin, petunidin and mal- vidin (Faria et al., 2011; Tavares et al., 2016), which are responsible for fruit pigment and are also thought to have antioxidant and anticancer bioactivity (Clifford, 2000; Nile and Park, 2014). Con- suming these anthocyanin-rich fruits are described as a strategy to prevent cardiovascular diseases, cancer and neurodegenerative diseases (Oliveira et al., 2012). dx.doi.org/10.1016/j.indcrop.2016.04.055 http://www.sciencedirect.com/science/journal/09266690 http://www.elsevier.com/locate/indcrop http://crossmark.crossref.org/dialog/?doi=10.1016/j.indcrop.2016.04.055&domain=pdf mailto:igbranco@assis.unesp.br dx.doi.org/10.1016/j.indcrop.2016.04.055 2 ops an f m f p i t i i c a l f i a s a e a p t e V j v t e f t p o a S a p s p 2 2 c q g a 2 a S b s a o s ( b i t m 26 I.G. Branco et al. / Industrial Cr However, most studies published to date have essentially ocused on raw foods, despite the fact that the human diet includes ainly cooked and processed foods. The domestic consumption of ruits may be either fresh or processed, including handmade frozen ulp, juice, jams, jellies and others. Industrial processing typically ncludes a heat treatment, such as drying, sterilisation and/or pas- eurization, in order to destroy pathogenic microorganisms and nactivate enzymes, in order to increase shelf-life and availabil- ty of the product for consumption. However, such processing an drastically affect phenolic content and, as a consequence, the ntioxidant capacity and anticancer bioactivities of foods formu- ated from these raw materials. For these reasons, anthocyanin-rich ruits, with few exceptions, have been poorly explored by the food ndustry, because they are with changes in temperature, pH, oxygen nd light (Turfan et al., 2011). Anthocyanins are the most sensitive, ince they are also affected by the presence of metal ions, enzymes, scorbic acid, and sulphur dioxide (Cavalcanti et al., 2011; Turfan t al., 2011). The majority of studies found in the literature correlate the nti-proliferative activities and extracts of fruits in vitro with their olyphenolic compounds, but such studies have not examined he role of thermal treatment (Medina et al., 2011; Leite-Legatti t al., 2012; Neri-Numa et al., 2013; Zhang et al., 2013; Rascón- alenzuela et al., 2015). Therefore the functional properties of ambolan and the growing market demand for fruit pulps with fla- or and colour, the pulp of this fruit shows high potential for use in he food processing industry, since this potential remains under- xplored. Pasteurization is a heat treatment widely used in the ruit juice industry and the evaluation of changes/losses during this hermal processing is very important, because the use of high tem- eratures may result in a significant decrease in the concentration f bioactive compounds and consequently reduction of biological ctivity. In this context, considering the therapeutic potential of . cumini pulp and the potential benefits for the human diet, we imed to evaluate the influence of pasteurization during pulp-fruit rocessing on polyphenol levels in S. cumini pulp content. We then ought to correlate our findings with antioxidant and in vitro anti- roliferative activity. . Materials and methods .1. Chemical reagents The following reagents were purchased from Sigma Chemi- al Co.: 2,2-diphenyl-1-picrylhydrazil (DPPH), Folin-Ciocalteau and uercetin. Aluminium chloride hexahydrate, potassium acetate and allic acid salts were obtained from Synth, as were solvents and cids. .2. Processing and pasteurization of S. cumini pulp Jambolan fruits (Syzygium cumini (L.) Skeels) were collected t Paulista State University (UNESP), Campus of Assis—São Paulo tate, during January and February 2011. Five kilograms of jam- olan fruit were harvested from three plants (clones), then anitised with chlorinated water (25 ppm). The pits were removed, nd the pulp was processed in a domestic processor. The pulp btained was distributed in polyethylene bags and subsequently tored at −18 ◦C until the time of application. The jambolan pulp 100 g each) was submitted to pasteurization by heating in a water ath at 70 ◦C for 5 min with stirring, followed by immediate cool- ng in an ice bath and storage at −18 ◦C. The pulp, with or without hermal treatment, was submitted to analyzes of pH, total acidity, oisture and soluble solids as described by AOAC (2000). d Products 89 (2016) 225–230 2.3. Extract preparation Extracts were prepared according to Barreto et al. (2009), with some modifications. For every 10 g of jambolan pulp, 40 mL of methanol/water (8:2) was added. The solution was stirred for one hour. The mixture was then filtered in a Buchner funnel and washed using small portions of methanol. This step was per- formed two more times, using 20 mL of an 8:2 methanol/water solution. The filtrates were transferred to a volumetric flask, and the final volume was adjusted to 100 mL. Extract levels of DPPH, flavonoids and phenolic compounds were analyzed. For HPLC and anti-proliferative activity analyzes, the extract obtained was then concentrated through a rota-evaporator at 40 ◦C and lyophilised for 72 h. 2.4. Quantitative analysis of phenolic content Total phenolic content was estimated using the Folin-Ciocalteau colorimetric method, based on the procedure of Singleton and Rossi (1965). Quantitative measurements were performed based on a standard calibration curve generated with 100, 200, 300, 400 and 500 mg/L of gallic acid in 80% methanol. Total phenolic content was expressed as gallic acid equivalents (GAE) in grams per 100-g sample (mg GAE/100 g). Total flavonoid content was determined using the methodol- ogy proposed by Chang et al. (2002). The extracts (0.5 mL) were mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminium chlo- ride hexahydrate, 0.1 mL of 1 M potassium acetate and 2.8 mL deionised water. After 40-min incubation at room temperature, the absorbance of the reaction mixture was measured at 415 nm. Results were expressed as milligrams of quercetin equivalents (QE) per 100 g of sample (mg QE/100 g). The standard curve for quercetin was obtained with a concentration range of 0–50 mg/L. Radical scavenging activity was performed according to Rufino et al. (2010). First, a DPPH methanolic solution (0.06 mM) was pre- pared. Then, 100 �L of fruit extract were added to 3.9 mL of DPPH solution. The decrease in absorbance at 515 nm was monitored once each minute until stabilisation. Antioxidant capacity was expressed as the sample concentration required to reduce the original amount of DPPH radicals by 50% (EC50), and values were expressed as g fruit/g DPPH. Total anthocyanin content was determined according to Fuleki and Francis (1968a,b): extraction with ethanol and HCl (1.5 N) (85:15) solution, followed by mixture filtration and analysis in a spectrophotometer at 535 nm. Results were expressed as mil- ligrams of cyanidin 3-glucoside (cyd 3-glu) equivalent per 100 g of fresh weight. 2.5. Analysis by HPLC High-performance liquid chromatography coupled with ultraviolet-visible diode-array detection (HPLC-DAD/UV–vis) was performed in a Dionex UltiMate 3000HPLC system (Dionex, Idstein, Germany) equipped with an UltiMate 3000 Pump, Ulti- Mate 3000 Autosampler Column Compartment, UltiMate 3000 Photodiode Array Detector and Chromeleon software. A reversed phase Acclaim® 120 column (C18, 5 �m, 120 Å, 4.6 mm × 250 mm) was used for these experiments. The column temperature was held at 40 ◦C for chromatographic separation. After 10 min of re- equilibration, the column was ready for a new injection. Phenolic acids and flavonols are usually detected at wavelengths ranging from 210 to 320 nm. The injection volume was 10 �L (250 mg/mL in 40% ethanol). The mobile phase was a gradient of 1% phosphoric acid aqueous solution (A) and methanol (B). The gradient was as follows: 0–2 min, 0–15% B; 2–5 min, 15–25% B; 5–10 min, 25–30% B; 10–15 min, 30–35% B; 15–25 min, 35–50% B; 25–30 min, 50–60% ps and Products 89 (2016) 225–230 227 B s c m C i i s 2 o N ) n f ( ( ( 0 m w p a c ( fi i fi w c t c t ( 2 f o 9 T C 8 3 S l d t A t n l M l t l p c u Table 1 Physical-chemical, phenolic compounds and anti-radical activity determined in jambolan pulp with or without pasteurization (fresh matter). Without Pasteurization With Pasteurization pH 4.15a ± 0.20 4.12a ± 0.30 Moisture (%) 86.07a ± 0.003 84.76b ± 0.001 Soluble Solids (◦Brix) 17.80a ± 0.20 19.00b ± 0.17 Total phenolics (mg GAE/100 g) 206.95a ± 1.11 221.83b ± 0.60 Total anthocyanin (mg cyd 3-Glu/100 g sample) 213.00a ± 1.02 195.00b ± 0.72 Total flavonoids (mg QE/100 g) 25.29a ± 0.22 29.45b ± 2.15 Antioxidant capacity EC50 (gfruit/gDPPH) 53.25a ± 2.93 83.16b ± 9.78 Results expressed as mean ± standard deviation; Different letters indicate signifi- cant difference between means in the same line (Tukey’s test p ≤ 0.05); GAE: gallic acid equivalent; cyd 3-glu: cyanidin 3-glucoside; QE:quercetin equivalent; EC50: I.G. Branco et al. / Industrial Cro ; 30–35 min, 60–80% B; 35–45 min, 80–100% B. The flow rate was et at 1 mL/min. The phenolic acids (gallic acid, chlorogenic acid, affeic acid, p-coumaric acid, ferulic acid) and flavonols (rutin, yricetin, quercetin and kaempferol) standards (Sigma Chemical o.) were applied. Stock solutions of all standards were prepared n methanol. For the HPLC analysis, phenolic compounds were dentified by comparing their retention times with those of pure tandards (Granato et al., 2011). .6. In vitro anti-proliferative activity Anti-proliferative activity was screened using the methodol- gy described by the Developmental Therapeutics Program at CI/NIH (Monks et al., 1991; available at http://dtp.nci.nih.gov/ . A non-tumour cell line, VERO (epithelial cells of monkey kid- ey), and the following human tumour cell lines were obtained rom the National Cancer Institute (Frederick, MA, USA): U251 glioma), UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES ovarian-expressing phenotype multiple-drug resistance), 786-0 renal), NCI-H460 (lung, non-small cell), PC-3 (prostate), OVCAR- 3 (ovarian), and K562 (leukaemia). Stock cultures were grown in edium containing 5 mL RPMI 1640 (GIBCO BRL) supplemented ith 5% foetal bovine serum (FBS, GIBCO) at 37 ◦C with 5% CO2. A enicillin:streptomycin mixture (1000 �g/L:1000 U/L, 1 mL/L) was dded to the experimental cultures. Cells in 96-well plates (100 �L ells × well−1) were exposed to extracts in DMSO (Merck)/RPMI 0.25, 2.5, 25, and 250 �g × mL−1) at 37 ◦C, 5% CO2 in air for 48 h. The nal DMSO concentration (less than 0.2%) did not affect cell viabil- ty. Before (T0 plate) and after sample addition (T1 plates), cells were xed with 50% trichloroacetic acid. The level of cell proliferation as determined by spectrophotometric quantification (540 nm) of ellular protein content using the sulphorhodamine B assay. Using he concentration-response curve for each cell line, the GI50 (con- entration that produces 50% growth inhibition) was determined hrough non-linear regression analysis using ORIGIN 8.5® software OriginLab Corporation) (Vendramini-Costa et al., 2010). .7. Statistical analysis The results were expressed as mean values ± standard deviation rom two extraction replicates, each run in triplicate. The analysis f variance (ANOVA) was determined using SAS software (version .2, SAS Institute, Cary, N.C., USA). The means were compared using ukey’s test. Statistical significance was set at a level of p < 0.05. orrelation and regression analyzes were performed using ORIGIN .5® software (OriginLab Corporation). . Results and discussion Our results regarding the pH and total soluble solid levels of the yzygium cumini pulp are compatible with those reported in the iterature (Mussi et al., 2015; Tavares et al., 2016). Previous reports escribed S. cumini as an acidic fruit with pH ranging from 3.29 o 4.04 and soluble solid content ranging from 11.4 to 11.76 Brix. cidity is an advantage in S. cumini pulp, because intensive heat reatment is not necessary, as bacteriological development does ot take place at these pH levels. In this study, we found higher evels of soluble content (about 18 Brix) than those described by ussi et al. (2015). Highly soluble content is important, because ess sugar will need to be added and less time will be required for he water to evaporate. These factors require the expenditure of ess energy, resulting in a more economical process with increased roduct yield (Pereira et al., 2012). Therefore, S. cumini is a good andidate for industrial processing (Table 1). Differences in phenol, anthocyanin and flavonoid levels are sually related to variations in the methodologies employed in Concentration of antioxidant required to reduce the original amount of free radicals by 50%. sample extraction, reaction conditions, and other factors that affect fruit composition. Total phenolic content was higher in this study as compared to the results reported by Rufino et al. (2010) (185 mg/100 g fresh matter) and Faria et al. (2011) (148.3 mg/100 g of fresh matter). Similarly, total flavonoid content (Table 1) was much higher in this study as compared to the results reported by Benherlal and Arumughan (2007) (7 mg/100 g fresh matter) but lower when compared to the results reported by Faria et al. (2011) (91.2 mg of cathechin equivalent/100 g sample). Sultana and Anwar (2008) found total flavonol (kaempeferol, quercetin, myricetin) values of 0.24 mg/100 g dry matter and quercetin levels of 0.12 mg/100 g dry matter in jambolan pulp. The antho- cyanin content in our experiments was higher than the results obtained by Benherlal and Arumughan (2007) (134 mg cyanidin- 3-glucoside/100 g fresh weight) and by Rufino et al. (2010) (93.3 mg/100 g fresh matter) but lower than that obtained by Mussi et al. (2015) and similar to levels reported by Faria et al. (2011) (211 mg/100 g). The next step will be to analyze differences in the phenolic com- position profile induced by the pasteurization of S. cumini pulp. These values should then be compared to those obtained with- out thermal treatment. In this study, pasteurization did not affect Syzygium cumini pulp pH and increased total soluble solid content (about 6.7%), as shown in Table 1. Total phenolic and flavonoid levels showed an increase after pasteurization (about 7.2% and 16.4%, respectively) with a reduc- tion in anthocyanin content (about 8.5%). Decreases in total phenolic and flavonoid levels (especially anthocyanins) induced by changes in temperature are well described in the literature. Brownmiller et al. (2008) reported a 43% decrease in total antho- cyanin content in blueberry purees after pasteurization. Turfan et al. (2011) evaluated the stability of anthocyanins after pas- teurization (95 ◦C/10 min) during the processing of pomegranate anthocyanins (Punica granatum L., cv. Hicaznar) and verified a loss of 8–14%. Patras et al. (2009) reported that anthocyanin and phe- nol levels in strawberry and blackberry purees were also affected by thermal processing (70 ◦C/2 min), which showed that pasteur- ization reduced flavonoid levels in fruit purées. Zoric et al. (2014) noted that the duration of heating as well as the temperature used impacted anthocyanin degradation more than that of other phe- nols. Mussi et al. (2015) described a reduction in anthocyanin con- tent (60–70%) when processing S. cumini residue, but antioxidant activity levels still ranged from 93 to 97%. The authors did not cor- relate this trend with activity levels for other phenolic compounds. In this study, total anthocyanin and antioxidant activity levels were reduced after pasteurization (about 8.5% and 56%, respectively) http://dtp.nci.nih.gov/ http://dtp.nci.nih.gov/ http://dtp.nci.nih.gov/ http://dtp.nci.nih.gov/ http://dtp.nci.nih.gov/ http://dtp.nci.nih.gov/ 228 I.G. Branco et al. / Industrial Crops and Products 89 (2016) 225–230 Table 2 Quantification and chromatographic and spectroscopic characteristics of non-anthocyanic phenolic compounds from jambolan pulp obtained by HPLC-DAD. Compounds tR (min) � (nm) Without pasteurization (mg/Kg of fresh weight) With pasteurization (mg/Kg of fresh weight) Gallic acid* 6.71 272 28.04a ± 0.93 44.04b ± 1.90 Chlorogenic acid* 10.12 280 205.60b ± 6.01 93.08a ± 2.51 Ferulic acid* 18.45 280 7.81 ± 0.23 n.d. Rutin* 19.88 370 5.96 ± 0.34 n.d. Myricetin 27.33 215, 338 35.40b ± 0.85 23.56a ± 0.79 Quercetin 31.60 370 4.52a ± 0.22 5.16b ± 0.28 Total 287.33b ± 5.98 165.84b ± 4.78 * Note: Results in mean ± standard deviation. Means on the same line followed by different letters are significantly different (p < 0.05); n.d.: not detected; tR: Retention time, �: Wavelength. Table 3 Antiproliferative activity (GI50, �g/mL) of doxorubicin and jambolan extracts against human cell lines. 2 u m a 7 4 o h q doxorubicin (positive control) 0.05 ± 0.04 <0.025 <0.025 0.18 ± 0.01 0.025 <0.025 <0.025 0.22 ± 0.01 <0.025 without pasteurization > 250 <0.25 >250 1.21 ± 0.17 >250 >250 >250 >250 >250 with pasteurization > 250 > 250 > 250 193.5 ± 7.71 3.8 ± 2.2 >250 53.7 ± 39.0 >250 >250 2 = U251 (glioma, Central Nervous System); u = UACC-62 (melanoma); m = MCF-7 (breast adenocarcinoma); a = NCI-ADR/RES (ovary, multidrug resistance phenotype); 7 = 786- 0 (kidney); 4 = NCI-H460 (lung, non-small cells adenocarcinoma); o = OVCAR-3 (ovary); h = HT-29 (colon); q = HaCaT (keratinocyte human, immortalized normal cell). ithout ( a c l o d fl i I b a c a Fig. 1. Chromatographic profile of jambolão pulp w Table 1). Therefore, we suggest that flavonoids and phenolic acids re as important as anthocyanins for the free radical scavenging of S. umini pulp. Some studies about the antioxidant potential of pheno- ic compounds in fruits or foods have demonstrated the existence f synergistic or antagonistic effects among the various antioxi- ants present in the food matrix; thus, activity not only depends on avonoids and/or phenolic structure but also on the environment n which said compounds are found (Palafox-Carlos et al., 2012; oannou et al., 2012). Even so, S. cumini pulp pasteurization remains advantageous, ecause the process induces only limited anthocyanin loss, which llows the pulp to be used as a functional ingredient in several pro- essed products that require the incorporation of fruit pulp, such s preserves, juices, nectar, ice cream, and yogurt. However, the (top) and with (bottom) pasteurization at 280 nm. medium must be slightly acidic to preserve the characteristic colour of the fruit. For the HPLC-DAD analysis, phenolic assessments were carried out by UV–vis analyzes, by comparing the retention times (tR) for available standards and by considering information from the lit- erature. Three major peaks on the chromatogram shown in Fig. 1 were identified as the anthocyanins. The first peak, with a retention time of 9.4 min (UV absorption at 275 and 522.2 nm), may repre- sent delphinidin 3-diglucoside. The second peak, with a retention time of 10.3 min (UV absorption at 274 and 524 nm), likely indi- cates the presence of delphinidin 3,5-diglucoside. The third peak at 13.4 min (UV absorption at 275 and 525 nm) suggests the existence of petunidin-3,5 diglucoside. These results are similar to those described by Faria et al. (2011), who found five types of anthocyanin ps an a v w t f ( a t F i 1 m a l o H t A d t t f o a a i w p t a s s w m p g c t c c o n c c a l a p c F i l n 4 c t a p I.G. Branco et al. / Industrial Cro glycones in S. cumini pulp: delphinidin, cyanidin, petunidin, mal- idin and peonidin. Comparisons of jambolan pulp measurements ith the retention times of authentic standards allowed us to iden- ify phenolic acids (gallic acid: 6.71 min, chlorogenic acid: 10.1 min, errulic acid: 18.4 min) and flavonoid (rutin: 19.9 min). Myricetin 27.3 min) and quercetin derivatives (31.6 min) were also detected nd confirmed by UV absorption (215 and 338 nm, 370 nm, respec- ively). These results (Table 2) are similar to those described by aria et al. (2011). In Table 2, the results of HPLC quantification show that pasteur- zation increased gallic acid levels by 57% and quercetin levels by 4%, but there was a significant decrease in chlorogenic acid and yricetin levels (about 54% and 33%, respectively). Neither ferulic cid nor rutin was detected after pasteurization. The increase in gal- ic acid after pasteurization may be directly related to the cleavage f covalent bonds and the release of this molecule to the medium. owever, the mechanism underlying the decrease in rutin con- ent that accompanied the increase in quercetin remains elusive. t first, the increase in quercetin might seem to be related to the ecrease in rutin glucose, but quercetin is less resistant to increased emperature than is rutin (Ioannou et al., 2012). Ioannou et al. (2012) also described, in their revision, the fact hat it is not easy to dissociate the effect of thermal processing from ood matrix effects, because the degradation of flavonoids is not nly a function of temperature and the duration of heating; it may lso depend on other parameters such as pH, the presence of oxygen nd the presence of other phytochemicals in the medium. Regard- ng pH, the degradation of rutin and quercetin is enhanced under eakly alkaline and neutral reaction conditions. Notably, S. cumini ulp is acidic. Alternately, the process could be oxidative, because he presence of high concentrations of oxygen induces quercetin nd rutin degradation, while the absence of oxygen has the oppo- ite effect. However, Ioannou et al. stated that other phytochemicals uch as chlorogenic acid in the medium may play a protective role, hich could explain the medium presence of quercetin and maybe yricetin, because the latter compound is found in S. cumini pulp. In this work, the anti-proliferative activity of the tested com- ounds was expressed as the concentration that produced 50% cell rowth inhibition or a cytostatic effect (GI50, mg/mL−1) for each ell line (Table 3). For S. cumini pulp, optimal results with regard to selec- ivity and cytostatic effects on growth inhibition among the ells tested before pasteurization were found for the melanoma ell line (UACC-62, GI50 < 0.25 mg/mL), followed by the resistant varian cell line (NCI–ADR/RES, GI50 = 1.21 �g/mL), where sig- ificant cytostatic activity was observed. After pasteurization, S. umini presented a weak inhibitory effect on the growing ovary ell line (OVCAR-3, GI50 = 53.7 �g/mL) and significant selectivity nd cytostatic effects on the inhibition of growing kidney cell ines (786-0, GI50 = 3.7 �g/mL) (Table 3). These differences in the nti-proliferative profile may be explained by chemical changes romoted by heat treatment as indicated by total phenolic, antho- yanin and flavonoid levels (Table 1) and HPLC analysis (Table 2, ig. 1). Although the pulp had high selectivity for those cell lines, t showed weak anti-proliferative activity against other cancer cell ines and HaCat cells; i.e., pulp did not affect the proliferation of ormal cells, making it an excellent candidate nutraceutical food. . Conclusion These findings suggest that pasteurization can be applied suc- essfully to S. cumini pulp. The pH as well as total soluble solid, otal phenol and flavonoid levels were increased. Despite the over- ll reduction in anthocyanin content and antioxidant activity in asteurised as compared to non-pasteurised pulp, pasteurised pulp d Products 89 (2016) 225–230 229 demonstrated robust in vitro anti-proliferative activity in kidney cells (786-0) and weak in vitro anti-proliferative activity in ovarian cells (OVCAR-3), without affecting the proliferation of normal cells (HaCat cells). Therefore, S. cumini pulp pasteurization is advantageous: with limited anthocyanin loss, fruit colour was preserved. 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Skeel... 1 Introduction 2 Materials and methods 2.1 Chemical reagents 2.2 Processing and pasteurization of S. cumini pulp 2.3 Extract preparation 2.4 Quantitative analysis of phenolic content 2.5 Analysis by HPLC 2.6 In vitro anti-proliferative activity 2.7 Statistical analysis 3 Results and discussion 4 Conclusion References