Free Radicals and Antioxidants

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1 Free Radicals and Antioxidants 3 (2013) 30e34 Contents lists available at SciVerse ScienceDirect Free Radicals and Antioxidants journal homepage: Original article Antioxidant activity of garlic using conventional extraction and in vitro gastrointestinal digestion Anjali Bhatt, Vinayak Patel * Laboratory of Foods and Nutrition, P G Department of Home Science, Sardar Patel University, Vallabh Vidyanagar , Gujarat, India article info abstract Article history: Received 14 February 2013 Accepted 18 March 2013 Available online 28 March 2013 Keywords: ABTS Radical Scavenging Ability DPPH Radical Scavenging Ability Ferric Reducing Antioxidant Power Reducing Power Assay Total Phenol Content Introduction: Garlic is well known for its health protective abilities. Many studies have also proven garlic as an oxidative stress fighter with unique antioxidant potential. These studies have extracted raw garlic in conventional manner i.e. using organic solvents. Such antioxidant capacities cannot be well implicated for health purposes. Methods: This study deals with measurement of antioxidant capacity of raw as well as cooked garlic extracted by chemical as well as physiological method (in vitro gastrointestinal digestion). The Total antioxidant capacity was measured by methods like DPPH Radical Scavenging Ability, ABTS Radical Scavenging Ability, Ferric Reducing Antioxidant Power and Reducing Power Assay. Total Phenol was also evaluated. Results: Results show a wide difference between the antioxidant capacity of conventional and physiological extracts. The in vitro digested extracts of raw garlic show highest antioxidant capacity of all raw and cooked garlic extracts. Loss of phenolic compounds and antioxidant potential on cooking can also be clearly observed in both chemical and physiological extracts. Conclusion: It can be thus concluded that the physiological method of antioxidant extraction is more applicable and reliable than the conventional chemical extraction methods that do not resemble the biological behavior of antioxidants. Copyright Ó 2013, SciBiolMed.Org and Phcog.Net, Published by Reed Elsevier India Pvt. Ltd. All rights reserved. 1. Introduction Excess production of oxygen radical species such as hydrogen peroxide, superoxide anion radical, and the hydroxyl radical are thought to cause damage in cells. 1 The oxidative damage to cells is one of the factors causing many diseases, including atherosclerosis, diabetes and cancer. 2 Garlic (Allium sativum) has been considered to be one of the best disease-preventive foods. Dietary foods contain a wide variety of free radical scavenging antioxidants. 3 Garlic is composed mainly of fructose-containing carbohydrates and sulfur compounds. According to Banerjee et al, (2002), 4 garlic possesses antiproliferative properties. A number of investigations have Abbreviations: ABTS, 2,2 azinobis (3eethylbenzothiazoline-6-sulfonic acid) diammonium salt; ABTSRSA, ABTS Radical Scavenging Ability; DPPH, 1,1-diphenyl- 2-picryl-hydrazyl; DPPHRSA, DPPH Radical Scavenging Ability; FRAP, Ferric Reducing Antioxidant Power Assay; RPA, Reducing Power Assay; TEAC, Trolox Equivalent Antioxidant Capacity; TPTZ, 2,4,6etris (2-pyridyl)esetriazine; Trolox, 6- hydroxy-578-tetra methyl-chromane-2 carboxylic acid. * Corresponding author. Tel.: þ91 (02692) , þ91 (02692) ; fax: þ91 (02692) addresses: anjalivithlanibhatt@gmail.com (A. Bhatt), patelvh2009@ gmail.com (V. Patel). reported that garlic extract has a wide range of health benefits, e.g., against cancer and cardiovascular disorders 5 and as an antioxidant. 6,7 Numerous studies have been found reporting the antioxidant compounds in garlic as well as the antioxidant capacity of garlic. These studies extract raw garlic which is seldom consumed so. Food processing steps such as dehulling, peeling, thermal processing, mashing, etc. contribute to degradation and loss of phenolic compounds. 8,9 Also, we know that phenolic compounds mainly exist as glycosides linked to various sugar moieties or as other complexes linked to organic acids, amines, lipids, carbohydrates, and other phenols. Cooking sets the phenolic compounds free from these linkages to make them more bioaccessible. Moreover, garlic is extracted in organic solvents or their mixtures. The enzymatic treatments hydrolyze starch and protein, which may favor the release of polyphenols. The biological properties of antioxidants depends on the release of phenolic compounds from the food matrix during the digestion process (bioaccessibility) and may differ quantitatively and qualitatively from those produced by the chemical extraction employed in most studies. 10 Thus this study deals with analysis of both conventional as well as physiological extracts of raw and cooked garlic /$ e see front matter Copyright Ó 2013, SciBiolMed.Org and Phcog.Net, Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

2 A. Bhatt, V. Patel / Free Radicals and Antioxidants 3 (2013) 30e Materials and methods 2.1. Chemicals Pepsin (P-7000), Pancreatin (P-1750), Lipase (L-3126), Bile Extract Porcine (B-8631), a-amylase (A-3176), Amyloglucosidase (A-7095), ABTS (A-1888), DPPH (D-9132), Catechin (C-1251), Vanillin (V-2375), Rutin (R-5143), Gallic acid (G-7384) and TPTZ (T- 1253) were purchased from Sigma Aldrich-Germany and Troloxe was purchased from Fluka Sample preparation Experiment was done in two duplicate batches with two separate purchases in the same season. Garlic was purchased from the local market, peeled and finely pound. For cooking, 50 g of peeled garlic was pressure cooked without direct addition of water for 10 min. This cooked sample was cooled and pound like the raw sample. Further, it was extracted along with raw sample as stated below Chemical extraction 900 mg of raw and cooked garlic sample was extracted twice in 80% acidic methanol (ph set 2.0 with 1 N HCl) by shaking at room temperature for 45 min. Supernatants were filtered and centrifuged and volume was made up to 30 ml with the solvent. All samples were transferred to Eppendorf tubes and stored at 20 C for antioxidant determination Extraction by in vitro gastrointestinal digestion 900 mg of raw and cooked garlic sample was used for in vitro gastrointestinal digestion. The digestive enzymatic extraction was carried out by using the in vitro procedure previously described by Serrano et al, (2007). 10 Samples were successively incubated with digestive enzymes to simulate digestion in the small intestine. A control of sample was also incubated similarly with buffers without addition of enzymes. Sample was incubated with pepsin (0.6 ml of a 300 mg/ml solution in a buffer of 0.2 M HCl KCl, ph 1.5, 40 C, 1 h), pancreatin (3 ml of a 5 mg/ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6 h), lipase (6 ml of a 7 mg/ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6 h), bile extract porcine (6 ml of a 17.5 mg/ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6 h) and a-amylase (3 ml of a 120 mg/ml solution in 0.1 M tris-maleate buffer, ph 6.9, 37 C, 16 h). Then, the samples were centrifuged (15 min, 6000 rpm) and supernatants were collected. Residues were washed twice with 5 ml of distilled water, and all supernatants were combined. Each supernatant was incubated with 300 ml of amyloglucosidase for 45 min at 60 C. Volume of all samples was made up to 30 ml. All samples were transferred to Eppendorf tubes and stored at 20 C for antioxidant determination. Both chemical and digestive extracts (control and enzymatic) were used to determine the antioxidant capacity Total Phenol estimation FolineCiocalteu method 11 was used to determine the total phenol content of the chemical and physiological extracts. Different aliquots of known concentration of gallic acid were taken as standard Ferric Reducing Antioxidant Power Total antioxidant capacity of the chemical and physiological extracts for FRAP was determined by using the method of Benzie and Strain (1999). 12 Different aliquots of Trolox were treated as standard and results were expressed in terms of TEAC (mg of Trolox Equivalent/100 g) Reducing Power Assay This assay was performed as suggested by Oyaizu (1986). 13 Different aliquots of Trolox were treated as standard and results were expressed in terms of TEAC (mg of Trolox Equivalent/100 g) DPPH Radical Scavenging Ability The antioxidant activity of the extracts, on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical, was determined by the method described by Brand-Williams et al, (1995). 14 The percent inhibition and IC 50 was calculated and results were expressed in terms of TEAC (mg of Trolox Equivalent/100 g) ABTS Radical Scavenging Ability The radical scavenging ability of was determined using the modified ABTS radical decolorization assay. 15 The percent inhibition was calculated and results were expressed in terms of TEAC (mg Trolox Equivalent/100 g) Statistical analysis Differences between variables were tested for significance by using a one-way analysis of variance, DUNCAN using the level significance of p 0.05 by SPSS. 3. Results Many studies measuring the antioxidant capacity of garlic have been found. Usually, different organic solvents and their mixtures are used for extraction of antioxidant compounds. This extraction method does not imply to the physiological absorption. Thus in this study, the bioavailable antioxidant capacity of garlic is measured by simulation of gastrointestinal conditions. In physiological extraction, raw and cooked garlic were digested in vitro with enzymatic treatments. A control of sample was also incubated similarly with buffers without addition of enzymes. This can clearly show the difference between antioxidant capacity of chemical and physiological extracts. Control shows the degree of activity of enzymes. Also, comparison between raw and cooked can be made. Table 1 shows the Total Phenolic Content of different garlic extracts. The Total Phenolic Content of chemically extracted raw garlic was 67.5 mg GAE/100 g. Bozin et al, (2008) 6 extracted garlic in 80% methanol and found that Total Phenol Content of garlic was 50 mg GAE/100 g. The chemically extracted cooked garlic suffered a loss of 90% in phenolic content. Park et al, also found similar results on heating of garlic. However, the extraction of garlic in physiological conditions helps better extraction of phenolic compounds. The control raw garlic extract had mg GAE/100 g of TPC whereas enzymatically extracted raw garlic had mg GAE/ 100 g of TPC which was around 80% more than the chemically extracted raw garlic. Also, the enzymatically extracted cooked garlic showed better TPC than the chemical counterpart. Only 13e14% loss of TPC can be observed on cooking in enzymatic extracts.

3 32 A. Bhatt, V. Patel / Free Radicals and Antioxidants 3 (2013) 30e34 Table 1 Total Phenol Content, FRAP and RPA of different garlic extracts. TPC (mg GAE/100 g) FRAP (mg TE/100 g) RPA (mg TE/100 g) ME Raw b b b 18.9 ME Cooked a a a 8.5 DE R c c c 9.7 DE Raw e e e 28.3 DE Cooked d d d 19.0 F Value * * * Values are mean S D of four observations where ME ¼ Methanolic Extract, DE ¼ Digested Extract, R0 ¼ Raw Control, GAE ¼ Gallic Acid Equivalent, TE ¼ Trolox Equivalent. *Within column, values with the different following superscript letter differ significantly from each other (p 0.05). Table 2 Radical Scavenging Ability of different garlic extracts. DPPHRSA (mg TE/100 g) ABTSRSA (mg TE/100 g) ME Raw d c 4.7 ME Cooked b b 7.4 DE R a a 3.8 DE Raw e d 3.9 DE Cooked c c 1.6 F Value 0.164* 0.239* Values are mean S D of four observations where ME ¼ Methanolic Extract, DE ¼ Digested Extract, R0 ¼ Raw Control, GAE ¼ Gallic Acid Equivalent, TE ¼ Trolox Equivalent. *Within column, values with the different following superscript letter differ significantly from each other (p 0.05). The Ferric Reducing Antioxidant Power of different garlic extracts is depicted in Table 1. Raw chemically extracted garlic had mg TE/100 g of FRAP. Gorinstein et al, (2009) 17 studied the FRAP of raw garlic with the help of acidic aqueous methanol and found mm TE/g of antioxidant activity i.e mg TE/100 g. Raw chemically extracted garlic had almost 30% higher FRAP than cooked chemically extracted garlic which had mg TE/100 g FRAP. The enzymatically extracted garlic had significantly higher (55%) FRAP than the chemically extracted raw garlic. Also, on cooking 20% lower FRAP was observed in the enzymatically extracted garlic than the raw. The FRAP of garlic shares a positive and strong relationship with TPC (Fig. 1). The Reducing Power of the enzymatically extracted raw garlic was the highest (i.e mg TE/100 g) among all the garlic extracts. The chemically extracted raw garlic and the cooked enzymatic extract had respectively 62% and 19% lower Reducing Power than the raw enzymatic extract. Also, the chemically extracted cooked garlic had 82% lower Reducing Power than the raw chemical extract which had mg TE/100 g of Reducing Power. The control physiological extract of raw garlic had mg TE/100 g of Reducing Power which was lower than both chemical and enzymatic extracts of raw garlic. Radical Scavenging Ability of different garlic extracts is shown in Table 2. Though DPPHRSA follows similar pattern as FRAP i.e. enzymatically extracted garlic has higher antioxidant capacity than the chemical extract, the difference between the two as well as raw and cooked garlic is not significant. Chemically extracted raw garlic had mg TE/100 g of DPPHRSA whereas enzymatically extracted raw garlic had merely 6% higher DPPHRSA than the chemical extract. ABTS Radical Scavenging Ability of enzymatic extract of raw garlic was higher (47.2 mg TE/100 g) than the chemical extract which had 39.9 mg TE/100 g of antioxidant capacity. Gorinstein et al, (2009) 17 studied the ABTSRSA of raw garlic with the help of acidic aqueous methanol and found mm TE/g of antioxidant activity i.e mg TE/100 g. Cooked chemical extract suffered a loss of 22.8% in antioxidant capacity than the raw. Similarly, the enzymatically extracted cooked garlic had 19.8% lower ABTSRSA than the raw enzymatic extract which had 47.2 mg TE/100 g of ABTSRSA whereas the raw control had 24.5 mg TE/100 g of ABTSRSA. Fig. 1 depicts strong and positive correlation between Total Phenolic Content and antioxidant capacity measured by ABTSRSA, FRAP and RPA. This shows that the antioxidant capacity is dependent on the phenolic content of garlic. The in vitro digestive extracts showed considerably higher antioxidant capacity than the chemical extracts in all the parameters. 4. Discussion All antioxidant activity determinant parameters show almost similar trend in antioxidant activity of different extracts of garlic. In vitro gastrointestinal digestion gives extracts with much higher antioxidant potential than the conventional procedure. This suggests release of phenolic compounds during digestion and that the implication of antioxidant values by extraction using organic solvents may not prove true. When phenolic compounds are exposed to in vitro digestion, various enzymes transform them into different structural forms. These structures possess different chemical properties and functions 18 Phenolic compounds are present in Fig. 1. Relationship between Total Phenol Content and Total antioxidant capacity of different garlic extracts. GAE ¼ Gallic Acid Equivalent, TE ¼ Trolox Equivalent.

4 A. Bhatt, V. Patel / Free Radicals and Antioxidants 3 (2013) 30e34 33 bound form with proteins and other biomolecules and are gradually released during the hydrolysis process in the digestive system. 19 The amount of nutrients and phytochemicals absorbed during digestion is governed by the physical properties of the food matrix which affects the efficiency of physical, enzymatic and chemical digest. Many studies have reported antioxidant potential of different food stuffs after gastrointestinal digestion. 20e22 Jimenez and Saura-Calixto, (2005) 23 have stated that the antioxidant capacity of foods may be underestimated in the literature because the extraction solvents usually used do not allow a complete release of antioxidant compounds and additionally nonextractable polyphenols with a high antioxidant capacity are ignored. Similarly, this experiment clearly shows the difference between the conventional extraction and gastrointestinal digestion. In the DPPHRSA, the difference between the raw and cooked garlic samples in both chemical and physiological extracts was lower than other antioxidant determinant parameters (i.e. 10% and 13% respectively). The composition of phenolic compounds as well as other antioxidant compounds may be responsible for the difference. Also, the solvent used for extraction and processing conditions add to the factors affecting the antioxidant potential of the food. According to Ryan and Prescott (2010), 18 when phenolic compounds are exposed to in vitro digestion, they are transformed into different structural forms and possess different chemical properties and functions. These different properties and functions may give different antioxidant activity results as evaluated by different methods. So, evaluation of antioxidant capacity measurement by more than one method is recommended by many authors. 24e26 On the other hand, cooking leads to considerable loss of phenolic compounds. Both chemical as well as enzymatic extracts of cooked garlic had lower antioxidant potential than their counterparts. This shows depletion of phenolic compounds due to heat. Studies that support decrease in antioxidant activity after cooking have been found. 27e29 Gorinstein et al, (2008) 30 evaluated different bioactive compounds from garlic before and after various heat treatments and confirmed heat destruction of phenolic compounds. They observed the differences in the protein profile during processing. The protein profile is dependent on the time temperature combinations which in turn are responsible for different physical properties like texture, color, matrix softening, and increased extractability. These physical changes are the possible reasons why bioactive compounds, antioxidant activities, and proteins in garlic changed after cooking. Heat processing promotes polymerization of phenolic compounds to form brown-colored macromolecules 18 which may also be responsible for the drop in antioxidant activity on cooking. The release of phenolic compounds during digestion and the loss of antioxidant compounds due to heat are clearly indicated in the results. Most studies evaluating antioxidant potential do not compare raw and cooked forms of food whereas they are not consumed raw. Thus, by evaluating conventional and gastrointestinal extracts of both raw and cooked samples, this study gives complete results of garlic antioxidant potential. Difference between raw and cooked form as well as conventional and gastrointestinal extraction can be clearly seen. 5. Conclusion Results of this study thus prove that mere extraction by organic solvents may not be sufficient for the determination of antioxidant capacity. Also, the quantity and quality of antioxidant compounds extracted by organic solvents may not reflect their bioavailability. Such conventional extraction procedures may prove misleading for assessment of the antioxidant potential of foods. By extracting the vegetables with both methods and estimating their antioxidant potential and compounds with different methods, we can conclude that conventional method of extraction using organic solvents does not imply the actual physiological conditions. Conflicts of interest All authors have none to declare. References 1. Wickens AP. Ageing and the free radical theory. Respir Physiol. 2001;128: 379e Osawa T, Yoshida A, Kawakishi S, Yamashita K, Ochi H. Protective role of dietary antioxidants in oxidative stress. In: Cutler RG, Packer L, Bertram J, Mori A, eds. Oxidative Stress and Aging. Basel: Birkhauser; 1995:367e Shahidi F, Naczk M. Food Phenolics, Sources, Chemistry, Effects, Application. Lancaster: Technomic Publishing Co. Inc.; 1995:75e Banerjee SK, Maulik M, Mancahanda SC, Dinda AK, Gupta SK, Maulik SK. Life Sci. 2002;70:1509e Bhagyalakshmi N, Thimmaraju R, Venkatachalam L, Murthy KNC, Sreedhar RV. Nutraceutical applications of garlic and the intervention of biotechnology. Crit Rev Food Sci Nutr. 2005;45:607e Bozin B, Mimica-Dukic N, Samojlik I, Goran A, Igic R. Phenolics as antioxidants in garlic (Allium sativum L., Alliaceae). Food Chem. 2008;111:925e Gorinstein S, Leontowicz M, Leontowicz H, et al. Supplementation of garlic lowers lipids and increases antioxidant capacity in plasma of rats. Nutr Res. 2006a;26:362e Howard LA, Wong AD, Perry AK, Klein BP. b-carotene and ascorbic acid retention in fresh and processed vegetables. J Food Sci. 1999;64:929e Hunter, Fletcher JM. The antioxidant activity and composition of fresh, frozen, jarred and canned vegetables. Innov Food Sci Emerg Technol. 2002;3: 399e Serrano J, Goni I, Saura-Calixto F. Food antioxidant capacity determined by chemical methods may underestimate the physiological antioxidant capacity. Food Res Int. 2007;40:15e Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of FolineCiocalteau reagent. Meth Enzymol. 1999;299:152e Benzie I, Strain J. Ferric radical antioxidant power assay; direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Meth Enzymol. 1999;299:15e Oyaizu M. Studies on product of browning reaction produced from glucose amine. Jpn J Nutr. 1986;44:307e Brand-Williams M, Cuvelier, Berset C. Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol. 1995;28:25e Re, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26:1231e Park JH, Park YK, Park E. Antioxidative and antigenotoxic effects of garlic (Allium sativum L.) prepared by different processing methods. Plant Foods Hum Nutr. 2009;64:244e Gorinstein S, Seo Park Y, Heo B, et al. A comparative study of phenolic compounds and antioxidant and antiproliferative activities in frequently consumed raw vegetables. Eur Food Res Technol. 2009;228:903e Ryan L, Prescott SL. Stability of the antioxidant capacity of twenty-five commercially available fruit juices subjected to an in vitro digestion. Int J Food Sci Tech. 2010;45:1191e Hsu CL, Hurang SL, Chen W, Wenig YM, Tseng CY. Qualities and antioxidant properties of bread as affected by the incorporation of yam flour in the formulation. Int J Food Sci Tech. 2004;39:231e Goni I, Serrano J, Saura-Calixto F. Bioaccessibility of b-carotene, lutein and lycopene from fruits and vegetables. J Agric Food Chem. 2006;54:5382e Gawlik-Dziki U, Dziki D, Baraniak B, Lin R. The effect of simulated digestion in vitro on bioactivity of wheat bread with tartary buckwheat flavones addition. LWT e Food Science and Technology. 2009;42:137e Akillioglu HG, Karakaya S. Changes in total phenols, total flavonoids, and antioxidant activities of common beans and pinto beans after soaking, cooking, and in vitro digestion process. Food Sci Biotechnol. 2010;19: 633e Perez-Jimenez J, Saura-Calixto F. Literature data may underestimate the actual antioxidant capacity of cereals. J Agric Food Chem. 2005;53:5036e Day AJ, Williamson G. Biomarkers for exposure to dietary flavonoids: a review of the current evidence for identification of quercetin glycosides in plasma. Br J Nutr. 2001;133:105e Felgines C, Talavera S, Gonthier MP. Strawberry anthocyanins are recovered in urine as glucuro- and sulfo conjugulates in humans. J Nutr. 2003;133: 1296e Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans I. Review of 97 bioavailability studies. Am J Clin Nutr. 2005;81:230e242.

5 34 A. Bhatt, V. Patel / Free Radicals and Antioxidants 3 (2013) 30e Gorinstein S, Leontowicz H, Leontowicz M, Drzewiecki J, Najman K, Katrich E. Raw and boiled garlic enhances plasma antioxidant activity and improves plasma lipid metabolism in cholesterol-fed rats. Life Sci. 2006b;78: 655e Gorinstein S, Leontowicz M, Leontowicz H, et al. Dose-dependent influence of commercial garlic (Allium sativum) on rats cholesterol-containing diets. J Agric Food Chem. 2006c;54:4022e Roy MK, Takenaka M, Tsushida T. Antioxidant potential, antiproliferative activities, and phenolic content in water-soluble fractions of some commonly consumed vegetables: effects of thermal treatment. Food Chem. 2007;103: 106e Gorinstein S, Leontowicz H, Leontowicz M, et al. Comparison of the main bioactive compounds and antioxidant activities in garlic and white and red onions after treatment protocols. J Agric Food Chem. 2008;56:4418e4426.

6 PRAJÑÂ - Journal of Pure and Applied Sciences Vol. 20: (2012) ISSN EVALUATION OF ACTUAL ANTIOXIDANT CAPACITY OF JAMUN (SYZYGIUM CUMINI) USING THE IN VITRO GASTROINTESTINAL MODEL Anjali Bhatt and Vinayak Patel* *P G Department of Home Science, Sardar Patel University, Vallabh Vidyanagar, , Gujarat, India ABSTRACT Many scientific evidences have proven that diets rich in antioxidant compounds which occur particularly in plant foods lower the risk of developing lifestyle diseases. Jamun (Syzygium cumini) is a good source of anthocyanin and other phenolic compounds. The pulp of the Jamun berry contains antioxidants like anthocyanins, delphinidin, petunidin and malvidin-diglucosides, which impart the fruit its bright purple color. Abundant literature exists on the content of different phenolic compounds present in fruits and vegetables and their antioxidant capacities measured from the extracts using different polarity solvents based on in vitro methods. These features may differ quantitatively and qualitatively when fruits and vegetables are digested in the human gastrointestinal tract. Also, other antioxidant compounds may be adversely or positively affected by the ph, temperature, and other factors during the digestion process. The present study involves the measurement of the antioxidant potential of the fruit jamun using the in vitro digestion model. Keywords: Syzygium cumini, Antioxidant activity, Phenolic compounds, Anthocyanin INTRODUCTION Free radicals have been implicated in the etiology of a large number of major diseases. They can adversely alter many crucial biological molecules leading to loss of form and function. Such undesirable changes in the body can lead to disease conditions. Antioxidants can protect against the damage induced by free radicals acting at various levels. There are epidemiological evidences correlating higher intake of foods with antioxidant abilities to lower the incidence of various human morbidities or mortalities. Dietary and other components of plants form major sources of antioxidants. Phenolic compounds such as catechins, phenolic acids, flavonoids, proanthocyanidins and anthocyanins have exhibited a range of biological effects including antibacterial, antiviral, anti-inflammatory, antithrombotic and vasodilatory actions [1-3]. They also exert pronounced antioxidant and free radical-scavenging activities [4-8]. The traditional Indian diet, spices and medicinal plants are rich sources of natural antioxidants. Higher intake of foods with functional attributes including a high level of antioxidants in functional foods is one strategy that is gaining importance in advanced countries and is making its appearance in our country. Jamun fruit (Syzygium cumini) is a good source of phenolic compounds and anthocyanin. Jamun contains the widest variety of anthocyanidin groups: delphinidin, cyanidin, petunidin, peonidin, and malvidin, all present as diglucosides [9-11]. These impart a bright purple color to the fruit. The anthocyanin rich edible part of Jamun is comparable with that of blueberry, blackberry and blackcurrant, whose nutraceutical properties are well documented, suggesting the potential nutraceutical value of Jamun fruit. Anthocyanins in these fruits are reported to be powerful antioxidants and stability studies showed that they are stable up to 6 months in dry pulps [12]. Anthocyanins (cyanidin glucosides] have been shown to protect cell membrane lipids from oxidation [13]. Also, hypoglycemia [14], anti-inflammatory [15] neuropsychopharmacological [16], anti- bacterial [17], anti-hiv [18] and anti-diarrhoeal [19] effects of Jamun plant has been reported earlier. Some cyanidins are many times more powerful antioxidants than tocopherols [20]. *Corresponding author: patelvh2009@gmail.com Bertuglia et al, [21] showed that anthocyanin supplements effectively inhibited inflammation and subsequent blood vessel damage and maintained the integrity of vascular micro capillaries in the animal model. Health benefits associated with anthocyanin intake include reduced risk of coronary heart disease [22], protection against obesity and hypoglycemia [23], memory enhancement [24] and protection of fetal brain tissue [25]. Abundant literature exists on the content of different phenolic compounds and anthocyanin present in fruits and vegetables and their antioxidant capacities measured from these extracts using different polarity solvents. These features may differ quantitatively and qualitatively when fruits and vegetables are actually digested in the human gastrointestinal tract. Also, antioxidant compounds may be adversely or positively affected by the ph, temperature, and other factors during the digestion process. According to the literature reviewed, no study has reported the antioxidant potential of Jamun using the in vitro digestion model so far. The present study was thus carried out to compare the antioxidant activity of Jamun based on chemical and physiological extracts. MATERIALS AND METHODS Materials: Wholesome, ripe Jamun were selected and purchased from the local market of Anand and were used for chemical and in vitro enzyme based digestive (physiological) extraction. Chemicals : Pepsin (P-7000), Pancreatin (P-1750), Lipase (L-3126), Bile Extract Porcine (B-8631), á- Amylase (A-3176), Amyloglucosidase (A-7095), ABTS [2,2 Azinobis (3 ethylbenzothiazolin-6-sulfonic acid) diammonium, salt] (A-1888), DPPH (2,2-Diphenyl-1- picryl-hydrazyl) (D-9132), Catechin (C-1251), Vanillin (V-2375), Rutin (R-5143), Gallic acid (G-7384) and TPTZ (2,4,6 Tris (2-pyridyl) s triazine) (T-1253) were purchased from Sigma Aldrich-Germany and Trolox (6- Hydroxy-578-tetra methyl-chromane-2 carboxylic acid) was purchased from Fluka. Sample preparation: Jamuns were washed thoroughly, air dried and the edible portion was collected and homogenized for pulp preparation.

7 68 PRAJÑÂ-Journal of Pure and Applied Sciences, Vol. 20: (2012) December Extraction: Extraction of jamun was done by two methods (a) chemical (methanolic) extraction (ME) and (b) in vitro enzyme based digestive (physiological) extraction which involved a control (PC) and an enzymatic extract (PE). Control (PC) was extracted in similar buffers as enzymatic extraction as mentioned below but without the addition of enzymes. (a) Chemical extraction: About 900 mg of pulp was extracted in 80% methanol which was acidified to ph 2.0 with 1N HCl by shaking at room temperature for 45 minutes. Supernatants were centrifuged and volume was made up to 30 ml with the solvent. These samples were stored at -20 C for antioxidant activity determination. (b) Extraction using in vitro gastrointestinal digestion: About 900 mg of Jamun pulp was used for in vitro gastrointestinal digestion. The digestive enzymatic extraction was carried out by using the in vitro procedure previously described by Serrano et al, [26]. Samples were successively incubated with digestive enzymes to simulate digestion in the small intestine. About 900 mg of sample was incubated with pepsin (0.6 ml of a 300 mg/ml solution in a buffer of 0.2 M HCl- KCl, ph 1.5, 40 C, 1 h, Merck 7190), pancreatin (3 ml of a 5 mg/ ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6h, Sigma P-1750), lipase (6 ml of a 7 mg/ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6 h, Sigma L-3126), bile extract porcine (6 ml of a 17.5 mg/ml solution in 0.1 M phosphate buffer, ph 7.5, 37 C, 6 h, Sigma B-8631) and á-amylase (3 ml of a 120 mg/ ml solution in 0.1 M tris-maleate buffer, ph 6.9, 37 C, 16 h, Sigma A-3176). Also, all samples were digested with similar buffers without enzyme addition which was referred as control (PC) so as to evaluate the effect of enzyme addition. Then, the samples were centrifuged (15 min, 6000 rpm) and supernatants were removed. Residues were washed twice with 5 ml of distilled water, and all supernatants were combined. Each supernatant was incubated with 300 ìl of amyloglucosidase (Sigma A- 7095) for 45 min at 60 C. Supernatants were filtered and centrifuged and volume was made up to 30 ml. All samples were transferred to eppendorf tubes and stored at -20 C for antioxidant determination. Both chemical and digestive enzymatic extracts were used as test samples to determine the antioxidant capacity and polyphenol content. Total Phenol: Folin Ciocalteu method [27] was used to determine the total phenol content of the chemical and physiological extracts. Different aliquots of known concentration of gallic acid were taken as standard. Flavonoid: Different aliquots of concentrated sample were taken and volume was made up to 5 ml with distilled water [28]. Different aliquots of known concentration of Rutin was treated as standard. Anthocyanin: Anthocyanin was measured by the ph differential method suggested by Rapisarda, 2000 [29]. Concentration of anthocyanin was calculated by the equation considering the molecular mass and molar absorptivity of cyanidin-3-glucoside chloride after calculation of the difference of absorbance at 510 nm between ph 1 and ph 4.5 solutions. Flavonol: Different aliquots of concentrated samples were taken and volume was made up to 500 µl with methanol [30]. Catechin was treated as standard. Determination of Antioxidant Capacity: The antioxidant capacity was measured by four different methods namely FRAP, DPPHRSA, ABTSRSA and RPA. For each method, a calibrated Trolox curve was standardized and results were expressed in terms of TEAC (mg of Trolox equivalents/100g.) The antioxidant activity of the extracts to scavenge the stable DPPH radical was determined by the method described by Brand-Williams et al. [31]. The radical scavenging capacity of different wheat extracts was determined using the modified ABTS radical decolorization assay [32]. FRAP and RPA were determined by using the method of Benzie and Strain [33] and Oyaizu [34] respectively. Statistical analysis: Experiment was done in duplicate batches with two separate purchases in the same season. Four observations of two different experiments were analyzed statistically. Differences between variables were tested for significance based on a one-way analysis of variance, DUNCAN based on level of significance (p 0.05) by using SPSS RESULTS AND DISCUSSION There are very limited studies conducted on Jamun fruit for its chemical composition and biological activities. Antioxidant capacity of different Jamun fruit parts has been reported by Benherlal and Arumughan, [35] by ethanolic extraction but no reports have been found on the effect of gastrointestinal digestion on the antioxidant property of Jamun. However, the effect of digestion on the anthocyanin levels and antioxidant levels of wine and raspberries have been reported by Mc Dougall et al, [36] and on chokeberry by Bermudez-Soto et al, [37]. So, in the present study, the investigators compared the antioxidant activity of Jamun fruit by the chemical method based on methanolic extraction and the in vitro method based on gastrointestinal model referred to here as the enzymatic method. The total phenolic content (TPC) of the methanolic extract (ME) was found to be mg GAE/100g. Kaur and Kapoor, [38] found 215mg/100g of TPC in Jamun whereas Benherlal and Arumughan, [35] found 390 mg/100g of TPC in Jamun pulp extracted in ethanol. Luximon-Ramma et al, [39] extracted Jamun in acetone/water (70:30 v/v) which showed a TPC of mg gallic acid/100g fresh weight. The physiologically digested extracts varied significantly among themselves. Control Jamun (digested without enzyme addition) (PC) had mg GAE/100g whereas the enzymatic extract (PE) had % higher TPC than the chemical extract. This rise may be due to the release of phenolic compounds from carbohydrates, proteins and other molecules as a result of enzymatic action during digestion. The flavonol content followed a similar pattern as the TPC which is shown in Figure 1. The ME showed a µg catechin eq/g of flavonol. In physiologically digested samples, PC showed µg catechin eq/g while the PE showed µg catechin eq/g i.e. 149% higher flavonol than the chemical extracts. TPC and flavonol content of different jamun extracts is depicted in Figure 1. Figure 2 depicts the flavonoid and anthocyanin content of different Jamun extracts. The flavonoid content of the ME was found to be mg rutin eq/100g. Luximon-Ramma et al, [39] found 13.5 mg quercetin/ 100g in acetone /water extracts of Jamun. In the physiological extracts, the flavonoid content was found to

8 2012 Bhatt and Patel - Antioxidant Capacity of Jamun 69 decrease significantly. The PC ( %) and PE ( %) had lesser flavonoid content than the ME i.e mg rutin eq/100g. The decrease in flavonoid content may be a result of decrease in the anthocyanin content. The ME in the present study was found to have 4.45 mg cyanidin- 3 glycoside eq/100g of anthocyanin content whereas Benherlal and Arumughan, [35] reported the anthocyanin content in the Jamun pulp to be Fig 1: Total Phenol and Flavonol content of different Jamun extracts 7 mg/100g. Anthocyanin content of the PC in the present study was 2.93 mg cyanidin- 3 glycoside eq/100g which was lower by % and the PE was 3.93 mg cyanidin- 3 glycoside eq/100g which was lower by % when compared to the ME. The decrease in anthocyanin levels after digestion may be due to the increase in ph on pancreatin addition. The pancreatin is dissolved in phosphate buffer with ph 7.5 for 6 hours. Anthocyanins exist in equilibrium as four molecular species; the colored basic flavylium cation and three secondary structures; the quinoidal bases, the carbinol pseudobase and the chalcone pseudobase forms. At ph 2 or below, the flavylium cation form predominates but as the ph is raised towards neutrality; the colorless chalcone pseudobase begins to dominate. Chalcone formation is also favored by elevated temperatures and prolonged exposure may enhance degradation between the B and C rings resulting in the destruction of the anthocyanin chromophore [40, 41]. Strack and Wray, [40] reported that anthocyanins in red wine were stable to gastric conditions whereas there was a small loss in the TPC. However, after pancreatic digestion, the total anthocyanins were very poorly recovered compared to the bulk phenols. The ph shift to >7.5 in the pancreatic/small intestine digestion was the main factor in the irreversible breakdown of the anthocyanins. Mc Dougall et al, [36] studied the stability Fig 2: Flavonoid content and Anthocyanin content of different Jamun extracts of anthocyanin from red wine and found that anthocyanins were stable to gastric digestion but after the pancreatic digestion, the total anthocyanin were negatively affected due to the increase in ph. Other studies also support the breakdown of anthocyanins after pancreatic digestion. Bermudez-Soto et al, [37] studied the stability of polyphenols in chokeberry subjected to in vitro gastric and pancreatic digestion and found a loss in antioxidant capacity after in vitro digestion. Ryan et al, [42] reported a similar degradation of anthocyanin and consequential loss of antioxidant capacity of commercially available fruit juices of cranberry, red grape and pomegranate when subjected to an in vitro digestion. The presence of bile salts does not interfere with the total polyphenol and flavonoid assays but interferes with the assay used to quantify the total anthocyanin concentration in the digested samples [36]. Anthocyanins can form insoluble complexes with particulates and can also bind to components of the pancreatin/bile salts mixture [43]. The antioxidant capacity of Jamun measured by FRAP, ABTSRSA and RPA is represented in Figure 3. FRAP value of the chemical extract was found to be mg TE/100g. Luximon-Ramma [39] found the FRAP value of Jamun to be 1.6 mmol Fe (II) /100g. On physiological digestion, reduction was seen. PC extract showed % lesser FRAP value than the ME extract Fig 3: Ferric Reducing Antioxidant Power, ABTS Radical Scavenging Ability and Reducing Power Assay of different Jamun extracts Fig 4: Relationship between Anthocyanin content and Total Antioxidant Capacity of different Jamun extracts

9 70 PRAJÑÂ-Journal of Pure and Applied Sciences, Vol. 20: (2012) December while the PE showed % lesser FRAP value than the ME. Luximon-Ramma, [39] measured the ABTSRSA of Jamun and found 1.5 mmol TE/100g whereas in the present study the ME had mg TE/100g which is equal to 7.88 mmol TE/100g. The PC had mg TE/100g and PE had mg TE/100g which was % and % lower respectively than the chemical extract (ME) of Jamun. The Reducing Power of ME of Jamun was found to be mg TE/100g. It was reduced in both PC (-36.17%) and PE (-16.51%) as compared to the ME. Benherlal and Arumughan [35] reported lower Reducing Power of Jamun as compared to ascorbic acid. Figure 4 shows the relationship between Anthocyanin content and Total Antioxidant Capacity of different Jamun extracts. Best correlation was found between anthocyanin and FRAP although all parameters showed positive and strong correlation with anthocyanin. The reduction in the antioxidant capacity of physiological extract measured by the three different methods may be a result of the decrease in anthocyanin content of Jamun and this is confirmed in the present study. CONCLUSION From the results, it is concluded that the chemical extraction method does not imply the actual antioxidant capacity of Jamun and also that the antioxidant capacity of Jamun is dependent on the flavonoid especially anthocyanin content of Jamun. ACKNOWLEDGEMENT We thank University Grants Commission, Delhi for awarding first author of this research article by Research Fellowship in Sciences to Meritorious Student under BRS. REFERENCES [1] Di Carlo, G., Mascolo, N., Ice, A.A. and Capasso, F. (1999) Old and new aspects of a class of natural therapeutic drugs. Life Sci, 65: [2] Duthie, G.G., Duthie, S.J. and Kyle, J.A.M. (2000) Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev 13: [3] Middleton, E. and Kandaswami, C. (1994) The impact of plant flavonoids on mammalian biology: implication for immunity, inflammation and cancer. In The Flavonoids: Advances in Research since 1986 (Ed by Harborne JB), Chapman and London, Hall, pp [4] Asgary, S., Naderi, G.H., Sarrafzadegan, N., Ghassemi, N., Boshtam, M., Rafie, M. and Arefian, A. (1999) Anti-oxidant effect of flavonoids on hemoglobin glycosylation. Pharm Acta Helv 73: [5] Bestwick, C.S. and Milne, L. (2001) Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells. Biochem Biophys Acta 1528: [6] Bahorun, T., Trotin, F., Pommery, J., Vasseur, J. and Pinkas, M. (1994) Antioxidant activities of Crataegus monogyna extracts. Planta Med 60: [7] Bahorun, T., Gressier, B., Trotin, F., Brunet, C., Dine, T., Luyckx, M., Vasseur, J., Cazin,M., Cazin, J.C. and Pinkas, M. (1996) Oxygen species scavenging activity of phenolic extracts from hawthorn fresh plant organs and pharmaceutical preparations. Arzneim- Forsch Drug Res 46: [8] Bahorun, T. Trotin, F. and Vasseur J. (2002) Polyphenol production in Crataegus tissue cultures (hawthorn). In Biotechnology in Agriculture and Forestry: Medicinal and Aromatic Plants XII (Ed by Nagata T and Ebizuka Y), Springer, Berlin, pp [9] Veigas, J.M., Narayan, M.S., Laxman, P.M., Neelwarne, B. (2007) Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of Syzygium cumini skeels. Food Chem 105: [10] Brito, E.S.D., Araújo, M.C.P.D., Alves, R.E., Carkeet, C., Beverly A. Clevidence, And Janet A. Novotny. (2007) Anthocyanins Present in Selected Tropical Fruits: Acerola, Jambolão, Jussara, and Guajiru. J. Agric. Food Chem., 55: [11] Sharma, B., Viswanath, G., Salunke, R. and Roy, P. (2008) Effects of flavonoid-rich extract from seeds of Eugenia jambolana (L.) on carbohydrate and lipid metabolism in diabetic mice. Food Chem., 110: [12] Banerjee, A., Dasgupta, N. and De, B. (2005) In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chem 90: [13] Tsuda, T. (1998) Dietary cyanidin 3-O-beta-Dglycoside increases in vivo oxidative, resistance of serum in rats. Lipids 6: [14] Chopra, R.N., Chopra, I.C., Handa, K. L., Kapur, L. D. (1958) In Chopra's Indigenous drugs of India, 2nd ed. UN Dhar and Sons Pvt. Ltd., Calcutta, pp [15] Chaudhuri, A.K.N., Pal, S., Games, A. and Bhattacharya, S. (1990) Anti-inflammatory and related actions of Syzigium cummini seed extract. Phytotherapy Research 4: [16] Chakrabarty, D., Mahapatra, P.K., Chaudhuri, A.K.N. (1985) A Neuro psychopharmacological study of Svzigium cummini. Planta Medica 2: [17] Bhuiyan, M.A., Mia, M., Rashid, M.A. (1996) Antibacterial principles of the seeds of Eugenia jambolana. Bangladesh J Biol; 25: [18] Kusumoto, I.T., Nakabayashi, T., Kida, H., Miyashira, H., Hattari, H., Namba, T., Shimotohno, K. (1995) Screening of various plant extracts used in ayurvedic medicine for inhibitory effects on human immunodeficiency virus type I (HIV-I) protease. Phytotherapy Research; 12: [19] Indira, G., Mohan, R. (1993) Jamun Fruits, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad [20] Rice-Evans, C.A. (1995) The relative antioxidant activities of plant derived polyphenolic flavanoids. Free Radicals; 22:

10 2012 Bhatt and Patel - Antioxidant Capacity of Jamun 71 [21] Bertuglia, S., Malandrino, S. and Colantuoni, A. (1995) Effect of Vaccinium myrtillus anthocyansides on ischemia reperfusion injury in hamster cheek pouch microcirculation. Pharmacol Res 31: [22] Sumner, M.D., Elliot-Eller, M., Weidner, G., Daubenmeier, J.J., Chew, M.H., Marlin, R., Raisin, C.J., Ornish, D. (2005) Effects of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease. Am. J. Cardiol. 96: [23] Jayaprakasam, B., Olson, L.K., Schutzki, R.E., Tai, M.H., Nair, M.G. (2006) Amelioration of obesity and glucose intolerance in highfat- fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas). J. Agric. Food Chem. 54:1, [24] Andres-Lacueva, C., Shukitt-Hale, B., Galli, R.L., Jauregui, O., Lamuela-Raventos, R.M., Joseph, J.A. (2005) Anthocyanins in aged blueberry-fed rats are found centrally and may enhance memory. Nutr. Neurosci. 8: [25] Loren, D.J.; Seeram, N.P.; Schulman, R.N.; Holtzman, D.M. (2005) Maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury. Pediatr. Res. 57: [26] Serrano, J., Goni, I., and Saura-Calixto, F. (2007) Food antioxidant capacity determined by chemical methods may underestimate the physiological antioxidant capacity Food Res. Int. 40:1, [27] Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M. (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods Enzymol 299: [28] Zhishen, J., Mengcheng, T., Jianming, W., (1999), Determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals, Food Chem., 64: [29] Rapisarda, P., Fanella, F. and Maccarone, E., (2000) Reliability of Analytical Methods for Determining Anthocyanins in Blood Orange Juices. J. Agric. Food Chem., 48:2249. [30] Yermakov, A.I., Arasimov, V.V. and Yarosh, N.P. (1987), Methods of Biochemical Analysis of Plants. John Wiley and Sons, New York. [31] Brand-Williams, M., Cuvelier and C. Berset, (1995), Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie 28: [32] Re, N., Pellegrini, A., Proteggente, A., Pannala, M., Yang and Rice-Evans, (1999), Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology. 26: [33] Benzie, I., Strain, J. (1999), Ferric Radical Antioxidant Power Assay; Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Method Enzymology. 299: [34] Oyaizu, M. (1986), Studies on product of browning reaction produced from glucose amine. Jap J Nutr 44: [35] Benherlal, P.S. and Arumughan, C. (2007), Chemical composition and in vitro antioxidant studies on Syzygium cumini fruit. J Sci Food Agric 87: [36] Mcdougall, G.J., Dobson. P., Smith, P., Blake A. and Stewart, D. (2005), Assessing potential bioavailability of raspberry anthocyanins using an in vitro digestion system. J. Agric. Food Chem. 53: [37] Bermudez-Soto, M.J., Toma s-barbera n, F.A. & Garcý a-conesa, M.T. (2007). Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion. Food Chemistry, 102: [38] Kaur, C. and Kapoor, H.C. (2005). Antioxidant activity of some fruits in Indian diet. Acta Hort. (ISHS) 696: [39] Luximon-Ramma, A., Bahorun, T. and Crozier (2003), Antioxidant actions and phenolic and vitamin C contents of common Mauritian exotic fruits. Journal of the Science of Food and Agriculture. 83, 5: [40] Strack, D. and Wray V. (1993) Anthocyanins. In: J.B. Harborne, Editor, Methods in Plant Biochemistry, Plant Phenolics. Vol. 1, Academic Press, London pp [41] Clifford M.N. (2000) Anthocyanins nature, occurrence and dietary burden, J. Sci. Food Agric. 80: [42] Ryan, L. & Stuart, L. Prescott. (2010). Stability of the antioxidant capacity of twenty-five commercially available fruit juices subjected to an in vitro digestion. International Journal of Food Science and Technology, 45: [43] Bridle, P., Timberlake C.F. (1997) Anthocyanins as natural food colours - Selected aspects. Food Chem., 58: