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Evaluation of Antioxidant and Cytotoxic Activity of AlcoholicBeverages from Topinambour by in vitro and ex vivo Tests

OCTAVIAN VASILE POP1,2, ADRIAN VAMANU2, ANDREA ERDELYI POP1,2, EMANUEL VAMANU2*, SULTANA NITA3

1 Hypericum Impex, 3A Gutinului Str., 435100, Baia Sprie, Romania2 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnology, 59 Marasti Blvd, 011464,Bucharest, Romania3 National Institute of Chemical-Pharmaceutical Research-Development – ICCF Bucharest, 112 Vitan Road, 031299, Bucharest,Romania

A number of alcoholic beverages were produced from topinambour (Helianthus tuberosus) in order todetermine the antioxidant potential by using in vitro methods (total antioxidant activity and reducing power)and ex vivo (inhibition of erythrocytes hemolysis and lipid peroxidation). It has also been determined thecytotoxic effect on HCT 8 cells. The total phenolic content confirmed the applied in vitro and ex vivo assays,thus resulting high values in distillates containing extracts of wild berries, with a maximum for the formula6 (BF 6), of 64.6 ± 1.20 mg gallic acid / mL. Cromatographic analysis of polyphenolcarboxilic acids showedthe presence of gallic acid (104.66 ± 0.10 mg / 100 mL) and of homogentisic acid (189.9 ± 0.02 mg / 100mL), in the same formula mentioned above. Instead, formula 8 (BF 8), which contains a distillate of apple,presented a significant amount of homogentisic acid (389.02 ± 0.04 mg / 100 mL). The results showed thatadditions of taste correctors (cinnamon and wild berries), which are masking the unpleasant taste of stevia,has as outcome a formula with a high content of bioactive compounds.

Keywords: wild berries, topinambour,antioxidant activity, cytotoxicity

* email@emanuelvamanu.ro; Tel.: (+40)742218240

In the recent years identifying some natural sources ofobtaining alcoholic beverages with high biological activitiesis becoming more and more intense. The addition of somemedicinal plant extracts, for example, represents the mosteffective substrate, because the resulting drinks haveexquisite taste and aromas. It will also result in a productwith a high content of biologically active compounds whichare effective in degenerative and cardiovascular diseasesand even in diabetes. These are intended to be analternative to coffee and medicinal herbal tea [1]. The bigdisadvantage comes from the ethanol intake, whichincreases the symptoms in people suffering fromneurodegenerative disorders.

These beverages are effective due to the proposedformulas, to the degree of ingredient processing and to thestability of bioactive components. The process of digestioncauses, in this case, an inactivation of a significant portionof the bioactive compounds, and also a transformation intoother compounds with a smaller molecular weight [2].From here, results the impact and stability that the bioactivecomponent (mainly phenolic compounds) has to establisha composition with high bioavailability [1]. The purpose ofthis study was to determine the antioxidant potential ofsome natural alcoholic beverages, correlated with achromatographic analysis of the phenolic acids that arepresent. The tests were carried out by in vitro and ex vivomethods.

Experimental partChemicals

All chemicals and reagents were purchased from SigmaAldrich GmbH (Steinheim, Germany). All other unlabelledchemicals and reagents were of analytical grade.

Preparation of alcoholic beverages from topinambour.Tubers of topinambour, called also Jerusalem artichoke(Helianthus tuberosus) were well cleaned of the soil andwashed with water under pressure. They were autoclaved

for one hour at 120 - 1300C, and then milled to obtain ahomogenous paste. The decoction is obtained fromtopinambour supplemented with starch from corn / applefruit in a ratio of 1: 1.

The decoction with starch was heated up to 500C, pH 6- 6.5 and Spritase HiTaA17105L (0.4 mL / kg) was added.The mixture was maintained at 500C for 30 min withconstant stirring. Subsequently, glucoamylase Spritase GA14400 (0.7 mL / kg) was added. In the final stage DanstilA at a dose of 25 g / 100 L decoction was added and thetemperature was maintained at 25-320C. The fermentationlasts for 3-5 days. The liquid undergoes a triple distillationand the final product had an alcoholic strength of 60-65degrees.

The apple decoction was heated up to a temperature of800C, and then cooled to the fermentation temperature,300C, whereupon the selected yeast - Danstil A (25 g / 100L decoction) were added. The temperature was maintainedat 25-320C. The fermentation lasts for 6-7 days, and thedistillation process was the same as described for the firstvariant.

In the final stage were obtained distillates supplementedwith natural extracts in a ratio of 10-15%, sweetened withstevia 1%, then filtered through the filter with plates. Theanalyzed extracts had the following composition: BF 1topinambour and apple distillate; BF 2 topinambour, appleand cinnamon distillate; BF3 topinambour and starchdistillate; BF 4 topinambour, starch and cinnamondistillate; BF 5 topinambour, starch, momordica and steviadistillate; BF 6 topinambour, starch, wild berries and steviadistillate; BF 7 topinambour, apple, momordica and steviadistillate; BF 8 topinambour, apple, wild berries and steviadistillate. As a control a distillate from topinambour (BF 9)aged for four years in oak barrel was used.

Determination of bioactive compoundsThe total phenolic and flavonoids contents of ethanolic

extracts were determined using Folin-Ciocalteu reagent

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and the aluminium chloride colorimetric method,respectively [3-5].

The determination of polyphenol carboxylic acids, andflavones was performed by HPLC (ELITE – LaChrom, withDAD detector) and was presented in a previous study [6].

In vitro Determination of Antioxidant ActivityTotal Antioxidant Activity

\Total antioxidant Activity (TAA) of beverages wasdetermined by the method reported by Khan et al., 2013[7] with little modifications. 0.1 mL of samples was mixedwith 0.6 mL of reaction mixture containing 0.6 M sulphuricacid, 28 mM sodium phosphate and 1% ammoniummolybdate into the Falcon tubes. The Falcon tubes wereincubated at 93°C for 10 min to complete the reaction, in aMemmert. The absorbance was measured at 695 nm usinga Helios λ spectrophotometer. Ascorbic acid and quercetin(1 mg/mL) were used as standards. % TAA was calculatedusing the following equation, % TAA = [(AC – AS)/AC)×100,where AC is the absorbance of the control, and AS is theabsorbance of the sample [8-10].

Reducing powerFerric ion reducing power was evaluated by the capacity

to convert Fe3+ to Fe2+ [9], measuring the absorbance at700 nm with a Helios λ spectrophotometer (Thermo FisherScientific, Inc., USA) [11]. Cupric ion reducing power wasevaluated by the copper (II)-neocuproine [Cu (II)-Nc]reagent as the chromogenic oxidizing agent, measuringthe absorbance at 450 nm with a Helios ëspectrophotometer (Thermo Fisher Scientific, Inc., USA)[12]. Quercitin and ascorbic acid (both at 1 mg/mL) wereused as standards.

Ex vivo testsInhibition of erythrocytes hemolysis by 2,22 -azobis-2-

amidinopropane dihydrochloride (AAPH). It wasdetermined by the method reported by Borra et al., 2013[13] with little modifications. Chicken blood (provided bySilvia Muscalu household, Berceni Village, Prahova County,Romania) was heparinised and centrifuged at 2000 × g,for 10 min. The sediment was washed with phosphatebuffer, pH 7.4. The reaction mix consisted of 0.2 mL sample,0.2 mL AAPH 100 mM and 0.2 mL erythrocyte suspension.The reaction mixture was incubated for 3 h , at 370C, diluted20 times with phosphate buffer and centrifuged at 1000 ×g, for 10 min. The absorbance was measured at 540 nmusing a Helios λ spectrophotometer [9]. Ascorbic acid andquercetin (1 mg/mL) were used as standards. Erythrocyteshemolysis inhibition was calculated using the followingequation, % Inhibition = [(AC – AS)/AC)×100, where AC isthe absorbance of the control, and AS is the absorbance ofthe sample [8,10].

Inhibition of lipid peroxidationIt was determined by the method reported by Ferreira et

al., 2009 [14] with little modifications. Chicken brain(provided by Silvia Muscalu household, Berceni Village,Prahova County, Romania) was homogenized with Tris-HCl buffer, pH 7.4 and centrifuged at 3000 × g, 10 min. Thereaction mix consist of 0.1 mL supernatant, 0.2 mL sample,0.1 mL FeSO4 10µM, 0.1 mL ascorbic acid 0.1 mM andkeept at 370C for 1 h. The reaction was stopped by theaddition of 0.5 mL trichloroacetic acid 28% and 0.38 mLthiobarbituric acid 2%. The mixture was then inserted at800 C for 20 minutes into a water bath Memmert WNB 7,centrifuged at 3000 × g, 10 min and the absorbance wasmeasured at 540 nm using a Helios λ spectrophotometer

[9]. Ascorbic Acid and quercetin (1 mg/mL) was used asstandard. Lipid peroxidation inhibition was calculated usingthe following equation, % Inhibition = [(AC – AS)/AC)×100,where AC is the absorbance of the control, and AS is theabsorbance of the sample [8,10].

Cytotoxicity assay in vitroHCT 8 cells from the collection of MICROGEN Center

(Center for Research, Education and Consulting inMicrobiology, Genetics and Biotechnology) [15] weregrown in 75 cm2 tissue culture flasks using RPMI mediumsupplemented with 10% fetal bovine serum (FBS) (Sigma)[16]. Cells were incubated at 37°C in 5% CO2 in a humidifiedatmosphere until a 80% confluence. On 80% confluencecells were trypsinized and seeded onto (105 cell/ well) 100-well microplate (honeycomb plate for Bioscreen C MBR)at 100µL per well, and incubated in anaerobic atmospherewith 5% CO2 at 37°C, for 24 h, and then treated with theextracts at various concentrations. After 24 h, 48 h and 72h of treatment, 50 µL of 1 mg/mL MTT solution were addedto each well, and further incubated for 4 h. The cells ineach well were then solubilized with DMSO (100 µL foreach well) and the optical density (OD) was recorded at580 nm with Bioscreen C MBR [17]. Mitomycin-C (1 mg/mL) was used as standard. % Cytotoxixity was calculatedusing the following equation, % Cytotoxicity = [(AC – AS)/AC)×100, where AC is the absorbance of the control, andAS is the absorbance of the sample [8,10].

Statistical analysisAll parameters for antioxidant activity was assessed in

triplicate, and the results were expressed as mean ± SDvalues of 3 observations. The mean values and standarddeviation were calculated with the EXCEL program fromMicrosoft Office 2010 package.

Results and discussionsIn vitro antioxidant activityTotal Antioxidant Activity

The total antioxidant activity of different formulas ofalcoholic beverages is shown in figure 1. The distillateswhich were derived from the fermentation of topinambourshowed an antioxidant activity at least comparable to theone of the controls (ascorbic acid). BF 6 presented a similarvalue, averaging at 65 ± 0.7%, but lower than that of thesecond standard (quercetin) with about 7± 0.15%. Theremaining samples had values higher by 2-3% comparedto that of the quercetin. An exception was BF 9, which hada TAA value of 84 ± 1.50%, with over 15% higher comparedwith quercetin (1 mg / mL). If normally TAA value increaseswith increasing the concentrations of product in the testedsample [7], in the case of distillates the stability of thephenolic component represents the key element in theexpression of the antioxidant potential. Also, following theaging process (4 years) TAA of the distillates oftopinambour is a high one, averaging around 75 ± 0.08%,similar to that expressed by ascorbic acid and by BF 5,which contains momordica and was sweetened withstevia .

Reducing power evaluationThe evaluation of power reduction is shown in figure 2,

along with the effect on iron and copper ions. The resultsrevealed a very low specificity relative to the copper ion inextracts BF 1 - BF 4. However, the presence of wild berryextract increased the value of power reduction, especiallyin relation to the copper ion (CUPRAC method). The valuewas even higher than that of BF 9 with approximately 10±

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0.30%. The distillates supplemented with momordica andwild berries showed a similar capacity of Fe3+ ion reduction(p <0.01) that varied by about 2%. The values were onaverage by 8 ± 0.20% lower. It was noted that the value ofBF 9 sample was comparable to that of quercetin (1 mg /mL) as regards binding to the copper ion. For the ferric ionreducing power the samples supplemented with naturalextracts had values similar to the control sample (BF 9)and with approximately 10% higher than the quercetin.

Ex vivo antioxidant activityEx vivo tests showed a characteristic behavior for each

extract, generally directly related to the presence of certainclasses of compounds with antioxidant effect. First, therehas been noticed a high activity, of 50%, for BF 3, wherethe presence of flavonoid amount was higher comparedto BF 1, BF 2 and BF 4 (table 1). AAPH radical is responsiblefor generating an oxidative stress upon the lipid and proteincomponents of the erythrocyte membrane. This processleads to the installment of red cell haemolysis [18]. Again,the presence of wild berries extract was superior to that ofmomordica, the BF 5 sample having a low activity, of 27 ±

0.60%, similar to the samples P3 and P4 (fig. 3). Instead,the control BF 9 exhibited a lower average value than thesamples BF 6 - BF 8, with a maximum of 30% (sampleP8).

In both ex vivo tests it was noticeable the BF 8 sample,by having an equal inhibitory activity, and also an activity ofinhibiting lipid peroxidation in sample BF 5, with a value ofaround 85%. The latter was in contrast with the protectioncapability against the erythrocyte hemolysis (fig. 3). SampleBF 7 had a similar inhibitory activity with the two controls,quercetin and ascorbic acid, of around 60 ± 3.00%. Thesamples without wild berry extract and stevia showed thefollowing inhibitory activity, in descending order: BF 3>BF 1> BF 4> BF 2.

Cytotoxicity effectThe analyzed distillates showed a cytotoxic activity

exceeding 45 ± 0.22%, except the samples BF 1 - BF 4,which had very low values, of below 10%, against HTC 8cell line (data not shown). BF 6 and BF 7 had approximatelyequal values, of 77.50 ± 0.01%. The resistance of the cellline to the first four distillates matched the absence of tannin

Fig. 2. Reducing power of different formulas ofalcoholic beverages

Table 1 CONTENT OF BIOACTIVE

COMPOUNDS IN ALCOHOLICBEVERAGES FROM

TOPINAMBOUR

Fig. 1. Total antioxidant activity of different formulasof alcoholic beverages

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among the biological active molecules. The results of theaged distillate (of 4 years) expressed a cytotoxic effect ofapproximately 61.70 ± 0.09%, which was similar to thatof mitomycin-C, at a concentration of 1 mg / mL. Theseresults were with 20% lower than the maximum measuredactivity (extract BF 6), of 76.16 ± 0.1%.

Compounds with antioxidant effectThe total phenolic content confirmed in vitro and ex vivo

determinations, resulting in high values in the distillatesthat contained extracts of wild berries, with a maximumfor BF 6, of 64.6 ± 1.20 mg gallic acid / mL (table 1). Thelow level of phenols which was ascertained in BF 5 and, inparticular, in BF 7, is supported by previous studies [19].This content also brought about a non-specific behavior, inparticular in correlating the ex vivo assays. It should alsobe taken into account that the results of Folin-Ciocalteu’sReagent method were influenced by a number of non-phenolic compounds or by the resulting secondarymetabolites [20]. Thus, the values were not positivelycorrelated with the activities shown in figure 1 - 3

From the point of view of flavonoid content, thetopinambour distillates had a high total amount offlavonoids, in particular the BF 4 sample. From the samplessupplemented with momordica or wild berries, BF 6 samplewas highlighted, with 103.93 ± 0.10 of mg quercitin

equivalent / mL. This value was more than three timeshigher than the value recorded in the sample BF 8 (table1).

Following the chromatographic analysis, BF 6 and BF 8had the largest quantities of some polyphenolcarboxylicacids, which corresponded to the presence of wild berriesin their composition (table 2). In BF 6, gallic acid (104.66± 0.10 mg / 100 mL) and homogentisic acid (189.9 ± 0.02mg / 100 mL) were ascertained. Conversely, in BF 8 asignificant amount of homogentisic acid (389.02 ± 0.04mg / 100 mL - fig. 4, peak 1) was present, while theremaining acids were in low amounts (below 1 mg / 100mL -fig. 4, peak 2 & 3) or were missing. The remainingsamples displayed low values of polyphenolcarboxylicacids, BF 1 - BF 4 samples showing the lowest presence ofthese compounds. An exception was BF 4, whichregistered a quantity of 14.34 ± 0.03 mg / 100 mL gallicacid.

The analyzed beverages, based on a topinambourdistillate, have proven a significant biological activity,consistent to the content of bioactive compounds of thesupplements that enriched the taste and aroma. [20] Wildberries present in BF 6 and BF 8 showed biological activitiesevidenced by in vitro and ex vivo studies, mainly due to thepolyphenolcarboxylic acids content (table 2). Gallic acid,a compound commonly found in bioactive extracts was

Table 2CROMATOGRAPHIC

ANALYSIS OFPOLYPHENOLCARBOXILIC

ACIDS

Fig. 3. Ex vivo tests of different formulas of alcoholicbeverages

Fig. 4. Cromatographic analysisof polyphenolcarboxilic acids

for sample BF 8

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the least stable, being identified only in three of theanalyzed formulas. Instead, the caffeic acid was presentin all samples, although its value has not exceeded 1 mg /100 mL. The stability of the formulations was a high one intime, not showing significant losses (p <0.05), theappearance being a clear and constant one.

ConclusionsCompared to other beverages containing a high amount

of anthocyanins, the BF 6 and BF 8 samples have a verylow content of ascorbic acid, linked to long-term stability.Had it been present, the ascorbic acid would have led toloss of biological activity, as a result of the degradation ofthe anthocyanin component. These results are consistent,according to studies by Hernandez-Herrero, MJ Frutos,2015, to a color stability of the product, and the antioxidantactivity is also directly influenced by the presence of theseflavonoids in wild berries, along with tannins [21].

Such beverages, containing natural bioactivecompounds, sweetened with stevia, can be consumedeven by people with diabetes. Cinnamon and wild berriesmask the unpleasant taste of stevia, resulting in aformulation containing a high level of bioactive compounds,and therefore the target groups can have a normal lifestyle.These innovative formulas are not encouraging theconsumption of alcohol, but represent an attempt to showthat a small amount of alcohol can have beneficial effectson the organism, by an intake of antioxidants with highstability, due to the provision of valuable secondarymetabolites.

Acknowledgments: This research was financed through the projectPNCDI II UEFISCDI – Human Resources, Theme 102/2015 (http://www.robiomush.ro) and by the doctoral programme of the UASVMBucharest, Romania.

References1. FERRUZZI, M.G., Physiol. Behav., 100, no. 1, 2010, p. 33.2. VAMANU, E., PELINESCU, D., AVRAM, I., NITA, S., BioMed Res. Int.,2013, Article ID 289821, 2013, p. 1.3. VAMANU, E., Pak. J. Bot., 45, no. 1, 2013, p. 311.

4. LING A.L.M., MD. YASIR, S., MATANJUN, P., BAKAR M.F.A., Int. J.Pharm. Phytopharm. Res., 2015, accepted paper.5. VAMANU, E., NITA, S., Rev. Chim.(Bucharest), 65, no. 3, 2014, p.372.6. VAMANU, E., PELINESCU, D., AVRAM, I., NITA, S., BioMed Res. Int,2013, Article ID 289821, 2013, p. 1.7. KHAN, M.A., RAHMAN, A.A., SHAFIQUL, I., KHANDOKHAR, P., PARVIN,S., ISLAM MD B., HOSSAIN, M., RASHID, M., SADIK, G., NASRIN S.,MOLLAH, M N.H., ALAM, AHM K., BMC Res. Notes, 6, no. 24, 2013, p.1.8. MIGUEL, M., BOUCHMAAA, N., AAZZA, S., GAAMOUSSI, F., LYOUSSI,B., Fres. Environ. Bull., 23, no. 6, 2014, p. 1.9. OWUSU, J., MA, H., WANG, Z., AFOAKWAH, N. A., ZHOU, C., AMISSAH,A., J. Food Biochem., 39, 2015, p. 91.10. KHLIFI S., HACHIMI Y.E.L., KHALIL A., ES-SAFI N., ABBOUYI A.E.l.,Indian J. Pharmacol., 37, no. 4, 2005, p. 227.11. MARTINS, N., BARROS, L., SANTOS-BUELGA, C., SILVA, S.,HENRIQUES, M., FERREIRA, I.C.F.R., Food Chem., 167, 2015, p. 131.12. KARAKOCA, K., OZUSAGLAM, M.A., CAKMAK, Y.S., ERKUL, S.K.,EXCLI J.,12, 2013, p. 150.13. BORRA, S.K., GURUMURTHY, P., MAHENDRA, J., JAYAMATHI, K. M.,CHERIAN, C. N., CHAND, R., J. Med. Plants Res., 7, no. 36, 2013, p.2680.14. FERREIRA, I.C.F.R., AIRES, E., BARREIRA, J.C.M., ESTEVINHO,L.M., Food Chem., 114, no. 4, 2009, p. 1438.15. STANZIALE, S.F., PETROWSKY, H., JOE, J.K., ROBERTS, G.D.,ZAGER, J.S., GUSANI, N.J., BEN-PORAT, L., GONEN, M., FONG, Y.,Surgery, 132, no. 2, 2002, p. 353.16. BLACHE, P., VAN DE WETERING, M., DULUC, I., DOMON, C.,BERTA, P., FREUND, J.N., CLEVERS, H., JAY, P., J. Cell. Biol., 166,no. 1, 2004, p. 37.17. CAO, P., LIANG, Y., GAO, X., LI, X.-M., SONG, Z.-Q., LIANG, G.,Molecules, 17, 2012, p. 13631.18. BARROS, L., FERREIRA, M.-J., QUEIRO´S, B., FERREIRA, I.C.F.R.,BAPTISTA, P., Food Chem., 103, 2007, p. 413.19. POP, A .E., VAMANU, A., POP, O.V., VAMANU, E., Rev.Chim.(Bucharest), 66, no. 10, 2015, p. 1687.20. GIRONÉS-VILAPLANA, A., MENA, P., GARCIA-VIGUERA, C.,MORENO, D.A., LWT - Food Sci. Technol., 47, 2012, p. 279.21. HERNANDEZ-HERRERO, J.A., FRUTOS, M.J., 2015, Food Chem.,173, 2015, p. 495

Manuscript received: 14.01.2016