Extraction and determination of total polyphenols and antioxidant capacity of red coffee (Coffea arabica L.) pulp of wet processing plants

Abstract

Background

Coffee waste disposal from wet processing units is increasing with an increase in coffee consumption throughout the world. This not only has a limitation in terms of environmental pollution, but also high waste volume is an opportunity to extract essential bioactive compounds and antioxidants for food and pharmaceutical industries. The aim of this paper was to investigate the effects of extraction solvents and coffee cherry pulps of different varieties in terms of their yield potential for total polyphenols and antioxidant capacity.

Results

Results show that coffee cherry pulps from different varieties extracted by different solvents showed significant effects. Among the studied varieties, coffee cherry pulp from Ababuna variety was found to be superior in total polyphenols (1809.9 mgGAE/gm) content and antioxidant capacity (70 % inhibition). This value was followed by pulp from the Dessu variety. But the pulp from variety 741, extracted using aqueous ethanol solvent, showed inferior results for both total polyphenol content and antioxidant capacity. Among the solvents studied in this work, aqueous methanol responded better for pulps of all varieties except Dessu which responded better for aqueous acetone solvent.

Conclusions

Red coffee cherry pulp discarded as a waste from wet coffee-processing plants can be used as a raw material to extract important value-added products like polyphenols and other antioxidants. These compounds can be used as biological additives in food and pharmaceutical industries to fight chronic diseases and aging. Furthermore, the study shows the possibility of extracting value-added bioactive compounds from biological wastes.[Figure not available: see fulltext.]

Geremu et al. Chem. Biol. Technol. Agric. (2016) 3:25 DOI 10.1186/s40538-016-0077-1

Extraction anddetermination oftotal polyphenols andantioxidant capacity ofred coee (Coea arabica L.) pulp ofwet processing plants

Melkayo Geremu1, Yetenayet Bekele Tola1* and Abrar Sualeh2

Background

Bioactive compounds are value-added products, justifying their isolation from industrial wastes [1]. These residues, in fact, could be an alternative source for obtaining natural antioxidants, which are considered completely safe in comparison with synthetic antioxidants [2, 3]. Coee is one of the copious beverages, and the worldwide production is over 105million tons annually [4]. Industrial processing of coee cherry is done to isolate coee beans by removing pulp and mucilaginous parts from

cherries. To get beans for roasting, coee is subjected to wet (washed) and dry (unwashed) processing. In the former case, red coee cherry pulp is removed mechanically within a short period of harvesting time to produce better quality coee beans. For every 2tons of coee cherries processed, nearly 1 ton pulp is generated [5]. Most of the coee pulp generated during wet processing goes without any treatment directly to huge waste disposal sites or river streams. Toxic compounds from disposed fermenting cherries leach to the environment and pollute sources of water in the coee-producing regions. Such ways of waste disposal have been the major environmental health challenges in coee-growing and -processing

*Correspondence: [e-mail]; [e-mail]

1 Jimma University College of Agriculture and Veterinary Medicine, PO. BOX 307, Jimma, EthiopiaFull list of author information is available at the end of the article

2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/

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Web End =publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.

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areas. Moreover, such unsafe ways of waste disposal have greatly aected the terrestrial and aquatic biota [6].

So far, dierent attempts have been made to use the pulp from wet processing plants. However, a study showed that coee pulp cannot be used as an animal feed, mainly due to its toxic components such as caffeine, mineral salts, amino acids, tannins, phenols and other polyphenols [7]. On the contrary, it is used to make compost to enrich soil fertility or as a mulch material to prevent evaporation of moisture and to control weed growth. New aspects concerning the use of this waste as a raw material to extract important bioactive components is the other aspect to minimize environmental pollution through the value addition process. Because of their high value nature and less cost of extraction methods, extraction and use of bioactive compounds from waste pulp as a food additive are gaining interest [8, 9]. Coee pulp as a by-product contains associated bioactive compounds like polyphenols with good antioxidant properties, which endow additional health benets. Studies [10, 11] also indicated the possibility of extraction of total polyphenol compounds and antioxidants from coee pulp to manage coee waste. However, the eect of varieties and extraction solvents on the total polyphenols and antioxidant capacity has not yet been studied. Therefore, the study aimed to investigate the eect of variety and extraction solvents on total polyphenols and antioxidants capacity of coee cherry pulp of wet processing stations.

Methods

Experimental materials andsample preparation

Coee pulp samples from Ababuna (hybrid of 741Dessu), pure line varieties of 741, Dessu and 74110 were obtained at full maturity and ripening stages from

Jimma Agricultural Research Center, Ethiopia. Ababuna was included purposefully for two reasons. First, due to its high yielding capacity, coee cherry pulp from this variety will be high in the future during massive production. The second reason was to investigate the combined eect of the two lines on the total polyphenol and antioxidant capacity of cherry pulp from the hybrid. Before the pulping process, coee cherries were sorted to remove over-ripe, deteriorated and bruised ones. Pulping was done on the same date of harvesting using a manually operated coee pulper. The pulp was drained of excess water, blotted on tissue paper and stored at 18C for further investigation.

Extraction ofthe sample

The coee cherry pulp sample was extracted according to Saewan etal. [12] and Jagtap and Bapat, [13]. For extraction purpose, three solvents (aqueous 80 % methanol, 80% acetone and 80% ethanol) were used. Teng coee pulp was mixed with 100ml of the respective solvent and

ground for 3 min in a homogenizer and transferred to conical ask. The ground samples were extracted using the maceration technique by soaking the samples in the solvents for 24 h at room temperature, followed by ltration using Whatman No. 1 lter paper. The ltered extract was used to determine the total polyphenol content and antioxidant capacity.

Determination oftotal polyphenol content

Total polyphenol content (TPC) of coee pulp extract was determined using the FolinCiocalteu method [14]. FolinCiocalteu reagent, a mixture of phosphotungstic (H3PW12O40) and phosphomolybdic (H3PMo12O40) acids, is reduced to blue oxides of tungsten (W8O23) and molybdenum (Mo8O23) during phenol oxidation. This reaction occurs under alkaline condition provided by sodium carbonate. The intensity of blue color reects the quantity of polyphenol compounds, which can be measured using a spectrophotometer [15]. Gallic acid was used as a standard and the total polyphenols were expressed as mg/g gallic acid equivalents (GAE) from the calibration curve (R2 = 0.996) using gallic acid. Gallic acid (0.5 g)

was accurately weighed into a 10 ml volumetric ask, dissolved in 10 ml absolute methanol and the solution was made up to 100ml with 80% of the same solvent. For standard curve 0, 1, 2, 3, 4 and 5ml of standard solutions were added into a 100ml ask and diluted to give 0, 50, 100, 150, 400 and 500mg/l of gallic acid. Then, 0.5ml of each sample was introduced into test tubes and mixed with 2.5 ml of a tenfold dilute FolinCiocalteu reagent and 2ml of 7.5% sodium carbonate. The tubes were covered with aluminum foil and allowed to stand for 30min at room temperature before the absorbance was read at 765 nm using UV/Vis spectrophotometer (T80, China). The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained.

Determination oftotal antioxidant capacity

Free radical scavenging assay or DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was done as per the method of Hemalatha et al. [16] with minor modication. One ml solvent extract of the samples was taken in a test tube and 2ml of 0.1mM DPPH was added. The mixture was shaken well and incubated at room temperature in the dark for 30min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517nm (UVVis spectrophotometer, T80, China). All the experiments were performed in triplicate and the meanSE values were reported. The scavenging activity was calculated from the control sample using the following equation:

Radical scavenging activity (%) =

~Ac At/As Ac

100,

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where Ac=the absorbance of the control, At=the absorbance of the test solution, As=the absorbance of the standard solution, and the IC50 value was also calculated from the graph of the percentage DPPH free radical scavenging activity versus concentrations of the samples.

Experimental design anddata analysis

The experiment was laid out in two-factors factorial arrangement in complete randomized design in triplicate. Factors were coee cherry pulp of four varieties and three types of extraction solvents as indicated above. ANOVA of the collected data were analyzed using Minitab computer software program (Version 16.0, Inc. Pennsylvania, USA). Means separation was carried out using Tukeys test for means showing signicant dierence at p<0.05.

Results anddiscussion

Total polyphenol content ofcoee cherry pulp

Polyphenols are secondary metabolites that are produced by plants to protect themselves against plant diseases and insects, but they play important roles in human health in protecting against a number of diseases related to oxidative stress and free radical-induced damage [17]. In this study, considerable variation (p 0.001) was observed among the type of solvents used and pulps tested. The amount of total polyphenols recovered from dierent coee pulp extracts varied from 1809.9 to 489.5mg GAE/g. Coee pulp from Ababuna exhibited the highest polyphenol (1809.9 GAE/g) content when its cherry pulp was extracted with 80% methanol. However, the lowest (489.5mg GAE/g) value was obtained from a cherry pulp of 741 extracted with 80% ethanol. When pulp sources were compared, Ababuna responded better to all solvent types as compared to other varieties except for the considerable eect of acetone on Dessu (Table1). Since Ababuna is a hybrid of 741 and Dessu, the heterosis eect resulted in creating not only high-yielding hybrid vigor variety, but also superior variety in terms of total polyphenol content. However, the two pure parent lines as well as genotype 74110 and 741 exhibited lower polyphenol values, except Dessu (Table1). This result is also in agreement with Gorinstein et al. [18] who reported that the total phenol content and antioxidant potential were signicantly higher in grapefruits hybrid than parent lines. As it is known in plant pathology, plants mainly produce phenolic compounds as a means to defend themselves against diseases and insect pests [19]. Therefore, disease or stress resistance of the hybrid may be associated with such type of metabolic boost which has a great signicance in improving the bioactive content of plants for the benet of human health.

When extraction solvents are considered, they show a signicant eect on the yield of total polyphenols

extracted. Methanol in all cases except acetone for Dessu showed a signicant eect in extracting better total polyphenol yield. The total polyphenol content of the methanol extracts varied from 900.6 mg GAE/g (741) (wb) to 1809.9 mg GAE/g (Ababuna) (Table 1). This is in agreement with Siddiq et al. [20]; they reported that 80% methanol is known to be an efficient and widely used solvent system to extract natural antioxidative components, especially phenolics, from plant materials. This might be because the methanol water mixture has high polarity and thus has greater efficacy toward extraction of polar phytochemicals such as phenolics and avonoids. Furthermore, methanol is the most suitable solvent in the extraction of polyphenol compounds due to its ability to inhibit the reaction of polyphenol oxidase that causes the oxidation of phenolics and its ease of evaporation compared to water [21]. Turkmen et al. [22] also reported that solvents with dierent polarity had signicant eect on polyphenol content and antioxidant activity. Similar to Ababuna, 74110 and 741 showed better extraction of total polyphenols when treated with the same solvent. However, Dessu responded better when it was treated with 80% acetone. This shows that methanol might not serve as a good extraction solvent for all varieties, and hence varietal preference for suitable type of solvent should be checked for better extraction efficiency. But from the current study, considering the concentration of total polyphenols extracted, the extraction efficiency of methanol>acetone>ethanol.

For the purpose of this study, polyphenol yield potential (in percent) of pulps and extraction potential of solvents were compared after taking Ababuna and methanol as a standard pulp variety and solvent type, respectively. As indicated in Table1 (column 3), 741 showed the lowest polyphenol yield potential, even though it has a significant boosting eect when combined with Dessu. Variety 74110 exhibited almost the same result with 741, but Dessu performed better than these two varieties. Therefore, a hybridization program of coee not only resulted in better hybrid vigor varieties for quantitative traits like yield, but also oers an opportunity to extract bioactive components from wastes to use in the food and pharmaceutical industries.

Antioxidant activity ofcoee cherry pulp

An antioxidant is a molecule stable enough to donate an electron to a rampaging free radical and neutralize it, thus reducing its capacity for cell damage. These anti-oxidants delay or inhibit cellular damage mainly through their free radical scavenging property [23]. The DPPH scavenging assay was done for all four coee cherry pulps and three solvent extracts, taking ascorbic acid as

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Table 1 Interaction eect ofvariety andsolvents onpolyphenol contents ofcoee cherry pulp andpercent yield

Types ofvariety as source ofpulp

Types ofextraction solvent

Total polyphenol content (mg GAE/g)*SE

Total polyphenols (%) ofvarieties ascompared toAbabuna taking methanol asa standard solvent

Ababuna Acetone 1434.3 3.49d 79.2**

Ethanol 1603.8 3.04c 88.6 Methanol 1809.9 1.33a 100.0

Average 1616 89.3

Dessu Acetone 1709.0 1.02b 94.4

Ethanol 1448.4 1.01d 80.0 Methanol 929.1 2.64f 51.3

Average 1362.2 75.3

74110 Acetone 662.1 0.65i 36.6

Ethanol 664.8 0.99i 36.7 Methanol 993.5 0.68e 54.9

Average 773.5 42.7

741 Acetone 707.4 1.32h 39.1

Ethanol 489.5 3.98j 27.0 Methanol 900.6 3.68g 49.8

Average 699.2 38.6

CV (%) 8.8

*Mean values followed by the same letter in the column are not signicantly dierent at p<0.05

**Values calculated taking 1809.9 of Ababuna and methanol as a base to compare with others

a standard antioxidant. The interaction eect of solvents and coee pulps showed highly signicant (p < 0.001) eects on DPPH scavenging capacity. The percentage of DPPH free radical scavenging activity of dierent types of coee pulps as aected by the type of extracting solvent is shown in Fig. 1. In the methanolic extract, the scavenging activity ranged from 17.3 % (741) to 70.2 % (Ababuna). In the acetone extract, the scavenging activity ranged from 9.8% (74110) to 50% (Dessu). In the ethanolic extract, the scavenging activity ranged from 7.5% (741) to 37.3 (Ababuna).

The methanol extract from the hybrid of Ababuna showed the highest antioxidant activity (70.2%), which is in line with its high polyphenol content. This shows the potency of the extract from the hybrid for use as a good source of antioxidants. The lowest scavenging activity (7.5 and 10%) was measured from 80% ethanol extract of 741 and 80% acetone extract of 74110 varieties. However, the scavenging activity of the standard (ascorbic acid) (93%) was higher as compared to the dierent coffee pulp extracts. The results of this study are also in line with the reports of Murthy and Naidu [4], who reported the percentage free radical scavenging activity of coee pulp to be 65%. The discrepancy might be due to the difference in the type of coee pulp and extraction solvent used, since they used aqueous iso-propanol (60%) as the extraction solvent.

The extracts that perform the highest antioxidant activity (Fig.1) have the highest concentration of polyphenols (Table1). Polyphenols are very important plant constituents, because of their scavenging ability on free radicals due to their hydroxyl groups. This is in good agreement with Sultana and Anwar [24] who reported that, with the increase in concentration of polyphenol compounds, DPPH free radical scavenging capacity increases and thus antioxidant activity increases. Therefore, the polyphenol content of plants may contribute directly to their anti-oxidant action [25]. Another case extract from the Dessu variety showed higher percentage of radical scavenging

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Table 2 Interaction eect of variety and solvents on the DPPH scavenging assay IC50 value (mg/ml)

Variety Solvents IC50 value

(mg/ml)*SE

Ababuna Acetone 4.4 0.05c

Ethanol 2.4 0.05b Methanol 0.4 0.01a

Dessu Acetone 1.1 0.03ab

Ethanol 4.1 0.17c Methanol 8.4 0.72e 74110 Acetone 15.8 0.10h

Ethanol 15.4 0.16h Methanol 6.2 0.39d 741 Acetone 13.6 0.11g

Ethanol 22.6 0.16i Methanol 11.3 0.11f

CV (%) 5

*Mean values followed by the same letter in the column are not signicantly dierent at p<0.05

activity in acetone. This is also in accordance with other studies [26, 27] that reported that 80% acetone was the most suitable solvent for the extraction of DPPH scavenging compounds from barley and legumes.

The inhibition concentration (IC50) value for the DPPH scavenging assay was calculated for all the four solvent extracts of coee pulps. IC50 value is dened as the concentration of antioxidant required for 50% scavenging of DPPH radicals. It is a parameter widely used to measure antioxidant activity of biological and non-biological compounds. A smaller IC50 value corresponds to a higher antioxidant activity of the plant extract [28], since it requires less concentration to achieve the same value. In the present study, the lowest IC50 values of 0.

4 mg/ml was obtained when a hybrid of Ababuna was extracted with methanol, which indicated its powerful free radical scavenging ability followed by the acetone extract of the Dessu pulp (1.1mg/ml) (Table2). According to this study, to achieve 50% equal inhibition of free radicals, the required concentration by 74110 and 741 is 15.5 and 28.3 times more as compared to the extract of Ababuna, respectively. The highest IC50 value (22.6mg/

ml) was obtained when 741 was extracted with ethanol, which indicated its least potent antioxidant activity. Due to higher values of total polyphenols and antioxidant activity of methanolic extracts except acetone in the case of Dessu, the lowest IC50 value was also estimated when methanol was used as a solvent. Azlim Almey and others [29] showed that the dierence in IC50 values between methanolic and ethanolic extracts might be due to the intrinsic properties of plant compounds and their interaction with the solvents used. Even though a methanolic

extract of Ababuna showed higher antioxidant activity, however, statistically, it was not dierent (p>0.05) from the acetone extract of Dessu. In this study, the IC50 values of cherry pulp from hybrid and other coee varieties has been determined for the rst time, and hence no literature data are available to compare our results with information from other literature.

Conclusions

Coee is one of the hot beverages consumed copiously every day throughout the world by millions of people. Even though the quality coee is determined by the type of coee variety, premium quality coee comes from washed coee of wet processing units than unwashed coee from dry processing methods. However, coee cherry pulping, fermentation and washing of parchment in the wet processing method discharges million tons of fresh coee cherry pulp to the environment. But as a waste utilization strategy and value addition process, pulps discarded into the environment can be used to extract valuable ingredients for use as an additive in the food and pharmaceutical industries. The current study showed that total polyphenols, antioxidant capacity and free radical inhibition concentration varied among coffee pulps from dierent varieties and the type of solvent used for extraction. Waste pulp from Ababuna exhibited better total polyphenol content, antioxidant capacity and IC50 value when 80% methanol was used as an extraction solvent. Among solvent types, the same solvent showed better extraction efficiency for all pulps except Dessu. Therefore, extraction of such type of valuable bioactive compounds from coee pulp discarded as a waste could add value to improve the economic value of coee pulp waste. Furthermore, coee cherry pulp can be used as an alternative source to extract polyphenols and antioxidant compounds for the benet of human health.

Authors contributions

MG carried out most of the research works and wrote the paper, YBT was involved in developing the concept and part of the research activities as well as in editing the manuscript, AS was involved in facilitating research work and editing the manuscript. All authors read and approved the nal manuscript.

Author details

1 Jimma University College of Agriculture and Veterinary Medicine, PO. BOX 307, Jimma, Ethiopia. 2 Ethiopian Institute of Agricultural Research, Jimma Agricultural Research Center, POX 192, Jimma, Ethiopia.

Acknowledgements

The authors thank the Jimma Agricultural Research Center for supplying coee pulps and the Jimma University College of Agriculture and Veterinary Medicine of Ethiopia and PHMIL project of Global Aairs of Canada for nancial support to conduct this research.

Competing interests

The authors declare that they have no competing interests.

Received: 15 May 2016 Accepted: 7 July 2016

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References

1. Moure A, Cruz JM, Franco D, Dominguez JM, Siniero JMJ, Dominguez H, et al. Natural antioxidants from residual sources. Food Chem. 2001;72:1458.

2. Chen C, Pearson AM, Gray JI. Eects of synthetic antioxidants (BHA, BHT and PG) on the mutagenicity of IQ-like compounds. Food Chem. 1992;43(3):17783.

3. Kahl R, Kappus H. Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E. Zeitschrift fur Lebensmittel-untersuchung und-forschung. 1993;196(4):32938.

4. Murthy PS, Naidu MM. Recovery of polyphenols antioxidants and functional compounds from coee industry by-products. Food Bioprocess Technol. 2012;5(3):897903.

5. Roussos S, Aquihuatl MA, Trejo-Hernndez MR, Perraud IG, Favela E, Ramakrishma M, Raimbault M, Viniegragonzlez G. Biotechnological management of coee pulp-isolation, screening, characterization, selection of caeine-degrading fungi and natural microora present in coee pulp and husk. Appl Microbiol Biotech. 1995;42:75662.

6. Navia PDP, Velasco MRDJ, Hoyos CJL. Production and evaluation of ethanol from coee processing by-products. Vitae. 2011;18(3):28794.

7. Braham JF, Bressani S. Coee pulp: composition, technology and utilization. International Development Research Center, Ottawa, Canada. 1979: IDRC-108e.

8. Oreopoulou V, Tzia C. Utilization of plant by-products for the recovery of proteins, dietary bers, antioxidants, and colorants. In: Oreopoulou V, Russ W, editors. Utilization of by-products and treatment of waste in the food industry. New York: Springer; 2007. p. 20932.

9. Arvanitoyannis IS, Tserkezou P. Wheat, barley and oat waste: a comparative and critical presentation of methods and potential uses of treated waste. Int J Food Sci Technol. 2008;43(4):694725.

10. Torres-Mancera MT, Cordova-Lpez J, Rodrguez-Serrano G, Roussos S, Ramrez-Coronel MA, Favela-Torres E, Saucedo-Castaeda G. Enzymatic extraction of hydroxycinnamic acids from coee pulp. Food Technol Biotechnol. 2011;49:36973.

11. Pushpa S, Murthy MR, Manjunatha G, Sulochannama M. Extraction, characterization and bioactivity of coee anthocyanins. Eur J Biol Sci. 2012;4(1):139.

12. Saewan N, Atisakul K, Mana T, Jimtaisong A, Kittigowittana K. Antioxidant capacity of dierent levels of ripeness and parts of Coea arabica extracts. Pure and applied chemistry international conference. 2012.

13. Jagtap UB, Bapat VA. Antioxidant activities of various solvent extracts of custard apple (Annona squamosa L.) fruit pulp. Nutrafoods. 2012;11(4):13744.

14. Singleton VL, Orthofor R, Raventos RML. Analysis of total phenols and other oxidation substrates and antioxidants by means of FolinCiocaltau reagent. Methods Enzymol. 1999;299:15278.

15. Conforti F, Statti G, Uzunov D, Menichini F. Comparative chemical composition and antioxidant activities of wild and cultivated Laurus nobilis L. leaves and Foeniculum vulgare subsp. piperitum (Ucria) coutinho seeds. Biol Pharm Bull. 2006;29(10):205664.

16. Hemalatha S, Lalitha P, Arulpriya P. Antioxidant activities of the extracts of the aerial roots of Pothos aurea (Linden ex Andre). Der Pharma Chemica. 2010;2(6):849.

17. Vladimir-Kneevi S, Blaekovi B, Bival tefan M, Babac M. Plant polyphenols as antioxidants inuencing the human health. Phytochemicals as nutraceuticalsglobal approaches to their role in nutrition and health; 2012. p. 15580.

18. Gorinstein S, Zachwieja Z, Katrich E, Pawelzik E, Haruenkit R, Trakhtenberg S, Martin-Belloso O. Comparison of the contents of the main antioxidant compounds and the antioxidant activity of white grapefruit and his new hybrid. LWT-Food Sci Technol. 2004;37(3):33743.

19. Lattanzio V, Lattanzio VM, Cardinali A. Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects. Phytochem Adv Res. 2006;661:2367.

20. Siddiq A, Anwar F, Manzoor M, Fatima A. Antioxidant activity of dierent solvent extracts of Moringa oleifera leaves under accelerated storage of sunower oil. Asian J Plant Sci. 2005;4(6):6305.

21. Yao LH, Jiang YM, Datta N, Singanusong R, Liu X, Duan J, Raymont K, Lisle A, Xu Y. HPLC analyses of avanols and polyphenols acids in the fresh young shoots of tea (Camellia sinensis) grown in Australia. Food Chem. 2004;84(2):25363.

22. Turkmen N, Sari F, Velioglu YS. Eects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and FolinCiocalteu methods. Food Chem. 2006;99(4):83541.

23. Halliwell B, Murcia MA, Chirico S, Aruoma OI. Free radicals and antioxidants in food and in vivo: what they do and how they work. Crit Rev Food Sci Nutr. 1995;35(12):720.

24. Sultana B, Anwar F. Flavonols (kaempferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants. Food Chem. 2008;108(3):87984.

25. Tosun M, Ercisli S, Sengul M, Ozer H, Polat T, Ozturk E. Antioxidant properties and total phenolic content of eight Salvia species from Turkey. Biol Res. 2009;42(2):17581.

26. Zhao H, Dong J, Lu J, Chen J, Li Y, Shan L, Gu G. Eects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.). J Agric Food Chem. 2006;54(19):727786.

27. Xu BJ, Chang SKC. A comparative study on phenolic proles and antioxidant activities of legumes as aected by extraction solvents. J Food Sci. 2007;72(2):S15966.

28. Maisuthisakul P, Suttajit M, Pongsawatmanit R. Assessment of polyphenols content and free radical scavenging capacity of some Thai indigenous plants. Food Chem. 2007;100(4):140918.

29. Azlimalmey AA, Ahmedjalalkhan C, Syedzahir I, Mustaphasuleiman K, Aisyah MR, Kamarulrahim K. Total polyphenols content and primary anti-oxidant activity of methanolic and ethanolic extracts of aromatic plants leaves. Int Food Res J. 2010;17(4):107783.