344 Advances in Environmental Biology, 7(2): 344-348, 2013 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

Determination of Antioxidant Capacities and Biochemical Compounds of Berberis vulgaris L. Fruits

Muttalip Gundogdu

Yüzüncü Yıl University, Agriculture Faculty, Department of Agricultural Biotechnology, 65080 Van, Turkey

Muttalip Gundogdu, Determination of Antioxidant Capacities and Biochemical Compounds of Berberis vulgaris L. Fruits

ABSTRACT

Research on biochemical properties of Berberis vulgaris which has diverse usage areas has increased the popularity of this plant. In this study, antioxidant properties, organic acid contents and phenolic compound contents of Berberis vulgaris fruits naturally grown in Gevaş Region (Van Province/Turkey) were determined. With respect to organic acids, malic acid content was found as 1.862 g/kg, citric acid content as 1.253 g kg-1, tartaric acid content as 0.702 g kg-1 and succinic acid content as 0.086 g kg-1 in the study. The content of chlorogenic acid was the highest among phenolic compounds which was found as 0.752 g kg-1. The antioxidant capacity of the barberry fruits was determined as 8.731 µmol TE g-1 in the study. Due to their high antioxidant capacity and content of biochemical compounds, barberries are considered as valuable fruits.

Key words: Berberis vulgaris, antioxidant, organic acids, phenolic compounds.

Introductıon metal ions and hence, prevent their oxidation catalyzing effects [4,20]. Proportion of total acid Wild type barberries (Berberis vulgaris L.) in content to sugar content is an indicator of fruit Gevaş Region (Van Province/Turkey) are used in this maturity. Organic acid profile is also used for study. The barberry fruit belongs to Berberidaceae verification of purity [16,20]. Recent research have family and is native to Asia and Europe. The proven anticancer effects of some flavonoids and barberry is a thorny shrub that can grow to 1 – 3 m hence, there is an increasing demand for fruits [5,9,25,2,21,1]. Barberry fruits are pink in colour and containing antocyanidin and antocyanin [23]. about 10 mm in length. The plant is a long and red Phenolic compounds exist in fruits and coloured shrub. Turkey is one of the 5- 6 countries vegetables at varying levels, but they cause diverse where the plant is native. It contains alkaloids problems during the processing of these products including berbamine, berberine and berberrubine. (especially in fruit juice production). Phenolic Besides, it has antioxidant, antitumour and compounds have a determinant role in taste antibacterial activities [10,11]. Berberis vulgaris formation and they contribute to astringency and fruits are essential components of human nutrition. bitterness. Anthocyanins are phenolic compounds The roots of the fruit are used to improve appetite, that are responsible for colour formation of fruits and stimulate immune systemi and lower fever and are vegetables. Phenolic compounds have an important generally marketed with the name "amberparis role in fruit juice processing industry since they roots". Due to its appealing flowers and fruits, it is increase the turbidity and sedimentation of drinks grown for ornament. The fruits of many Barberry such as fruit juices and wines [8]. species are important nutrients for birds. Sometimes Anatolia is an important region for its rich the fruits are used in making jam and marmalade. biodiversity and fruit gene resources due to the The roots produce a yellow dye and the hard yellow advantages brought by ecological factors, woods are used in making furniture [3]. The topographical structure and geopolitical significance. antioxidant properties, organic acid and vitamin Anatolia is particularly important for the content of the plant are effective in treatment of investigation and sustainment of wild fruit types, various health problems [13]. about which limited number of research exists. Since acids in fruits are oxidized quickly in Recent research has suggested that Berberis vulgaris human metabolism,they do not have any negative fruits have an important role for human health and effects on human body. The fruit salts have alkaline nutrition. Due to the limited number of studies on properties and have important role in human nutrition biochemical properties of Berberis vulgaris, the fruit [20]. Organic acids form complexes with heavy is not well recognized among people. Hence, this Corresponding Author Muttalip Gundogdu, Yüzüncü Yıl University, Agriculture Faculty, Department of Agricultural Biotechnology, 65080 Van, Turkey Tel.: +90 432 2251331; E-mail : [email protected] 345 Adv. Environ. Biol., 7(2): 344-348, 2013 study is deemed important for contributing to the Extraction and determination of phenolic popularity of the fruit as well as to the world compounds: literature. In this study, antioxidant properties, organic acid contents and phenolic compound The phenolic compounds were determined using contents of Berberis vulgaris fruits naturally grown the HPLC separation method described by in Gevaş Region (Van Province/Turkey) were Rodriguez-Delgado et al. [19]. About 500 g of determined. samples were fragmented and 25 g from each sample was transferred to centrifuge tubes. The samples Materials and Methods were mixed homogenously then diluted 1:1 with distilled water and centrifuged at 15,000×g for 15 Plant Material: min. The supernatantwaspassed through 0.45µm membrane filter (Millipore Millex-HV Hydrophilic The study was conducted in Gevaş Region of PVDF, Millipore, USA), then injected into HPLC Van Province, Turkey. Firstly, the spots of naturally system (gradient). The chromatographic separation in grown Berberis vulgaris fruits were identified. The Agilent 1100 series HPLC took place in DAD fruits were harvested at maturity phase. The study detector (Agilent, USA) with 250 mm×4.6 mm, 4 µm was performed in 2010-2011 period and the averages ODS column (HiChrom, USA). The following of two-year data were taken. The harvested fruits solvents in water with a flow rate of 1ml/min and were immediately taken into sample cups to prevent 20_l injection volume were used for spectral moisture loss, transferred to laboratory and stored at measurements at 254, and 280 nm: as mobile phase -20 C0 until analyses. The samples were subjected to solvent A, methanol–acetic acid–water (10:2:88) and analyses as quickly as possible. Solvent B, methanol–acetic acid–water (90:2:8).

Chemicals: Extraction and determination of total antioxidant activity: In the present study, chemicals with analytical purity were used. Organic acid standards (citric acid, For the standard trolox equivalent antioxidant tartaric acid, malic acid, succinic acid), phenolic acid capacity (TEAC) assay, TEAC extract was prepared: standards (gallic acid, catechin, caffeic, chlorogenic ABTS was dissolved in acetate buffer and prepared acid, o-coumaric acid, p-coumaric acid, ferulic acid, with potassium persulfate, as described by Rice- syringic, vanillic, quercetin, rutin, and pyrocatechin) Evans et al. [18] and Ozgen et al. [15]. The mixture were obtained from Sigma–Aldrich (St. Louis, MO, was diluted in acidic medium of 20mM sodium USA). The other chemicals were obtained from acetate buffer (pH 4.5) to an absorbance of Merck (Darmstadt, Germany). 0.700±0.01 at 734nm for longer stability [15]. For the spectrophotometric assay, 3ml of the ABTS+ Extraction and determination of organic acids: solution and 20 µl of fruit extract were mixed and incubated for 10 min and the absorbance was For organic acid extraction, the method by determined at 734 nm determined after 6 min from Bevilacqua and Califano (1989) was modified. About mixing. 500 g of samples was fragmented and 25 g from each sample was transferred to centrifuge tubes. The 10 Results and Dıscussıon ml of 0.009N H2SO4 was added to the samples and the samples were homogenized with Heidolph Silent Wild type barberries (Berberis vulgaris L.) exist Crusher M, Germany. Then, the samples were mixed in the study location, Gevaş Region (Van for an hour with a shaker (Heidolph Unimax 1010, Province/Turkey) which have not been subjected to Germany) and centrifuged at 15,000×g for 15 min. any agricultural procedures (fertilization, pruning, The supernatant were passed through coarse filter soil cultivation etc.). In this study, antioxidant paper, then twice in 0.45 µm membrane filter properties, organic acid contents and phenolic (Millipore Millex-HV Hydrophilic PVDF, Millipore, compound contents of Berberis vulgaris L. fruits USA), and last in the SEP-PAK C18 cartridge. The have been determined. In terms of organic acid concentration of organic acids was determined by content, malic acid was identified to be the HPLC using an Aminex column (HPX-87H, predominant organic acid with a content of 1.862 g 300mm×7.8mm, Bio-Rad) fitted on an Agilent 1100 kg-1. Malic acid was followed by citric acid with a series HPLC G 1322 A, Germany) (Bevilacqua and content of 1.253 g kg-1. Organic acid distribution of Califano, 1989). Organic acids were detected at 214 the examined Berberis vulgaris fruits was identified and 280nm wavelengths. As the mobile phase, as M>C>T>S (Table 1, Fig 1). In their study, 0.009N H2SO4 were passed through 0.45 µm filter Hanachi and SH [13] reported a malic acid content of membrane. 116.03 µg 100g-1. Organic acids are determinants in many physiological processes in fruits (taste formation, maturity etc.) and they are also important

346 Adv. Environ. Biol., 7(2): 344-348, 2013 for human health [7,20,12]. Proportion of total acid analyses, but can not totally be prevented. Therefore, content to sugar content is an indicator of fruit changes and reactions in fruit physiologies affect the maturity. The content level of acids determines the level of organic acid content. Additionally, species taste of fruits and while lower contents of acids are characteristics and environmental factors are also associated with sweet taste, higher contents of acids known to influence the level of organic acid content are associated with sour tastes in fruits. It should be [17]. kept in mind that loss of organic acids can be minimized during harvesting, preservation and

Table 1: TEAC and organic acids contents in Berberis vulgaris L. Organic acids ve TEAC Values* Malic acid (g/kg) 1.862 ± 0.055 Citric acid (g/kg) 1.253 ± 0.037 Tartaric acid (g/kg) 0.702 ± 0.024 Succinic acid (g/kg) 0.086 ± 0.008 TEAC (mmol TE/l) 8.731 ± 0.185 *Mean ± standart error of mean (n= 3).

Fig. 1: Chromatograms of organic acid content of the fruits of Berberis vulgaris. Peaks; 1: Citric acid, 2: Tartaric acid, 3: Malic acid, 4: Succinic acid.

The antioxidant capacity of the barberry fruits be the predominant phenolic compound in Berberis was determined as 8.731 mmol TE l-1 in the study. In vulgaris fruits. The phenolic compound distribution their study, Zovko Koncic et al. [26] determined the of the examined fruits was identified as antioxidant contents of Crni Lug originated Berberis Cl>Ca>G>Caf>R>O-C>S>P>V>F>P-C>Q (Table 2, vulgaris fruit by DPPH and antioxidant contents of Fig 2). the roots, branches and leaves were found as 1292.86 In a study conducted in Seydişehir Region µg ml-1, 691.57 µg ml-1 and 65.09 µg ml-1, (Konya Province/Turkey), the total phenolic respectively. Surweswaran et al. [22], reported that compound content of Berberis vulgaris fruits was Berberis aristata roots contain 3.59 mmol 100g-1 reported as 789.32 mg 100g-1 and total antocyanin antioxidants. This study suggested that antioxidant content was reported as 931.05 mg kg-1 [1]. In their content of Berberis vulgaris fruits is significantly study on Skard originated Berberis vulgaris fruits, higher compared to that of other fruits. Zovko Koncic et al. [26] determined total phenolic With respect to phenolic compounds, Berberis compound contents of the roots, shoots and leaves as vulgaris fruits were found to contain 0.752 g kg-1 10.34 mg g-1, 12.53 mg g-1 and 52.54 mg g-1, chlorogenic acid, 0.218 g kg-1 catechin and 0.132 g respectively. Polyphenol oxidase (PPO) catalyzes kg-1 gallic acid. Chlorogenic acid was determined to oxidation of phenolic compounds which results in

347 Adv. Environ. Biol., 7(2): 344-348, 2013 darkening of fruits and vegetables [8]. Although and storage of products, which is explained by phenolics and antocyanins are sources of natural temperature, pH, oxygen and water activity antioxidants, they are not totally stable in processing [14,24,1].

Table 2: Phenolic compounds contents in Berberis vulgaris L. (g/kg). Phenolic compounds Values* Phenolic compounds Values* Gallic acid 0.132 ± 0.011 Ferulic acid 0.020 ± 0.001 Catechin 0.218 ± 0.017 O-coumaric 0.042 ± 0.001 Chlorogenic acid 0.752 ± 0.027 Prothocatechuic 0.023 ± 0.001 Caffeic 0.095 ± 0.001 Vanillic 0.022 ± 0.001 Syringic 0.032 ± 0.001 Rutin 0.081 ± 0.001 P-coumaric 0.014 ± 0.000 Quercetin 0.011 ± 0.000 *Mean ± standart error of mean (n= 3).

Fig. 2: Chromatograms of phenolic compounds content of the fruits of Berberis vulgaris. Peaks; 1: Gallic, 2: Protocatechuic, 3:Catechin, 4: Chlorogenic, 5: Vanillic, 6: Caffeic, 7: Syringic, 8: P-coumaric, 9: Feulic, 10: Rutin, 11: O-coumaric, 12: Quercetin.

Conclusıon: suggest that Berberis vulgaris fruits have an important potential for their antioxidant activities. Organic acids and phenolic compounds are Therefore, wider recognition and consumption of this determinants in many physiological activities in fruit will have benefits for human health. Many fruits. Phenolic compounds are particularly important studies on this fruit have rather focused on the for human health and nutrition due to their determination of total phenolic and acid contents. antioxidant properties. The findings of the study This study, on the other hand, aimed to specifically

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