Anal Chem Ind J, Volume: 18( 1)
Partial Purification of CAT and Study Anti-Oxidant Activity of Grape Seeds Vitis vinifera
- Farhan LO , Department of Chemistry, College of Science for Women, Baghdad University, Iraq, Tel: +964773644 3447; E-mail: [email protected]
Received: February 15, 2017; Accepted: February 11, 2018; Published: February 16, 2018
Citation: Farhan LO, Shwayel FW, Talal GK. Partial Purification of CAT and Study Antioxidant Activity of Grape Seeds Vitis vinifera. Anal Chem Ind J. 2018;18(1):130
Grape (Vitis vinifera L.) is the chief fruit crop in the world. It is one of the fruits which are grown from ancient time; Grapes contain about 80% water, so it is delicious, lowâÂÂcalorie snack or dessert. During making juice from a grape, high quantities of the grape pulp, seeds, and skin will be as food for animals. Hence, the study was aimed to Measure of MDA, TAC, CAT and Vit C in different types of grape, and Partial purification of CAT from Red Globe. TAC, MDA, Vitamin C, Total protein and cat had been measured in seven kinds (Christmas rose, Italia, emperor, Muscat of Alexandria, early Muscat, red Globe, and black rose) of grape seeds and cat enzyme was purified from Red Globe grape seeds. The results showed that the Christmas rose, red globe and black rose have the highest vitamin C content and Muscat of Alexandria has the lowest vitamin C content from this analysis. While Muscat of Alexandria has the highest MDA content and Emperor has the lowest MDA content from this analysis, and Early Muscat has the highest TAC content and Black Rose has the lowest TAC content from this analysis. In addition purification of Cat from Red Globe grape seeds by gel filtration Sephadex G-100 and EDTE-Sepharose.
Grape seeds; Antioxidants; MDA; Vit C
The compounds which are able to inhibit the oxidation of different biomolecules are known as Antioxidants. These Antioxidants help in repairing the damages caused to the body tissues caused by oxidation processes. Various studies have focused on importance of using antioxidants from natural sources so as to be used as healthy additives and potential antioxidant in our daily food intake habits .
There are two great groups of antioxidants in living cells: enzymatic antioxidants and non-enzymatic antioxidants. These groups are divided into a few divisions. The separation of enzymatic antioxidants will be essential and secondary enzymatic barriers . The essential resistance has three vital chemicals that stop the development of kill free radicals : glutathione peroxidase, which gives two electrons to make less peroxides by selenols arrangement and furthermore expels peroxides as potential substrates for the Fenton response; catalase, which transforms hydrogen peroxide into water and sub-atomic oxygen. Catalase is viewed as a standout amongst the most critical and productive cancer prevention agents which is known today, so one particle of catalase believers 6 billion atoms of hydrogen peroxide .
Catalase (EC 184.108.40.206) is one of the strongest catalysts known. The reactions of catalyses are important to life. Dismutation of two molecules of H2O2 is catatalysed by using this enzyme. This powerful oxidizing agent has potentially harmful to water and molecular oxygen. In plants, H2O2 that generated during mitochondrial electron transport can be scavenged by catalase .
Grape (Vitis vinifera) is one of the biggest fruit crops of the world grape seed contains about 40% fiber, 16% oil, 11% proteins, and 7% complex phenols, in addition to sugars, mineral salts, etc. In addition, grape seeds have rich sources of monomeric phenolic compounds, like (+) Catechins, (-) Epicatechin, (-) Epicatechin-3-o-gallate, and dimeric, trimeric and tetrameric procyanidins, which contain antimutagenic and antiviral effects .
Several flavonoids with a phenolic nature are found in functional ingredients of grape seeds. These flavonoids include monomeric flavanols, dimeric, trimeric and polymeric procyanidins, and phenolic acids . Various cancer types, cardiovascular diseases and several dermal disorders can be treated with the antioxidant activity of grape seeds phenolic compounds because they are closely associated with this activity [7,8].
The objective of this study to measure of MDA, TAC, CAT and Vit C in different type of grape, and partial purification of CAT from grape seeds.
Materials and Methods
We collected seven kinds of grapes from local market in Baghdad in July 2016. We separated the peels from the seeds, then we dried the seeds by the air for one week, and we grinded the dried seeds by the blender, in this step the samples are ready to use.
Preparation of extracts
One gram of every specimen has been homogenized in a mortar and pestle with 5 mL super cold extraction buffer (0.1 M potassium phosphate buffer, Ph-7.8).
The homogenate has been centrifuged at 18000 g for 30 min at 4°C and after that supernatant has been sifted through paper. The supernatant part was utilized as an unrefined concentrate for the measure of antioxidant parameters and enzyme activity. The antioxidant capacity of each illustration was assessed by the strategy delineated by Benzieand Strain . Malondialdehyde (MDA) substance was controlled by the thiobarbituric destructive reaction . Protein obsession was controlled by the technique for Lowery . Vitamin c was measured in serum according to Wills, and. Wimalasiri strategy . The activity of Catalase (CAT) was settled at 25°C as said by Aebi . The reaction mix contained 40 mm H2O2 in a 50 mm phosphate 22 support pH-7.0, and 0.1 ml pure enzyme of 3 ml which is the total volume. Catalase (CAT) development was surveyed by lessened in absorbance of H2O2 at 240 nm.
Partially purified cat was done by gel filtration chromatography utilizing Sephadex G-75 segment. The section was stuffed to 1.0 × 90 cm in a glass segment and equilibrated with 0.1M phosphate cradle [Ph-7]. Total proteins and action of chemical were resolved . Dynamic division recognized for catalase movement by observing the abatement in A240. The parts were gathered and pooled for ensuing filtration in DEAE-cellulose section ( 2 × 10 cm) that had been equilibrated with phosphate Buffer (pH 7).
Results are imparted as mean values 7 S.D. Correlation of the method for three estimations utilizing a noteworthiness level of P<0.05 was achieved by one-route investigation of change (ANOVA) utilizing the State graphics Computer System, variant 5.1.
Results and Discussion
A sum of 7 sorts of grape seeds (Christmas rose, Italia, Emperor, Muscat of Alexandria, Early Muscat, Red Globe, and Black rose) were contemplated. TAC, MDA and Vitamin C are substance of the seven sorts of seeds grape are arranged in Table 1. The outcomes demonstrated that the Christmas rose, Red Globe and Black rose have the largest vitamin C substance and Muscat of Alexandria has the least vitamin C content from this investigation. While Muscat of Alexandria has the largest MDA substance and Emperor has the least MDA content from this analysis, and Early Muscat has the largest TAC substance and Black Rose has the least TAC content from this investigation. As shown in Table 1.
|Kind of seeds grape||TAC
[mean ± SD]
(umol/L) [mean ± SD]
[mean ± SD]
|Christmas rose||0.42 ± 0.029||0.40 ± 0.01||15.58 ± 0.03|
|Italia||0.47 ± 0.002||0.41± 0.02||11.50 ± 0.05|
|Emperor||0.44 ± 0.01||0.25 ± 0.01||8.25 ± 0. 13|
|Muscat of Alexandria||0.55 ± 0.01||0.54 ± 0.01||2.13 ± 0.01|
|Early Muscat||0.59 ± 0.01||0.35 ± 0.10||9.80 ± 0.15|
|Red Globe||0.33 ± 0.01||0.39 ± 0.01||14.95 ± 0.05|
|Black Rose||0.09 ± 0.011||0.34 ± 0.01||15.73 ± 0.27|
Table 1: Mean and standard deviation of TAC, MDA and Vitamin C from different kinds of seeds grape.
In the most recent decade, countless of assays are directed to amount total antioxidant capacity in nourishment and organic lattices. Each of them has its own attributes; there are variances in the free radical making framework, atomic target, end point, active, natural grid, home in lipo-and hydrophilic compartment and physiological significance. The impact of every single significant parameter can't be assessed utilizing just a single test convention .
It was proposed that the electron giving limit, mirroring the lessening force of bioactive mixes, is connected with cell reinforcement action. Antioxidants can be reductants, and inactivation of oxidants by reduction's can be portrayed as redox responses in which one response species types is decreased to the detriment of the oxidation of the other . Each value is replicated three times in the experiments [mean ± SD].
There are many reviews that arrangement with Antioxidants Parameters, for example, vitamin C content in Fruits and Vegetables sources, since report alludes to vitamin C which was taken from Cabbage it has got 17.416 (mg/10 g), 1.868 (mg/10 g) from Sour Orange and 4.337 from (mg/10 g) Orange [16 ].
Antioxidants help cells be safe from the harming effects of ROS, such as superoxide, singlet oxygen, hydrogen peroxide, hydroxyl radicals, peroxyl radicals, and peroxy nitrite. A few ROS, for example, superoxide and hydrogen peroxide, are commonly made in cells as by-consequences of biochemical reactions as indicating molecules .
ROS activity can be limited by antioxidant botanicals obviously. The dynamic constituents found in a few Antioxidant herbs which react clearly with ROS by scavenging and decreasing ROS activity . Catalase (EC.220.127.116.11) is a tetrameric heme containing enzyme found in every single vigorous living being is known to assume a key part in ensuring cells against oxidative stress .
There are many reviews that arrangement with Antioxidants Parameters, for example, CAT activity from various sources, since report alludes to Catalase activity which was taken from Jatropha curcas sources it has 173.2 (U/mg) and 26.03 for Centella asiatica. In vigorous life forms and plants, oxygen is a fundamental component yet it can be diminished and frame ROS to be a peril and restricting variable in development and advancement of plants . Catalase activity in various sort of seeds grape is given in Table 2. High catalase Specific activity has been seen in the seeds concentrate of Red Globe (0.192 Units/mg ) In perspective of its high Specific action in Red Globe, preparatory reviews have been done on the extractability, soundness and properties of the crude enzyme.
|Kind of seeds grape||Cat activity Unit/ml
[mean ± SD]
|Total protein (mg/ml)
[mean ± SD]
|24.63 ± 0. 631||153.075 ± 5.13||0.161|
|Italia||18.11± 0.28||105.06 ± 10.00||0.172|
|Emperor||14.41± 0.85||103.755 ± 8.04||0.138|
|Muscat of Alexandria||2. 78 ± 0.75||53.59 ± 7.52||0.051|
|Early Muscat||18.49 ± 0.15||105.12 ± 6.10||0.175|
|Red Globe||12.21± 1.155||104 ± 7.79||0.192|
|Black Rose||15.72 ± 0.62||126.10 ± 2.01||0.124|
Table 2: Mean and standard deviation of cat activity, total protein and Specific activity from different kinds of seeds grape.
Each value is replicated three times in the experiments [mean ± SD]. In this study catalase was purified from Red Globe grape. The data pertaining to the purification of catalase from Red Globe seeds has been summarized in Table 3.
|Purification step||Activity (Unit/ml)||Total
|Fold purification||Recovery (%)|
Table 3: Purification summary of catalase from Red Globe grape seeds.
The enzyme eluted as a solitary species types in the underlying Sephadex G-100 chromatography venture as demonstrated Fig. 1. Extra purging of the enzyme was accomplished by gel filtration on EDTE-Sepharose Fig. 2. Table 3 compresses the consequences of every progression of the catalase cleaning. The chemical was purified around 7.12- fold, with a last specific activity of 11.02 U/mg.
The peak specific activity of catalase was 11.02 U/mg was watched for Red Globe grape seeds. There was ascending in specific activity in every purification step . were watched the ascent of specific enzyme activity in each purification step Catalase is in partially purified from Banana Peels, and Van Apple [golden delicious], to 6.85, 8.7 crease with a yield of 40% and 11%, individually , to 6.85, 8.7 crease with a yield of 40% and 11%, individually [22,23].
On the premise of results acquired from various 7 sort of grape seeds (Christmas rose, Italia, head, Muscat of Alexandria, Early Muscat, red Globe and dark rose ) a decent wellspring of sound compound, red globe grape seeds great hotspot for catalase catalyst. These discoveries warrant additionally examines on the cleaning of cell reinforcement compound from different sorts of grape seeds on the grounds that the seeds of grape may helpful in the avoidance of sickness in which free radicals are involved.
- Msaada K, Jemia MB, Salem N, et al. Antioxidant activity of methanolic extracts from three coriander (Coriandrum sativum L.) fruit varieties. Arabian J Chem. 2013; 12(11).
- Carocho M, Ferreira IC. A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem Toxicol. 2013 ;51:15-25.
- Shebis Y, Iluz D, Kinel-Tahan Y, et al. Natural antioxidants: Function and sources. Food Nutrition Sci. 2013;4(6):643.
- Tayefi-Nasrabadi H, Dehghan G, Daeihassani B, et al. Some biochemical properties of catalase from safflower (Carthamus tinctorius L. cv. M-CC-190). Afr J Agric Res. 2011;6(23): 5221-6.
- Kim SY, Jeong SM, Park WP, et al. Effect of heating conditions of grape seeds on the antioxidant activity of grape seed extracts. Food Chem. 2006;97(3):472-9.
- Guendez R, Kallithraka S, Makris D. Determination of low molecular weight polyphenolic constituents in grape (Vitis vinifera sp.) seed extracts: Correlation with antiradical activity. Food Chem. 2005;89:1-9.
- Li H, Wang X, Li P, et al. Comparative study of antioxidant activity of grape (Vitis vinifera) seed powder assessed by different methods. J Food Drug Anal. 2008;16(6).
- Saleh N, Nadia H. The protective effect of grape seed extract on cardiotoxicity induced by doxorubicin drug in male rats. Advances in Bioscience and Biotechnology. 2014;5:1078-1089.
- IFF B, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Anal Biochem. 1996;239:70-6.
- Bailly C, Benamar A, Corbineau F. Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seed as related to deterioration during accelerated aging. Physiol Plant. 1996; 97:104-10.
- Lowery H. Rosebough protein measurement with the folin phenol. J Biol Chem. 1951;193: 265-75.
- Wills R, Wimalasiri P, Greenfield H. Liquid chromatography, microfluorometry, and dye-titration determination of vitamin C in fresh fruit and vegetables. Assoc Off Anal Chem. 1983;66:1377-9.
- Aebi H. Catalase in vitro. In: Methods in enzymology. Academic Press, USA. 1984;105:121-6.
- Pulido R, Saura-Calixto F. Calix to 1 contribution of beverages to the intake of lipophilic and hydrophilic antioxidants in the Spanish diet. Eur J Clin Nutr. 2003;57:1275-82.
- Aliyu AB, Ibrahim MA, Musa AM, et al. Free radical scavenging and total antioxidant capacity of root extracts of Anchomanes difformis Engl (Araceae). Acta Pol Pharm. 2013;70(1):115-21.
- Mohammed QY, Hamad WM, Mohammed EK. Spectrophotometric determination of total Vitamin C in some fruits and vegetables at Koya Area?Kurdistan Region/Iraq. J Kirkuk Univ?Sci Stud. 2009;4.
- Al-Sowayan NS, Mahmoud NH. The protective effect of grape seed extract on cardiotoxicity induced by doxorubicin drug in male rats. Advances in Bioscience and Biotechnology. 2014;5(14):1078-89.
- Stangl V, Lorenz M, Stangl K. The role of tea and tea flavonoids in cardiovascular health. Molecular nutrition and food research. 2006;50(2):218-28.
- Chaudière J, Ferrari-Iliou R. Intracellular antioxidants: From chemical to biochemical mechanisms. Food Chem Toxicol. 1999;37(9):949-62.
- Madhavi DL, Deshpande SS, Salunkhe DK. Food antioxidants: Technological, toxicological and health perspectives. CRC Press. 1995.
- Loewen PC, Switala J, Smolenski M, et al. Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli. Biochem Cell Biol. 1990;68(7):1037-44.
- Farhan LO, Mustafa SA, Hameed AS. Partial purification and characterization of catalase from banana peels. 2016.
- Kim L, Eum D, Yoon WC. Human liver catalase cloning, expression and characterization of monoclonal antibodies. Mol cells. 2003;15:381-86.