Scholarly article on topic 'Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower'

Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Wesen Adel Mehdi, Faridah Yusof, Layla O. Farhan, Atheer Awad Mehde, Raha Ahmed Raus

Abstract Objective The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower. The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources. In the present work, we study several biochemical parameters in the pulp and peel of sunflower. Methods Pulp and peel of sunflower was extracted, antioxidant enzymes and non-enzymatic antioxidant were measured. Alkaline protease was measured and purified from pulp in sunflower. Results High carbohydrate concentration, beta-carotene, catalase and ascorbate peroxidase activities, free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower. Whereas, MDA and ceruloplasmin activities were high in the pulp of sunflower. Conclusions The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses. Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

Academic research paper on topic "Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower"

Accepted Manuscript

Levels of Antioxidant Enzymes and Alkaline Protease from Pulp and Peel of Sunflower

Wesen Adel Mehdi, Faridah Yusof, Layla O. Farhan, Atheer Awad Mehde, Raha Ahmed Raus

PII: S2221-1691(17)30492-6

DOI: 10.1016/j.apjtb.2017.05.002

Reference: APJTB 487

To appear in: Asian Pacific Journal of Tropical Biomedicine

Received Date: 7 May 2016

Revised Date: 30 June 2016

Accepted Date: 2 January 2017

Please cite this article as: Mehdi WA, Yusof F, Farhan LO, Mehde AA, Raus RA, Levels of Antioxidant Enzymes and Alkaline Protease from Pulp and Peel of Sunflower, Asian Pacific Journal of Tropical Biomedicine (2017), doi: 10.1016/j.apjtb.2017.05.002.

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Title: Levels of Antioxidant Enzymes and Alkaline Protease from Pulp and Peel of Sunflower

Authors: Wesen Adel Mehdi1, Faridah Yusof2, Layla O. Farhan1, Atheer Awad Mehde2'3*, Raha Ahmed Raus2

Affiliations:

'Department of Chemistry, College of Sciences for Women, University of Baghdad, 10071, Iraq

2Department of Biotechnology Engineering, College of Engineering, International Islamic University Malaysia, 50728 Kuala Lumpur

3Department of Technical medical Analysis ,College of Health and Medical Technology, Middle Technical University, Baghdad, 10047, Iraq

Keywords:

Pulp sunflower

Antioxidant enzymes

Free radical scavenging capacity

Free flavonoid content Alkaline protease

*Corresponding author: Atheer Awad Mehde, PhD, Department of Biotechnology Engineering, College of Engineering, International Islamic, University Malaysia. Department of Technical medical Analysis, College of Health and Medical Technology, Middle Technical University, Baghdad, Iraq.

E-mail: atheerawod74@gmail.com The journal implements double-blind peer review practiced by specially invited international editorial board members.

This manuscript included 2 tables and 4 figures.

Article history: Received 7 May 2016

Received in revised form 30 Jun 2016

Accepted 2 Jan 2017

Available online

ABSTRACT

Objective: The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower. The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources. In the present work, we study several biochemical parameters in the pulp and peel of sunflower.

Methods: Pulp and peel of sunflower was extracted, antioxidant enzymes and non-enzymatic antioxidant were measured. alkaline protease was measured and purified from pulp in sunflower.

Results: High carbohydrate concentration, beta-carotene, catalase and ascorbate peroxidase activities, free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower. Whereas, MDA and ceruloplasmin activities were high in the pulp of sunflower.

Conclusions: The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses. Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

1. Introduction

Sunflower (Helianthus annuus L.) is important for its oil. It is grown under dry land conditions and, several studies reported that substantial yield performance decreases under water stressed conditions[i]. It is grown mainly in many countries in the world especially in Russia, Argentina, China and France, which are the highest global producers[2].

Different condition causes several variations in plant metabolism like osmotic stress and formation of reactive oxygen species (ROS). The ROS production is removed by an antioxidant compounds and antioxidant enzymes like ascorbate peroxidase (APX) ,catalase (CAT) and superoxide dismutase (SOD)[3,4] .The ROS contribute mainly in the initiation and development of lipid peroxidation through induce oxidative stress[5]. In a cell enzyme SOD in the first line of defense against reactive oxygen species[6]. The SOD detoxifies superoxide anion to hydrogen peroxide and molecular oxygen by catalyzing the dismutation, which acts as a metalloprotein to work in the reactions in the mitochondria ,cytosoland chloroplasts[6].The 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical which has an unpaired valence electron at one atom of nitrogen[7].The DPPH has been widely used for antioxidant capacity screening and estimation due to its clear reaction mechanism, solvent compatibility and the technical simplicity of its assays which requires no special equipment[7]. Phenolics are compounds possessing aromatic rings with hydroxyl groups. When are in diet, these compounds provide health benefits associated with reduced risk of chronic disease[8].

Enzyme hydrolysis is commonly using for development of functional properties of food proteins[9]. Proteases are protein-digesting enzymes which are categorized depending on the optimal operating pH; neutral ,alkaline and acidic proteases, it can be found in all living creatures[io].Proteases play a main role in biotechnology and are generally used in the tanning industry, in bioremediation processes ,in the manufacturing of biological detergents, pharmaceutical industry and peptide synthesis[ii].

The objective of current study is to investigate several antioxidant parameters in peel and pulp of sunflower, as well

as to extract, purify and characterize of alkaline protease of pulp sunflower.

2. Materials and Methods

One gram sample was homogenized with 5 mL coldly extraction buffer (0.1 M potassium phosphate buffer, pH 7.8).The homogenate was separated by centrifugation at 4°C for 30 minute.Supernatant was used as a crude extract for measurement of several biochemical parameters.

The activity of catalase was measured according Pereira et al method[i2]. The H2O2 concentration was determined according to Rummun et al. [13]. Measurement of ascorbate peroxidase activity (APX) was conducted according to Nakano and Asada method[i4].The activity of ceruloplasmin oxidase was measured according the modified method of Rice[i5].Malondialdehyde (MDA) content was determined by Bailly et.al.[i6].Beta carotene concentration was measured according to Santra method[i7]. Estimation of total phenols content (TPC) using folin is based on the reaction between phenol and oxidizing agent phosphomolybdate[i8].

The free radical scavenging capacity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used to determine the antioxidant activity[8].Flavonoid contents in the extract was determined using colorimetric method[19].SOD activity [U/mg (protein)] was determined using the spectrophotometric method[20].

The reducing sugar was measured according to McDonald and Chen[21]. Measurement of fatty acid was determined according to Velikova et al[17]. The protease activity was measured according to McDonald and Chen[21]. Protein concentration was determined by Lowery method[22].

To purity alkaline protease from the crude extract, 50% saturation of ammonium sulphate were utilized and placed in ice bath at 0°C and kept at 4°C for overnight. The resulting suspension after ammonium sulphate precipitation was centrifuged at 10,000g for 30 min at 4°C,followed by dialysis in dialysis sac overnight at 4°C using

sodium bicarbonate. Gel filtration chromatography was used to partially purify protease. The column was packed with Sephadex G-100 (120X2) cm in a glass column and equilibrated with 0.1 M Tris-HCl buffers (pH 10)[23]. Enzyme activity and protein of purified alkaline protease were measured.

To characterize the enzyme, several independent experiments were carried out as the following. Alkaline protease reaction was conducted at optimum condition using different concentration of casein as a substrate [0.50, 0.10, 0.15, 0.25, 0.40, 0.50, 0.60, and 0.70] mg/mL. The data obtained were used to plot Lineweaver-Burk graph to calculate Km and Vmax values for alkaline protease.

The effect of temperature on enzyme was conducted by carrying out the enzyme reaction at different temperatures[25, 30, 35, 40, 45, 50, 55, 60, and 65]. The pH optimum was assessed by different pH[7,8,9,9.5,10,11,12]for alkaline protease . The activation energy (Ea.), also free energy (AG*), enthalpy (AH*), and entropy (AS*) of the transition state were determined. The thermodynamic factors of the transition state were calculated from Arrhenius plot.

Statistical analysis of data was performed by SPSS [version 21.0]. The significance difference between mean values was carried out by student T-Test.

3. Results

The results showed higher concentration of fatty acid ,carbohydrate, H2O2, P-carotene, protein, TPC, MDA and activity of APX, CAT, free radical scavenging capacity and free flavonoid content in peel, while ceruloplasmin, and protease activity were lower in peel when compared with pulp ,(Table1) .The result showed no significant different in SOD activity (Table 1). The result exhibited a significant low level of alkaline protease activity in peel compared with the pulp (Table1).

The result showed one peak by gel filtration separations (figure 1).The specific activity of the enzyme was

increased in 4.73 folds than the activity in initial extract (Table 2) for sample.

The maximum activity of the enzyme was obtained by using [0.40 mM] of casein, (Figure 2) .A linear relationship was shown before reaching Vmax at 31.45 U/mL and Km value of [0.142 mg/mL].

The current result indicated that maximum enzyme activity was at pH 10 (Figure3A), and the maximum incubation temperature at 50 °C, (Figure 3B).

Arrhenius plot was used to calculate thermodynamic factors of the transition by plotting ln K+1 values against (1/T) values,(Figure 4). A linear relationship was obtained with the activation energy of 16.29 KJ/mol and also free energy (AG*) was 95.61 KJ/mol, enthalpy (AH*) was 13.60 KJ/mol, and entropy (AS*) was253.90 J/mol.K of the transition state were determined

4. Discussion

In many countries, production of sunflower seeds is for the development of oil and also for their use as toppings in snacks. Sunflower seeds are excellent sources of energy and are often utilized for livestock feed sources of energy and are often utilized for livestock feed[24] .Plants have two methods for detoxifying the H2O2 produced [25] .CAT removes ROS produced in different stress conditions and avoid oxidant damage[26]. In seed germination, the increase of MDA contents in endosperms and cotyledons suggest that lipid peroxidation increases through germination process [27].

The antioxidant activity of flavonoids results from the combination of their iron chelating activity and their ability to scavenge ageing-inducing free radicals. Flavonoids can inhibit oxidases such as cyclooxygenase, lipooxygenase , NADPH oxidase and xanthine oxidase , thus preventing the in vivo formation of ROS and organic hydroperoxide. Furthermore, it has been found that flavonoids stimulate enzymes with well-known antioxidant

properties, such as CAT and SOD[28]. The present study shows peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants .

An enzyme with low Km has more affinity for its substrate. Previous studies showed that maximum activity was achieved when casein was used as a substrate [29, 30].

The velocity of enzyme-catalyzed reactions depends on pH. Enzymes have pH optimum and frequently give bell-shaped curves of velocity against pH, even though other shapes. Our results agreed with other studies of different sources [31, 32]. Temperature is a main factor for maximum activity of enzyme as well as for numerous industrial applications; relatively high thermo stability is desirable for characteristic of an enzyme [33]. Previous study showed a maximum activity at 40°C while, further increase in temperature leads to decrease the activity and showing 80% loss in activity at 70°C[30],while another study described maximum enzyme activity of alkaline protease from Aspergillus niger at 45°C[34].

Characterization and environmental friendly potential application of alkaline protease from pulp in sunflower (H. annuus) were studied. The purified enzyme showed maximum activity of 31.45U/mL with its corresponding Km value of 0.142 mg/mL. The specific activity and substrate affinity of alkaline protease from pulp in sunflower is greater than those of other reported; therefore, it is concluded that it may be potentially useful for industrial purposes. We concluded that peel in sunflower are strong radical scavengers, also it might be sources of natural antioxidant for medicinal and commercial uses

Conflict of interest statement

We declare that we have no conflict of interest.

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Table 1

The mean of biochemical parameters in pulp and peel in sunflower.

Parameters

Pulp(Mean peel(Mean

Fatty acid [mg/100gm]

28.59±3.15

31.48±1.85

Carbohydrate [gm./100 mL]

2.57±0.45*

3.47 ±0.37

[^mol./gm. FW] 3.12±0.46

3.28±0.52

ß-carotene [ppm] 6.98±1.05* 8.22±1.13

Protein [mg/mL] 1.57±0.15 1.74±0.25

TPC [^gm/gm] 2.19±0.43* 4.68±0.31

MDA [mmol/gm] 0.030±0.003* 0.012±0.0001

Ceruloplasmin Oxidase[mg/dL] 9.42±1.11* 6.98±0.79

CAT [U/mL] 245.2±22.25** 216.9±29.5

APX [U/mL] 9.56±1.05** 14.89±1.32

Free radical scavenging capacity [%] 63.14±1.05* 69.30±1.23

Free flavonoid content [^g/g] 555.33±28.22* 569.41±20.05

SOD [U/mg] 4.86±1.08 4.52±1.00

Protease [U/mL] 29.31±0.35*** 4.25±0.10

* P < 0.05; ** P < 0.01 and *** :P < 0.001.

Table 2

Purification summary of an alkaline protease from pulp in sunflower

Purification Stage Volume Activity Total Total S.A[U/mg] Yiel Folds of

[ml] U/mL Activity[U] Protein[mg] protein d Purification

Enzyme Crude 12 31.10 373.2 21.9 17.04 100 1

Ammonium Sulfate 10 22.5 225.00 11.55 19.48 60.2 1.14

Precipitation 9

Dialysis 5 18.50 92.50 3.30 28.03 24.7 1.64

Sephadex G-100 2 24.6 49.2 0.61 80.66 13.1 4.73

Figure legend:

Figure 1 .Atypical elution profile for the chromatography alkaline protease Figure 2. Determination of Km (A) and Vmax (B) in partial purified protease. Figure 3. Effect of pH (A) and temperatures (B) in alkaline protease activity. Figure 4. Arrhenius plot.

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