|Year : 2020 | Volume
| Issue : 2 | Page : 86-90
Studies on In vitro antioxidant analysis of cucumeropsis mannii (melon) seed
H A Enemor Victor, Chinenye Enoch Oguazu, Mbamalu C Linda
Department of Applied Biochemistry, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria
|Date of Submission||11-Apr-2020|
|Date of Decision||16-Jun-2020|
|Date of Acceptance||17-Jul-2020|
|Date of Web Publication||11-Feb-2021|
Dr. Chinenye Enoch Oguazu
Department of Applied Biochemistry, Nnamdi Azikiwe University, Awka, Anambra State
Source of Support: None, Conflict of Interest: None
Introduction: Cucumeropsis mannii (melon seed) is grown as a source of oil and can be dried and ground for use as soup condiment in Nigeria. It has been said to contain antioxidants. The crop holds significant nutritional value rich in essential amino acids, vitamins, and minerals. The seed is an excellent vegetable protein and is ideal for battling nutritional debilitations. Aims: In this study, the in vitro antioxidant activities of the ethanolic extract of melon seeds were analyzed. Materials and Methods: The C. mannii (melon) seeds were dried, and the sample was ground with a corona manual grinder into a fine powder and was stored in an airtight container. Sample extraction was performed by soaking 20 g of the ground sample in 100 ml of 70% ethanol and placed in a shaker (HY-4A multipurpose oscillator) for 1 h. Analysis for percentage of free radical scavenging activity (RSA), reducing power activity, inhibition of lipid peroxidation, hydrogen peroxide scavenging activity, and antioxidant enzyme activity was done using standard methods. Statistical Analysis: The results were presented as mean ± standard deviation, and analysis was carried out using the Student's t-test at 95% confidence level considered at P = 0.05. Results: The result obtained showed that melon seed had an EC50 free RSA of 2382.69 mg/ml, reducing power activity of 16,660 mg/ml, while lipid peroxidation showed EC50 of 473.832 mg/ml. The catalase (0.12985 ± 0.00 U/mg), superoxide dismutase (0.0032095 ± 0.00U/mg), and H2O2 scavenging activity showed high activities. Conclusions: C. mannii (melon seed) contains active antioxidants that prove that it is of essential nutritional value.
Keywords: Antioxidants, Cucumeropsis mannii (melon seed), lipid peroxidation, reducing power activity, scavenging activity
|How to cite this article:|
Victor H A, Oguazu CE, Linda MC. Studies on In vitro antioxidant analysis of cucumeropsis mannii (melon) seed. Niger J Exp Clin Biosci 2020;8:86-90
|How to cite this URL:|
Victor H A, Oguazu CE, Linda MC. Studies on In vitro antioxidant analysis of cucumeropsis mannii (melon) seed. Niger J Exp Clin Biosci [serial online] 2020 [cited 2021 Jun 14];8:86-90. Available from: https://www.njecbonline.org/text.asp?2020/8/2/86/309172
| Introduction|| |
In recent times, the incidence of various chronic diseases has been on the increase. Consequently, there has been increasing interest in local and neglected food evidently rich in various antioxidants as a means of preventing and/or controlling the development and advancement of chronically debilitating conditions. Natural antioxidants present in foods have had increased interest among consumers and the scientific community due to its nutritional and epidemiology evidence and the significant increase in the occurrence of chronic disease.
Cucumeropsis mannii [Figure 1], belonging to the Cucurbitaceae family, is a climber that grows in wet humid climate, mostly in the eastern and southwestern region of Nigeria where it is grown as a source of oil and can also be obtained in shelled [Figure 2] or unshelled forms in the markets. It is often dried and ground for use as soup condiment or other delicacies in Nigeria. It is commonly and generally referred to as Egusi in Igbo language. Melon (Cucumis melo L.) is one of the most consumed and exported fresh seeds. It has high economic value, and it is cultivated in several regions due to its adaptability to different soil types and climates. The seeds are processed as soup condiments and oil products and are also eaten individually as a snack either as whole toasted seeds or as fried cake. This crop also holds significant nutritional value rich in essential amino acids, vitamins, and minerals. The seed is an excellent source of vegetable protein and is ideal for battling nutritional debilitations. Soups made from the seeds of this crop compliments the starch and grain diet of most Africans. The report of proximate analysis earlier carried out on the kernel of the melon seed [Figure 2] and [Figure 3] revealed that it had significant concentrations of oil (44%), protein (30%), carbohydrate (10%), ash (4%), fiber (3%) as well as certain fatty acids such as linoleic acid (64.9%), oleic acid (12.4%), stearic acid (11.8%), and palmitic acid (10.9%)., Vitamins thiamin, niacin, B1, and B2 are also prevalent in the seed as well as many micronutrients. Notable minerals include phosphorus, as the largest mineral component, with potassium, magnesium, manganese, sulfur, calcium, iron, and zinc. The bulk of carbohydrates are starch and soluble sugars. Melon is the perfect complement to the largely starch-rich grain diet of Nigeria, providing a high-protein and high-energy concentrate. The amino acid content of melon seed proteins makes it a sufficient vegetable protein. There is potential for these seeds as a critical tool for interventions in diseases such as marasmus and kwashiorkor. The seed contains every important macro- and micronutrient in quantities ideal for nutrition. Just 100 g of seed daily provides essential fatty acid, amino acid, and Vitamin E requirements. Despite the crops obvious advantages, C. mannii remains an underutilized tool for nutritional intervention. In this study, the antioxidant potentials of the C. mannii seed were determined through the in vitro assay for its active components: free radical scavenging activity (RSA), reducing power activity, inhibition of lipid peroxidation, hydrogen peroxide scavenging activity, superoxide dismutase (SOD) activity, and catalase activity.
| Materials and Methods|| |
Sample collection and preparation
The melon seeds were obtained from a second market in Ifite Awka, Anambra State, Nigeria. The dried sample was ground with a corona manual grinder into a fine powder and was stored in an airtight container till further use.
Twenty grams of the ground sample was soaked in 100 ml of 70% ethanol and placed in a shaker (HY-4A multipurpose oscillator) for 1 h. It was then allowed to stand for 24 h at room temperature. The mixture was filtered through Whatman paper No. 4, and the filtrate was evaporated at 78°C using a water bath (Techmel and Techmel, 420, USA). The dried residue was weighed and reconstituted in 70% ethanol at a concentration of 10 mg/ml and stored at 4°C in a refrigerator till further use.
Diphenyl-1-picrylhydrazyl scavenging activity
The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was used for the determination of free RSA of the ethanolic extract of the sample according to the method of Ebrahimzadeh et al. Exactly, 0.3 ml of 6 different concentrations of the extract (0, 200, 400, 600, 800, and 1000 μg/ml) was mixed with 2.7 ml of ethanolic solution of DPPH (100 μM) in test tubes. The mixture was shaken and kept in the dark for 60 min. The absorbance was taken at a wavelength of 517 nM using spectrophotometer. Butylated hydroxyanisole (BHA) was used as standard. The percentage of scavenging activity was calculated using the formula:
% radical scavaging ativity %RSA = [(ADPPH - As)/ ADPPH] ×100
where As is the absorbance of the test solution with the sample and ADPPH is the absorbance of DPPH solution. The EC50 (concentration of sample at 50% RSA) was calculated from the graph of %RSA against the sample concentration.
Reducing power capacity
The reducing power was determined according to the method of Barros et al. This method is based on the principle of increase in the absorbance of the reaction mixture.
Exactly, 2.5 ml of 6 different concentrations of the ethanolic extract of the sample (0, 200, 400, 600, 800, and 1000 μg/ml) was mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid (TCA) was added, and the mixture was centrifuged at 1000 rpm for 8 min. The upper layer (5 ml) was mixed with 5 ml of deionized water, followed by the addition of 1 ml of 0.1% ferric chloride. The absorbance was measured at 700 nM. The graph of absorbance at 700 nM against the extract concentrations was plotted. BHA was used as a standard antioxidant.
Inhibition of lipid peroxidation activity
This was determined by the method of Barros et al. Determination of the extent of inhibition of lipid peroxidation was carried out using a homogenate of a goat brain. The brain from a goat of weighing 70 kg was purchased from Kwata Animal Slaughter House at Awka, Anambra State. The brain was dissected and homogenized with pestle and mortar in an ice-cold Tris-HCL buffer (pH 7.4, 20 mM) to produce 50% w/v brain homogenate which was centrifuged at 3000 rpm for 10 min. An aliquot (0.1 ml) of the supernatant was incubated with 0.2 ml of the sample extract at six different concentrations (0, 200, 400, 600, 800, and 1000 μg/ml) in the presence of 0.1 ml of 10 μm ferrous sulfate and 0.1 ml of 0.1 nm ascorbic acid at 37°C for 1 h. The reaction was stopped by the addition of 0.5 ml of 28% TCA, followed by the addition of 0.38 ml of 2% thiobarbituric acid (TBA). The mixture was then heated at 80°C for 20 min. After centrifugation at 3000 rpm for 10 min to remove the precipitated protein, the color intensity of the malondialdehyde-TBA complex in the supernatant was measured by its absorbance at 532 nM. The inhibition ratio (%) was calculated using the following formula:
Inhibition ratio (%) = [(A - B) / A] ×100%
where A and B were the absorbance of the control and the compound solution, respectively. The extract concentration providing 50% lipid peroxidation inhibition (EC50) was calculated from the graph of antioxidant activity percentage against the extract concentrations. BHA was used as the standard.
H2O2 scavenging activity
This was determined by the method of Burkill. The extracts of six different concentrations (0, 200, 400, 600, 800, and 1000 μg/ml) were dissolved in 3.4 mL of 0.1 M phosphate buffer (pH 7.4) and mixed with 600 μl (0.6 ml) of H2O2 (40 mM) prepared in 0.1 M phosphate buffer (pH 7.4). The absorbance value of the reaction mixture was recorded at 230 nm. Using BHA as standard, the percentage of H2O2 scavenging was calculated from the formula:
% Scavenged [H2O2] = [(As - Ac) / As] x 100.
Sample preparation: 1 g of the ground sample was extracted in ice-cold 0.1 M phosphate buffer (pH 7.4) and centrifuged at 1500 rpm for 10 min. The supernatant was used for the enzyme activity assay.
SOD activity was determined by the method of Sun and Zigma. This is based on its ability to inhibit the auto-oxidation of epinephrine determined by the increase in absorbance at 480 nm. The reaction mixture (3 ml) containing 2.95 ml 0.05 M sodium carbonate buffer pH 10.2, then 0.02 ml of liver homogenate, and 0.03 ml of 0.3 mM adrenaline in 0.005N HCL was used to initiate the reaction. The reference cuvette contained 2.95 ml buffer, 0.03 ml of substrate (epinephrine), and 0.02 ml of water. Enzyme activity was calculated by measuring the change in absorbance at 480 nm for 3 min.
Catalase activity determination
The catalase activity was determined according to the method described by Burkill by measuring the decrease in absorbance at 240 nm due to the decomposition of H2O2 in an ultraviolet recording spectrophotometer. The reaction mixture (3 ml) contained 0.1 ml of sample extract in phosphate buffer (50 mM, pH 7.0) and 2.9 ml of 30 mM H2O2 in phosphate buffer pH 7.0. An extinction coefficient for H2O2 at 240 nm of 40.0/M/cm was used for the calculation. The specific activity of catalase was expressed as moles of H2O2 reduced per minute per mg protein.
| Results|| |
The results were presented in graphs as mean ± standard deviation, and analysis was carried out using the Student's t-test at 95% confidence level considered at P = 0.05 and uploaded as multimedia files.
| Discussion|| |
The result obtained for free RSA of C. mannii, as shown in [Figure 4], revealed that the EC50 of C. mannii seed was 2,382.69 mg/ml. The reducing power activity result of C. mannii seed [Figure 5] revealed EC50 of 16,660 mg/ml, indicating that its reductive capabilities are higher than that of the standard (BHA). The lipid peroxidation result [Figure 6] showed that the EC50 of C. mannii seed is 473.832 mg/ml. This implies that the sample inhibits lipid peroxidation better than standard (BHA of EC50: 394.7 mg/ml). The H2O2 scavenging activity [Figure 7] depicts that C. mannii seed showed a degree of activity; thus, it is capable of scavenging hydrogen peroxide. [Figure 8] shows the result for catalase and SOD activity; although catalase activity appeared to be higher than SOD activity, the melon seed showed activity in the two antioxidant enzymes.
|Figure 4: Free radical scavenging activity of Cucumeropsis mannii seed (melon seed)|
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|Figure 6: Inhibition of lipid peroxidation of Cucumeropsis mannii seed (melon)|
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|Figure 7: Hydrogen peroxide scavenging activity of Cucumeropsis mannii seed (melon). Antioxidant enzyme assay of Cucumeropsis mannii seed (melon). Superoxide dismutase activity. Catalase activity|
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|Figure 8: Bar chart showing catalase and superoxide dismutase activities|
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| Conclusion|| |
From the above discussion, it was clear that the C. mannii seed has a high antioxidant activity. Seed extracts can be applied in food technology, pharmacology, and medicine studies.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]