Comparison of Antioxidant activity Total Phenolic, and Total Flavonoids content of Butanolic extracts from Haloxylon scoparium and Traganum nudatum
Messaouda Allaoui, Mohammed Lakhdar Belfar*, Zaoui-Djelloul Daouadji*
University of KASDI Merbah, Ouargla 30000, Algeria.
*Corresponding Author E-mail: maneldaouadji@yahoo.fr, mbelfar@gmail.com
ABSTRACT:
The plant is a full-fledged pharmaceutical industry because it contains hundreds or even thousands of active substances, such as antioxidants, phenolic acids, polyphenols, and flavonoids. These compounds scavenge free radicals and thus inhibit the oxidative mechanisms that lead to degenerative diseases such as atherosclerosis, cancer, and tissue damage in rheumatoid arthritis.
Free radicals with one or more unpaired electrons are generated during normal and pathological cellular metabolism. Reactive oxygen species (ROS) readily react with free radicals to become free radicals. ROS are various forms of activated oxygen, including free radicals such as superoxide anionic radicals (O-2) and hydroxyl radicals (OH.), as well as non-radical (H2O2) and single oxygen forms1O27-9.
The objectives of the present study were to determine the antioxidant activity, total phenolic content and total flavonoid content of the extracts from two Saharian Chenopodiaceae: Haloxylon scoparium and Traganum nudatum.
MATERIALS AND METHODS:
Preparation of Extract:
The aerial parts of H. scoparium were collected from Ghardaia (Barienne region)-located in the northern of the Algerian desert- in November 2012. The aerial parts of T. nudatum were collected from Touggourt (gamaa region) in April 2013. The plants were identified by Pr. Abdelmadjid Chehma from Ouargla University and a voucher specimen (MA4 and MA5), were deposited at the Chemistry Department, University of Ouargla. The plant materials were dried under shade and then ground and stored in closed container away from light and moisture.
The extracts were prepared by soaking 200 g of the plant powder in a solution of EtOH/H2O [70/30] for 24H. The procedure was repeated three times and the filtrates were combined before being evaporated under reduced pressure. The resulting extracts were diluted with distilled water and left for a whole night. The filtrates were then subjected to extraction by various solvents with increasing polarity (petroleum ether, dichloromethane, ethyl acetate, and butanol). The organic phases were separated and evaporated.
Determination of total Phenolic:
The total phenolic content (TPC) of the obtained extracts was determined by Folin–Ciocalteu colorimetric method. The extract samples (0.1ml of different dilutions) were mixed with Folin Ciocalteu reagent (1.5ml, 1:10 diluted with distilled water) for 5 min and aqueous Na2CO3 (1.5 ml, 6٪) were then added. The mixture was allowed to stand for 90 min and the phenols were determined by colorimeter at 725nm. The standard curve was prepared (0.03-0.3mg/mL) solutions of gallic acid in methanol. The total content of phenolic compounds in the extract in gallic acid equivalents (GAE) was calculated by the following formula:
Where:
T: Total content of phenolic compounds, milligram per gram of plant extract, in GAE.
C: The concentration of gallic acid established from the calibration curve, milligram per milliliter.
V: The volume of extract, milliliter.
M: The weight of extract, gram10-14.
Determination of total Flavonoids:
Aluminum chloride colorimetric method was used for flavonoids determination. Briefly, a volume of 0.5mL of AlCl3 ethanol solution (2%) was added to 0.5mL of extract. After one hour at room temperature, the absorbance was measured at 420nm. The analysis was performed in triplicate A yellow color indicated the presence of flavonoids. Extract samples were evaluated at a final concentration of 0.1mg/mL. Total flavonoids content (TFC) was calculated as quercetin equivalent (mg/g) using the following equation based on the calibration curve: y = 30,493x + 0,0914R² = 0,999, where x is the absorbance and y is the quercetin equivalent (mg/g)15-19.
DPPH scavenging test:
The radical scavenging activity of the butanolic extract was determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, and Ascorbic acid was used as standard in (0.04-0.4mg/mL) solution., and butylated hydroxyl toluene (BHT) were used for comparison or as a positive control. Substantially a solution of 0.25mM of DPPH in methanol was prepared and 1.5ml of this solution was mixed with 1.5ml of extract in methanol containing 0.05 mg of extract. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. Different known antioxidants. The absorbance was measured at 517nm against a blank (methanol) in a UV-Vis spectrophotometer. Measurements were taken at least in triplicate. DPPH radical’s concentration was calculated using the following equation:
Where:
Ac: Absorbance of control
As: Absorbance of sample solution.
IC50: Value is the concentration of the sample required to scavenge 50% DPPH free radical20-25.
Reducing power assay:
The reducing power of different extracts was measured according the method reported by Hinneburg et al. (2006). After adding 1mL of each extract at different concentrations with 2.5mL of 0.2 M phosphate buffer at pH = 6.6 and 2.5mL of a 1% potassium ferricyanide solution, the obtained mixture was incubated for 20 min at 50°C, and then 2.5mL of 10% trichloroacetic acid was added to stop the reaction. The mixture was centrifuged at 3000rpm for 10 min. 2.5mL of supernatant was mixed with 2.5mL of distilled water and 0.5mL of FeCl3 (0.1%) and the absorbance was measured spectrophotometrically at 700nm. Increase in absorbance of the reaction mixture was interpreted as increase in reducing activity of the extract and the results were compared with ascorbic acid which was used as a positive control. The percentage of reduction compared to standard (ascorbic acid) was calculated using the formula26, 29:
Where:
Ac: Absorbance of standard at maximum concentration
As: Absorbance of sample
3. RESULTS AND DISCUSSIONS:
Determination of total phenolic contents:
Total phenol compounds, as determined by folin Ciocalteu method, are reported as gallic acid equivalents by reference to standard curve (y = 4,0914x + 0,0719, R² = 0,995). The total flavonoid content was expressed as quercetin equivalents (RE) in milligram per gram of dry material. The calibration curve of quercetin to determine flavonoid content was shown in Figure2: (y = 30,493x + 0,0914, R² = 0,999).
Figure 1. Calibration curve of standard gallic acid for determination of total phenolics
Figure 2. Calibration curve of standard Quercetin for determination of total flavonoid content.
The values of TPC and TFC extracted from the calibration curves represented in the following figure:
Figure 3. Graphical representation of the results of quantitative estimation of phenolic and flavonoids compounds in Butanolic extracts.
The results represented in figure 3 showed that Butanolic extract of Haloxylon scoparium had a higher TPC and TFC than Butanolic extract of Traganum nudatum. The total flavonoid content results were entirely synchronous with those of the total phenolic. It was successfully shown that samples with high level of phenolic content also contain flavonoids in great amount.
DPPH scavenging test:
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method was used to evaluate the free radical scavenging ability of butanolic extracts, the radical scavenging activity of the compounds can be measured as a decolorizing effect following the trapping of the unpaired electrons of DPPH, the parameter used to measure the radical scavenging activity is IC50 value, defined as the concentration of antioxidant required for 50% scavenging of DPPH radicals in this specified time period, the smaller IC50 value, the higher antioxidant activity of the extract. The percent inhibition of the DPPH radical as a function of the antioxidant concentrations is shown in Figure 4.
Figure 4. (a) Antioxidant activity of Ascorbic acid, (b) Antioxidant activity of BHT (c) Antioxidant activity of Butanolic extract of Haloxylon scoparium and (d) Antioxidant activity of Butanol extract of Traganum nudatum
The results represented by the curves in figure 4 reveal that the percentage inhibition of DPPH radical was found to be increased with concentration. The IC50 values of different samples extracted from this curves represented in the following figure.
Figure 5. Graphical representation of the results of test radical scavenging activity.
As presented in Figure 5, the IC50 values show the following decreasing order:
But T. nudatum > But H. scoparium > BHT > Ascorbic acid, this means that butanolic extract of Haloxylon scoparium had higher activity compared to butanolic extract of Traganum nudatum.
According to the results, the antiradical activity of plant extracts, evaluated by simple tests, was only due to a production of secondary metabolites. If we compare the contents of total phenols and flavonoids in butanolic extracts, it appears that the extract, which is richer in polyphenols or flavonoids, has a higher antioxidant power30.
Reducing power assay:
The reducing power (RP) of Butanolic extracts was determined by direct electron donation in the reduction of ferricyanide [Fe(CN)6] -3 to ferrocyanide [Fe(CN)6] -4. The product was visualized by addition of free Fe+3 ions after the reduction reaction, by forming the intense blue color complex, (Fe+3)4[Fe+2 (CN-)6]-3, and quantified by absorbance measurement at 700 nm (Riberio et al., 2008). Figure 6 shows the reductive capabilities of the two extracts compared to ascorbic acid.
Figure 6. Curves of Reducing Power assay.
Figure 6 represents the reducing capacity of ascorbic acid, and butanolic extracts. This capacity increases as a function of the concentration of the extracts. The extract could reduce the most part of Fe+3 ions, whereby the color of the test solution, which was yellow, transforms to various hues of green and blue, based on the extent of the reducing power of the samples. Increased absorbance of the reaction indicated increased reducing power31.
The efficiency of iron reduction is inversely proportional to the EC50 value.It is found that butanolic extracts have reducing capacity exceed the reducing capacity of ascorbic acid.
The results of this study were accordance with the study of Allaoui et al.32 applied to ethyl acetate extracts of both for the same two plants, where a higher antioxidant activity, total phenolic content and total flavonoid content were exhibited by the ethyl acetate extract of Haloxylon scoparium. In comparison, we found that all studied extracts have significant antioxidant activity, the value of which varies from one extract to another; According to the difference in the type of compounds present in each extract separately, the highest antioxidant activity was recorded in the Haloxylon scoparium plant because it contained the maximal TPC and TFC compounds. By comparing the extracts among themselves for each plant separately, we note that ethyl acetate extract is more effective than butanolic extract32.
CONCLUSION:
On the basis of the overhead results, it can be concluded that a higher antioxidant activity, total phenolic content and total flavonoid content were exhibited by the Butanolic extracts of Haloxylon scoparium. The phytochemical study aiming to separate the active principles and to elucidate the mechanism of action of this extract is the subject of ongoing investigation in our group.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
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Received on 25.04.2022 Modified on 28.06.2022
Accepted on 11.08.2022 ©AJRC All right reserved
Asian J. Research Chem. 2022; 15(5):345-350.