Evaluation of Total Phenolic, Total Flavonoid Content And Antioxidant Activity of Rhus Vulgaris

Background: Rhus vulgaris is rich in various classes of polyphenols and flavonoids that act as free radical scavengers and reduce oxidative stress and cure various harmful human diseases. The plant is a traditionally known medicinal plant which is used against a number of diseases including cancer. Methods: The current investigation points towards the investigating quantitative phenolic contents, flavonoid contents and the free radical scavenging activity & antioxidant activity of Rhus vulgaris extract in n-hexane, acetone, and 80% of aqueous methanol. The phenolic contents were estimated by Folin – Ciocalteu procedure and gallic acid as a reference molecule whereas the total flavonoid contents were determined by using aluminum chloride and catechin. In addition, the dilution serial method was used to evaluate the leaves extract of rhus vulgaris , and the DPPH (2,2-diphenyl-1-picrylhydrazyl) method was utilized to assess the above-mentioned extracts against oxidative stress. Results: The result revealed that the ranges of total phenolic content from 5.82 ± 4.6 to 83.15 ± 7.6 mg GAE/g of the dry weight of extract, expressed as gallic acid equivalents. The total flavonoid concentrations were varied from 2.21 ± 7.34to 23.47 ± 4.87 mg CE/g, expressed as catechin equivalents. Antioxidant activity of extracts was expressed as the concentration of DPPH radical’s inhibition ranges from 1.2 ± 0.32 to 22.86 ± 3.71 mg AAE/ g. Conclusion: The 80% aqueous methanolic extract of Rhus vulgaris showed the highest phenolic and flavonoid contents and strong antioxidant potential and it could be used as antibiotics for different curable and incurable diseases.


Introduction
Phenolic compounds are associated with a high number of biological activities and one with special interest is their antioxidant capacity and may help to protect the cells against the oxidative damage caused by free radicals [1]. Antioxidants are compounds that can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction. Synthetic antioxidants are widely used, but their use is being restricted nowadays because of their toxic and carcinogenic side effects [2].
Sumac is the common name for a genus (Rhus) that contains over 250 individual species of flowering plants in the family Anacardiaceae [3]. Traditional species of Rhus used to manage several ailments including influenza, wounds, diarrhea, abdominal pain, indigestion, diabetes, malaria, rheumatism, aching gums, toothaches, swollen legs, dog bites, peptic ulcer, kidney stones, skin eruptions, bruises, and boils [5,6]. R. vulgaris in Ethiopia is used to treat various diseases according to Ethno-botanically or Ethno-pharmacological studies? For instance, it is used in the treatments of diarrhea, gonorrhea, inflammation, evil eye, wound, and lung TB in the Amhara region [7,8].
Previous studies on phytochemical investigation of the stem bark of Rhus vulgaris revealed the presence of secondary metabolites such as tannins, saponins, flavonoids, terpenoids, glycosides, alkaloids, and phenol. R. vulgaris methanolic extract (1000 mg/kg) showed greater antiinflammatory activity compared to indomethacin (10 mg/kg), the standard anti-inflammatory drug, with a decrease in inflammation for up to 90 min. The dichloromethane, ethyl acetate, and aqueous extracts of R. vulgaris stem bark, root, and leaves have exhibited moderate to toxic toxicity against brine shrimp with LC50 values ranging from 3.55 μg/ml to 734.06 μg/ml while cyclophosphamide, the positive control, demonstrated an LC50 value of 15.28 μg/ml [9]. The other reports describe isolations of new biflavonoids on the genus Rhus. Such as agathisflavone, amentoflavone, hinokiflavone, rhus flavanone, and succedanea flavone have been sourced from Rhus species and evaluated for activity against a range of pathologically significant viruses [10,11].
The present work to conduct a quantitative analysis of the antioxidant activity, total phenolic content, and total flavonoids in hexane, acetone, and 80% aqueous methanolic extracts of different varieties of Rhus vulgaris leaves and to correlate the total phenolic contents and flavonoid contents with the antioxidant activities as such comparative antioxidant study of these different varieties of the genus Rhus has been reported.

Plant materials
Fresh and healthy leaves of R. vulgaris were collected from a local farm in the Region of Amara; Central Gondar city in February 2020. The plant was taxonomically identified by the Department of Biology (Botany), University of Gondar. The plant was washed thoroughly with tap water to remove dust particles from the leaves. The collected plant material was dried under shade at room temperature for 10 days. Then the dried leaves were powdered using an electrical grinder uniformly and kept in a dry & cool place for further use.

Extraction procedure
The exhaustive maceration was used to extract Rhus vulgaris plant materials by soaking 1 g of powdered samples sequentially into 25 ml of n-hexane, acetone, and 80% aqueous methanol in an enclosed glass bottle with occasional shaking at room temperature for 24 hrs. Then the extracts were filtered through a Whatman filter paper No. 1. The extracts were concentrated using a rotary evaporator then stored at -4ºC for further usage.

Determination of total phenolic content
The concentration of total phenol present in Rhus vulgaris extracts was determined by Folin-Ciocalteu (FC) reagent method described by Munro et al. [1].
In brief, (0.50) ml of each of the following, namely: 80% aqueous methanol solution, acetone, and n-hexane were poured into the three different test tubes. Then, 3ml of distilled water and 250μL of Folin-Ciocalteu reagent were added and hand shaken. After 5 minutes, 1 ml of 7.5 % sodium carbonate was added. It was allowed to incubate for 90 min at room temperature. The intense blue color was developed due to the reaction between the phenols and Folin-Ciocalteu reagent. After incubation, absorbance was measured at a 760 nm spectrophotometer using a UV/Visible spectrophotometer. The amounts of total phenolic compounds in the Rhus vulgaris extract were determined using equation 1.
The total phenolic content was calculated from the calibration curve using gallic acid as a standard and the results were expressed as milligram of gallic acid equivalents per gram dry weight of extract (mg GAE/g DW). All of the studied samples were tested in triplicate for each extract and the mean values of absorbance were reported. The same method was repeated for the standard molecule of gallic acid, and then the required calibration curve was constructed ( Figure   1). Where: • TPC = the total content of phenolic compounds, mg/g plant extract in GAE • C = concentration of gallic acid obtained from the curve (mg/L) • V= the volume of the sample solution (L) • m= mass of extract in grams.

Determination of total flavonoid contents
Catechin was used as a standard to express total flavonoid contents of samples as mg catechin equivalent per gram of dry weight (mg CE/g of DW) [12]. The amounts of total flavonoid in the extract were determined as follows: Briefly, an aliquot (0.50 ml) of each plant extract was added to a 10 ml volumetric flask containing 4 ml of distilled water. To this flask, 0.3 ml of 5% NaNO2 was added. After 5 min of incubation, 0.3 ml of 10% AlCl3 was added. At the 6 th min, 2 ml of 1 M NaOH was added, and finally distilled water was added till the mark. An Orange yellowish color was developed. After 10 min of incubation, the absorbance was measured at 510 nm using a UV-vis spectrophotometer. Each sample was tested in triplicate. The total flavonoid contents in the extract were determined using equation 2. where: • TFC = the total content of flavonoids compounds, mg/g plant extract in CE • C = concentration of catechin obtained from the curve (mg/L) • V = the volume of the sample solution (L) • m = mass of extract in grams.

Determination of antioxidant activity-DPPH
The 2, 2-diphenyl-1-picryl-hydrazyl radical (DPPH) radical scavenging assay is an easy, rapid and sensitive method to screen the antioxidant activities of plant extracts. Several methods are available for the determination of free radical scavenging activity but the assay employing the stable DPPH has received great attention owing to its ease of use and convenience [13].
A solution of 0.1 mM DPPH in 80% of aqueous methanol was prepared, and 1.

Total polyphenols
The results obtained from the assay were expressed as means ± standard deviation of triplicate analyses and are presented in The total phenolic content in n-hexane extract, acetone extract, and 80% of aqueous methanolic extract was 5.82 ± 4.6 mg GAE/g, acetone, extract 43.23 ± 6.98 mg GAE/g, and 83.15 ± 7.6 mg GAE/g respectively. The highest value of total phenol was obtained in the aqueous methanol extract.
The amount of total phenolic content in R. vulgaris samples collected from Rhus vulgaris was influenced significantly by extracting solvent (p < 0.05), and the contents were varied within the range of 5.82 to 83.15 mg of GAE/100 g in dry weight for n-hexane to 80% aqueous methanol, respectively. Among the solvents, 80% aqueous methanol was the most efficient extracting solvent for TPC, followed by acetone, and n-hexane, indicating that the TPC extracted in ginger was higher in polar solvents compared with less polar solvents ( Table 1).
The amount of total phenol was done based on the standard calibration plot of gallic acid. It was constructed in the concentration range of (12.5-200) mg/L and the coefficient of determination (R 2 ) was found to be 0.9992 (Figure 1). Based on the standard plot of gallic acid (y=0.0116x+0.0414). Absorbance =760 nm

Concentration vs Absorbance
Phenolic compounds are known as powerful antioxidants due to their ability to scavenge free radicals, singlet oxygen, and superoxide radicals. The radical scavenging activity is attributed to hydroxyl groups replacing the aromatic ring of the phenolic components [14]. Various investigations have linked a high phenolic diet to the prevention of several cardiovascular, metabolic, infectious, and cancer diseases due to their pharmacological and biological activities [15].
The high phenolic content recorded in this study in the presence of methanol fraction is consistent with the research in [16], which reported that the highest phenolic content in the methanol extract of R.vulgaris. The result is responsible for powerful antioxidants due to their ability to scavenge free radicals, singlet oxygen, and superoxide radicals. Other studies also reported phenolic contents in plant extracts. The methanolic extract of leaves, stem, and fruit of the Rhus pentaphylla were 71.16, 141.79, 76.88, and Rhus tripartite 64.13, 108.83, and 75.16 respectively in (mg GAE g −1 DW) [17]. On the contrary, the ethanolic extracts of Rhus javanica from leaves (51.91±0.01), steam (28.5 ± 0.03), greenish fruit (53.66 ± 0.01), and blackish-grey fruit (32.74 ± 0.03) using Gallic acid standards [18] is less than the value obtained in this study. This difference could be the difference in the species, environmental (e.g., season, temperature, light, soil), and genetic factors that determine the variation of the antioxidant activity of these compounds.
This finding indicates that 80% of aqueous methanolic extract of R. vulgaris can be used as a suitable candidate in the manufacturing of natural antioxidants and synthetic antibiotics. In line with previous studies, the current study found a strong positive correlation between antioxidant activities of plant extracts and total phenolic content, which implies that the studied plant's phenols are the major contributor to the antioxidant activity in these extracts.

Total flavonoids
The total content of flavonoids in 80% of methanol, acetone, and n-hexane extract of R. vulgaris was determined from the regression equation of the calibration curve (, R 2 = 0.9987) and expressed as milligrams of catechin equivalents (CE). In the case of flavonoids, high content was observed in 80% of aqueous methanol extracts (23.47 ± 4.87 CE/100g of DW) and acetone extracts (12.56 ± 0.12), whereas n-hexane has the lowest content (2.21 ± 7.34 CE/100g). It was observed that the effect of solvents on TFC is similar to that on TPC.

Total antioxidant activity
The calibration curve was plotted as percentage inhibition versus different concentrations of ascorbic acid and the value of the absorbance obtained corresponding to concentrations. The percentage inhibitions against concentration are given in Figure 3. Ascorbic acid equivalent antioxidant capacity is expressed as mg of AAE per 100g of Rhus vulgaris leaves extracts.
The free radical-scavenging activity of R. vulgaris leaves extract, measured by DPPH assay, is shown in Table 1. It was observed that DPPH free radical-scavenging activity of the extract (1.2, 4.63, and 22.86 mg AAE/g n-hexane, acetone, and 80% aqueous methanol respectively) due to the presence of a relatively higher value of total phenol and total flavonoid contents as shown in Figure 3. Its percent inhibition was increased with increasing concentration. This means, as the concentration increased, the DPPH radical scavenging activity by the extract was also increased.
The concentration and absorbance of the sample are inversely related. The absorbance was decreased as the concentration of the sample increased. Except for n-hexane and acetone extracts, there were significant differences (p < 0.05) of DPPH radical scavenging abilities between all extracts. C18H12N5O6, M=394.33). The assay is based on the measurement of the scavenging capacity of antioxidants towards it. As shown in Fig.4 the odd electron of a nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine [20].
DPPH is a stable free radical which presents a deep purple color and a strong absorption band in the range of 517 nm. In the presence of antioxidant compounds, DPPH can accept an electron or a hydrogen atom from the antioxidant scavenger molecule, to be converted to a more stable DPPH molecule. As the reduced form of DPPH is pale yellow, it is possible to determine the antioxidant activity by studying the change of color spectrophotometrically. The greater the free radical scavenging capacity of an antioxidant compound, the more reduction of DPPH and the less purple color there is in the sample [21].   reporting substantial antioxidant activity plant parts from this species. They have displayed good antioxidant activity because they contain phenols, flavonoids, and tannins [16].

Conclusion
In the present study, the phenolic, flavonoid, and antioxidant activity was evaluated from Rhus vulgaris. Rhus vulgaris possessed high phenolic and flavonoid content and exhibited good antioxidant activity by DPPH. It is noticed that the highest concentration of phenolic compounds in the extracts were obtained using solvents of high polarity; 80% aqueous methanolic extract manifested greater power of extraction for phenolic compounds from Rhus vulgaris. The high contents of phenolic compounds and significant linear correlation between the values of the concentration of phenolic compounds and antioxidant activity indicated that these compounds contribute to the strong antioxidant activity. The results of this work indicated that Rhus vulgaris, when a proper extraction solvent is established, could serve as a medicine against free-radicalassociated oxidative damage. further investigations on the different mechanisms shall be studied further.