Evaluation of Total Phenolic, Flavonoid contents, and Antioxidant activity of Rhus vulgaris leaves extracts

doi:10.21203/rs.3.rs-2650283/v1

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Evaluation of Total Phenolic, Flavonoid contents, and Antioxidant activity of Rhus vulgaris leaves extracts

https://doi.org/10.21203/rs.3.rs-2650283/v1

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Background

Rhus vulgaris is rich in various polyphenols and flavonoids that act as free radical scavengers, reduce oxidative stress, and cure various harmful human diseases. Traditionally, the plant is used to treat various diseases including cancer.

Methods

This study involved extraction using the solvents hexane, acetone, and 80% aqueous methanol, phytochemical screening, and antimicrobial testing. Using the spectrophotometric techniques of Folin-Ciocalteu and aluminum chloride, respectively, the plant's total phenolic and flavonoid contents were determined. The extracts' activity was assessed with the help of the (DPPH) radical scavenging assay.

Results

The extracts mostly contain alkaloids, phenols, flavonoids, glycosides, tannins, steroids, carbohydrates, and anthraquinones. Compared to the standard, the methanol extracts showed better inhibitory zones. Results revealed that total polyphenols and flavonoid contents were in the range of 5.82 ± 0.6–83.23 ± 0.6 mg GAE/100 g and 2.21 ± 0.34–23.47 ± 0.87 mg CE/100 g, respectively. Leaves extracts of R.vulgaris was found to have higher antioxidant activities ranging from 0.756 ± 0.8 to 131.56 ± 0.6 mg AAE/g sample.

Conclusion

The R. vulgaris 80% methanolic extract displayed the highest phenolic and flavonoid concentrations as well as a powerful antioxidant capacity. It could be used as an antibiotic for different curable and incurable diseases.

Rhus vulgaris

Total phenolic contents

Total Flavonoid contents

Antioxidant activity

Reactive oxygen species, such as singlet oxygen, superoxide ion, hydroxyl ion, and hydrogen peroxide, are highly reactive, toxic molecules, which are generated normally in cells during metabolism[1]. Free radicals contribute to more than a hundred disorders in humans including degradation of proteins, lipids, enzymes, and DNA by covalent binding and lipid peroxidation, atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer, diabetes mellitus. It causes many diseases such as cancer, diabetes, atherosclerosis, and many chronic diseases[2].

Antioxidants function by protecting cells within the human body at various levels. They do so by preventing the formation of free radical species, intercepting radical chained reactions, transforming existing free radicals into less harmful molecules, and repairing oxidative damage[3]. Although synthetic antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) are available commercially and widely used [1, 5], their continuous use has been linked to adverse effects [4]. In recent times, the use of synthetic antioxidants has been restricted due to their toxic and carcinogenic consequences [6].

The Rhus genus, commonly known as sumac, comprises over 250 flowering plant species in the Anacardiaceae family[7], predominantly found in tropical, subtropical, and temperate regions worldwide. The name "sumac" originated from the Syriac word "sumaga," meaning red. These non-agricultural plants have been utilized by indigenous people for medicinal and other purposes, indicating their potential for commercial application without competing with food production[8]. Traditional Rhus species have been employed to treat various ailments, including influenza, wounds, diarrhea, stomach pain, indigestion, diabetes, malaria, rheumatism, gum and toothaches, swollen legs, dog bites, peptic ulcers, kidney stones, skin eruptions, bruises, and boils[9, 10]. In Ethiopia, R. vulgaris is used to manage various diseases according to ethnobotanical and ethno-pharmacological research, such as diarrhea, gonorrhea, infections, the evil eye, wounds, and lung tuberculosis in the Amhara region[11, 12]. In Kenya, this plant’s stem bark is used to treat malaria [7]. In Tanzania, Fresh leaves are burned and the ash is used for oxytocic action, and externally, the ash is applied for the treatment of scabies. The plant is used to stop diarrhea, wounds, gonorrhea, and infertility, and to ease delivery[13]. In Uganda, this plant is used to treat diarrhea, malaria, hemorrhoids[14], Yellow fever, Cough, Malaria, Gastro-intestinal disorder, toothaches, Syphilis, Immunity booster, Smallpox, Swollen lymph, and Prevention diseases [15], in Uganda more preferable chewing-sticks over synthetic toothbrushes [16]. In Kenya, the roots, leaves, and fruits are used as the treatment of cancers like Stomach, skin, and breast cancer[17].

Many studies are investigating different kinds of fruits, vegetables, and plant extracts as an antioxidant for reducing the risk of diseases caused by free radicals[2]. The recent study aimed to Evaluation of the antimicrobial activity and safety of Rhus vulgaris (Anacardiaceae) extracts. The Result revealed tannins, saponins, flavonoids, terpenoids, glycosides, alkaloids, and phenol from stem bark in methanol extracts R.vulgaris. And further studies of R. Vulgaris methanolic extract (1000 mg/kg) confirmed more anti-inflammatory pastime compared to indomethacin (10 mg/kg), the same old 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 in opposition to brine shrimp with a Lethal Concentration of 50% of LC₅₀ values starting from three [18]. Another researcher reported that, revealed biflavonoids in the genus Rhus such as agathisflavone, amentoflavone, hinokiflavone, Rhus flavanone, and succedanea flavone have been sourced from Rhus species and evaluated for interest against a variety of pathologically substantial viruses [19, 20].

Therefore, the objectives of the present study were to determine the total phenolic and flavonoid content and evaluate the antioxidant activity in hexane, acetone, and 80% aqueous methanolic extracts of R.vulgaris leaves and to correlate the total phenolic contents and flavonoid contents with the antioxidant activities as such comparative antioxidant look at of these special kinds of the genus Rhus has been studied. An easy, rapid, and sensitive method for the antioxidant screening of plant extracts is a free radical scavenging assay using 1,1-diphenyl-2-picryl hydrazine (DPPH) stable radical spectrophotometrically.

Study area description

The study was conducted on selected traditional plants collected from Takusa Woreda, which was situated in the Central Gondar Zone of the Amhara regional state (Fig. 1). Geographical location 12° 1′ 56.64″ N, 36° 56′ 47.76″ E.

Chemicals and reagents

All the chemicals and reagents used in the present study were of analytical grade. The chemicals and reagents included: Methanol (98%), acetic anhydride, ferric chloride solution, concentrated hydrochloric acid, ammonia, Mayer’s reagent, Benedict’s reagent, Sodium bicarbonate, ammonia, Ninhydrin, Molisch’s reagent, Millon’s reagent, lead acetate, Folin-Ciocalteu reagent, gallic acid, ascorbic acid, sodium hydroxide, anhydrous sodium carbonate, and anhydrous aluminum chloride, which were purchased from Loba Chemie (Mumbai, India); whereas acetone, n-hexane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium nitrite and catechin were purchased from Merck (Mumbai, India).

Apparatus and equipment

Electrical grinder (IAK–WERKE, Germany), electronic balance (Bosch, Germany), Whatman No. 42 filter paper (110 mm), graduated pipettes, micropipettes (Mumbai, India), refrigerator (Hitachi LR902T, USA), orbital shaker (GEMMY Orbital Shaker, VRN-480, Taiwan), lyophilizer (Scientz-10N, Ningbo Science Biotechnology Co., Ltd., China), rotary evaporator (Bibby RE200, Sterilin Ltd., UK) and UV/Vis spectrophotometer (Sanyo SP75, UK) were used in the study.

Collection of Plant materials

Fresh mature whole leaves of R. vulgaris. Were collected in February 2020 from the Region of Amara; Central Gondar city, Ethiopia. The plant was taxonomically identified by Mr. Getenet Chekole at the Department of Biology (Botany), University of Gondar. A voucher specimen (Id 001-20219) was deposited at the herbarium of Botanical Science, Department of Botany, University of Gondar. The collected leaves were washed thoroughly with tap water, followed by distilled water. Here, the leaves samples were first dried at room temperature for ten days under a plastic cover to avoid dust contamination, powdered using an electric grinder, and kept in clean polyethylene plastic bags until extraction.

Extraction procedure

The dried and ground powdered root sample (1 g) was extracted with 25 mL of n-hexane, acetone, and 80% aqueous methanol, respectively, by maceration using an enclosed glass bottle with occasional shaking at room temperature for 24 hrs. Then, the extract was filtered through Whatman No. 42 (110 mm) filter paper, and its volume was adjusted with n-hexane, acetone, and 80% methanol. Finally, the filtrate was stored in a refrigerator at 4°C.

Preparation of standard solutions

The gallic acid standard was prepared by dissolving gallic acid in 80% methanol; 7.5% Na₂CO₃, 5% NaNO₂, 10% ACl₃, and 1M NaOH solutions were prepared with double distilled water. Catechin and ascorbic acid standard solutions were also prepared with methanol.

Preliminary Phytochemical Screening

Phytochemical screening was carried out to assess the qualitative chemical composition of crude extracts using commonly employed precipitation and coloration to identify the major natural chemicals groups such as alkaloids, phenolic compounds, glycosides, carbohydrates, flavonoids, saponins, terpenoids, anthraquinones, tannins, steroids, amino acids, coumarins, and proteins. General reactions in these analyses revealed the presence or absence of these compounds in the crude extracts tested. Crude extracts of the plants previously prepared and stored in a refrigerator were used for the phytochemical tests [21–25].

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. [26]. Briefly, 0.50 mL of each extract was added into a test tube containing 3 mL of distilled water and 0.25 mL of Folin-Ciocalteu reagent and shaken. After 5 min in the dark, 1 mL of 7.5% Na₂CO3 was added and incubated at room temperature for 90 min in the dark. The reagent blank was also parallelly prepared using distilled water. The absorbance was measured against the prepared reagent blank at 760 nm using a double-beam UV/Vis spectrophotometer. The total phenolic content was calculated from the calibration curve using gallic acid as a standard and the results were expressed as milligrams 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 total phenolic contents in all the samples were calculated by using the formula (equ.1):

TPC = \(\frac{C.V.DF}{m}\)……………………………………………. (1)

Where TPC = the total content of phenolic compounds, mg/g plant extract in GAE dry extract, C = concentration of gallic acid obtained from the curve (mg/L) V = volume of extract in mL, m = mass of extract in grams, and DF = Dilution factor

Determination of total flavonoid contents

Catechin was used as a standard to determine the total flavonoid contents of samples as mg catechin equivalent per gram of dry weight (mg CE/g of DW) [27]. A 1000 µg/mL stock solution was prepared by dissolving 100 mg of catechin in 100 mL of 80% methanol and then diluted to get the concentrations 50, 100, 150, 300, and 500mg/L.

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 each volumetric flask containing 4 mL of distilled water. To each test tube, 0.3 mL of 5% NaNO₂ was added. After 5 min of incubation, 0.3 mL of 10% AlCl₃ was added. After 1 min, 2 mL of 1 M NaOH was added to each test tube and the volume was adjusted to 5 mL with distilled water. A yellow coloration of the mixture indicated the presence of flavonoids. After 10 min, the absorbance of the resulting solution was measured at 510 nm. Catechin was used as a standard to express the total flavonoid contents of samples as mg catechin equivalent per 100 g of sample (mg CE/100 g sample). All the samples were analyzed in triplicate. The total flavonoid contents in the extract were determined using Eq. 2.

TPC = \(\frac{C.V.DF}{m}\)……………………………………………. (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, and DF = Dilution factor.

Determination of antioxidant activity-DPPH

The 2, 2-diphenyl-1-picryl-hydroxyl 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 [28].

A mass of 0.02 g of DPPH was dissolved with a small amount of 80% methanol in a 500 mL volumetric flask. After the dissolved DPPH, the flask was filled up to the mark with 80% methanol. For the samples of each extraction prepare different concentrations, a (0.05, 0.1, 0.4, 0.8, 1.6, and 2.4 µg/mL). Afterward, the volume of each solution was adjusted to 4 mL with n-hexane, acetone, and 80% aqueous methanol, and the incubation was made for 30 min at room temperature in a dark cupboard. The control was prepared by mixing 3 mL of methanol and 2 mL of DPPH solution. In addition, a stock solution of ascorbic acid was prepared by dissolving 50 mg of ascorbic acid in 100 mL of methanol. Generation of the calibration curve was achieved by preparing different concentrations, viz. 1, 2.5, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg/L, from the stock solution. To each test tube, 2.4 mL of standard ascorbic acid and 1.6 mL of DPPH solutions were added; then the test tubes were covered by aluminum foil and incubated in the dark for 30 min. A mixture of 2.4 mL of n-hexane, acetone, and 80% aqueous methanol and 1.6 mL of DPPH was prepared as a blank solution. The reaction mixture was kept in the dark for 30 min. Finally, the absorbance was measured at 517 nm using a UV-vis spectrophotometer with slight modification. The percentage of DPPH radical scavenging activity was determined using the formula (equ 3):

% inhibition =\(\frac{{(\text{A}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}- {\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}) \text{x} 100 \text{\%}}{{\text{A}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}}\)…………………………. (3)

Where A_control is the absorbance of DPPH solution without extract and A_sample is the absorbance of a sample with DPPH solution. Sample concentration giving 50% inhibition was estimated as IC₅₀ value using the dose inhibition curve in linear range by plotting the extract concentration versus the corresponding scavenging activity. Measurements were performed in triplicates. The results were expressed as mg ascorbic acid equivalent per gram of sample (mg AAE/g sample).

Evaluation of the antibacterial activity

Antibacterial activities of R.vulgaris extracts were tested against four strains of bacteria using the agar well diffusion method. The test cultured bacteria were swabbed on the top of the pre-leveled media (about 45-50ml) and allowed to dry for 10 minutes. The sterilized well borer (6mm diameter) was used to bore holes on the plates and 100 mg/ml of each extract (n-hexane, chloroform, and methanol) was introduced in the holes in triplicates by using Gentamicin disc (30 mcg/disc) as positive antibiotic control. The Petri dishes were then incubated at 37\(℃\) for 24 hrs. At the end of the incubation period, the antibacterial activity of each extract and positive antibiotic control recorded the average inhibition zone in millimeters.

Statistical analysis

All statistical analyses were undertaken using SPSS Version 16. Differences at p < 0.05 were considered statistically significant.

Phytochemical screening of the Leaves Extracts of Rhus vulgaris

The phytochemical analysis of each crude extract of Rhus vulgaris revealed the presence of pharmacologically useful classes of secondary metabolites. Alkaloids, Glycosides, Steroids, Anthraquinones, and Carbohydrates were present in all extracts. Phenols, Flavonoids, Tannins, Coumarins, and Proteins were present only in methanol extracts while Terpenoids, Saponins, and Amino acids were absent in all the extracts summarized in Table 1.

Table 1

Phytochemical constituents of the leaves extract of Rhus vulgaris.
Phytochemical constituent	Hexane	Chloroform	Methanol
Alkaloids	+	+	++
Phenols	-	-	++
Glycosides	+	+	++
Flavonoids	-	-	++
Terpenoids	-	-	-
Steroids	+	+	+
Carbohydrates	+	+	+
Anthraquinones	+	+	+
Proteins	-	-	+
Coumarins	-	-	+
Tannins	-	-	+
Saponins	-	-	-
Amino acid	-	-	-

Key: (+) = Present, (-) = Absent

Antibacterial Activity of the Crude Extracts against Bacterial Strain

The antibacterial property of Rhus vulgaris extract using different solvents showed varying degrees of response towards the selected pathogens (Table 2). In this work, three samples were tested by the above method. The results showed the crude methanol extract was strongly active while the pure n-hexane and chloroform extract was not active at all against the S.typhi and K.pneum pathogen.

Table 2

Antibacterial efficacy of extracts against pathogens
Extract	Inhibition zone(mm) against
Extract	S.aureus	S.typhi	K.pneum	E.coli
MeOH	15	14	11	13
CHCl₃	7	0	0	7
n-hexane	3	0	0	2
Gentamycine	18	17	14	15
DMSO	0	0	0	0

In this work, two samples were tested by the above method. The results showed the crude methanol extract was strongly active while the pure n-hexane and chloroform extract was not active at all against the S. Typhi and K. pneum pathogens. The chloroform and n-hexane extract of S. aureus and E. coli are active. The outcome of this study has shown that the leaves extract of Rhus vulgaris possesses inhibitory potential against S. aureus while S. Typhi and K. pneum were partially resistant to their activity.

Generally, the extracts showed greater antibacterial activity against gram-positive as compared to gram-negative bacteria. Concerning individual pathogens, the methanol extract showed greater inhibition than chloroform and n-hexane extract.

Total phenol and total flavonoid Contents

In the present study, the total phenolic, flavonoid contents, and antioxidant activities of three different extracts of R.vulgaris were determined. TPC and TFC were calculated by

extrapolation from the calibration curve prepared from gallic acid, catechin, and ascorbic acid concentrations, respectively. Table 3 displays the TPC, TFC, and AAE of three different extracts of R.vulgaris.

Table 3

Extraction yield, TPC, TFC, and antioxidant activity of n-hexane, acetone, and 80% of methanol in the leaves extract of *Rhus vulgaris (*mean ± SD, n = 3).
Extracts	TPC (mg GAE/g of DW)	TFC (mg CE/g)	DPPH Inhibition ( mg AAE/ g)
n-hexane extract	5.82 ± 0.6	2.21 ± 0.34	0.756 ± 0.8
Acetone extract	43.23 ± 0.98	12.56 ± 0.12	1.10 ± 0.1
80% aqueous methanol extract	83.15 ± 0.6	23.47 ± 0.87	131.56 ± 0.6

Key: - GAE = gallic acid equivalent; CE = Catechin Equivalent; TFC = total flavonoid content; TPC = total phenol content; AAS = Ascorbic Acid Equivalent; DPPH = 2, 2-diphenyl-1-picryl-hydroxyl radical.

The results of total phenolic contents were obtained from the regression equation of the calibration curve of gallic acid Y = 0.0116x + 0.0414, R² = 0.9992) shown in Fig. 2. The Values are expressed in gallic acid equivalents (GAE) for phenols shown in (Table 3).

The study showed that the plant extracts had a significant amount of total phenolic and flavonoid content. Among the extracts, the methanol extract of R. vulgaris had the highest total phenolic content, which was measured to be 83.15 ± 0.6 mg GAE/g. Additionally, the methanol extract had the highest total flavonoid content, followed by the acetone and hexane extracts. The concentration of total phenolic and flavonoid content may vary depending on the polarity of the solvents used in the extraction process. Phenolic compounds are known for their strong antioxidant properties as they can neutralize free radicals, singlet oxygen, and superoxide radicals by replacing the aromatic ring of the phenolic components with hydroxyl groups [29]. Numerous studies have linked a high phenolic diet to the prevention of cardiovascular, metabolic, infectious, and cancer diseases due to the pharmacological and biological activities of phenolic compounds [30].

The high phenolic content recorded in this study in the presence of methanol fraction is consistent with the research in [31], which reported 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. Recent studies aimed to the evaluation of phenolic contents in this the Rhus family plant extracts. The methanolic extracts 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) [32]. Another report also revealed an evaluation of total phenolic contents in ethanolic extracts of Rhus javanica from leaves (51.91\(\pm\)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 [33] all reports were 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% 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 and positive correlation between the antioxidant activities of plant extracts and total phenolic content, which implies that the plant’s phenols are the major contributor to the antioxidant activity in these extracts.

Total flavonoids Contents

The flavonoid content of R. vulgaris 80% methanol, acetone, and n-hexane extracts was determined by calculating the milligrams of catechin equivalents (CE) using the regression equation of the calibration curve (R² = 0.9987), shown in Fig. 3. The results revealed that the 80% aqueous methanol and acetone extracts contained high flavonoid contents of 23.47 ± 4.87 CE/100g DW and 12.56 ± 0.12 CE/100g DW, respectively, while n-hexane had the lowest content of 2.21 ± 7.34 CE/100g DW. Additionally, it was observed that the solvent used had a similar effect on both total flavonoid content (TFC) and total phenolic content (TPC).

Flavonoids are naturally occurring polyphenol compounds with benzo-pyrone structures. The flavonoid content was determined by the aluminum chloride method. Aluminum chloride forms acid-stable complexes with the C-4 keto groups and either of the C-3 or C-5 hydroxide groups of flavones and flavanols [34].

In this study, the researchers compared the total flavonoid content of R. vulgaris with that of R. javanica from different parts of the plant, including leaves, stems, and fruits. The results showed that R. javanica leaves had the highest total flavonoid content, with a concentration of 17.78 ± 0.002 mg/L 1g of extract, while R. javanica greenish fruit had the lowest concentration, with only 11.3 ± 0.02 mg/ 1 g of extract. R. javanica stem had a slightly higher concentration of flavonoids compared to the greenish fruit, with a concentration of 5.06 ± 0.004mg/ 1 g of extract. These findings indicate that the total flavonoid content varies significantly among different parts of the same plant species [33]. Interestingly, other studies have also reported similar variations in flavonoid content among different species of Rhus. The species R. tripartite and R. pentaphylla exhibited significantly different flavonoid contents in their methanolic extracts of leaves, stem cortex, and fruit methanol extracts. Specifically, R. tripartite had the highest flavonoid content in its leaves, with 58.91 mg RE g^− 1 DW, while R. pentaphylla had the highest flavonoid content in its leaves, with 62.91 mg RE g^− 1 DW. The stem cortex and fruit extracts of R. pentaphylla also exhibited significant amounts of flavonoids, with 5.88 and 10.41 mg RE g^− 1 DW, respectively[32].

Dpph Radical Scavenging Activity

The antioxidant activity of R.vulgaris was assessed using the DPPH assay, with ascorbic acid serving as the standard. For this analysis, various extract solutions and ascorbic acid solutions were subjected to different concentrations and incubated at room temperature. The spectrophotometer was then used to measure the absorbance of each solution. The scavenging percentage of free radicals at various concentrations, as well as the IC₅₀ values for each extract and ascorbic acid, were computed and are presented in Table 3 and Fig. 4.

Table 1 displays the DPPH assay results for the free radical-scavenging activity of R. vulgaris leaves extract. The extract's DPPH free radical-scavenging activity (0.756 ± 0.8, 1.10 ± 0.1, and 131.56 ± 0.6mg AAE/g n-hexane, acetone, and 80% aqueous methanol, respectively) was observed to increase with increasing concentration. The methanol extract was found to be more potent in terms of antioxidant activity due to its higher total phenol and total flavonoid contents, as shown in Table 3. As the concentration increased, the percent inhibition by the extract also increased. The sample's concentration and absorbance were inversely related, with the absorbance decreasing as the sample concentration increased. With the exception of the n-hexane and acetone extracts, there were significant differences (p < 0.05) in the DPPH radical scavenging abilities between all the extracts.

The high level of antioxidant activity in the methanol extract could be attributed to the presence of phytochemicals like flavonoids, polyphenols, and tannins. Previous studies have also supported the fact that various plant-derived compounds, such as tannins, saponins, flavonoids, terpenoids, glycosides, alkaloids, and phenol, have significant antioxidant properties[18].

This study aimed to determine the inhibitory effect of the extracts at different concentrations. Table 4 shows the percentage of inhibition of the 80% aqueous methanol, acetone, and n-hexane extracts, while Fig. 4 illustrates the relationship between the percentage of inhibitory activity and concentration.

Table 4

% Inhibition of aqueous 80% methanol, acetone, and n-hexane extracts
Conc.(mg/L)	% Inhibition
Conc.(mg/L)	aqueous 80% methanol	Acetone	n-hexane
0	0	0	0
0.05	37.82383	40.41451	48.4456
0.1	78.23834	88.34197	89.89637
0.2	78.75648	90.67358	92.48705
0.4	77.72021	91.45078	94.55959
0.8	78.49741	94.04145	97.40933

DPPH method was developed by Blois (1958) with the viewpoint to determine the antioxidant activity in a like manner by using a stable free radical 2,2-diphenyl-1- picrylhydrazyl (DPPH; C₁₈H₁₂N₅O₆, M = 394.33). The assay is based on the measurement of the scavenging capacity of antioxidants towards it. As shown in Fig. 5 the odd electron of a nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine [35]. DPPH is a stable free radical that 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 of the sample [36].

Previous studies have shown that the plants of the Rhus genus, namely Rhus typhina and Rhus javanica, have been used for their medicinal properties. That was reported that the DPPH free radical-scavenging activity of the extract increased with increasing extract concentration. In particular, the methanol extracts of Rhus typhina fruit showed 48.91%, 60.89%, and 93.79% inhibition at 10, 20, and 50 mg/ml, respectively [37]. In another report, the ethanolic extracts of Rhus javanica were tested for DPPH scavenging activity, and the IC₅₀ values were found to be 157.699, 561.046, 189.31, and 239.276 for Rhus leaves, stem, greenish fruit, and blackish-grey fruit, respectively, using quercetin standards [33]. Our results corroborate those of Odongo et al. (2017), who reported that plant parts of this species have significant antioxidant activity due to the presence of phenols, flavonoids, and tannins [31].

In the present investigation, extractions from Rhus vulgaris leaves powder have been carried in hexane, acetone, and methanol solvents. Phytochemical screening of the extracts revealed the presence of Alkaloids, Glycosides, Steroids, Anthraquinones, Carbohydrates, Phenols, Flavonoids, Tannins, Coumarins, and Proteins. The plant possesses a high amount of total phenolic and total flavonoid contents. DPPH scavenging activity showed that the plant possesses high antioxidant properties. Among all three extracts, the concentration of methanol extract was the highest (131.56 ± 0.6mg AAE/g). The methanol extract was found to be the most potent antibacterial agent to inhibit the growth of S. aureus and S.typhi. Besides, the acetone and n-hexane extracts of Rhus vulgaris are found to be slightly less than the killing of S.aureus and E.coli but also no antibacterial effect on K.pneumoniae and S.typhi with the reference of Gentamicin antibiotic. Hence, the Rhus vulgaris used in this study can be suggested as a suitable source of natural antioxidants. Further investigations on the different mechanisms shall be studied.

Acknowledgment

The support of the University of Gondar and Jigjiga University and individuals is greatly appreciated

Ethics approval and consent to participate

All methods are carried out according to the institution’s guidelines and regulations.

Authors’ contributions

AA - Study design, Literature search, data collection, data analysis, data interpretation, writing the manuscript, GG - Research supervision, Study design, editing manuscript, DS – Research supervision, editing the manuscript, ML- Research supervision, Provision of reagents and materials used in the study. All authors read and approved the final manuscript.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Funding

Funding is not applicable to this study.

Availability of data and materials

All data generated and analyzed are included in this research article.

Baba SA, Malik SA. Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume. J Taibah Univ Sci. 2015;9:449–54. 10.1016/j.jtusci.2014.11.001.
Ullah S, Hyun CG. Evaluation of total flavonoid, total phenolic contents, and antioxidant activity of strychnobiflavone. Indonesian J Chem. 2020;20:716–21. 10.22146/ijc.44331.
Kumar S, Sandhir R, Ojha S. Evaluation of antioxidant activity and total phenol in different varieties of Lantana camara leaves. BMC Res Notes. 2014;7:1–9. 10.1186/1756-0500-7-560.
Kumar SV, Kumar SP, Rupesh D, et al. Journal of Chemical and Pharmaceutical Research preparations. J Chem Pharm Res. 2011;3:675–84.
Wadkar KA. .Magdum. Evaluation of total phenolic content, flavonoid content and antioxidant activity of stem bark of Bridelia retusa (Linn.) spreng. Nat Prod. 2009;5:167–70. 10.1002/9780470034590.emrstm0340.
Plaza CM, De Torres LED, Lücking RK, et al. Antioxidant activity, total phenols and flavonoids of lichens from venezuelan andes. J Pharm Pharmacogn Res. 2014;2:138–47.
Mohamed, Khedr FG, Mohammed EI. Phenolic Compounds, Antioxidant and Antibacterial Activities of Rhus flexicaulis Baker. 2019; 12:17–21
Rayne S, Mazza G. Biological activities of extracts from sumac (Rhus spp.): A review. Plant Foods Hum Nutr. 2007;62:165–75. 10.1007/s11130-007-0058-4.
Njoroge PW, Opiyo SA. Antimicrobial Activity of Root Bark Extracts of Rhus natalensis and Rhus ruspoli. Basic Sci Med. 2019;8:23–8. 10.5923/j.medicine.20190802.01.
Alam P, Parvez MK, Arbab AH, et al. Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita. Pharm Biol. 2017;55:1450–7. 10.1080/13880209.2017.1304428.
Gebeyehu G, Asfaw Z, Enyew A, et al. Ethnobotanical study of traditional medicinal plants and their conservation status in Mecha Woreda. Int J Pharmaceuticals Health Care Res. 2014;02:137–53.
Seble WY, Zemede A, Ensermu K. Ethnobotanical study of medicinal plants used by local people in Menz Gera Midir District, North Shewa Zone, Amhara Regional State, Ethiopia. J Med Plants Res. 2018;12:296–314. 10.5897/jmpr2018.6616.
Ramathal DC, Ngassapa OD. Medicinal plants used by Rwandese traditional healers in refugee camps in Tanzania. Pharm Biol. 2001;39:132–7. 10.1076/phbi.39.2.132.6251.
Tabuti JRS, Lye KA, Dhillion SS. Traditional herbal drugs of Bulamogi, Uganda: Plants, use and administration. J Ethnopharmacol. 2003;88:19–44. 10.1016/S0378-8741(03)00161-2.
Okullo JBL, Omujal F, Bigirimana C, et al. Ethno-Medicinal Uses of Selected Indigenous Fruit Trees from the Lake Victoria Basin Districts in Uganda. J Med Stud. 2014;2:78–88.
Odongo CO, Musisi NL, Waako P, et al. Chewing-stick practices using plants with anti-streptococcal activity in a Ugandan rural community. Front Pharmacol. 2011;2:1–5. 10.3389/fphar.2011.00013.
Ochwangi DO, Kimwele CN, Oduma JA, et al. Medicinal plants used in treatment and management of cancer in Kakamega County, Kenya. J Ethnopharmacol. 2014;151:1040–55. 10.1016/j.jep.2013.11.051.
Mutuku A, Mwamburi L, Keter L, et al. Evaluation of the antimicrobial activity and safety of Rhus vulgaris (Anacardiaceae) extracts. BMC Complement Med Ther. 2020;20:1–12. 10.1186/s12906-020-03063-7.
Abegaz BM. Novel phenyl anthraquinones, isoflurano naphthoquinones, homoisoflavonoids, and biflavonoids from African plants in the genera Bulbine, Scilla, Ledebouria, and Rhus. Phytochem Rev. 2002;1:299–310. 10.1023/A:1026066626537.
Mdee LK, Yeboah SO, Abegaz BM. Rhus chalcones II-VI, five new bichalcones from the root bark of Rhus pyroides. J Nat Prod. 2003;66:599–604. 10.1021/np020138q.
Arsule CS, Sable KV. Preliminary Phytochemical Analysis of Euphorbia hirta Linn. Leaves. Int J of Life Sciences. 2017;5:746–8.
Nataraj ND. Preliminary Phytochemical Screening of Solanum Trilobatum (L.) Young Leaves. Int Res J Pharm. 2014;5:80–2. 10.7897/2230-8407.050216.
Vimalkumar CS, Vilash V, Krishnakumar NM. Comparative Preliminary Phytochemical Analysis of Ethanolic Extracts of Leaves of Olea Dioica Roxb, Infected with The Rust Fungus Zahouania oleo Cummins and Non Infected Plants. J Pharmacogn Phytochem. 2014;3:69–72.
Ayoola GA, Coker HAB, Adesegun SA, et al. Phytochemical Screening and Antioxidant Activities of Some Selected Medicinal Plants Used for Malaria Therapy in Southwestern Nigeria. Trop J Pharm Resear. 2008;7:1019–24.
Suhrulatha D, Savithramma N. Screening of Selected Medicinal Plants for Secondary Metabolites. Middle-East J Sci Res. 2011;8:119.
Ezez D, Tefera M. Effects of Solvents on Total Phenolic Content and Antioxidant Activity of Ginger Extracts. J Chem 2021; 2021: 1–5. doi:10.1155/2021/6635199
Haile K, Mehari B, Atlabachew M, et al. Phenolic composition and antioxidant activities of cladodes of the two varieties of cactus pear (opuntia ficus-indica) grown in Ethiopia. Bull Chem Soc Ethiop. 2016;30:347–56. 10.4314/bcse.v30i3.3.
Abebe M, Abebe A, Mekonnen A. Assessment of antioxidant and antibacterial activities of crude extracts of verbena officinalis Linn root or Atuch (Amharic). Chem Int. 2017;3:172–84.
Farahmandfar R, Esmaeilzadeh Kenari R, Asnaashari M, et al. Bioactive compounds, antioxidant and antimicrobial activities of Arum maculatum leaves extracts as affected by various solvents and extraction methods. Food Sci Nutr. 2019;7:465–75. 10.1002/fsn3.815.
Khalid A, Shadid, Ashok K, Shakya RR, Naik N, Jaradat, Husni S, Farah NaeemShalan. NoomanA.Khalaf AndGhalebAO. Phenolic Content and Antioxidant and Antimicrobial Activities of Malva sylvestris L., Malva oxyloba Boiss., Malva parviflora L., and Malva aegyptia L. Leaves Extract Khalid. J Chem 2021; 2021: 1–10. doi:10.1016/j.indcrop.2013.09.002
Odongo E, Mungai N, Mutai P, et al. Antioxidant and anti-inflammatory activities of selected medicinal plants from western Kenya. Afr J Pharmacol Ther. 2017;6:178–82.
Itidel C, Chokri M, Mohamed B, et al. Antioxidant activity, total phenolic and flavonoid content variation among Tunisian natural populations of Rhus tripartita (Ucria) Grande and Rhus pentaphylla Desf. Ind Crops Prod. 2013;51:171–7. 10.1016/j.indcrop.2013.09.002.
Anggraini NB, Elya B, Anti-elastase. Antioxidant, Total phenolic and Total Flavonoid Content of Macassar Kernels (Rhus javanica L) from Pananjung Pangandaran Nature Tourism Park- Indonesia. J Nat Remedies. 2020;20:61–7. 10.18311/jnr/2020/24240.
Kamtekar S, Keer V, Patil V. Estimation of phenolic content, flavonoid content, antioxidant and alpha amylase inhibitory activity of marketed polyherbal formulation. J Appl Pharm Sci. 2014;4:61–5. 10.7324/JAPS.2014.40911.
Kedare SB, Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Technol. 2011;48:412–22. 10.1007/s13197-011-0251-1.
Carmona-Jiménez Y, García-Moreno MV, Igartiburu JM, et al. Simplification of the DPPH assay for estimating the antioxidant activity of wine and wine by-products. Food Chem. 2014;165:198–204. 10.1016/j.foodchem.2014.05.106.
Kossah R, Zhang H, Chen W. Antimicrobial and antioxidant activities of Chinese sumac (Rhus typhina L.) fruit extract. Food Control. 2011;22:128–32. 10.1016/j.foodcont.2010.06.002.

No competing interests reported.

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Evaluation of Total Phenolic, Flavonoid contents, and Antioxidant activity of Rhus vulgaris leaves extracts

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Material And Methods

Study area description

Chemicals and reagents

Apparatus and equipment

Collection of Plant materials

Extraction procedure

Preparation of standard solutions

Preliminary Phytochemical Screening

Determination of total phenolic content

TPC = \(\frac{C.V.DF}{m}\)……………………………………………. (1)

Determination of total flavonoid contents

TPC = \(\frac{C.V.DF}{m}\)……………………………………………. (2)

Determination of antioxidant activity-DPPH

Evaluation of the antibacterial activity

Statistical analysis

Results And Discussion

Antibacterial Activity of the Crude Extracts against Bacterial Strain

Total phenol and total flavonoid Contents

Total flavonoids Contents

Dpph Radical Scavenging Activity

Conclusion

Declarations

References

Additional Declarations

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