INTRODUCTION:

From the dawn of human civilization, medicinal plants have been used as remedial agents because medicinal plants serve as the most privileged source for the majority of population of the world. Human populations the world over have been consistently aware about plants for the treatment of various diseases and for their proper use.

Medicinal plants are the organisms that are naturally supplied with chemical compounds having high remedial properties in the advancement of physiological action of synthetic stimulants that are beneficial to humans. Medicinal plants have an antique origin for their use basically in the form of beverages and extracts in different cultures around the world¹. Medicinal plants contain a significant amount of phenolic compounds that are of great interest because these compounds are related with various activities; the most significant is antioxidant activity, which is important in preventing oxidative stress. Inconsistent production of antioxidants by the cells due to the production of free radicals in excess is the main reason due to which oxidative stress happens².

Zanthoxylum armatum, also called winged prickly ash, is a species of plant in the Rutaceae family. Zanthoxylum armatum is found in hot valleys of subtropical to temperate Himalayas (Kashmir to Bhutan), north-east India and Pakistan, Myanmar, Thailand, China, Bangladesh, Bhutan, Japan, North and South Korea, North Taiwan, Philippines and Sumatra³. The different parts of the plants: leaves, fruits, stem, bark, seeds have been used in various domestic medicinal systems as carminative, antipyretic, appetizer, stomachic, toothache, dyspepsia^4,5,6. Zanthoxylum armatum DC improves speech in kids and increases saliva secretion⁷. Various pharmacological studies have been demonstrated that a broad group of chemical compounds containing alkaloids, flavonoids, lignins, coumarins, phenols, terpenoids etc., have been found in this plant which are responsible for various biological activities like antioxidative, antimicrobial, antiviral, hepato-protective, insecticidal and anticancerous⁸. Manjula et al (2020) have compared antimicrobial and synergistic potential of extracts of leaves of Zanthoxylum armatum DC. from different geographical regions of Himachal Pradesh, India⁹. Gas chromatography-mass spectrometry analysis of the essential oil resulted in the identification of 38 components, of which linalool (62%) and limonene (18.1%) were the major components⁹. Therapeutic potential of Z. armatum extract is directly related to total phenolic and flavonoids contents. Today the interest subject of research beneficial for the pharmaceutical purposes are the active metabolites especially obtained from herbs. Natural products procured specifically from plant sources have the efficiency to reduce oxidative stress by acting as antioxidants, therefore, in this work, variation in the total content of phenolics and flavonoids compounds and antioxidant activity in different extracts of leaves of Z.armatum collected from different geographical regions of Himachal Pradesh was evaluated. Also, correlation between different parameters such as phenolic content, flavonoid content, DPPH and FRAP assay was analyzed.

MATERIALS AND METHODS:

Chemicals and solvents:

The chemicals such as 2, 2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), were obtained from Sigma-Aldrich Co. LLC, Mumbai. The solvents such as chloroform, methanol, butanol and dimethyl sulfoxide (DMSO) used were procured from Loba Chemie Pvt. Ltd., Mumbai.

Sample Collection and Extract preparation:

Fresh leaves of Z. armatum were collected from six districts of Himachal Pradesh i.e. Hamirpur, Mandi, Bilaspur, Solan, Shimla and Sirmour in the months of May-June 2017. The leaves were carefully washed with tap water followed by 70% ethanol and then allowed to dry in shade. After drying, the leaves were grind to fine powder using an electric grinder. The dried powder was then used for preparation of extracts using different solvents (i.e. methanol, n-butanol, chloroform and aqueous). Cold maceration technique was used to prepare extracts¹⁰ and extracts were allowed to evaporate at 40ºC. The dried extracts were stored in air tight bottles at 4ºC till further use.

Quantification of total phenolic contents:

The total phenolic content of various solvent extracts of Z. armatum leaves collected from different regions were determined by using Folin-ciocalteau reagent method¹¹ using gallic acid as a standard. The concentration of phenolic compounds was calculated from calibration curve of gallic acid (10 to 50µg ml^-1) and expressed in terms of gallic acid equivalents (GAE) per gram of the extract.

The total content of phenolic compounds in the plant extracts was calculated using the following equation:

c X V

C= -------------

Where ‘C’ is total content of phenolic compounds in mg g^-1 plant extract in GAE, ‘c’ is the concentration of gallic acid estimated from the calibration curve (mg ml^-1), ‘V’ is the volume of extract in ml and ‘m’ is the weight of crude plant extract in grams.

Quantification of total flavonoid content:

The total flavonoid content of various solvent extracts of Z. armatum leaves collected from different regions were determined by using aluminium chloride (AlCl₃) method ¹². Rutin was used as standard. The flavonoid content was calculated from standard curve of rutin (10 - 50μg ml^-1) and expressed as rutin equivalents (RE) per gram of the extract.

The total content of flavonoid in the extract was calculated by using the following equation:

c X V

C= -------------

Where ‘C’ is total content of flavonoid compounds in mg g^-1 plant extract in GAE, ‘c’ is the concentration of rutin estimated from the calibration curve (mg ml^-1), ‘V’ is the volume of extract in ml and ‘m’ is the weight of crude plant extract in grams.

In vitro antioxidant activity in various solvents extracts of leaves of Z. armatum:

Antioxidants play a defensive role against reactive oxygen species toxicities by the prevention of the reactive oxygen species construction, by scavenging reactive metabolites and converting them to less reactive molecules. Extracts of leaves of Z. armatum were dissolved at a concentration of 1mg ml^-1 and then diluted in order to prepare different concentrations for antioxidant assay. The antioxidant potential of Z. armatum was determined by two different methods¹³. Antioxidant activity of extracts was determined in terms of IC₅₀ value (half maximal inhibitory concentration) indicating how much of a particular drug or other substance is required to inhibit a given biological process. Lower the value of IC₅₀, higher will be the antioxidant activity.

DPPH (2, 2-Diphenyl-1-picrylhydrazyl) radical scavenging activity:

DPPH radical scavenging activity of the different extracts of Z. armatum leaves collected from different geographical regions were measured by using method¹⁴ with some modifications. The capability of scavenging DPPH radical was calculated using the following equation:

A (control) - A (sample)

% DPPH radical =------------------------------ x 100

scavenging activity A (control)

Where A (control) is absorbance of control and A (sample) is absorbance of sample.

FRAP (Ferric Reducing Antioxidant Power) assay:

The method earlier described to measure the ability to reduce ferric ions¹⁵. A positive reference standard used was Ascorbic Acid. The antioxidant capacity based on the ability to reduce ferric ions of extract was calculated from the linear calibration curve of FeSO₄ (2.5-20μM) and expressed as μmol FeSO₄ equivalents per gram of extract.

Statistical analysis:

Each sample was analyzed individually in triplicates and the results were then presented as the mean values along with their standard deviations. Total phenolic content, total flavonoid content and half maximal inhibitory concentration (IC₅₀ values) of the antioxidant assay were calculated by linear regression analysis method.

RESULTS AND DISCUSSION:

Determination of total phenolic content in various solvents of Z. armatum leaves:

The phenolic compounds are one of the most effective antioxidative constituent that contributes to the antioxidant activity in medicinal and food plants and it is important to quantify the total phenolic content. The higher contents of phenols and flavonoids in the plants are mainly responsible for strong antioxidant properties ^16,17. In the present study, total amount of phenolic content was calculated from standard curve of gallic acid (y = 0.008x + 0.007; R²=0.999). Among all the solvent extracts of different locations, methanol extract (9.26 ± 1.13mg g^-1 GAE) of leaves samples collected from Sirmour showed higher phenolic content followed by extract of Sirmour (8.70±0.3 mg g^-1GAE) and n-butanol extract of Bilaspur (8.28±0.9mg g^-1 GAE). Least phenolic content was found in aqueous extract of Solan (2.98±0.7 mg g^-1 GAE) and Hamirpur (2.29 ± 0.11mg g^-1 GAE) (Fig. 1). In contrast to our study,¹⁸analyzed the phenolic and flavonoid content in ethanolic and n- Hexane extracts of leaves, bark and fruits of Z. armatum and found that highest phenolic contents were found in ethanolic extract of fruit (21.68±0.44mg g^-1) followed by ethanolic extract of bark (16.48±1.33mg g^-1) and leaf (11.66±0.33mg g^-1). The amount of TPC in methanol extract of leaves was also quantified by^19,20. In contrast to our results, they showed higher amount of TPC in methanolic extract of Z. armatum leaves i.e. 13.1±0.8 mg g^-1 GAE, 89.2±0.59 mg g^-1 GAE, respectively. Enormous amount of phenols (140.71mg GAE g^-1) in methanolic extract of Z. armatum leaves were also reported²¹.

Fig. 1: Total phenolic content in different extracts of leaves of Z. armatum. Gallic acid was used as standard and phenolic content was expressed as GAE (Gallic acid equivalents).

Determination of total flavonoid content in various solvents of Z. armatum leaves:

Total flavonoid content was calculated from standard curve of rutin (y = 0.006x + 0.060; R²=0.992) where ‘x’ was the absorbance and ‘y’ was RE expressed as mg g^-1. Total flavonoid content in different solvent extracts of Z. armatum leaves was given in Fig. 2. It was observed that total flavonoid content was higher in chloroform extract of Solan (10.7 ± 0.29 mg g^-1 RE) and Bilaspur (10.15 ± 0.77 mg g^-1 RE), while least amount of flavonoid were present in aqueous extract of Hamirpur (1.18 ± 0.34 mg g^-1 RE) and Bilaspur (0.93 ± 0.31 mg g^-1 RE) (Fig. 2). In contrast to our study,¹⁸ analyzed the flavonoid content in ethanolic and n- Hexane extracts of leaves, bark and fruits of Z. armatum and found that highest flavonoid content was found in ethanolic extract of fruit (22.8 ± 1.33 mg g^-1) followed by ethanolic extract of bark (18.33 ± 1.22 mg g^-1) and leaf (13.68 ± 0.66 mg g^-1). Flavonoid content of 12.7± 0.8 QE g^-1was also reported in methanolic extract of leaves ^[19]. In contrast to our report, ^[20] reported higher amount of total flavonoid content in methanolic extract of Z. armatum (63.8 ± 0.34 mg g^-1) which was also in agreement with the results of²¹ showing enormous amount of flavonoid content (88.53 mg of Quercetin per gram of extract) in methanolic extract of Z. armatum leaves.

(A)

Fig. 2: Total flavonoid content in different extracts of leaves of Z. armatum. Rutin was used as standard and flavonoid content was expressed as RE (Rutin equivalents).

In-vitro antioxidant activity of leaves of Z. armatum collected from different geographical regions of Himachal Pradesh:

Antioxidant characteristics are evaluated by different methods, but the proper antioxidant property of the extracts cannot be provided by one single method. So, it is necessary to characterize the extract by different mechanisms of antioxidants^22,23. Antioxidant activities of different extracts of Z. armatum leaves were analysed at different concentrations (10-50 µg ml^-1) using DPPH radical scavenging assay and FRAP assay. Antioxidant activities were expressed in terms of IC₅₀ and lower the IC₅₀ value, higher will be the antioxidant potential.

Among the antioxidant activity screening methods the DPPH assay has been proven to be most sensitive method and it is fast and easy²⁴. Half maximal inhibitory concentration (IC₅₀) of different extracts of leaves of Z. armatum collected from different regions with DPPH assay was shown in table 1 and Fig. 3. Highest DPPH radical scavenging was shown by chloroform extract of Sirmour (9.61±4.02µg ml^-1) followed by n-butanol extract of Sirmour (17.47±2.3µg ml^-1). Least DPPH radical scavenging was observed with n-butanol extract of Mandi (156.16±4.8µg ml^-1), and aqueous extract of Hamirpur (152.15±2.1µg ml^-1) (Fig. 3). In FRAP assay, extracts also showed a significant variation in antioxidant potential in different extracts of leaves of Z. armatum from different regions of Himachal Pradesh (Fig. 4, Table-2). Highest FRAP activity was observed with methanol extract of Shimla (55.92±2.0µM Fe (II) equivalents) followed by aqueous extract of Sirmour (58.35±2.9 µM Fe (II) equivalents) and methanol extract of Sirmour (63.88 ± 3.9µM Fe (II) equivalents). The least FRAP activity was found with n-butanol extract of Mandi (415.8±1.4 µM Fe (II) equivalents) and n-butanol extract of Bilaspur (265.23 ± 1.9 µM Fe (II) equivalents) (Fig. 4).

DPPH radical scavenging of methanolic leaf extract (78.39 %) as compared to that of standard (83.50%) at 250μg ml^-1 was also reported²⁴. Study conducted on essential oil and methanol extract of Z. armatum leaves showed that % DPPH radical scavenging was higher in methanolic extract (IC₅₀- 0.067 ± 0.002 mg ml^-1), while ethanolic extract showed (IC₅₀- 6.04 ± 0.08 mg ml^-1). There are several other reports on antioxidant nature of essential oil from leaves of Z. armatum^25,26,27,28. However, antioxidant activity plants depend upon the amount of phenols and flavonoids ²⁹.

Figure 3: DPPH radical scavenging activity of standard and four different extracts of Z. armatum leaves collected from different regions of Himachal Pradesh. Each point represents the values obtained from three independent experiments (mean ± standard deviation).

Figure 4: (FRAP) Ferric reducing antioxidant power activity of standard and four different extracts of Z. armatum leaves collected from different regions of Himachal Pradesh. Each point represents the values obtained from three experiments, performed in triplicates (mean ± standard deviation).

Table 1: Half maximal inhibitory concentration (IC₅₀) of different extracts of leaves of Z. armatum collected from different districts using DPPH and FRAP assay

Assay	Type of Extract	Hamirpur	Mandi	Bilaspur	Solan	Shimla	Sirmour	Ascorbic acid
DPPH^*	Methanol	69.73 ± 0.6	93.02 ± 6.0	72.25 ± 3.6	126.63± 4.2	61.39 ± 4.3	117.06 ± 6.5	17.30 ± 1.24
	n-Butanol	83.42 ± 5.6	156.16 ± 4.8	127.87 ± 6.3	130.25 ± 5.1	144.35± 4.9	17.47 ± 2.3
	Chloroform	86.57 ± 2.8	77.48 ± 6.6	102 ± 4.7	67.16 ± 3.9	139.72 ± 4.7	9.61 ± 4.02
	Aqueous	152.15 ± 2.1	87.68 ± 6.8	102.38 ± 4.1	109.29± 4.7	75.13 ± 5.5	58.69 ± 6.0
FRAP^**	Methanol	128 ± 6.7	100 ± 2.6	114.71 ± 3.3	91.41 ± 6.9	55.92 ± 2.0	63.88 ± 3.94	116.57 ± 8.7
	n-Butanol	205 ± 1.8	415.8 ± 1.4	265.23 ± 1.9	218.21 ± 1.0	72.10 ± 4.98	160.72 ± 3.75
	Chloroform	128.58 ± 4.4	114.23 ± 1.0	75.99 ± 2.7	103.62 ± 2.3	76.85 ± 4.7	105.06 ± 3.6
	Aqueous	89.9 ± 4.5	97.9 ± 2.7	154.14 ± 4.2	122.42 ± 2.9	140.92± 0.4	58.35 ± 2.9

Values are expressed as mean ± standard deviation of three independent experiments.

*-µg ml^-1, **-µM Fe (II) equivalents.

Table 2: Pearson correlation matrix of between total phenolic content, total flavonoid content, DPPH and FRAP in different extracts of leaves of Z. armatum

Assay	Chloroform				Methanol				n-Butanol				Aqueous
Assay	TPC	TFC	DPPH	FRAP	TPC	TFC	DPPH	FRAP	TPC	TFC	DPPH	FRAP	TPC	TFC	DPPH	FRAP
TPC	1	.569*	-.341	.208	1	.061	.398	-.471*	1	.316	-.271	.555*	1	-.012	-.681**	-.656**
TFC		1	-.205	-.369		1	-.339	-.616**		1	.126	.044		1	-.520*	.063
DPPH			1	-.580*			1	-.224			1	.140			1	.326
FRAP				1				1				1				1

** Significant at the 0.01 level (p < 0.01); TPC- Total phenolic content, TFC – Total flavonoid content.

CONCLUSIONS:

The present study showed that all the extracts of Z. armatum leaves possess antioxidant activity. Amount of phenols and flavonoid content varies in different solvents and also in different geographical regions within same solvent, indicating the effect of solvent on solubility of compounds and effect of environmental conditions on phytochemical composition of plants. However, the present study confirms also the utilization of Z. armatum leaves as source of natural antioxidant compounds.

ACKNOWLEDGEMENT:

The authors acknowledge Shoolini University, Solan, for providing infrastructure support to conduct the research work. Authors also acknowledge the support provided by Yeast Biology Laboratory, School of Biotechnology, Shoolini University, Solan, India.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 30.04.2020 Modified on 12.06.2020

Research J. Pharm. and Tech. 2021; 14(4):2270-2276.

DOI: 10.52711/0974-360X.2021.00401