wjpmr, 2021,7(2), 186-192 SJIF Impact Factor: 5.922 WORLD JOURNAL OF PHARMACEUTICAL Research Article Nishat et al. World Journal of Pharmaceutical and Medical Research AND MEDICAL RESEARCH ISSN 2455-3301 www.wjpmr.com Wjpmr

EVALUATION OF TOTAL POLYPHENOLS, FLAVONOIDS AND ANTIOXIDANT ACTIVITY OF MYRICA ESCULENTA BUCH-HAM.EX D.DON: FRUITS

Dr. Nishat Anjum1* and Y. C. Tripathi2

1Science Department, Ras Bihari Bose, Subharti University, Dehradun-248007. 2Chemistry and Bioprospecting Division, Forest Research Institute, PO New Forest, Dehradun – 248006, Uttarakhand, India.

*Corresponding Author: Dr. Nishat Anjum

Science Department, Ras Bihari Bose, Subharti University, Dehradun-248007.

Article Received on 16/12/2020 Article Revised on 06/01/2021 Article Accepted on 26/01/2021

ABSTRACT

Background: Fruits of Myrica esculenta belongs to Family Myricaceae, Fruits have been used to treat a variety of

diseases for thousands of years. It is widely used in folk medicine to treat; a lot of research has been finding natural antioxidants of fruits of Myrica esculenta. The present study was aimed to evaluate the total polyphenol and antioxidant activity of Myrica esculenta. Methods: The present study was standard methods of phytochemicals. Total phenolic content the various extracts was determined spectrometrically using a modified Folin-Ciocalteu

method and antioxidant efficacy by following DPPH radical scavenging protocol, total flavonoid content by reported method. Results: The results showed that Myrica esculenta of Phytochemical screening the presence of alkaloids, steroids, terpenoids, flavonoids, phenolics, tannins, saponins, glycosides, carbohydrate, protein and amino acids in different extracts of Myrica esculenta fruits. Fruits were recorded methanol extract maximum

number of chemical constituents. High polarity solvent was present of flavonoids and phenolic constituents mostly found in extract. The total phenolic content (7.12±0.42 mg GAE/g of extract), total flavonoid content (4.54±0.017

mg QE/g of extract) and antioxidant activity (IC50, 55.00±0.341 µg/ml) were found highest in methanol extract. Conclusions: The present study established fruits of Myrica esculenta as rich sources of phenolic compounds and

natural antioxidants.

KEYWORDS: Myrica esculenta, Fruits, Phytochemicals, Phenolics, Flavonoids, Antioxidant activity.

1. INTRODUCTION is an evergreen dioecous tree, it is shade giving, medium size tree and it traditionally eats whole-seed and all Oxygen through excessive production of reactive oxygen fruits. All the parts of M. esculenta plant have huge species (ROS) can potentially cause cellular damage. nutritional and therapeutic importance. Fruits are used Oxidative stress is an important contributor to the for syrup, jams, pickels and preparation for refreshing pathophysiology of a variety of human pathological drinks.[9,10] Kafal is a naturally occurring antioxidant, it is conditions including cardiovascular dysfunction, widely used in folk medicine to treat ailments such as atherosclerosis, inflammation, carcinogenesis, drug cough, chronic bronchitis, ulcers, anaemia, fever, toxicity, reperfusion injury and neurodegenerative diarrhea and ear, nose and throat disorders.[11] M. diseases .[1] Plant contains many phytochemical that are esculenta has been studied for its chemical property and useful sources of natural antioxidant, such as phenolic a number of phytochemicals of differet type has been diterpenes, flavonoids, tannins and phenolic acid.[2,3] isolation and characterization from various parts of Polyphenols, especially flavonoids are generally known plants. Recently reported pharmacognostical and as the antioxidant agent in plant extracts.[4] phytochemical activity of M. esculenta steam.[12,13]

However, no systematic phytochemical and biological Myrica esculenta belongs to Family Myricaceae, Plant studies have so far been reported on fruits of the plant. species of this genus are distributed in china, Taiwan, The aim of study, therefore, was evaluation of total Japan, Western Highland of Cameroon, North America, polyphenols, flavonoids and antioxidant activity, Nepal and India.[5-8] Myrica esculenta is an important phytochemistry M. esculenta of fruits by DPPH radical medicinal plant in the sub-tropical Himalayas tracks upto scavenging assay. an altitude of 1200 to 2000m and flowering of M. esculenta is February-April and fruiting May-June, distributed to thoughtout commonly known as box berry, fruits are sweet and local name is Kafal or Kaphal. Kafal

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Nishat et al. World Journal of Pharmaceutical and Medical Research

2. MATERIALS AND METHODS replicated three times. It is based on the results of qualitative phytochemical screening, petroleum ether 2.1 Chemicals and Reagents (60-800), chloroform, ethylacetate, acetone and methanol All the chemicals and reagents used for the analytical extracts of Myrica esculenta fruits were selected for works were of laboratory grade and refer to Merck and evaluation of TPC and TFC and antioxidant activity. SD fine-chem limited. DPPH (1, 1-diphenyl-2- picrylhydrazyl), Folin-Ciocalteu reagent, sodium 2.5. Evaluation of total phenolic content (TPC) carbonate, aluminium chloride were all purchased from Total polyphenolic content (TPC) based on the results of Merck, USA. Specified detecting reagents like qualitative phytochemical analysis, petroleum ether (60- Dragendorff’s reagent, FeCl (Ferric choride), Ehrlich 3 800), chloroform, ethylacetate, acetone and methanol reagent, Ninhydrine etc. (for qualitative analysis) extracts were selected for estimation of total phenolic depends upon the nature and class of chemical content. The concentration of phenolics in the extracts constituents. Some reagents were freshly prepared for was determined using spectrophotometic method.[21] qualitative phytochemical screening of fruits extracts. Most active extract solution of concentration of 1mg/ml Some of the reagents were prepared during the was used in the analysis. Methanol solution of the extract experiments while some of the readymade available was used the analysis. Mixture was prepared by mixing reagents were used for qualitative detection. 0.5ml of methanol solution of extract; 2.5ml of 10% Spectrophotometer UV/Vis double beam (Chemito 1700, FolinCiocalteu reagent dissolved in the water and 2.5ml Thermo Fisher) was used for spectrophotometric 7.5% Na CO3. Blank test tube prepared containing 0.5ml determination of TPC (Total phenolic content), TFC 2 methanol, 2.5ml 10% Folin-Ciocalteu reagent dissolved (Total flavonoid content) and free radical scavenging in water and 2.5ml of 7.5% of Na CO . The solution was activity. 2 3 thereafter incubated at room temperature for 45min. The

absorbance was determined using spectrophotometer at 2.2. Plant materials λ = 765nm. The sample was prepared in triplicate for Fresh fruits of Myrica esculenta diseases free were max each analysis and the mean value of absorbance was collected from outskirts of Chail Chowk, Mandi, obtained. The same procedure was repeated for the Himachal Pradesh (H.P.), India. The fruits were standard solution of Gallic acid or calibration line was identified and authenticated by Systematic Botany made. Measured absorbance, the concentration of Section of Department of Botany, FRI. A voucher phenolics was read (mg/ml) from the calibration line; specimen has been preserved in the Chemistry & then the content of phenolics in extracts was expressed in Bioprospecting Division, FRI for future reference. The terms of Gallic acid equivalent (mg of GAE/100mg of collected fruits were cleaned properly under running tap extract). Over all the experiments were replicated thrice water to make them free from dust and then dried in and the data were presented as mean values ± standard shade at room temperature (250C). Dried fruits were deviation.[22, 23] stored in airtight bags at room temperature till future use.

2.6. Evaluation of total flavonoid content 2.3. Preparation of extracts Deviation The flavonoid content of chloroform, ethyl Dried Myrica esculenta fruits berries were successively acetate, acetone and methanol extracts was measured extracted with solvents, petroleum ether (60-800), based on methods [24]. Briefly, 0.5ml of sample was chloroform, ethylacetate, acetone, and methanol. The mixed with 1.5ml of methanol and then, 0.1ml of 10% exhaustive extraction was done by shaking the contents aluminium chloride was added, following by 0.1ml of at a regular interval of time till discolored solvents. potassium acetate and 2.8ml of distilled water. The Extracts so obtained were separately distilled under mixture was incubated at room temperature for 30 min. reduced pressure to obtained solvent free extracts and the The absorbance was measured by a spectrophotometer at extractive values were determined on dry weight basis λ = 415nm. The samples were prepared in triplicate with respect to the initial weight of dried and box berry max for each analysis and the mean value of absorbance was pulp taken. The resultant crude extracts were transferred obtained. The same procedure was repeated for the into airtight sample bottles and kept at 4 0C until they standard solution of quercetin (QE) and the calibration were used.[14] line was construed. The total flavonoids content in

extracts was expressed in quercetin equivalent (mg of 2.4. Analysis of Phytochemical screening QE/100mg of extract). The standard curve was prepared 1 gram of all extracts fraction was dissolved 100ml of by quercetin in different concentrations.[25] own mother solvents to make a stock stock solution of

1% (V/V) concentration. The test solution of all extracts 2.7. Determination of DPPH radical scavenging activity fraction were phytochemical screening to detect the To determine the radical scavenging capacity of the presence or absence. The chemical constituents were different extracts of Myrica esculenta a modified 1, 1- present in all extracts such as alkaloids, steroids, Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging terpenoids, phenolics, flavonoids, anthraquinones, assay adapted from.[26, 27] tannins, saponins, carbohydrates, proteins, amino acids

(FAA), etc. The extracts were tested qualitatively by standard methods.[15-20] All the qualitative tests were www.wjpmr.com │ Vol 7, Issue 2, 2021. │ ISO 9001:2015 Certified Journal │ 187 Nishat et al. World Journal of Pharmaceutical and Medical Research

DPPH (1, 1-Diphenyl-2-picrylhydrazyl) molecule is a regression analysis at a 95% significance level. P≤0.05 stable free radical with dark purple colour that decays in was identified as a significant difference. the presence of antioxidant, which can donate an electron to it. The change in absorbance is spectrometrically 3. RESULTS AND DISCUSSION measured at 517nm. Stock solutions of 1mg/ml. sixteen 3.1. Extractive value serial dilutions of the extract stock solution and one The yield of evaporated solvent of different extracts of blank sample (control) was made in methanol. 2ml of fruits Myrica esculenta was calculated as shown in prepared 1mg/ml stock solution of DPPH was added to following Table 1. each test tube. All the samples were thoroughly mixed and incubated in the dark at 28-30C. The absorbance was Table 1: Extractive value of M. esculenta fruits with read at 517nm after 25min. The percentage DPPH radical different solvents. scavenging was determined by the formula: Solvents Extractive value (%) Inhibition (%) = [(A control A sample) / (A control)] x 100 Petroleum Ether 3.01 Chloroform 6.32 A control is the absorbance of the control and A sample is the Ethyl acetate 6.01 [28] absorbance of sample extracts. Lower absorbance Acetone 24.56 values show higher free radical scavenging activity. Methanol 26.45 Ascorbic acid was used as a reference standard in different concentrations (µg/ml). The 50% inhibitory The yield of Myrica esculenta fruits exhaustively concentration value (IC50) is indicated as the effective extracted with solvents including Petroleum ether, concentration of the sample that is required to scavenge [29] chloroform, ethyl acetate, acetone, and methanol yielded 50% of the DPPH free radicals. Linear regression reported in above table 3.01%, 6.32%, 6.01%, 24.56%, analysis was carried out in SPSS and IC50 value was [30] 26.45% of the total extract respectively. The highest estimated from linear regression equations. yield of extract was recorded with methanol. (Table 1)

2.8. Statistical analysis 3.2. Preliminary Phytochemical screening Results were expressed as means ± standard deviations The result of the preliminary phytochemical screening (SD). Student t-test and variance (ANOVA) of one-way test of flavonoids, flavonol glycosides, alkaloids, amino was use by statistical analysis. Correlation between the acid, proteins, terpenoids, carbohydrates in the various IC50 and total phenolic content were established using extracts of Myrica esculenta as following in Table 2.

Table 2: Preliminary Qualitative phytochemical analysis of Myrica esculenta fruits Extracts.

Extracts Phytochemical Groups Petroleum Ether Chloroform Ethyl acetate Acetone Methanol Alkaloids - + + + + Steroids + + + + + Terpenoids + + + + Flavonoids - + + + + Phenolics - + + + + Tannins - - + + + Saponins + - - + + Glycosides - - - - + Carbohydrates - - - + + Proteins - - - - + Amino acid - - - - + (+) Present, (-) Absent

The result of the preliminary qualitative phytochemical phytochemicals. Futher work is needed to analysis the test of Myrica esculenta in various extracts presence of bond between phytochemical and various alkaloids, steroids, terpenoids, flavonoids, phenolics, pharmacological activities. tannins, saponins, glycosides, carbohydrates, proteins, amino acid in Table 2. 3.3. Total phenolic content (TPC) and Total flavonoid content (TFC) Assay Myrica esculenta is a role of pharmaceutical, anti- Total phenolic and flavonoid content in the various allergic, anti-histaminic.[31] and ethnomedicinal, extracts values of Myrica esculenta fruits are presented pharmacological activity of extracts.[32] The plant of in Table 3. kaphal is providing scientific explanation to the medicinal value of plant presence of different types of www.wjpmr.com │ Vol 7, Issue 2, 2021. │ ISO 9001:2015 Certified Journal │ 188 Nishat et al. World Journal of Pharmaceutical and Medical Research

Table 3: Total phenolic and flavonoid content in M. esculenta fruits extracts.

Extracts Total phenolic content (mg GAE/g extract) Total flavonoid content (mg QE/g extract) Chloroform 1.99±0.014 0.52±0.031 Ethyl acetate 3.54±0.022 1.45±0.046 Acetone 5.26±0.051 5.23±0.014 Methanol 7.12±0.042 4.54±0.017

Total phenolic and flavonoid content results are clearly Table 4: DPPH radical scavenging capacity (IC50) of reported in Table. 3. Methanol extract of MC fruits has Myrica esculenta fruits extracts. highest TPC and TFC as compared to other extracts. Extracts DPPH IC (µg/ml) TPC were reported as mg GAE/g of extract and TFC 50 were mg QE/g of extract. All the various extracts were Chloroform 154.23±0.243 analysis as per order Methanol˃Acetone˃Ethyl acetate Ethyl acetate 110.22±0.291 ˃Chloroform and the TPC and TFC values of the various Acetone 80.11±0.412 extracts methanol (7.12±0.042 mg/GAE and 4.54±0.017) Methanol 55.00±0.341 ˃Acetone (5.26±0.051 mg/GAE and 5.23±0.014 mg/QE) Vitamin C 31.12±0.212 ˃Ethyl acetate (3.54±0.022 mg/GAE and 1.45±0.046 Values are repotted as Mean ± SD for three analyses mg/QE) ˃Chloroform (1.99± 0.014 mg/GAE and 0.52±0.031 mg/QE). A stock solution of 1mg/ml solution of all extract in methanol was prepared. All Myrica esculenta fruits 3.4. Antioxidant assay (DPPH) extracts, however had vitamin C lower scavenging The results reported as IC50 µg/ml (50% inhibitory activity thus higher IC50 value (50% inhibition concentration) are followed in Table 4. Myrica esculenta concentration value). Myrica esculenta extracts IC50 fruits extracts were determine the scavenging capacity of value repotted in table 4. 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH) radical scavenging following DPPH protocol.[33] Myrica The relationship between DPPH (free radical scavenging esculenta fruits extract results repotted as IC50 µg/ml activity) and TPC (total phenolic content) was declared (50% inhibitory concentration) Table 4. by correlation analysis. Linear regression analysis was done by SPSS and IC50 values were repotted from linear regression equation in Fig. 1.

Fig. 1: Correlation between DPPH and TPC of Myrica esculenta fruits extracts.

Natural anti-oxidants have the ability to protect organisms from damage caused by free radical-induced oxidative stress and therefore can be used to fight a variety of ailments and diseases. The utility of antioxidant therapies in many diseases is well recognized. Phenolic compounds have capacity of reducing oxidative cellular damage caused by free radical.[34,35] The results repotted the Myrica esculenta fruits as a good source of natural antioxidants.

4. Phytochemistry Myrica esculenta has been analysied for its some number of important phytoconstituents of different types have been isolated and characterized from fruits of Myrica esculenta like Gallic acid, Myricitrin, Myricetin, Quercetin and β-Sitosterol.[36] www.wjpmr.com │ Vol 7, Issue 2, 2021. │ ISO 9001:2015 Certified Journal │ 189 Nishat et al. World Journal of Pharmaceutical and Medical Research

The structures of some important bioactive phytocontituents reported in Myrica esculenta plant fruits are present in Fig. 2 OH O OH OH

HO O OH

HO OH OH OH OH O Gallic Acid Myricetin

OH OH OH OH HO O OH HO O

OH CH3 O O OH O HO OH OH OH O Myricitrin Quercetin

CH3 H H CH H3C 3 H CH3 CH3 H CH3

H H HO β- Sitosterol

5. CONCLUSION Authors Contribution Statement Dr.Y.C.Tripathi conceptualized the work, designed all Based on the results aim of the study, conclusion of the experiments and guided analytical work, data analysis M. esculenta fruits are known for their antioxidants and and interpretation and drafting the manuscript. Ms. good pharmaceutical application. The study reported the Nishat Anjum carried out the experimental work, total phenolic and flavonoid contents (TPC and TFC) recorded data, evaluated the results and drafted the and antioxidant efficacy (DPPH) of different fruits manuscript. extract of M. esculenta. The total phenolic content of

Myrica esculenta fruits methanol extract has the highest ACKNOWLEDGEMENT TPC. Also, methanol extract of M. esculenta fruits highest antioxidant potency. Correlation reported Authors are grateful to the Director, Forest Research between TPC and DPPH (radical scavenging activity). Institute, Dehradun, India for affording necessary The phytochemical investigations have reported that M. facilities for carrying out the work. esculenta contains a number of different types of phytochemical compounds in different extracts of REFERENCES Myrica esculenta fruits which are key factors for the 1. Toyokuni S. Reactive oxygen species-induced various medicinal properties in view of the extensive molecular damage and its application in pathology. industrial application and market demand.[37-44] While the Pathol. Int., 1999; 49: 91-102. fruits of M. esculenta the present study were carried out 2. Lee J, Hwang W, Lim S. Antioxidant and anticancer to investigate antioxidant [45-51. The fruits of Myrica activities of organic extracts from Platycodon esculenta have traditionally been used in medicine. Since grandiflorum A. De Candolle roots. J. the study released that Myrica esculenta fruits have rich Ethnopharmacol., 2004; 93: 409-415. quantity of phenolic compound and thus antioxidant 3. Horax R, Hettiarachchy N, Islam S. Total Phenolic activity, therefore, it can be determined that Myrica contents and phenolic acid constituents in 4 varieties esculenta fruits potentially useful source of natural of bitter melons (Momordica charantia) and antioxidants. antioxidant activities of their extracts. J. Food Sci.,

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