PHYTOCHEMICAL SCREENING, TOTAL PHENOLICS, FLAVONOID CONTENT AND ANTIOXIDANT POTENTIAL OF DIFFERENT PARTS OF BETULA UTILIS D. DON FROM KASHMIR HIMALAYA

PHYTOCHEMICAL SCREENING, TOTAL PHENOLICS, FLAVONOID CONTENT AND ANTIOXIDANT POTENTIAL OF DIFFERENT PARTS OF BETULA UTILIS D. DON FROM KASHMIR HIMALAYA

Mohammad Saleem Wani ^{* 1}, Raghbir Chand Gupta ¹, A. H. Munshi ² and Saroj Kumar Pradhan ¹

Department of Botany ¹, Punjabi University Patiala, Patiala -147002, Punjab, India.

Department of Botany ², University of Kashmir, Srinagar - 190006, Jammu and Kashmir, India.

ABSTRACT: The aim of this study was to compare the phytochemical composition, total phenolic content (TPC), total flavonoids content (TFC) and antioxidant activity of aerial and underground parts of Betula utilis. Qualitative phytochemical screening of the three parts indicates the presence of alkaloids, flavonoids, phytosterols, tannins, terpenoids, phenols, proteins, amino acids, anthraquinone glycosides and steroids. Total phenolic content determined by Folin-Ciocalteu method ranged from 0.03 ± 0.003 to 33.6 ± 2.1, 0.34 ± 0.02 to 23.6 ± 0.67 and 0.08 ± 0.005 to 16.9 ± 1.9 mg/g of dry weight of roots, leaf and bark extracts, respectively, expressed as gallic acid equivalents. The total flavonoid contents as measured by aluminium chloride method ranges from 0.2 ± 0.003 to 7.3 ± 0.31, 0.3 ± 0.08 to 4.3 ± 0.08 and 0.3 ± 0.008 to 1.3 ± 0.03 mg/g of dry weight of leaves, bark and root extracts respectively, expressed as rutin equivalents. The results showed that TPC and TFC values were higher in methanol extract of root (33.6 ± 2.1) and leaves (7.3 ± 0.31). The antioxidant capacities in the forms of DPPH (2, 2-diphenyl-1-picrylhydrazyl) were evaluated by spectrophotometric methods. A direct correlation between total phenolic content of leaves and free radical scavenging activity was revealed (r = 0.72).

Betula utilis, Total phenolic content, Total flavonoid content radical scavenging activity, IC₅₀

INTRODUCTION: Phenolic compounds are common plant secondary metabolites with many physiological functions in plants, due to antioxidant properties that has positive effects for human health ^{1, 2}. Antioxidants play important roles in counteracting pathogenic processes related to cancer, cardio-vascular disease, muscular degeneration, cataracts and asthma, and can enhance immune function. Antioxidant defences shield the body from the adverse effects of free radicals generated as by-products of normal metabolism ².

In addition to anti-oxidative roles, phenolic compounds from different plants had been reported to have antimicrobial activity against various pathogenic micro-organisms ^{3, 4}. There is an increasing interest in medicinal plants as an alternative to synthetic drugs, especially against microbial agents because of the growth of antibiotic resistance ⁵. The search for new antimicrobial agents like phenolic compounds has subsequently turned out to be crucial.

A huge number of plants are well known in traditional medicine system for their medicinal and therapeutic potentials worldwide alike Betula utilis which is a deciduous tree belonging to the family Betulaceae. It is a moderate size tree that can grow up to 20 m in height in alpine Himalayas. The bark is shining, smooth, reddish white or white with horizontal lenticels. The outer bark is having numerous thin papery layers, exfoliating in wide horizontal rolls. Its bark is used in the treatment of various diseases such as wound healing, skin disinfectant, bronchitis, convulsions, leprosy and diseases of the blood and the ear ⁶. It is used as an antiseptic, carminatic and antibacterial agent ⁷. The purpose of this study is to evaluate the total phenolics content, total flavonoids content and anti-oxidant activity of B. utilis bark, leaves and roots extracts by using various solvents.

MATERIALS AND METHODS:

Chemicals: HPLC grade methanol, hexane, petroleum ether, ethanol, dichloromethane and chloroform were procured from the Merck. Specialties Pvt. Ltd. gallic acid, rutin, aluminium trichloride, DPPH (1, 1–diphenyl - 2 - picryl -hydrazyl), and ferric chloride were purchased from Sigma-Aldrich.

Plant Material: The aerial parts of the plant (bark and leaves) and roots were collected from Gulmarg at an altitude in the months of August-September 2016. The voucher specimen was deposited to the herbarium, Department of Botany, Punjabi University Patiala, India (accession no. = 61231). The aerial and underground parts of the plant were washed thoroughly with distilled water to clean the dust and topical fauna. Samples were air dried, powdered, sieved, weighed and stored in air tight container and subsequently referred to as powdered drug.

Extraction Procedure: Ten grams (10 g) of powdered plant materials were successively extracted by maceration with water, methanol, hexane, petroleum ether, ethanol, dichloromethane and chloroform for 72 h stirring. The macerates were filtered and concentrated using a rotary evaporator at 40-50 °C. The obtained extracts were stored at 4 °C until further use.

Phytochemical Analysis:  Phytochemical analysis of dichloromethane (DCM), hexane, petroleum ether, chloroform, ethanol, methanol and water extract was done for presence/absence of metabolites such as flavonoids, alkaloids, terpenoids, cardiac glycosides, saponins, phenolics, proteins, amino acids, anthraquinone glycosides, phytosterols, tannins and steroids ⁸ .

Determination of Total Phenolic content (TPC): The total phenolic content (TPC) was determined by the Folin-Ciocalteu method ^9, with some modifications. 0.5 ml of test assay was treated with 0.5 ml Folin-ciocalteu reagent (1N) and 20 % sodium carbonate solution sequentially. The sample is mixed over vortex. The reaction mixture was left for 30 minutes after mixing. The solution was maintained up to 12.5 ml with water. The absorbance was recorded at 765 nm wavelength. The content of phenolics in extracts was expressed in terms of gallic acid equivalent (mg of GA/g of extract).

Determination of Total flavanoid content (TFC): Total phenolic contents (TPC) in extracts were measured as described by Nadhiya and Vijaya-lakshmi ¹⁰. To 1 ml of extract (100 µg mL-1), 3 ml of methanol, 0.2 ml of 1 M potassium acetate, 0.2 ml of 10 % aluminium chloride and 5.6 ml of distilled water was added and left at room temperature for 30 minutes. Absorbance of the mixture was read at 420 nm using UV spectro-photometer. The content of flavonoids in extracts was expressed in terms of rutin equivalent (mg of RU/g of extract).

In-vitro Antioxidant Potential by DPPH Radical-Scavenging Activity: The ability of the plant extract to scavenge DPPH free radicals was assessed by the method of Shahat et al., 2015 with slight alterations ¹¹. The stock solutions of extracts were prepared in methanol to accomplish the concentration of 1 mg/ml. Dilutions were made to obtain concentrations of 200, 150, 100, 50 and 10µg/ml. Diluted solutions (1 ml each) were mixed with 1 ml of methanolic solution of DPPH in concentration of 1 mg/ml.

After 30 min incubation in darkness at room temperature (23 ^oC), the absorbance was recorded at 517 nm. Control sample contained all the reagents except the extract. Percentage inhibition was calculated using equation 1, whilst IC₅₀ values were estimated from the % inhibition versus concentration plot, using a non-linear regression algorithm. The data were presented as mean values ± standard error (n = 3).

RESULTS:

Preliminary Phytochemical Screening: Results obtained for qualitative screening of phyto-chemicals in stem bark and roots of B. utilis are presented in Table 1. Of the twelve phytochemicals screened for, all the twelve were found to be present in various solvent extracts. In all, more phytochemicals were found present in the stem bark than in the leaves and roots. Remarkably, cardiac glycosides, saponins, proteins, amino acids, anthraquinone glycosides, phytosterols, tannins and steroids were not present in roots but were present in stem bark. This suggests that the stem bark offers a wider array of phytochemicals than the root and leaves. From this analysis, methanolic extract of stem bark was found to have more constituents compared to leaves and root extracts.

TABLE 1: QUALITATIVE ANALYSIS OF THE PHYTOCHEMICALS IN BARK, LEAVES AND ROOTS

A*: Aqueous; M: Methanol: E: Ethanol; C: Chloroform; PE: Petroleum ether; H: Hexane; DCM: Dichloromethane

Total Phenolic and TFCs: Bark, leaves and root extracts using water and six different organic solvents (methanol, ethanol, dichloromethane, chloroform, petroleum ether and hexane) have been prepared to examine the free radical scavenging activity and contents of total phenols and flavonoid concentrations. Total phenolic content was estimated by using Folin-Ciocalteu reagent. Total phenolic content of the different extracts of B. utilis was solvent dependent and expressed as milligrams of gallic acid equivalents (GAE). Table 2 summarizes that total phenolic compounds in extracts varied widely, ranging from 0.07±0.002 and 33.6 ± 2.1 mg GA/g. In methanolic, extracts from roots, very high values of total phenolic contents are measured (33.6 ± 2.1 mg GA/g). In the analysis of results for the concentrations of total phenolic compounds in all root extracts, it is noticed that the highest concentration of phenolic compounds is in the extracts obtained using solvents of high and moderate polarity. The concentrations of phenols in the bark extract range from 0.08 ± 0.005 to 16.9 ± 1.9 mg GA/g.

In seven different leaves extracts, the highest content of phenols (23.6 ± 0.67 mg GA/g) is measured in the aqueous extract. In other extracts from leaves, the contents of phenolic compounds vary from 0.34 ± 0.02 to 23.6 ± 0.67 mgGA/g.

Flavonoids are the major components of the phenolic compounds. The concentration of flavonoids in plant extracts from leaves, bark and roots of B. utilis is determined using spectro-photometric method with AlCl₃. The summary of quantities of flavonoids identified in the tested extracts is shown in Table 3. Values designating concentration of flavonoids in the extracts of aerial and underground parts of plants are very uneven. The concentrations of flavonoids in plant extract range from 0.15 ± 0.003 to 7.23 ± 0.31 mg RU/g. The highest concentrations of flavonoids in leaves are measured in methanol, ethanol and water extracts, while DCM, chloroform, petroleum ether and hexane extracts are with small amounts of flavonoids. Concentration of flavonoids in the extracts from roots is smaller in the comparison to extracts from bark, but there is not much variation in the values. In extracts from bark the highest concentration of flavonoids is in methanolic and ethanolic extract.

TABLE 2: EFFECT OF DIFFERENT SOLVENTS ON EXTRACTION OF TOTAL PHENOLS FROM DIFFERENT PARTS OF B. UTILIS

*All analyses are the mean of triplicates ± standard error. Values are expressed as mg gallic acid/g of dry plant material

TABLE 3: EFFECT OF DIFFERENT SOLVENTS ON EXTRACTION OF TOTAL FLAVONOIDS FROM DIFFERENT PARTS OF B. UTILIS

All analyses are the mean of triplicates ± standard error. Values are expressed as mg rutin/g of dry plant material

DPPH Radical Scavenging Assay: It is notable that the antioxidant activity of plant extracts containing polyphenol components is because of the ability to be donors of hydrogen atoms or electrons and to catch the free radicals. The antioxidant proprieties of extracts were calculated in terms of their efficient IC₅₀ concentration corresponding to the sample concentration that reduced the initial DPPH• absorbance of 50 %. These IC₅₀ values are given in Table 4.

The obtained values for antioxidant activity range from 513.3 ± 6.9 to 43.9 ± 2.8mg/ml. The results revealed that methanolic extract of root has highest antioxidant action among all the extracts tested as denoted by the low IC₅₀ value of 43.9 ± 2.8mg/ml.

Correlation Analysis between TPC, TFC and Antioxidant Activity (DPPH): To establish the suitability, reliability and relationship amongst TPC, TFC and free radical scavenging activity, correlation analysis was performed. The correlation coefficients (R) for aerial and underground plant parts are given in Table 5. The total phenolic content showed strong positive correlation with leaves (R=0.7198). This suggests that phenols are the main compounds responsible for the antioxidant activity in B. utilis.

TABLE 4: ANTIOXIDANT (DPPH SCAVENGING) ACTIVITY OF INVESTIGATED PLANT EXTRACTS PRESENTED AS IC₅₀ VALUES (µg/ml)

TABLE 5: CORRELATIONS BETWEEN THE IC₅₀ VALUES OF ANTIOXIDANT ACTIVITIES AND PHENOLIC AND FLAVONOIDS CONTENT OF B. UTILIS

Each value in the table is represented as mean ± S.E (n = 3). *indicates significance at P < 0.05.

DISCUSSION: The preliminary phytochemical screening tests might be valuable in the detection of the bioactive principles and consequently may lead to the drug discovery and development. The phytochemical compounds detected are known to have therapeutic significance. For instance, alkaloids have been reported as powerful poison and many alkaloids derived from medicinal plants show biological activities like, anti-inflammatory ¹², antimalarial ¹³, antimicrobial ¹⁴, cytotoxicity, antispasmodic and pharmacological effects ¹⁵.

Similarly, steroids derived from plants are known to have cardio tonic effect and furthermore have antibacterial and insecticidal properties ¹⁶. These are very often used in medicines because of their their well-known biological activities. Tannins are known to have antibacterial ¹⁷, antitumor and antiviral activities ¹⁸. These work by precipitating microbial protein thus making nutritional protein inaccessible for them. Other phytochemicals called cardiac glycosides have been utilized to treat congestive heart failure and cardiac arrhythmia ¹⁹.

In the present study total phenolic content (TPC), total flavonoid contents (TFC) and free radical scavenging activity of B. utilis varied among the different plant parts extracted in various solvents. Phenolics or polyphenols are secondary plant metabolites that are most usually present in plants of high medicinal value. Phenolic compounds contribute to the antioxidant potential of plants by neutralizing free radicals and preventing de-composition of hydroperoxides into free radicals referred to have therapeutic uses such as anti-oxidant, anti-carcinogenic, antimutagenic activities ²⁰.

These are additionally known to decrease cardio-vascular risks ²¹. Besides, these have a metal chelation potential ²². Results of the assays for phenolics described in the present report, indicated a wide variation in the total phenolic content in the different extracts, showing the higher phenolic content in the methanol, which is in concurrence with Shukla et al., 2017 ²³. In the present study, we found the presence of higher amounts of phenolics in root part of the plant.

Also TPC in the bark was more as reported by Pandey et al., which varied from 0.41 mg/g to 14.47 mg/g gallic acid equivalent (GAE) ²⁴. The difference in the concentration of phenolics in the respective solvents is due to the polarity of the solvents used for extraction. Higher solubility of the phenols in polar solvents keeps higher concentration of these compounds in the extracts obtained by using the particular solvent during extraction ²⁵. In our present study the TPC in the bark was more as reported by Pandey et al., varied from 0.41 mg/g to 14.47 mg/g gallic acid equivalent (GAE) ²⁴.

Flavonoids are one class of secondary plant metabolites that are otherwise called Vitamin P. These metabolites are generally used in plants to produce yellow and other pigments which play an important role in the colors of plants. Flavonoids are easily ingested by humans and they seem to display important anti-inflammatory, anti-allergic and anti-cancer activities ²⁶. These are likewise strong antioxidants as these are capable of effectively scavenging the ROS due to their phenolic hydroxyl groups ²⁷. Higher amounts of flavonoids were recorded in the methanolic extract of leaf as compared to other plant parts which was also reported by Cao et al., in B. pendula ²⁸.

Concentration of flavonoids in the extracts from bark and roots is smaller in the comparison to extracts from leaves. These results are in accordance with the literature. Several diaryl-heptanoids, phenylbutanoids, lignans and phenol glycosides have been determined in wood, bark and stem of various birch species extracts ^{29, 30}, whereas flavonol derivatives were identified as the most abundant phenolics in birch leaves according to the results of previous studies on leaves from Betula sp. ³¹. Polar extracts (methanol, ethanol, and water extracts) showed more flavonoids than apolar extracts.

Various techniques are utilized to determine the radical scavenging effects of antioxidants. The DPPH method is a preferred technique since it is quick, simple and reliable and does not require a special reaction and device. DPPH is a stable, synthetic radical that does not deteriorate in water, methanol, or ethanol. The free radical scavenging activities of extracts depend on the capacity of antioxidant compounds to lose hydrogen and the structural conformation of these components ³². The DPPH free radical, which is at its maximum wavelength at 517 nm, can easily get an electron or hydrogen from antioxidant molecules to become a stable diamagnetic molecule ³³. Owing to the DPPH radical’s ability to bind H, it is considered to have a radical scavenging property.

A solution of DPPH radicals prepared in methanol is converted into DPPH-H (diphenylhydrazine) molecules in the presence of an antioxidant agent, as shown in the following equation. Discoloration occurs due to the decreasing quantity of DPPH radicals in the environment. The discoloration of the DPPH therefore reflects the radical scavenging activity of the analysed extract ³⁴.

In our present investigation, methanolic extracts exhibited a potent antioxidant activity, which is in accordance with Shukla et al., 2017 ²³. The hydroxyl radical scavenging activity of methanolic root extract was found to be higher than all the extracts which can be inferred from their IC₅₀ values in B. utilis. It was observed that the radical scavenging effect of the leaf extracts was positively correlated with their total amount of phenolic compounds (r = 0.7198). Poor correlations determined between the total phenols and radical scavenging capacity of the bark and root extracts, indicate to the synergistic or antagonist effects of molecules such as polyphenols and triterpenes present in the bark and roots of B. utilis ³⁵.

CONCLUSION: The results of the preliminary phytochemical screening of the plant extracts showed presence of alkaloids, flavonoids, phyto-sterols, tannins, terpenoids, phenols, flavonoids, proteins, amino acids, anthraquinone glycosides and steroids. Results of our study suggest the great value of the species B. utilis for use in pharmacy and phytotherapy. In light of this data, it could be concluded that this plant is natural sources of antioxidant substances of high significance. It is noticed that the highest concentration of phenolic compounds in the extracts were obtained with solvents of high polarity; the methanolic extract showed greater power of extraction for phenolic compounds from B. utilis. 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.

ACKNOWLEDGEMENT: We are highly thankful to the Department of Botany and Sophisticated Instrumentation Centre, Punjabi University Patiala for providing necessary facilities to carry out the HPTLC analysis. The first author thanks the DBT-IPLS (ref No. BT/PR 4548/NF/22/146/2012) for providing Junior Research Fellowship.

CONFLICT OF INTEREST: We declare that we have no conflict of interest.

REFERENCES:

1. Çalis¸kan O and Polat AA: Phytochemical and antioxidant properties of selected fig (Ficus carica) accessions from the eastern Mediterranean region of Turkey. Scientia Horticulturae 2011; 128(4): 473-78.
2. Nakilcioğlu E and Hışıl Y: Research on the phenolic compounds in sarilop (Ficus carica) fig variety. GIDA. 2013; 38(5): 267-74.
3. Megdiche-Ksouri W, Trabelsi N, Mkadmini K, Bourgou S, Noumi A, Snoussi M, et al.: Artemisia campestris phenolic compounds have antioxidant and antimicrobial activity. Industrial Crops and Products 2015; 63: 104-13.
4. Türkyılmaz M, Tağı Ş, Dereli U and Özkan M: Effects of various pressing programs and yields on the antioxidant activity, antimicrobial activity, phenolic content and colour of pomegranate juices. Food Chemistry 2013; 138: 1810-18.
5. Chandra H, Bishnoi P, Yadav A, Patni B, Mishra AP and Nautiyal AR: Antimicrobial resistance and the alternative resources with special emphasis on plant-based anti-microbials-A Review. Plants 2017; 6(2): 16.doi: 10.3390 /plants6020016

6. Safdar I, Bibi Y, Hussain M, Iqbal M, Saira H, Shaheen S, et al.: Review on Current Status of Betula utilis: An important medicinal plant from Himalaya. Research and Reviews: Journal of Botanical Sciences 2017; 6(2). e-ISSN: 2320-0189.
7. Mishra T, Arya RK, Meena S, Joshi P, Pal M, Meena B et al.: Isolation, Characterization and anticancer potential of cytotoxic triterpenes from Betula utilis PloS one 2016; 11(7): e0159430.
8. Gul R, Jan SU, Faridullah S, Sherani S and Jahan N: Preliminary phytochemical screening, quantitative analysis of alkaloids, and antioxidant activity of crude plant extracts from Ephedra intermedia Indigenous to Balochistan. The Scientific World Journal 2017.doi.org/10.1155/2017/5873648

9. Josipović A, Sudar R, Sudarić A, Jurković V, Matoša Kočar M and Markulj Kulundžić A: Total phenolic and total flavonoid content variability of soybean genotypes in eastern Croatia. Croatian Journal of Food Science and Technology 2016; 8(2): 60-65.
10. Nadhiya K and Vijayalakshmi K: Evaluation of total phenol, flavonoid contents and in vitro antioxidant activity of Benincasa Hispida fruit extracts. International Journal of Pharmaceutical Chemical Biological Sciences 2014; 4(2): 332-38.
11. Shahat AA, Ibrahim AY and Alsaid MS: Antioxidant capacity and polyphenolic content of seven Saudi Arabian medicinal herbs traditionally used in Saudi Arabia. Indian Journal of Traditional Knowledge 2015; 1(1): 28-35
12. Augusto LS, Josean FT, Marcelo S, Margareth FM, Petronio FA and Jose MB: Anti-inflammatory activity of alkaloids: an update from 2000 to 2010. Molecules 2011; 16(10): 8515-34.
13. Dua VK, Gaurav V, Bikram S, Aswathy R, Upma B, Dau DA, Gupta NC, Sandeep K and Ayushi R: Anti-malarial property of steroidal alkaloid conessine isolated from thebark of Holarrhena antidysenterica. Malaria Journal 2013; 12(1): 1-6.

14. Benbott A, Yahyia A and Belaidi A: Assessment of the antibacterial activity of crude alkaloids extracted from seeds and roots of the plant Peganum harmala Indian Journal of Natural Products and Resources 2012; 2(5): 568-73.
15. Thite SV, Chavan YR, Aparadh VT and Kore BA: Preliminary phytochemical screening of some medicinal plants. International Journal of Pharmaceutical Chemical Biological Sciences 2013; 3(1): 87-90.
16. Iqbal E, Salim KA and Lim LB: Phytochemical screening, total phenolics and antioxidant activities of bark and leaf extracts of Goniothalamus velutinus (Airy Shaw) from Brunei Darussalam. Journal of King Saud University-Science 2015; 27(3): 224-32.
17. Akiyama H, Fujii K, Yamasaki O, Oono T and Iwatsuki K: Antibacterial action of several tannins against Staphylococcus aureus. ‎Journal of Antimicrobial Chemo-therapy 2001; 48(4): 487-91.
18. Kumari M and Jain S: Review paper, Tannins: an anti-nutrient with positive effect to manage diabetes. Research Journal of Recent Sciences 2012; 1(12): 70-73.
19. Vladimir K and Ludmila M: Glycosides in medicine: the role of glycosidic residue in biological activity. Current Medicinal Chemistry 2001; 8(11): 1303-28.
20. Działo M, Mierziak J, Korzun U, Preisner M, Szopa J and Kulma A: The potential of plant phenolics in prevention and therapy of skin disorders. International Journal of Molecular Sciences. 2016; 17(2): 160.doi:  10.3390 /ijms 17020160

21. Yen GC, Duh PD and Tsai CL: Relationship between anti-oxidant activity and maturity of peanut hulls. ‎Journal of Agricultural and Food Chemistry 1993; 41(1): 67-70.
22. Rice-evans CA, Miller NJ, Bolwell PG, Bramley PM and Pridham JB: The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radical Research 1995; 22(4): 375-83.
23. Shukla S, Mishra T, Pal M, Meena B, Rana TS and Upreti DK: Comparative analysis of fatty acids and antioxidant activity of Betula utilis bark collected from different geographical region of India. Free Radicals and Anti-oxidants 2017; 7(1): 80-85.
24. Pandey N, Kumar A, Niranjan A, Lehri A, Meena B, Rana TS and Upreti DK: Biochemical composition of Betula utilis Don bark, collected from high altitudes of Indian Himalayas. Medicinal Plants International Journal of Phytomedicines and Related Industries 2016; 8(1): 33-39.
25. Mohsen SM and Ammar AS: Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chemistry 2009; 112(3): 595-98.
26. Kumar S and Pandey AK: Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal 2013.doi.org/10.1155/2013/162750

27. Cao G, Sofic E and Prior RL: Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radical Biology and Medicine 1997; 22(5): 749-60.
28. Raal A, Boikova T and Püssa T: Content and dynamics of polyphenols in Betula leaves naturally growing in Estonia. Records of Natural Products 2015; 9(1): 41-48.
29. Fuchino H, Konishi S, Satoh T, Yagi A, Saitsu K, Tatsumi T and Tanaka N: Chemical evaluation of Betula species in Japan. 2. Constituents of Betula platyphylla var japonica. Chemical and Pharmaceutical Bulletin 1996; 44(5): 1033-38.
30. Hiltunen E, Pakkanen TT and Alvila L: Phenolic compounds in silver birch (Betula pendula Roth) wood. Holzforschung 2006; 60(5): 519-27.
31. Lahtinen M, Lempa K, Salminen JP and Pihlaja K: HPLC analysis of leaf surface flavonoids for the preliminary classification of birch species. Phytochemical Analysis 2006; 17(3): 197-203.
32. Aksoy L, Kolay E, Ağılönü Y, Aslan Z and Kargıoğlu M: Free radical scavenging activity, total phenolic content, total antioxidant status, and total oxidant status of endemic Thermopsis turcica. Saudi Journal of Biological Sciences 2013; 20(3): 235-239.
33. Mohanasundari L and Suja S: Antioxidant and free radical scavenging activity of the mixture of ethanolic extracts of Alpinia speciosa and Alpinia calcarata Inter-national Journal of Pharmacy and Pharmaceutical Sciences 2016; 8(8): 164-70.
34. Molyneux P: The use of the stable free radical diphenyl picrylhydrazyl (DPPH) for estimating antioxidant activity. Journal of Science Education and Technology 2004; 26(2): 211-219.
35. Diouf PN, Stevanovic T and Boutin Y: The effect of extraction process on polyphenol content, triterpene composition and bioactivity of yellow birch (Betula alleghaniensis Britton) extracts. Industrial Crops and Products 2009; 30(2): 297-303.
    

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    How to cite this article:

    Wani MS, Gupta RC, Munshi AH and Pradhan SK: Phytochemical screening, total phenolics, flavonoid content and antioxidant potential of different parts of Betula utilis D. Don from Kashmir Himalaya. Int J Pharm Sci Res 2018; 9(6): 2411-17. doi: 10.13040/IJPSR.0975-8232.9(6).2411-17.

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