Quantification of Total Phenolic and Flavonoid content in leaves of Cucumis melo var agrestis using UV- spectrophotometer

 

Gopalasatheeskumar K¹, Ariharasiva Kumar G², Sengottuvel T³, Sanish Devan V⁴, Srividhya V⁵

¹Research Scholar, Department of Pharmacy, Annamalai University, Chidambaram, Tamil Nadu, India.

²Professor and Head of the Department of Pharmacology, KMCH College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore-641048, Tamil Nadu, India (Affiliated to The Tamil Nadu Dr. M.G.R. Medical University). ³Associate Professor, Department of Pharmacology, KMCH College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore-641048, Tamil Nadu, India (Affiliated to The Tamil Nadu Dr. M.G.R. Medical University). ⁴Department of Pharmacology, KMCH College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore-641048, Tamil Nadu, India (Affiliated to The Tamil Nadu Dr. M.G.R. Medical University).

⁵Assistant Professor, Department of Pharmacology, Excel College of Pharmacy, Pallakapalayam, Namakkal-

637303 (DT), Tamil Nadu, India (Affiliated to The Tamil Nadu Dr. M.G.R. Medical University).

 

ABSTRACT:

Plants and plant-derived products contain a wide range of phytochemicals, such as alkaloids, flavanoids, glycosides which are thought to have the medicinal properties. Cucumis melo var agrestis is belongs to the family of cucurbitaceae and well distributed in rural and coastal areas. This plant is consumed by rural people in Tamil Nadu, India. Phenolic and flavanoid contents plays the important role in antioxidant mechanism thereby cure many of the diseases. Therefore current research is aimed to quantification of total phenolic and flavanoid content present in leaves of CMA. Hydroalcoholic leaf extract of CMA (HALEC) was evaluated its phytochemicals using chemical tests and quantification of total phenolic content and total flavonoid contents by standard curve of Gallic acid and Quercetin using UV- Spectrophotometer. Our results revealed that HALEC has the phytochemicals like alkaloids, flavonoids, tannins, carbohydrates, saponins, glycosides, proteins and amino acids and quantitative tests confirms the presence of total phenolic content was 77.82 mg/g of extract calculated as Gallic acid equivalent and total flavonoid content was 30.06 mg/g of extract calculated as Gallic acid equivalent in HALEC. This research work is useful to animal study on leaf extract of CMA, Isolation of active principles and detection of exact mechanism.

 

 

 

INTRODUCTION:

Plants and plant-derived products contain a wide range of phytochemicals, such as alkaloids, flavanoids, glycosides which are thought to have the medicinal properties. Many of the herbals were reported as medicinal plants^1,2. In traditional medicine many of the countries used the herbal as therapeutic agents.

 

 

Now a day’s know the exact reason for using of medicinal plants is important in research. In that scenario extraction of the plant materials and identification of phytoconstituents plays the important role³. The phenolic and flavanoid contents were has potent antioxidant property, thereby which plays the important role in treatment of many of the diseases. The Cucumis melo var agrestis is belongs to the family of Cucurbitaceae. This plant was widely distributed in rural and coastal areas, this plant is commonly called as wild musk melon, Kachari, small guard, in Tamil this plant is known as Chukkan chedi and Mithukku vathal sedi⁴. In India many of the rural peoples from Tamil Nadu consuming this plant material for the nutritional and medicinal purpose. Some of the researchers have reported the antioxidant property, phytochemical screening, analgesic activity, anti-inflammatory, hypolipidemic activities^5,6. But still there are no reports/studies on the leaves of this plant material. Therefore current research is focused on study of phytochemical analysis and quantification of total phenolic and flavonoid content in leaves of CMA. And this research paper will be very helpful to the future researchers to study the leaves of CMA.

 

Materials and methods:

Plant collection and authentication:

The fresh leaves of the plant CMA was collected from Pungavarnatham village, Thoothukudi District, Tamil Nadu, India. Month of December 2017 and authenticated by Dr. S. Sankaranarayanan, Department of Medicinal botany, Government Siddha Medical College, Chennai, Voucher specimen No. GSMC/MB-87/18.

 

Preparation of plant material:

The collected leaves were cleaned, washed with distilled water, dried under sunshade in dark room, and powdered by using mechanical mixer. After size reduction leaves were sieved under sieve No. 40 and sieve No. 60, stored in airtight container at room temperature⁷.

 

Extraction of the plant material:

200g of finely powdered leaf powder was defatted with 1 L of petroleum ether in a soxhlet apparatus for 48h, obtained marc was further extracted with 1L of 60% methanol (600ml of methanol: 400ml of water) in soxhlet apparatus for 48h, obtained marc was again extracted with water by cold maceration method for 48 h. After extraction the extracts were separately concentrated by distillation and dried at room temperature until get viscous solid mass. The obtained crude extracts were weighed and stored at 4⁰C for the further analysis.⁸

 

Preliminary phytochemical analysis:

The obtained extracts were subjected to phytochemical evaluation and identified the various plant constituents present in the test sample qualitatively^9,10.

 

Quantification of total phenolics and flavonoids:

Estimation of total phenolics:

The total phenolic content of extracts was determined by Folin-Ciocalteau assay method^11,12. To an aliquot 100μl of extract (1mg/ml) or standard solution of Gallic acid (10, 20, 40, 60, 80, 100μg/ml) added 50μl of Folin-Ciocalteau reagent followed by 860μl of distilled water and the mixture is incubated for 5min at room temperature. 100μl of 20% sodium carbonate and 890μl of distilled water were added to make the final solution to 2ml.  It was incubated for 30min in dark to complete the reaction. The absorbance of the mixture was measured at 765 nm against blank.  Distilled water was used as reagent blank. The tests were performed in triplicate to get mean the values. The total phenolic content was found out from the calibration curve of Gallic acid and it was expressed as milligrams of Gallic acid equivalents (GAE) per gram of extract.

 

Estimation of total flavonoids:

The total flavonoid content of extracts was determined using Aluminium chloride colorimetric method. To an aliquot of 100μl of the extract or standard solutions of Quercetin (10, 20, 40, 60, 80, 100μg/ml) ethanol was added separately to make up the solution up to 2 ml^13,14. The resulting mixture was treated with 0.1 ml of 10% aluminium chloride, 0.1 ml of 1M potassium acetate and 2.8ml of distilled water. Mixed and allowed to stay at room temperature for 30 minutes. The absorbance was measured at 415 nm against blank, where a solution of 2ml ethanol, 0.1ml potassium acetate, 2.8ml distilled water and 0.1ml of aluminium chloride serve as blank solution. The total flavonoid content was determined from the standard Quercetin calibration curve and it was expressed as milligrams of Quercetin equivalents (QE) per gram of extract.

 

Results and Discussion:

Phytochemical analysis:

Results of qualitative phytochemical revealed that Hydroalcoholic leaf extract of CMA (HALEC) and Aqueous leaf extract of CMA (ALEC) have alkaloids, flavonoids, tannins, carbohydrates, saponins, glycosides, proteins and amino acids. Petroleum ether leaf extract of CMA (PELEC) have only of steroids and triterpenoids and absence of polysaccharides in all the extracts (Table 1).

Table 1: Preliminary phytochemical studies of plant extract of Cucumismelo var. agrestis

 

 

Figure 1: Standard graph of Gallic acid for the estimation of total flavonoid content

 

 

Figure 2: Standard graph of Quercetin for the estimation of total flavonoid content

 

Quantitative studies confirms the total Flavonoid content in HALEC and ALEC were found to be 30.06 mg/g and 20.82 mg/g of extract calculated as Quercetin equivalent and the total Phenolic content in HALEC and ALEC were found to be 77.82 mg/g and 21.83 mg/g of extract calculated as Gallic acid equivalent. From the results of qualitative analysis, HALEC has maximum phenolic and flavonoid content compared with ALEC. So that further studies were only performed for HALEC.

 

Phenolics and flavonoids normally scavenge the free radicals and play an essential role in prevention and therapy of cancer, and many more diseases by inducing antioxidant defense system, drug metabolizing enzymes, modulating diverse events in cellular level and inhibiting inflammation, hyperplasia, proliferation and oxidative DNA damage.

 

Conclusion:

From the results of this research was concluded that leaf extract of Cucumis melo var agrestis has higher concentration of phenolic and flavonoid content and these are the phenolic and flavonoid contents has the important medicinal property. This research work is useful to animal study on leaf extract of CMA, Isolation of active principles and detection of exact mechanism. 

 

AUTHOR CONTRIBUTION:

All authors contribute equal to this work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Patel DK, Kumar R, Laloo D, Hemalatha S. Natural medicines from plant source used for therapy of diabetes mellitus: an overview of its pharmacological aspects. Asian Pac. J. Trop. Dis. 2012; 239-250.

2.      Shokeen P, Anand P, Murali YK, Tandon V. Antidiabetic activity of 50% ethanolic extract of Ricinus communis and its purified fractions. Food Chem. Toxicol. 2008; 46(11):3458-3466.

3.      Mukherjee PK, Mukherjee K, Maiti K. Leads from Indian medicinal plants with hypoglycemic potentials. J. Ethnopharmacol. 2006; 106:1–28.

4.      Switi BG, Krishna Mohan G, Sandhya Rani M. Phytochemicals for Diabetes Management. Pharmaceutical Crops. 2014; 5(1):11-28.

5.      Alagar RM, Sahithi G, Vasanthi R, David B, Rao KNV, Selvakumar D. Study of phytochemical and antioxidant activity of Cucumis melo var. agrestis fruit. Journal of Pharmacognosy and Phytochemistry. 2015; 4(2):303-306.

6.      Gopalasathees Kumar K, Ariharasiva kumar G, Sengottuvel T et al. Confirmation of Best Method for Detection of Alpha Amylase Activity of Cucumis Melo Var Agrestis (Wild Musk Melon). J Adv Res Bio Chem Pharma 2019; 2(1): 1-3.

7.      Gopalasathees Kumar K, Parthiban S, Manimaran T, Boopathi T. Phytochemical Screening on Various Extracts (Benzene, Ethanolic and Aqueous) of Stem Parts of Zanthoxylum Rhetsa (Roxb.) Dc. ijupbs. 2017; 6(2):79-91.

8.      Karthishwa K, Mirunalini S, Dhamodhara G, Krishnaven M, Arulmozhi V. Phytochemical Investigation of Methanolic Extract of the Leaves of Pergulariadaemia. Journal of Biological Sciences. 2010; 10(3):242-246.

9.      Trease GE, Evans WC. Textbook of Pharmacognosy. 12th edn. Balliese Tindall and Company Publisher, London.1983 pp. 343-383.

10.   Gopalasathees Kumar K, Parthiban S, Manimaran T, Boopathi T. Phytochemical Screening on Various Extracts (Benzene, Ethanolic and Aqueous) of Stem Parts of Zanthoxylum Rhetsa (Roxb.) Dc. ijupbs. 2017; 6(2):79-91.

11.   Sharma A, Joshi N, Kumar R, Agrawal P, Prasad S. High performance liquid chromatographic analysis of phenolic compounds and their antioxidant properties from different cultivars of Cyamopsistetragonaloba (L.) Taub. Microchemical Journal. 2017; 133: 622-628.

12.   Andriyani R, Budiati T, Pudjiraharti S. Effect of Extraction Method on Total Flavonoid, Total Phenolic Content, Antioxidant and Anti-bacterial Activity of Zingiberis Officinale Rhizome. Procedia Chemistry. 2015; 16:149-154.

13.   Alves D, Maia de Morais S, Tomiotto-Pellissier F, Miranda-Sapla M, Vasconcelos F, Silva I et al., Flavonoid Composition and Biological Activities of Ethanol Extracts of Caryocarcoriaceum Wittm., a Native Plant from Caatinga Biome. Evid Based Complement Alternat Med. 2017; 2017:1-7.

14.   Chekroun E, Benariba N, Adida H, Bechiri A, Azzi R, Djaziri R. Antioxidant activity and phytochemical screening of two Cucurbitaceae: Citrulluscolocynthis fruits and Bryoniadioica roots. Asian Pacific Journal of Tropical Disease. 2015; 5(8):632-637.

 

 

 

Received on 16.10.2019         Modified on 25.10.2019

Accepted on 31.10.2019         © AJRC All right reserved