Phytochemical Screening and Antioxidant Potential of Syzygium cumini Leaves

Medicinal plants are the richest bio-resource of drugs in the traditional system of medicine, chemical entities for synthetic drugs, food supplements and pharmaceutical intermediates (Ncube et al., 2008). These are the local heritage with global importance and world is endowed with a rich wealth of medicinal plants (Suriyavathana et al., 2010). The non-nutritive secondary metabolites are known as phytochemicals that are naturally occurring in the medicinal plants and have defensive as well as disease preventive properties (Tan et al., 2010 and Suhas et al., 2014). The major secondary metabolites include alkaloids, carbohydrates, flavonoids, tannins, terpenoids and steroids (Edoga et al., 2005). Having a great pharmacological relevance, phytochemicals have been of great interest to human for long time. Plant based drug constitute a major share of medicine in India, China viz ayurveda, yoga, unani, siddha, homeopathy and naturopathy, except allopathy (Vaidya and Devasagagam, 2007). Beyond this pharmaceutical approach to plants, there is a wide tendency to utilize herbal products to supplement the diet, mainly with the intention of improving the quality of life and preventing the diseases (Joselin and Jeeva, 2014).
       
Syzygium cumini also known as Syzygium jambolanum and Eugenia cumini belongs to family Myrtaceae. It is commonly known as “Jamun” and is a traditional medicinal plant native to India having several medicinal properties such as hypoglycaemic, antidiarrhea, antibacterial and anti-HIV activity (Ravi et al., 2004). Leaves and barks of Syzygium cumini are known for their anti-inflammatory activity (Muruganandan et al., 2001). Its medicinal properties may be due to its ability to synthesize various phytochemicals. As a remedy for diabetes mellitus in many countries, S. cumini leaves have been used since ancient time (Texixeri et al., 2000). The plant possesses acetyl-oleanolic acid, triterpenoids, ellagic acid, isoquercetin, quercetin, kaempferol and myricetin in different concentrations (Rastogi and Mehrortra 1990). Keeping in view, wide medical applications of Jamun, the current experiment was designed to investigate the phytochemical compounds and antioxidant potential of ethyl acetate extract of Syzygium cumini leaves.

Material
 
Syzygium cumini (Jamun) leaves samples were acquired from the campus of Chaudhary Charan Singh Haryana Agricultural University, Hisar. The proposed study was conducted in Department of Chemistry, CCS HAU, Hisar during 2019-2021. The plant materials were brought and before processing, the materials were kept under the shade at room temperature. Taxonomical description of juman is presented in Table 1.
 

 
Methods
 
Prepartion of plant extract
 
Extraction plays an important role as it helps in the recovery of desired medicinally bioactive constituents from plants by using selective solvents and leaving out those non-desired with an aid of the solvents (Dhanani et al., 2017). The powdered sample of Jamun leaves was percolated by using conventional soxhlet apparatus using ethly acetate as solvent. The extracts was collected and kept for further studies.
 
Qualitative screening for phytochemicals
 
For the presence of phytoconstituents such as flavanoids, phenols, alkaloids, tannins, saponins, terpenoids and steroids, ethyl acetate extract of Syzygium cumini leaves was investigated. Standard methods suggested by Harborne (1998) Kokate (2001) were used for qualitaitive screening of phytochemicals.
 
Test for alkaloids (Mayer,s test)
 
1.36 gm of mercuric chloride and 5 gm of potassium iodide were dissolved in 60 ml, 10 ml distilled water, respectively. Prepared solvents were mixed and volume was made upto 100 ml using distilled water. To 1 ml of acidic solution of samples few drop of reagent was added. Formation of white or pale precipitates shows the presence of alkaloids.
 
Test for tannins (Lead-acetate test)
 
To a test tube containing a small amount of leaves extract, few drops of 1% lead acetate were added. Formation of yellow precipitate indicates the presence of tannins.
 
Test for saponins
 
In the test tube containing 50 ml leaves extract, a drop of sodium bicarbonate was added. The mixture was vigorously shaken and kept for two minutes. A honey comb like froth formation indicates the presence of saponins.
 
Test for flavonoids
 
To a test tube containing about 0.5 ml of alcoholic extract of sample, few drops of diluted HCl and small amount of Mg or Zn were added and the solution was boiled for 5 minute. Appearance of reddish pink colour supports the presence of flavonoids.
 
Test for phenols
 
To 1 ml of the alcoholic solution of the sample, 2 ml of distilled water followed by few drops of the 10% aqueous solution of ferric chloride were added. Development of blue or deep green colour indicates the presence of phenols.
 
Test for terpenoids
 
In a test tube containing 1 mL of extract, 2 ml of chloroform and 5-10 drops of concentrated H₂SO₄ were added and appearance of reddish brown colour suggests the presence of terpenoids in leaf sample.
 
Test for steroids
 
0.1 g of plant sample was dissolved in 2 ml of CHCl₃. H₂SO₄ was added carefully to form a lower layer. A reddish brown colour at the interface was an indicative of steroidal ring.
 
Proximate composition
 
1. Moisture content
2. Ash content
3. Crude fat content
4. Crude fiber content
5. Crude protein content
6. Total carbohydrate content
 
Determination of moisture content
 
Two gram of powdered leaves sample of Jamun in triplicate was taken and method of AOAC (1995) was used to calculate the percentage of moisture content.
 
Determination of ash content
 
Two grams of powdered sample of Jamun leaves was weighed in triplicates and transferred into previously ignited and weighed crucible which is placed for 2 hours in a muffle furnace. From the furnace it was transferred into a desiccator and allowed to cool and their weight was taken.
    
Determination of crude fat content
 
In a thimble, two gram of the dried powdered sample of jamun leaves was taken and placed in a soxhlet extractor. A 250 mL dried and pre-weighted round bottomed flask was connected to the soxhlet assembly and petroleum ether was added up to one and a half siphons. The assembly was heated and extraction was performed for 8 hr. After extraction, petroleum ether was evaporated and weight of the round bottomed flask with the residue was determined again. The crude fat (%) contents were calculated as follows:-
                                        
Determination of crude fibre content
 
Moisture and fat free three gram powdered sample of jamun leaves was taken. Thereafter, the percentage of crude fibre content was calculated by using modified method of Maynard (1970).
 
Determination of crude protein content
 
Micro-Kjeldahl method (AOAC, 1990) was used for the determination of nitrogen content. By multiplying % of nitrogen with 6.25 factor, crude protein was calculated.
 
Determination of total carbohydrates content
 
Total carbohydrates content was calculated by difference as follows:
   
Mineral analysis
 
The mineral content of Jamun leaves samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) after Microwave-assisted acid digestion.
 
Quantitative estimation of phytochemical parameters
 
Quantitative determination of tannin content
 
Vanillin-HCl method of Burns (1971) was used for estimation of tannin content as catechin equivalent.
 
Quantitative determination of alkaloid content
 
Method of Harborne (1973) was used for estimation of alkaloid content in Jamun leaves sample.
 
Quantitative determination of total phenolics
 
Analysis of total phenolics were done by the Folin-Ciocalteu method (Singleton and Rossi, 1965) and expressed as milligrams of Gallic acid equivalent per gram (mg GAE/g). 0.2 mL of extract was taken in a test tube and to adjust the optical density with in calibration limits were diluted with respective solvents.
 
Quantitative determination of total flavonoids
 
The amount of total flavonoids present was calculated using catechin as standard. Analysis of total flavonoids was done by aluminium chloride colorimetric assay, as explained by Marinova et al., (2005)., Using the standard curve, the total flavanoids contents in ethyl acetate extract was calculated and the results obtained are expressed as mg catechin equivalents per gram (mg CE/g).
 
Evaluation of DPPH free radical scavenging activity
 
Method of Hatano et al., (1988) with slight modifications was used for the evaluation of DPPH free radical scavenging activity.
 
Evaluation of total antioxidant capacity
 
Modified phosphomolybdenum method by Prieto et al., (1999) was used for the evaluation of total antioxidant capacity of Jamun leaves powder.
 
Statistical analysis
 
For statistical analysis, triplicates of each sample were taken and the resulting values are expressed as mean ± standard error (S.E.). To assess any significant differences between the means of sample, one way analysis of variances (ANOVA) were carried out in Online Statistical Analysis (OPSTAT). IC₅₀ values of DPPH free radical scavenging activity were calculated by regression analysis in Microsoft Excel 2016. All other measurements were also carried out in Microsoft Excel 2016.

Ethyl acetate extract of Syzygium cumini leaves was screened by qualitative analysis for the presence of various phytochemicals. The result of qualitative analysis is depicted in the Table 2.
 

 
In proximate composition, the  leaves part of jamun consist the moisture content (6.06%), crude fibre content (16.15%), ash content (5.25%), crude protein content (8.79%), crude fat (0.26) and total carbohydrates (63.49%).
 
The minerals (Fe, Mn, Zn and Cu) content was estimated and data is presented in Table 3. The mineral content in leaves part of Jamun, Fe (39.15 ppm), Mn (2.52 ppm), Zn (49.35 ppm) and Cu (2.05 ppm).
 

       
In chemical analysis, leaves part of Jamun contains the tannin content (1.59 mg CE/g) and alkaloid content (6.78%). In phytochemical parameters, the content of total phenolics and total flavonoids were estimated. Phenols are very important plant constituents because of their antioxidant activity. The antioxidant activities of the plant extracts are often explained by their total phenolics and total flavanoids content. The standard curve obtained using gallic acid for total phenol content determination was depicted in Fig 1. The total phenolics in ethyl acetate extract of Jamun leaves was (11.48 mg GAE/g) and the total flavanoid content in jamun leaves was calculated using standard curve of catechin as depicted in Fig 2. The total flavanoids content in ethyl acetate extract of jamun leaves was 5.17 mg CE/g.
 

 

       
The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating free-radical scavenging activities of antioxidants. The percentage of DPPH free radical scavenging activity kept on increasing when the concentration of ethyl acetate extracts of jamun leaves was increased (Table 4). Total antioxidant capacity estimation using phosho-molybdenum assay is based on the principle that antioxidants present in sample reduce the Mo (VI) to Mo (V). Mo (V) react with the phosphate group of sodium phosphate to form a green coloured complex i.e. Mo (V)- phosphate complex (phosphomolybdenum complex) in acidic medium which is measured using UV-Vis spectrophotometer. IC50 value for DPPH scavenging and phospho-molybdenum assay is 42.64 and 31.32 µg/mL, respectively was calculated using the quadratic regression equation for IC₅₀ value as depicted in Fig 3 and 4. The value suggests that ethyl acetate extract of Jamun leaves have appreciable antioxidant activity.
 

 

 

The result indicates the presence of medicinally important phytochemicals in the ethyl acetate extract of Syzygium cumini leaves. The present findings have a great importance in the field of dietary supplements, drugs and pharmaceutical companies. The present study suggests that Jamun leaves powder is a potential source of antioxidants enriched with phenolic and flavanoid content in the shade dried sample.

None.