INTRODUCTION:

Plants have been used as medicines since the beginning of humankind. Many plants are claimed to be medicinal plants due to their healing property. This is because of the presence of certain phytochemicals such as phenols, tannins, resins, terpenoids, saponins etc. Apart from medicines, the plant pigments are used as dyes. According to WHO report, 80% of people in the world are using plants as medicines for their primary purpose and it continues to increase globally.

Syzygiumcuminibelongs to Myrtaceae family, it is a slow growing species. It reaches upto 30meters and fruit has pulpish white inside it. Syzygiumcuminiis also called as jambal, jambolan, or jamun. This plant is notable for its curing properties and broadly used in ancient medicines¹.

The dye from the fruit could stain violet colour in the cell organelles of rat hepatic cells². The researchers had shown that the seeds of S.cumini contain good antidiabetic and anti-cancerous activity. The seed juice has proved to decrease the pressure of the blood by 34.6%. The seeds are affluent in antioxidant properties. The fruit contains natural carbohydrates and the gallic acid may support for its sourness. Anthocyanin may support for its dark purple colour. Itsseeds are widely used for numerous problems such as anti-inflammatory, hypolipidemic and it can curtail the sugar level and late experiments showed it provides protection to DNA against radiations³. Although the seeds of Syzygiumcumini were used as cardioprotective agents⁴. S.cumini shows sensitive to certain microorganisms like E.coli, S.aureus, P.aeruginosa, K.pneumoniae, E.faecalis^5-12.

Many plants having anti-cancerous property, S.cumini is one of the plants with anti-cancerous property which is approved by the national cancer institute¹³. Former studies have been found that flavonoids consist of anti-mutagenic effects¹⁴.

Table 1: The phytochemical constituents of S.cuminiseed extract

Phytochemical constituents	Ethanol extract
glycosides	+
steroids	+
Cardiac glycosides	+
phenols	+
Terpenoids	+
flavonoids	+
tannins	+
saponins	+
alkaloids	+

(+present,-absent)

Table 2: GCMS analytic result for major phytocomponents in ethanolic extract of S.cumini seeds.

RT	Name of compound	Molecular formula	Molecular weight	Peak area	Structure
12.25	1,6-OCTADIEN-3-OL, 3,7-DIMETHYL-, 2-AMINOBENZOATE	C₁₇H₂₃O₂N	273	2.261
17.39	PENTADECANOIC ACID, 14-METHYL-, METHYL ESTER	C₁₇H₃₄O₂	270	2.974
18.93	2,3-ANHYDRO-D-MANNOSAN	C₆H₈O₄	144	3.550
24.90	TETRATETRACONTANE	C₄₄H₉₀	618	4.482
25.55	HEXATRIACONTANE	C₃₆H₇₄	506	5.059
26.19	TETRATETRACONTANE	C₄₄H₉₀	618	6.681
26.94	HEXATRIACONTANE	C₃₆H₇₄	506	5.395
27.81	TRITETRACONTANE	C₄₃H₈₈	604	4.970
28.85	HEPTACOSANE, 1-CHLORO-	C₂₇H₅₅Cl	414	2.864

Figure 1: Different Bacteria and its zone of inhibition

Figure 2: The GCMS chromatogram of S.cumini seed ethanolic extract.

Figure 3: DPPH scavenging activity.

MATERIALS AND METHODS:

Collection of plant material and extraction:

The fruits are collected from a local shop in Salem which is situated in the southern part of India. The fleshy portion were removed and hard seeds were thoroughly cleaned with H₂Oand shade dried at 37^oC for a month. After drying the outer layer of the seeds were removed. The seeds are powdered into fine powder using a blender. 109g of seed powder were obtained after blending process.

Extraction:

The seed powder of 10g was packed within the filter paper and 200ml of 100% ethanol was taken as solvent. After that soxhlet’s extraction method was done at the temperature of 71^oC. After obtaining 14 cycles, the dark brown extract of S.cuminiwere obtained. The extract was concentrated to 15ml by using rotary evaporator^15,16.

PHYTOCHEMICAL SCREENING:

Glycosides:

To 3ml of ethanolic sample ,1ml DH₂O and aqueous NaOH were added. Its presence was confirmed when the yellow colour is formed ¹⁷.

Steroids:

The powdered seed sample of 100mg was dissolved in the mixture of 2ml of H₂SO₄+and chloroform. While mixing a reddish-brown colour was observed and this confirms its presence¹⁷.

Phenols

The ethanolicsample of 1ml and DH₂O of 2ml were mixed together, after that 3 drops of 10% FeCl₃ was added. The green colour formation points out the its presence ¹⁷.

Tannins:

The pulverized sample of 200mg and 10ml of DH₂O were mixed and boiled it for 5mins in a water bath. After boiling the hot mixture was filtered. Add few drops of 10 % FeCl₃to the sample. Its presence was confirmed when the dark blue precipitate is formed¹⁸.

Cardiac glycosides:

The pulverized sample of 100mg was mixed to the mixture of 1ml of CH₃COOH, then one drop of FeCl₃and 1ml of conc.H₂SO₄ solutions. A brown coloured ring is noticed while mixing shows its presence¹⁹.

Test for terpenoids:

The sample of 2ml was added to chloroform of 1ml. After that 1 ml of conc.H₂SO₄ was added. Its presence was confirmed when the reddish brown coloured precipitate is formed while mixing¹⁹.

Flavonoids:

TheDH₂O of 10ml was mixed to 1g of dehydrated seeds powdered sample and boiled in the water bath for 5mins. Few amount of NaOH (20%) was mixed to sample of 1 ml. By adding of acid, the colour change from yellow colour to colourless solution points out its presence.^[19]

Saponins:

The sample extract of 1ml and 10ml of DH₂O were mixed and boiled for 10 minutes in the water bath and then filtered while it is hot. And then, 7 ml of DH₂O and 3ml of filtrate were mixed and shaken forcefully. Its presence was confirmed when the frothingappeared¹⁹.

Alkaloids:

The powdered sample of 1g, 10 ml of HCL and water were mixed together and boiled in a water bath and then filtered. Ammonia was used to maintain pH between 6 to 7. The following reagents were added separately in small quantity to filtrate sample of 0.5 ml in individual test tubes. 10% tannic solution, Picric acid solution, Mayer’s reagent (Potassium mercuric iodide solution). After adding reagents, the samples were noticed for coloured precipitates or turbidity¹⁹.

Antimicrobial assay:

The antibacterial activity of S.cumini was analysed by disc diffusion method. The test microbes were Streptococcus agalactiae, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Clostridium perfringens, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Klebsiellapneumoniae, Aeromonashydrophila. These organisms were testedusingethanolic extract of S,cumini at different concentrations (20%, 40%, 60%, 80%, 100%). Each microorganism was injected in Sterilized Nutrient agar plates individually by spread plate method. The discs with extract were placed on the microbial culture. After 26 hours of incubation, the zone of inhibitions was measured.

GC-MS:

The sample was subjected to GC-MS analysis to quantify the number of molecules and its structures. The analysis was carried out using GC–MS (Perkin Elmer model: Clarus 680) and also it is equipped with mass spectrometer (Clarus 600 (EI) analyzed using (TurboMassver 5.4.2) software.Fused silica which is packed with Elite-5MS. At a constant flow rate about 1ml/min, carrier gas such as helium was used to separate the components. The temperature of the injector was adjusted to 260^oC while performing the experiment. The extract sample of 1µL was injected into the equipment the temperatures of the oven were 60^oC (2 mins); followed by 300^oC at the rate of 10^oCmin^-1; and 300^oC for 6mins. The conditions of the mass detector were: the temperature of transfer line was 240^oC; and ionization mode electron impact at 70eV, the duration time of scan interval is 0.2sec and scan interval is 0.1sec. The fragments from 40 to 600Da. The spectrum of componentswascorrespondingto the database of the spectrum of established components gathered in the GC-MS NIST library.

ANTIOXIDANT ASSAY

DPPH ASSAY:

The antioxidant capacity of S.cumini was identified through 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay. Equal volumes samples at different concentrations were mixed with 0.1mM of DPPH. Then the mixture was stored in dark place for 30mins. The colour change from violet to yellow indicated the presence of antioxidants. Quantification was calculated by absorbance. The absorbance was measured using spectrophotometer at 517nm against the blank. Each concentration was performed in triplicates. Ascorbic acid was used as the standard to compare with samples and IC50 (inhibitory concentration) was calculated for both sample and standard. The percentage of inhibition was calculated using the following formula:

% of Inhibition=[A₀-A₁/A₀] x 100

Where A₀ is absorbance of control (i.e, DPPH solution without sample) and A₁ is absorbance of sample or standard (i.e, DPPH solution with sample/standard)²⁰.

RESULT AND DISCUSSION:

The current experiment was performed by using the ethanolic extract of Syzygiumcumini. The results of phytochemical analysis disclose the existence of glycosides, steroids, cardiac glycosides, phenols, terpenoids, alkaloids, flavonoids, tannins, and saponins in the S.cumini. Nature is the source of flavonoids. Flavonoids belong to polyphenols family, it is consisting of many subclasses. So far 8000 flavonoid compounds were discovered. It possesses antioxidant behaviours, as it can able to eliminate the free radicals. Because of its antioxidant ability, it has anti-cancerous property²⁰. Cardiac glycosides are important agents in cardiac failure treatments and also it shows cytotoxic effects on HeLa cancer cell¹⁸.Saponins are considered as bio-surfactants and have the property of detergents²³ Tannins are the biomolecules found widely in plants. The recent studies illustrate that MRSA are sensitive to tannins and it can be used against MRSA²⁴. Terpenoids are contrary to diabetes^20.In our glycoside assay, the formation of yellow colour indicated the presence of glycosides in the sample. A study result indicates Glycosides were absent in S.cumini¹⁹.Another study details show glycosides are moderately present in S.cumini extract^20
.Most of the compounds with higher concentration are Flavonoids, alkaloids and phenols¹⁷.

GCMS results showed that long chained hydrocarbons exist majorly like tetratetracontane, hexatriacontane, tritetracontane. The graph reveals that 1,6-octadien-3-ol, 3,7-dimethy-,2-aminobenzoate is present in higher amount and other major compounds are Pentadecanoic acid,14-methyl-methyl ester, 2,3-anhydro-d-mannosan and heptacosane,1-chloro.

Most of the plants possess antibacterial activity. The consequence of antibacterial assay depicts Bacillus cereus showed highest sensitivity followed by Streptococcus agalactiae, Pseudomonas aeruginosa, Listeria monocytogenes shows high sensitivity and Clostridium perfringens showed less susceptible to the extract and remaining bacteria show the moderate level of sensitivity. Another study which is done with methanolic and water extract of S.cumini leaves reported that in the methanolic sample, Salmonella typhi showed highest sensitivity.Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa are the common bacteria in both experiments. However, these three bacteria showed the considerable level of sensitivity. And in aqueous sample, the bacteria which is common in both experiments are slightly sensitive in aqueous sample than methanolic sample and Salmonella paratyphi B showed the high degree of sensitivity in aqueous sample¹⁷.

Antioxidants are the molecules contrary to free radicals. Some free radicals may damage the tissues this causes ageing and even cancers. Such conditions can be controlled by antioxidants. The existence of antioxidants in the S.cumini was confirmed by DPPH assay. The IC₅₀ value have estimated about 0.21 for ascorbic acid(standard) and 0.8 for plant extract (sample). Another research which is performed with methanol solvent extract and its solvent fractions with chloroform, water, n-hexane and ethyl acetate of S.cumini, its conclusion of DPPH and FRAP assay exist as a proof for the presence of antioxidants in this plant²⁰.

CONCLUSION:

Based on the conclusions of the present and previous studies, Syzygium cumini seeds contains numerous phytochemicals and antioxidant property that can be used for the beneficial purpose and this may be useful in cancer treatments. Meanwhile, the biomolecules of seed were contrary to specific bacteria. Hence this may be used in drug development techniques against pathogens.

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Received on 19.04.2018 Modified on 29.05.2018

Research J. Pharm. and Tech 2018; 11(9): 4096-4100.

DOI: 10.5958/0974-360X.2018.00752.7