PHYTOCHEMICAL ANALYSES, ANTIBACTERIAL, IN VITRO ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF ETHANOLIC EXTRACT OF SYZYGIUM CUMINI (L.) SEED EXTRACT

PHYTOCHEMICAL ANALYSES, ANTIBACTERIAL, IN VITRO ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF ETHANOLIC EXTRACT OF SYZYGIUM CUMINI (L.) SEED EXTRACT

Joyita Banerjee and Narendhirakannan R.T.*

Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya University, (Karunya Institute of Technology and Sciences), Karunya Nagar, Coimbatore - 641 114, Tamil Nadu, India

ABSTRACT

Phytochemical constituents present in the aqueous and ethanolic extract of Syzygium cumini seeds were investigated which were found to be responsible for its medicinal efficacy. The major phytoconstituents present in S. cumini seeds were further determined by GCMS analysis which showed the presence of polyphenols, sesquiterpenes, n-alkanes. The antibacterial activity of ethanolic extract of S. cumini seeds were tested against common human pathogens by agar well diffusion method and minimum inhibitory concentration required to inhibit the growth of various pathogens were also evaluated. The seed extract was found to have high antibacterial activity. The total polyphenolic content of the ethanolic extract of S. cumini seed was determined by Folin-Ciocalteau method. The antioxidant activity of S. cumini seed ethanolic extract was evaluated by DPPH free radical scavenging assay, reducing power assay and total antioxidant capacity. S. cumini seed extract was found to have very high and potent antioxidant activity and might be due to the presence of high phenolic components in seed. The cytotoxicity assay of S. cumini seed ethanolic extract was carried out by trypan blue dye exclusion technique where the seed extract showed potential cytotoxic activity against cervical cancer cells.

Cytotoxicity assay

INTRODUCTION: From ancient times, medicinal herbs have been use in one form or another, under indigenous systems of medicine like Ayurveda, Sidha and Unani ¹. Herbal medicines are promising choice over modern synthetic drugs as they show minimum or no side effects and are considered to be safe ².

According to WHO, 80% of the world’s inhabitants problem should treated by medicinal herbal drug for their primary health care ¹. There are various medicinal plants which have significant medicinal values and are widely used in folklore medicine. The literature reveals that flavonoids, alkaloids, terpenes of plant derived products have received considerable attention in recent years due to their diverse pharmacological properties including cytotoxicity and cancer chemopreventive effects ³.

There are several factors which are responsible for causing various diseases and complications of the human health. In this present study, we have focused on the major two issues related to their complications on human health, multidrug resistant microorganisms and oxidative stress.

The present problem in the pharmaceutical industry is the wide prevalence of the multidrug resistant bacteria. The problem of microbial resistance of growing and the outlook for the use of antimicrobial drugs in the future is still uncertain ⁴.

So, there is a need to evaluate the plant as the source of potential chemotherapeutic agent, antimicrobial agent and their ethno medicinal use ⁵.

Another factor that inevitably affects the human health is the generation of free radicals. Free radicals such as Reactive oxygen species (ROS) are small, highly reactive, oxygen-containing molecules continuously in the cells as a consequence of both enzymatic and non-enzymatic reactions ⁶ and can react with and damage complex cellular molecules such as fats, proteins, or DNA ⁷. In low/moderate concentrations free radicals are involved in normal physiological functions but excess production of free radicals or decrease in antioxidant level leads to oxidative stress ⁸.

Oxidative stress is apparent in pathology associated with aging and many age-related chronic diseases, including atherosclerosis, diabetes mellitus, rheumatoid arthritis, and neurodegenerative diseases ⁹. There are synthetic antioxidants such as butylated hydoxyanisole (BHA), butylated hydoxytoluene (BHT), Propylgallate (PG), and test butylated hydroquinone, but owing to their side effects such as liver damage and carcinogenesis ^{10, 11}. Therefore, there is a need for isolation and characterization of natural antioxidant having less or no side effects, for use in foods or medicinal materials to replace synthetic antioxidants ¹².

Plants may possess pharmacological properties, but they may also be toxic or mutagenic. The toxic effects of most widely used medicinal plants are not well documented in the literature although one might expect plants used in traditional medicine over a long period to be safe ¹³. The severity of toxicity produced by any chemical is directly proportional to the concentration and the exposure time and this depends on the developmental stage of an organism and its physiological status ¹⁴.

Syzygium cumini (L.) is one such traditional medicinal plant of Indian origin, belonging to family Myrtaceae ^{2, 4, 15}. It is commonly known as Jamun (Hindi) ², Naaval (Tamil) ¹⁵, Java plum, Black plum, Jambul and Indian Blackberry ^{16, 17}. The medicinal value of S. cumini lies in its leaves, fruits, seed and bark. The seeds of S. cumini are widely considered to have anti-diabetic properties ^{2, 4}.

The fruits ¹⁸ and leaves ¹⁹ of S. cumini have unique anti-oxidant activity. Earlier studies showed that the seeds have significant anti-inflammatory ²⁰, anticancerous ³ and central nervous system ¹⁵ activities. In this present study, the ethanolic extract of S. cumini seed in regard to its antibacterial, in vitro antioxidant and in vitro cytotoxic activities were evaluated.

MATERIALS AND METHODS:

Chemicals and reagents: Nutrient broth, nutrient agar, 2,2-Diphenyl picrylhydrazyl (DPPH), trichloroacetic acid, ferric chloride, potassium ferricyanide, sodium phosphate, ammonium molybdate, trypan blue. All chemicals used were of analytical grade.

Collection of plant material and extraction: The seeds were collected from the Coimbatore area in the year 2010. The plants & seeds were identified and authenticated by Prof. V.S. Ramachandran, Dept. of Botany, Bharathiar University, Coimbatore. The seeds of the plant S. cumini were thoroughly washed and dried at 37^oC. The dried seeds were further pulverized into fine powder. 25gm of the powdered seed was taken for the extraction purpose in ethanol as the solvent, by using Soxhlet’s apparatus. This seed extract was used for studying the various antibacterial, in vitro antioxidant and cytotoxicity assays. All chemicals used were of analytical grade.

Phytochemical qualitative analyses: The aqueous and ethanolic extracts of the S. cumini seed were taken for carrying out various phytochemical analyses following the standard methods: Alkaloids with Mayer’s test, flavonoids with alkaline reagent test, carbohydrates with Molisch test, glycosides with Legal’s test, saponins using sodium bicarbonate, tannins using lead acetate, phytosterols with Salkowski’s test, phenols using ferric chloride, triterpenoids with Libermann Buchard test, anthroquinones using concentrated sulfuric acid, benzene and ammonia and amino acids with Ninhydrin test. These were identified by characteristic color changes using standard procedures ^{21, 22}.

GCMS analysis: The GCMS (Gas Chromatography Mass Spectroscopy) analysis of the S. cumini seed ethanolic extract was carried out at the South India Textile Research Association (SITRA), Coimbatore. The Gas Chromatography (GC) was carried out by using Thermo GC Trace Ultra Version 5.0 equipment with run time of 35:32 mins and the Mass Spectrometry (MS) was carried out by using Thermo MS DSQ II equipment.

Antibacterial assay: The antibacterial activities of S. cumini seed ethanolic extract against different gram positive and gram negative bacteria were carried out by Agar well diffusion method on nutrient agar plates ²³. Test organisms used were Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa. Different concentrations (50 mg/ml- 250 mg/ml) of seed extract were made and the zone of inhibition was calculated for each concentration. Antibiotics such as Kanamycin (30mcg), Norphoxacin (10mcg) and Cephalothin (5mcg) were used as the standards. The Minimum inhibitory concentration (MIC) of the sample was determined by Serial tube dilution technique ²⁴.

In vitro Antioxidant Assays:

Determination of Total Phenolic Content: The total phenolic content of ethanolic extract of S. cumini seed was determined by Folin-Ciocalteau method ^25,26. The total phenolic content was expressed in terms of Gallic acid equivalent (mg/g of dry mass).

DPPH Free Radical Scavenging Assay: 1ml of 0.1mM 2, 2-Diphenyl picrylhydrazyl (DPPH) in ethanol, was added to different concentrations of S. cumini seed extract. The reaction mixture was well shaken and incubated in dark for 30 mins. Absorbance was checked at 517 nm against a blank (ethanol). Ascorbic acid was taken as the standard. Lower the absorbance of the reaction mixture indicates higher percentage of scavenging activity. The percentage of inhibition or scavenging of free radicals was determined by the formulae;

% Inhibition = [(Absorbance _Control – Absorbance _Sample)/ Absorbance _Control] x 100,

Where control was prepared as above without extract ^27,28.

Reducing Power Assay: 500 μl of each concentration of seed extract was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (10g/l), then the mixture was incubated at 50º C for 20 minutes. 2.5 ml of trichloroacetic acid (100g/l) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml FeCl₃ (1g/l) and absorbance measured at 700nm in UV-Visible Spectrophotometer. Ascorbic acid was used as standard and phosphate buffer used as blank solution. Increased absorbance of the reaction mixture indicates stronger reducing power ^{29, 30}.

Total Antioxidant Capacity: 0.1 ml of different concentrations of seed extract were mixed in separate eppendorf with 1 ml of reagent solution (0.6 M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate; mixed in 1:1:1 ratio) respectively. The tubes were capped and incubated in a thermal block at 95°C for 90 min. After cooling to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank. Ascorbic acid was used as the standard and the total antioxidant capacity was expressed as equivalents of ascorbic acid ³¹.

Cytotoxicity Assay: The cytotoxicity activity of the seed ethanolic extract was done by Trypan blue dye exclusion technique ³². The tumor cells were procured by using HeLa Cervical cancer cell line, purchased from National Centre for Cell Science, Pune. The tumor cells were maintained in RPMI-1640 medium. Different concentrations of extracts were prepared diluting with RPMI-1640 medium and were added to 100 μl of cell suspension. After 3 hours of incubation, percentage of mortality was calculated using trypan blue (0.4%) under haemocytometer, control was prepared same as above except extract. The percentage of mortality was calculated using the formulae: Percent (%) of mortality = (number of non viable cells/ total number of cells) x 100.

Statistical Analysis: All the grouped data were statistically evaluated with SPSS/16 software. Hypothesis testing methods included Student’s t test followed by least significant difference (LSD) test. P values of less than 0.05 were considered to indicate statistical significance. Values are presented as the mean ± S.D. of each three replicates in each experiment.

RESULTS AND DISCUSSION:

Phytochemical Qualitative Analysis: A variety of herbs and herbal extracts contain different phytochemicals with biological activity that can be of valuable therapeutic index ³³. Table 1 shows the presence of various phytoconstituents in the aqueous and ethanolic extracts of S. cumini seed. The performed qualitative studies indicate the presence of various phytoconstituents except glycosides and anthroquinones in the ethanolic extract of S. cumini seed. These phytoconstituents might be responsible for different biological activities and medicinal importance of this seed.

TABLE 1: THE PHYTOCHEMICAL CONSTITUENTS OF S. CUMINI SEED EXTRACTS (+ PRESENT, - ABSENT)

GCMS analysis of S. cumini seed ethanolic extract: Figure 1 shows the GCMS chromatogram of the S. cumini seed ethanolic extract along with their retention time (RT). Table 2 shows the major phytocomponents present in the S. cumini seed along with molecular formula, molecular weight, peak area and structure. The GCMS chromatogram of ethanolic extract of S. cumini seed showed the presence of compounds such as alkanes, sesquiterpenes, polyphenols.

FIG. 1: THE GCMS CHROMATOGRAM OF S. CUMINI SEED ETHANOLIC EXTRACT

TABLE 2: GCMS ANALYTICAL REPORT FOR MAJOR PHYTOCONSTITUENTS IN ETHANOLIC EXTRACT OF S. CUMINI SEED

Antibacterial Assay: The antibacterial activities of S. cumini seed ethanolic extract was studied against two gram positive bacteria such as S. aureus and E. faecalis and three gram negative bacteria such as E. coli, K. pneumonia and P. aeruginosa  (table 3). The antibacterial activity of S. cumini seed extract was compared to the standard antibiotics (table 4). The seed extract showed to have potent antibacterial activity. The antibacterial effect was reported to be size and dose dependent.

TABLE 3: ANTIBACTERIAL ACTIVITY OF ETHANOLIC EXTRACT OF S. CUMINI SEED

Values expressed as mean ± standard deviation and was calculated by the zone of inhibition

TABLE 4: ANTIBACTERIAL ACTIVITY OF STANDARD ANTIBIOTICS

The minimum inhibitory concentration (MIC) represents the concentration of antimicrobial at which there is complete inhibition of growth of organism ³⁴. The MIC was determined by Serial tube dilution technique. The MIC value of the seed extract against E. coli, P. aeruginosa and E. faecalis was found to be 6.25mg/ml whereas against S. aureus and K. pneumoniae, MIC value was 3.13 mg/ml.

FIGURE 2: ANTIBACTERIAL ACTIVITY OF ETHANOLIC EXTRACT OF S. CUMINI SEED EXTRACT

 Total phenolic content: S. cumini is known to be very rich in gallic and ellagic acid polyphenol derivatives ^{35, 36}. The presence of high amount of polyphenols in the S. cumini seed was also evaluated from the estimation of total phenolic content of the seed extract by the Folin-Ciocalteau method, which was found to be 200.83±3.81 mg per gm of ethanolic extract of the S. cumini seed expressed as mg of Gallic acid equivalents. The phenolic compounds are very important plant constituents because of their antioxidant activities ³⁷.

DPPH Free Radical Scavenging Assay: Figure 3 shows the DPPH Free Radical Scavenging Assay of S. cumini seed ethanolic extract (Sc) where ascorbic acid (AA) was taken as standard. A freshly prepared DPPH solution exhibited a deep purple color with a maximum absorption at 517 nm. This purple color disappears when an antioxidant is present in the medium. Thus, antioxidants molecules can quench DPPH free radicals and convert them to a colorless product, resulting in a decrease in absorbance at 517 nm ³⁸. The DPPH free radical scavenging assay showed potent inhibitory capacity of S. cumini seed extract when compared with ascorbic acid. The percentage of inhibition of free radicals increased with increase in concentration of substrates.

FIGURE 3: DPPH FREE RADICAL SCAVENGING ASSAY OF S. CUMINI SEED ETHANOLIC EXTRACT

(Values are mean ± SD of three determinations)

Reducing Power Assay: Figure 4 shows the Reducing Power Assay of S. cumini seed ethanolic extract (Sc) where ascorbic acid (AA) was taken as standard. The reducing ability of a compound depends on the presence of reductants ³⁹ which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom ⁴⁰. Presence of reducers causes the conversion of the Fe3+/ferricyanide complex used in this method to the ferrous form. By measuring the formation of Perl’s Prussian blue at 700 nm, it is possible to determine the Fe2+ concentration ⁴¹. S. cumini seed extract was found to have very high reducing ability when compared to the standard and increased with increasing concentration of substrates.

FIG. 4: REDUCING POWER ASSAY OF S. CUMINI SEED ETHANOLIC EXTRACT

(Values are mean ± SD of three determinations)

Total Antioxidant Capacity: Figure 5 shows the Total antioxidant capacity of ascorbic acid (AA) and S. cumini seed ethanolic extract (Sc). The Total antioxidant capacity was calculated based on the formation of the phosphomolybdenum complex where the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate/Mo (V) complex with a maximal absorption at 695 nm ¹². The S. cumini seed extract was found to have very high total antioxidant capacity as compared to the standard.

FIG. 5: TOTAL ANTIOXIDANT CAPACITY OF S. CUMINI SEED ETHANOLIC EXTRACT

(Values are mean ± SD of three determinations)

Cytotoxicity Assay: Figure 6 shows the growth inhibitory and cytotoxic activity of S. cumini seed ethanolic extract against cervical cancer cells. The viable cells remained unstained and appeared transparent and bright. The non- viable cells became stained and appeared blue in color. The growth inhibition and cytotoxic activity of S. cumini seed extract was carried out by Trypan blue dye exclusion assay. The growth inhibitory and cytotoxicity assay was found to be dose dependent. The extract showed significant decrease in cervical cancer cell population. The extract which causes at least 50% growth inhibition can be counted as cytotoxic ⁴². The ethanolic extract of S. cumini seed, at a concentration of 0.5mg/ml showed more than 50% growth inhibition of cervical cancer cells.

FIG. 6: CYTOTOXICITY ASSAY OF S. CUMINI SEED ETHANOLIC EXTRACT

(Values are mean ± SD of three determinations)

CONCLUSION: This study suggests that the ethanolic extract of Syzygium cumini seed has potent antibacterial, antioxidant and cytotoxic activities. Phytochemical studies showed the presence of polyphenols, tannins, flavonoids, terpenoids etc., which are responsible for the medicinal value of S. cumini seed. This seed extract has high antibacterial activity and thus can be used against multidrug resistant bacteria and as an herbal medicine alternative to the antibiotics. The seed extract of S. cumini contains high amount of polyphenolic components which may be responsible for its potential antioxidant activity.

Since, free radicals are important contributors to various degenerative diseases such as cancer. The observed antioxidant properties of the seed extract of S. cumini might be useful for the development of newer and more potent and natural antioxidant and thus can be used as potential free radical scavengers and can be used against the various damages caused by free radicals. The high growth inhibitory and cytotoxic effect of S. cumini seed extract against cervical cancer cells and thus can be used as potential anticarcinogenic agents. The in vitro bioassays provide an introspective knowledge of antibacterial, free radical scavenging and anticancerous activities of S. cumini seed and thus can be further investigated for in vivo studies.

ACKNOWLEDGEMENT: The authors would like to express their gratitude to Dr. Paul Dinakaran, Chancellor, Dr. Paul P. Appasamy, Vice Chancellor, Dr. Anne Mary Fernandez, Registrar of Karunya University, for providing the necessary facilities for carrying out experiments. We extend our gratitude to SITRA, Coimbatore for carrying out the GCMS analysis and NCCS, Pune for providing with cancer cell line.

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