Antibacterial, Antioxidant analysis of Phytochemical Extracts derived from seeds of Syzygium cumini L. against Pathogenic Bacteria

 

Anupam Kumar1*, Anu1, Anand Mohan1, Neeta Raj Sharma1, Hasibur Rehman2

1Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India

2University of Tabuk, Tabuk

*Corresponding Author E-mail: anupam.kumar167@gmail.com

 

ABSTRACT:

Syzygium cumini   is a tropical fruit tree of great economic importance. The fruit is commonly known as jamun (Hindi), java plum, black plum, jambul and Indian blackberry. It is a large, evergreen widely distributed forest tree of India, Sri Lanka, Malaysia and Australia which is also cultivated for its edible fruits. The tree was introduced from India and tropical Asia to southern Africa for its edible and attractive fruits. S. cumini tree has proved to have medicinal properties against a number of diseases it has a high economic value for the application in the medicinal field its extract contain compound like flavonoids, alkaloids, glycosides, steroids, phenols, saponins, terpenoid, cardiac glycosides and tannins as the chemical class present in the extracts. In current research study, phytochemical extracts derived from seed of S. cumini for antimicrobial analysis and to explore its potential applications to develop green drug and as an alternative to antibiotics available to cure diseases without side effects. Phytochemical extracts were obtained by using water, methanol and acetone and various tests were performed to evaluate the presence of alkaloids and flavnoids with standard protocol; then biochemical characterization of these extracts were analyzed by using Fourier Transform Infrared (FT-IR) spectroscopy to evaluate the phytoconstiutents present in extracts. Then phytochemical extracts derived from seed extracts were examined for antimicrobial effects against various gram positive (Bacillus subtilis, Staphylococcus aureus, Bacillus cereus) and gram negative bacteria (Salmonella typhimurium, Salmonella enteric, E.coli) by disc diffusion method. After incubation period of 24 hrs at 370C zone of inhibition was measured and compared with standard antibiotic chloromphenicol and gentamycin. Antioxidant analysis was determined on the basis of the scavenging effect on the stable DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical activity and heavy metal absorption was also analyzed to detect the potential uses of these extracts as antioxidant. Methanolic extracts have shown more potent antimicrobial effects than water and acetone derived extracts. Terpenoids are well known for their antimicrobial, anti-inflammatory and anti-neoplastic activities, triterpenes and terpenoids have already been isolated. Saponins are reported to have antimicrobial, antiviral, antifungal, anti-inflammatory and haemolytic, hepatoprotective and anti- ulcer activities. Findings in current research studies can be implemented to develop green drug and nanoparticles for antimicrobial, anti-diabetic purposes and various immunomodulation studies to improve immunity of an individual.

 

KEYWORDS: Syzygium cumini L, Phytochemical extracts, FT-IR, antimicrobial.

 

 

 

INTRODUCTION:

The use of medicinal plants shows a high potential to treat as antimicrobial agents which generally used as phytochemical extract of the medicinal plant. Plants are available in various geographical distributions worldwide and people are using various parts of plants for food source and medicinal purposes since civilization of human beings. Many powerful drugs which help to fight against various diseases and enhance the immunity of an individual are generally plant derived and used in different parts of the world. The active metabolites which are present in many drugs is due to secondary metabolites; like alkaloids, flavanoids, saponins, tannins and steroids present in the plants which shows potential to fight against many pathogenic organisms and also to boost the immune system of human beings and other animals [1]. In developing countries basically people belong to rural areas want the cheaper medicines to cure diseases and infections and they are mainly depended on herbal medicines for their proper health [2]. There are so many medicinally important plants and their role to cure various diseases have been reported earlier and one of the most significant plants of interest for people is Syzygium cumini L. Since the history of Indian medicinal science S. cumini is reported as to cure many diseases. S. cumini L belongs to family Myrtaceae which is known by different names like jamun, java plum, jambul, and Indian blackberry. It is naturally available in many parts of India, Pakistan, Bangladesh, Sri Lanka, Malaysia and Australia. S. cumini is famous for edible fruit and different medicinal applications in entire tropical Asia to Southern African continents [1]. Various plant parts of S. cumini like; bark, leaves and seed extracts have observed and reported as anti-inflammatory [3, 4,], antidiabetic [5, 6], anti-diarrheal, antifungal and antibacterial effects [7,8,9]. Several medicinal spices and herbs contains great number of important natural antioxidants with different concentrations which has varying properties antioxidants can avoid food degradation by preventing rapid oxidation of lipids in food materials the most effective path to eliminate and diminish the action of free radicals which cause the oxidative stress is antioxidative defense mechanisms[10]. Phytochemical extracts of various parts (Leaves, Fruits etc) of S. cumini L have been reported also having antioxidant properties and increased scavenging activity have been confirmed against reactive oxygen species (ROS) [11, 12]. S. cumini L.  extracts have potential to develop as green drug to treat various diseases and infections.

 

Fig1: Shows the importance of S. cumini L

 

MATERIALS AND METHODS:

S. cumini L seeds were collected from local market of Phagwara city and Jalandhar city, Punjab, India. Samples were verified by experts of Botany in Department of Botany, LPU, Punjab. Further these seeds were processed with proper cleaning with tap water several times and followed by distill water wash thrice to avoid presence of any microbial cells. 150g Syzygium cumini seeds were taken in 200ml distilled water, Methanol. Acetone and extraction was done for 24hrs to get the clear solution. The above cooled content was filtered by using Whatman filter paper and then   the filtrate was used for phytochemical anaylsis and for the synthesis of nanoparticles (aqueous extract) and its antimicrobial activity [13].

 

Seeds were crushed properly and packed in muslin cloth and kept in Soxhlet apparatus for phytochemical extraction using water, ethanol and methanol as per standard protocol [14, 15]

 

Phytochemical screening:

Phytochemical tests were performed for the water, methanol and ethanol derived extracts.

 

Preliminary phytochemical analysis:

The extract was tested for the presence of bioactive compounds by using standard methods [16]. Biochemical analysis for flavonoids was performed by Shinoda test, for sterols evaluation Salkowaski test and Libermann-Burchard test was performed, for alkaloids presence Dragendorff’s test and Mayer’s reagent test was performed. Test for tannin was performed using ferric chloride and for saponins analysis foam test was performed. For carbohydrates evaluation in extract; Molisch’s test and Barfoed’s test were analyzed and for estimation of reducing sugar in extract Fehling’s test and Benedict’s test were performed. For glycosides evaluation pinch of extract treated with glacial acetic acid and few drops of ferric chloride solution was added and followed by addition of conc. H2SO4; formation of ring at the junction of two liquids indicated the presence of glycosides [17].

 

Antibacterial assay: Paper disc diffusion method:

The Muller Hinton Agar (4.1 g/100 ml) was taken and dissolved in 250 ml of distilled water flask. The medium were sterilized by using autoclaving then it was cool at room temperature. After that the medium was poured into the Petri plates. The discs were saturated with 50 µl of the extract of plants in different plates and allow being dried [18]. The discs were placed on MHA plate swabbed with the culture of microorganisms and incubated at 37oC for 24 hours. After incubation, the zones of inhibition around the discs were measured by ml scale. The experiment was replicated three times to confirm the reproducible results. The water methanol and acetone extract of Syzygium cumini L was used for antimicrobial activity. Agar-well diffusion methods were used to determine the antimicrobial activities for the plant extracts against micro organism. The   different bacterial strains were used and it was inoculated in a nutrient broth at 37oC for 24 hour in incubator [18].

 

Free radical scavenging assay:

The antioxidant activity using plant extracts was determined on the basis of the scavenging effect on the stable DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical activity. The ethanolic solution of DPPH (0.1 mM) 10ml was prepared. 5 test samples with varying extract volume of 200µl, 400µl, 600µl, 800µl and 1000µl were prepared. All the samples were made up to 2000µl by adding 1000µl 0.1mM DPPH solution and distilled water. The reaction mixture was shaken vigorously and incubated for 30min. The absorbance of samples was measured spectrophotometrically at 517nm. A blank sample with the same amount of ethanol and DPPH was taken. The radical scavenging activity was determined of the test samples, and the percentage of inhibition was determined [19].

 

Percent (%) inhibition of DPPH activity = [(AB – AA) / AB] x 100

 

Where AA is the absorbance values of the test And AB is the absorbance blank sample.

The graph between the percent inhibition and volume curve was drawn to determine the efficiency of plant extracts.

 

 

FTIR Measurements:

The different functional groups present in the extract were determined by the FT-IR studies. The dried extract was taken and the sample was prepared by using KBr then pellet was used for the FT-IR analysis.

 

Test organisms:

Bacillus subtilis (MTCC121), Bacillus subtilis (MTCC441) Salmonella enteric (MTCC1164), Staphylococcus aureus (MTCC96), Bacillus subtilis (MTCC1305), Salmonella typhimurium (MTCC3231) Staphylococcus   aureus (MTCC7443), Escherichia coli(MTCC40), Bacillus cereus  ( MTCC430), Bacillus cereus (MCC2086).

 

Antibiotics: Gentamycin, Chloromphenicol (Control)

 

Synthesis of Silver Nanoparticles:

30 ml of aqueous extract was added to 30 ml of aqueous solution of 20 mM silver nitrate for reduction of Ag+ ions and incubated at room temperature for 48 hrs. The silver nanoparticles were purified by repeated centrifugation of brown suspension at 10,000 rpm for 20 min followed by redispersion of the pellet of silver nanoparticles in deionized water and again centrifugation in the same way. Thus produced Ag-NPs were characterized and studied for their antimicrobial activity and catalytic activities [19].

 

Characterization of AgNP:

UV-Spectroscopy:

The reduction of Ag+ ions was monitored by measuring absorption of the reaction medium in a range of 400-450 nm wavelengths using a Shimadzu UV-2550 UV-Visible spectrophotometer with a cell of 1 cm path length.

 

FTIR Measurements:

The different functional groups present in the extract were determined by the FT-IR studies. The dried extract and synthesized AgNPs was taken and the sample was prepared by using KBr then pellet was made for the FT-IR analysis.

 

 

RESULTS AND DISCUSSION:

Phytochemical test Table1: Qualitative phytochemical evaluation of the water, methanol and acetone extracts of Syzygium cumini seeds

Phytoconstituents

Test performed/reagents used

Result

Water extract

Methanol extract

Acetone extract

Alkaloids

Mayer’s test

Cream color precipitate

+

+

+

Dragendorff’s test

Orange brown precipitate

+    +

+

+

Steroids   

Salkowski test

Red color

+

+

+

Flavonoids  

Flavonoids test

Yellow(NaOH) to colorless(HCl)

+

+

+

Tannins 

Ferric chloride

Dark green precipitate

+

+

+

Saponin

Saponin test

Foam formation

+

+

+

Glycosides

Molish test

No purple ring formation

-

-

-

Proteins and amino acids 

Biuret test

Violet color

+

+

+

Reducing sugar

Benedict test

No brick red color

-

-

-

 Fehling’s test

No brick red color

-

-

-

‘+’ indicates presence of the Phytoconstituents, ‘++’ indicates present in more quantity of the Phytoconstituents, ‘–’ indicates absence of the Phytoconstituents.

 

 

Table 2: Comparison of antibacterial activity test result of S. cumini L seed extract with control antibiotics Gentamycin and Chloromphenicol in mm (mean of triplets)

Microorganisms

Gentamycin

Water

Methanol

Acetone

Bacillus subtilis (121)

19.667

9.000

11.333

11.333

Salmonella typhimurium (3231)

13.667

3.667

10.333

6.333

Bacillus subtilis (441)

15.333

9.667

11.667

10.667

Salmonella enteric (1164)

13.667

8.667

10.667

6.333

Staphylococcus aureus(96)

15.333

4.667

10.333

6.667

Bacillus subtilis (1305)

15.333

9.000

10.333

6.333

Staphylococcus aureus (7443)

18.000

8.333

10.667

9.000

Escherichia coli(40)

17.333

7.333

9.667

8.667

Bacillus cereus ( MTCC430)

15.333

7.333

8.667

8.667

Bacillus cereus (MTCC2086)

16.333

8.000

9.667

8.667

 

Microorganisms

Chloramphenicol

Water

Methanol

Acetone

Bacillus subtilis (121)

17.000

9.000

11.333

11.333

Salmonella typhimurium (3231)

15.000

3.667

10.333

6.333

Bacillus subtilis (441)

16.667

9.667

11.667

10.667

Salmonella enteric (1164)

15.667

8.667

10.667

6.333

Staphylococcus aureus(96)

17.000

4.667

10.333

6.667

Bacillus subtilis (1305)

17.667

9.000

10.333

6.333

Staphylococcus aureus (7443)

18.000

8.333

10.667

9.000

Escherichia coli(40)

10.333

7.333

9.667

8.667

Bacillus cereus ( MTCC430)

13.000

7.333

8.667

8.667

Bacillus cereus (MTCC2086)

15.667

8.000

9.667

8.667

 

Fig 2a: FTIR analysis of Syzygium cumini seeds Water extract

 

 

Fig 2b: FTIR analysis of Syzygium cumini seeds AgNPs

The FTIR analysis of Syzygium cumini extract was done. The figure shows that the FTIR peaks at 3440.02 cm -1, 2923.44 cm -1, 1630.76 cm -1 and at around 1409.31 cm -1 in aqueous extract indicated the presence of functional group -OH, -COOH, -NH-amides.

 

Fig 3: Validation of antimicrobial activity of S. cumini L by analyzing standard deviation of triplets (Gentamycin as control)

 

Fig 4: Validation of antimicrobial activity of S. cumini by analyzing standard deviation of triplets (Chloromphenicol as control)

 

Fig 5: Synthesis of AgNPs UV spectroscopy of AgNPs derived from aqueous extract of S. cumini seeds

 

Fig 6: %inhibition of AgNPs derived from seed extract of Syzgium cumini and aqueous extract of Syzgium cumini (Antioxidant activity in presence of DPPH)

 

CONCLUSION

In the current research finding it can be concluded that seed extract of Syzygium cumini L has the potential to develop as antibacterial drug and can be explored as green herbal drug development. It has been also observed that extracts has potent antioxidant agents which can be used to treat various reactive oxygen species which may create fatal effects in humans. It is also suggested with current findings that phytoconstituents present in seed extracts of Syzygium cumini L can be analyzed to study as immune modulator agents against various diseases and anti- toxic agents. Methanol derived extracts shown more antimicrobial effects. Nanoparticles can be developed from seed extracts to evaluate the efficacy as antimicrobial agents, anti diabetic agents, anti inflammatory agents. Silver nanoparticles shown more antioxidant activities than aqueous extract it may be due to presence of silver ions in it.

 

ACKNOWLEDGEMENTS:

 Authors are thankful to School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab for providing laboratory facilities and other requirements.

 

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Received on 31.05.2017             Modified on 30.06.2017

Accepted on 04.07.2017            © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(8): 2017 951-958.

DOI: 10.5958/0974-360X.2017.00481.4