International Journal of Research ISSN NO:2236-6124

ANTIBACTERIAL ACTIVITY OF EXTRACTS OF AGIALITIS ROTUNDIFOLIA

1 2 P.Suneeta & Z.Vishnuvardhan

1. Research Scholar, Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh. 2. Professor, Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh.

Abstract : Leaf extracts of Aegialitis rotundifolia were prepared in Methanol, Ethyl acetate, Chloroform, and Acetone and their antibacterial activity was evaluated against Escherichia coli MTCC (727), Enterobacter aerogens MTCC (111), Klebsiella pneumonia (9544), Bacillus cereus (1272), Bacillus subtilis (441), Proteus mirabilis (9493), Pseudomonas aeruginosa (741) and Staphylococcus aureus (96) by agar well diffusion method. The leaf extracts in methanol and ethyl acetate demonstrated best antibacterial activity. The extracts that possessed antibacterial activity were further used for the determination of the Minimum Inhibitory Concentration (MIC) talking different concentrations viz., 100, 200, 300 and 400 µg/100 µl. The value of MIC was found to be ranged from 100 µg to 400 µg/100 µl for different test organisms. The present study reveals the potential of leaf extracts of Aegialitis rotundifolia.

Key words : leaf extracts, Aegialitis rotundifolia, Antibacterial activity.

1. Introduction Antibiotics since their introduction are one of the most important weapons in fighting against bacterial infections and have largely benefited humans. Many pathogenic organisms are developing plasmid mediated resistance to the prevailing drugs. Hence there is a need for novel natural compounds that can be obtained from the or microorganisms. , in particular, have been a source of inspiration for novel drug compounds since days immemorable. Plants serve as a reservoir of effective chemotheraputants and provide valuable sources of natural products in the control of several bacterial diseases. Many studies indicate that plants contain bioactive compounds such as peptides, glycosides, alkaloids, saponins, terpenoids, flavonoids etc., with antimicrobial activity against bacterial, fungal and viral infections (Jonathan et al., 2003, Khan et al., 2001, Perez et al., 2003). are usually found only in tropical climates, as they need consistently worm conditions for development and survival.

Volume VIII, Issue I, January/2019 Page No:789 International Journal of Research ISSN NO:2236-6124

They occur in approximately 112 countries and territories and are largely confined to the regions between 300 North and South of the equator (Bandaranayake 2002). Medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth. Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world (Rai et al., 2010). plant extracts have been used for centuries as a popular method for treating several health disorders. Plant derived substances have recently become of great interest owing to their versatile applications. Mangroves are biochemically unique, producing a wide array of novel natural products. Mangrove and mangrove associates contain biologically active antiviral, antibacterial and antifungal compounds (Varahalarao et al., 2009). The effects of mangrove extracts on some microorganisms including Shigella , Staphylococcus species, Pseudomonas species has been reported in some studies in the area of pharmacology (Abeysinghe et al., 2006, Ravikumar et al., 2010). Also different types of solvents including Ethanol, chloroform, ethyl acetate have been used for extraction (Abeysinghe et al., 2006). However, the antibacterial activity of mangrove plants has still not been studied as extensively as most other plant species following. Aegialitis rotundifolia is a mangrove tree belongs to . In the present study matured and young of Aegiatis rotundifolia were collected and extracted in different organic solvents according to their polarity to evaluate antibacterial activity against some MTCC bacterial cultures.

2. Materials and methods : 2.1 Plant material: The mature and young leaves of Aegialitis rotundifolia plants were collected from Nagayalanka mangrove forest, geographically located between latitude 150151-150 55 N and 800451- 810 00 t longitude in Krishna district, Andhra Pradesh, India. The plant materials were collected in new polythene bags and surface sterilized with 0.01% mercuric chloride solution. The leaves were chopped separately into small pieces and shade dried at room temperature for seven days.

2.2 Extraction: The extraction of leaves was carried out by using different solvents in their increasing order of polarity viz., Methanol, Ethyl acetate, Chloroform and Acetone by soaking the plant material in the respective solvents overnight at room temperature one after the other. (Choudhury et al., 2005). The contents of each flash were subjected to reflux below the boiling point of the respective solvents viz., Acetone (550C), Chloroform (770C), Ethyl Acetate (770C) and Methanol (650C) for 6- 8 hr in order to extract the active compounds into the solvent. Each extract was vacuum filtered and the filtrates were concentrated by vacuum distillation. The concentrated extracts were incubated at 370 for 3-4 days to facilitate complete evaporation of the volatile solvent leaving behind the dried plant extract. The dried plant extract of 100 mg each was dissolved in 100 µl of 1:10 diluted DMSO (in sterile distilled water) to obtain the final concentration of 10 mg / 100 µl (Nkere et al., 2005)

Volume VIII, Issue I, January/2019 Page No:790 International Journal of Research ISSN NO:2236-6124

2.3 Determination of antibacterial activity The bacterial strains viz., Escherichia coli MTCC 727, Enterobacter aerogens MTCC 111, Klebsiella pneumoniae 9544, Bacillus cereus 1272, Bacillus subtilis 441, Proteus mirabilis 9493, Pseudomonas aerugenosa 741 and Staphylococcus aureus 96 were used in the present study. The antibacterial activity of extracts against the bacteria was tested by agar well diffusion method (Okoli et al., 2005), and zones of inhibition were measured. Each experiment was performed in triplicate and the average value of inhibition and standard deviation were calculated. The zone of inhibition was compared with that of standard rifampicin concentration of 10 µg / 100 µl (Ahmad-rera et al., 2005).

2.4 Determination of MIC: Minimum Inhibitory Concentration (MIC) was determined by broth dilution assay method for the determination of MIC, the reconstituted extract in DMSO was serially diluted in Nutrient broth medium to get the concentrations of 100, 200, 300, 400 and 500 µg/ 100 ml.

3. Results and discussion: Ethyl acetate leaf extracts of 300 µg was not effective whereas 400 µg could produce upto 70% inhibition which may be considered as effective. Acetone and chloroform extracts have no effect on bacteria with reference to Klebsiella pneumoniae. Methanol leaf extract of 100, 200 µg had no considerable effect. 300 µg extract also had no impressive effect over bacteria. Whereas 400 µg could produce 16 mm zone of inhibition that falls on 72.1% which may be considered as effective. Ethyl acetate extract of 300 µg was not effective. 100 and 200 µg extract had no effect. Whereas 400 µg extract could also be not effective as it could produce only 16 mm inhibitory zone falling 63.6% which was not said to be effective in the antibacterial effect. Acetone and chloroform show effect on the bacteria.

On Enterobacter aerogens the effect of 100 µg and 200 µg methanol leaf extracts did not show good effect. 300 µg extract could produce 12 mm zone lies 75% of zone of inhibition whereas 400 µg could produce 14 mm zone lies on 87.2% on Staphylococcus aureus the effect of methanol 100 µg, 200 µg, of leaf extract could not prove to be effective. 300 µg could form the inhibitory zone ranging from 78.9 to 83.3%. The 400 µg leaf extract could be more effective in forming 100% inhibitory zone. Ethyl acetate extracts of 300 µg and 400 µg could result in forming 12 mm and 13mm ranging in 66.6 to 72.2% inhibitory zone. So higher concentration of 400 µg of ethyl acetate extract is considered to be promising in its antimicrobial effect. Low concentration of 100 and 200 µg had no effect on this organism. On Proteus mirabilis : Methanol leaf extract of 300 µg could produce 16 mm inhibitory zones come to 76% to 80% where as 400 µg has more effect that could produce 18 mm zone falling in 85 to 90% which was considered to be more promising.

Volume VIII, Issue I, January/2019 Page No:791 International Journal of Research ISSN NO:2236-6124

The lower concentrations of 100 and 200 µg could not bring that much of effect. 400 µg methanol leaf extract was very promising in producing the zone of inhibition of 90% so said to be more effective and to extent of 75% which can be treated as effective. On Bacillus cereus better effect was noticed by the concentrations of methanol leaf extracts of 300 µg and 400 µg, which could effect the bacteria ranging 15 to 17 mm i.e. 73.6 to 89.4% of zone of inhibition that could be considered as effective. Any how the higher concentration of methanol of 400 µg was very effective. Ethyl acetate effect was not very much considerative at 300 µg concentration whereas 400 µg leaf extract could be effective by showing 68.4% zone of inhibition but not proved be very effective as compared to methanol extract. Acetone and chloroform extracts have no effect over Bacillus cereus. The methanolic extract of 100 µg and 200 µg of A.rotundifolia did not show any promising effect on any of the bacteria taken, in producing zone of inhibition. The effect is better in higher concentration of 400 µg than 300 µg. Methanol and Ethyl acetate leaf extracts of 300 µg and 400 µg have been discussed in comparison to the effect of control in comparison to the effect of control Rifampicin and in percentage of formation of zone of inhibition the effect is discussed. There is no effect of Acetone and chloroform on the bacteria on E.coli both the concentration of 300 µg and 400 µg are almost similar in forming the inhibitory zone ranging in between 15 mm to 16 mm and 78.9% to 84.2% and was considerable. The Ethyl acetate leaf extracts could form 12 mm zone to the extent of 63.1% whereas 14 mm to the extent of 73.6% which was promising. So both methanol ethyl acetate leaf extracts of 400 µg could be promising in forming the inhibitory zone. On Bacillus subtilis ethyl acetate and methanol leaf extracts of lower concentration as well as 300 µg did not show promising effect as it falls 57 to 68% whereas methanol leaf extract of 400 µg could create 15 mm which was considered to be more promising. Ethyl acetate leaf extracts of 100, 200 µg had no effect whereas 300 µg also lies the pause and was also not effective. 400 µg could be effective in producing 81.2% zone of inhibition and was said to be very promising in its antibacterial activity. Acetone and chloroform extracts did not show any effect over the bacteria. With reference to Pseudomonas aeruginosa methanol extract of 400 µg was very promising than 300 µg in producing the zone up to 20 mm fall in 75% and this may considered to be effective. The Ethyl acetate extracts of all concentrations including 400 µg did not show effect as the zone of inhibition is limited to 50% only. Acetone and chloroform extract did not prove to be effective in formation of the inhibition zone. The antibacterial effect of the leaf extract of Aegialitis rotundifolia is methanol-400 µg. 100% over Staphycoloccus aureus followed by Proteus mirabilis and Bacillus cereus. The results are correlated with the work of Chowdary, S. et al., (2005). In the study of Padmakar and Ayyakanna (1977). S. aureus was the most susceptible bacterial pathogen followed by P.auriginosa was most resistant and the findings fall in line with these observations.

Volume VIII, Issue I, January/2019 Page No:792 International Journal of Research ISSN NO:2236-6124

The present study indicates the effect of Aegialitis rotundifolia on the bacteria could revealed the antibacterial effect. Further work is initiated to derive the phytochemicals and the active principle behind the antibacterial effect in the light of human disorder is needed. Economically feasible standard operating produces can be developed in preparing the extracts / fractions in large scale with reproducible antibacterial efficiency.

ANTIBACTERIAL ACTIVITY OF AGIALITIS ROTUNDIFOLIA, METHANOLIC LEAF EXTRACTS ON BACTERIA AT 100, 200, 300 AND 400 µg / ml CONCENTRATIONS.

FIGURE NO. 1 FIGURE NO. 2 Pseudomona aurugenosa Proteus mirabilis

FIGURE NO. 3 Staphylococcus aureus

Volume VIII, Issue I, January/2019 Page No:793 International Journal of Research ISSN NO:2236-6124

Table no. 1 : Effect of Agialitis rotundifolia leaf Extracts on Pathogenic Bacteria

µl = micro liters µg= micro gram *DMSO – Dimethyl Sulphoxide Each reading is an average of three replicates

Zone of inhibition (mm) Con. Enteroba Klebsiell Extrac of Staphylo cter a Escheric Bacillus Pseudomo t/ Std. the Bacillus coccus Proteus S. No. aerogene pneumon hia coli subtilis nas Antibi extra cereus aureus mirabilis s ia aeruginosa otic ct in

µg

100 9.3 9.6 9.6 9.6 10.0 10.3 10.6 12.6 200 10.6 11.0 11.3 12.0 12.0 12.6 14.0 18.3 1. Methanol 300 11.6 13.0 13.0 14.3 14.6 15.3 16.0 20.0 400 14.0 15.0 15.6 16.0 17.0 17.0 18.0 25.0

100 0 0 0 0 0 9.3 10.0 10.0 Ethyl 200 10.0 10.0 10.0 10.0 10.3 10.6 10.6 11.0 2. Acetate 300 10.0 10.0 11.3 12.0 12.0 12.0 12.6 13.0 400 13.0 13.0 13.0 13.3 14.0 14.0 14.3 15.0

3. Rifampicin 10.0 16.0 22.0 19.0 19.0 18.3 20.6 20.6 29.6

4. 100 0 0 0 0 0 0 0 0 DMSO* µl

Volume VIII, Issue I, January/2019 Page No:794 International Journal of Research ISSN NO:2236-6124

Volume VIII, Issue I, January/2019 Page No:795 International Journal of Research ISSN NO:2236-6124

Volume VIII, Issue I, January/2019 Page No:796 International Journal of Research ISSN NO:2236-6124

REFERENCES :

Abeysinghe P.D, Wanigatunge R.P and Pathirana R.N. Evaluation of antibacterial activity of different mangrove plant extracts. Ruhuna Journal of Science, 2006;1:104-112. Ahmad-reza S, Iranshahi M, Roohollah M, Hossein J, Ghodamerza Abbas S . Bioassay-guided isolation and identification of an antibacterial compound from Ferula persicavar perica roots. DARU 2005;13:17-9. Bandaranayake WM. Bioactivities, bioactive compounds and chemical constituents of mangrove plants. Wetlands Ecology and management,2002; 10:421-452. Choudhury S, Sree A, Mukherjee S.C, Patnaik P, Bapuji M. In vitro Antibacterial Activity of Extracts of Selected marine Algae and mangroves against fish pathogens. Asian Fisheries Science 2005;18 :285-94. Jonathan I, Yassin M, Chin C, Chen L, Sim N . Antifungal activity of the essential oils of nine zingiberaceae species. Pharmaceuti Bio. 2003;41:392-97. Khan M, Kihara, Ololoso A.D. Broad spectrum antibacterial activity of the leaves, stem and root barks of Myristica subabulata, Natural product sciences 2001; 7:7-12. Nkere CK, Ireogby CU. Antibacterial Screening of the root, seed and stem bark extracts of Picralima nitida. African Journal of Biotechnology 2005;4:522-6. Okoli S, Ireogbu C.U. In vitro antibacterial activity of Synclisa scabrida whole root extracts. African Journal of biotechnology 2005; 946-52. Padma Kumar, R and Ayyakkannu K. Antiviral Activity of marine Algae and mangroves. Third International marine Biotechnology conference Tromsoe (Norway) 1977;98-99. Perez, RM. Antiviral activity of compounds isolated from plants. Pharmaceut.Bio. 2003;41:105-107. Rai, P.S., Mehta, S., Gupta, R.K and Watal, G . A Novel Antimicrobial agents Trichosanthes diocia. International Journal of Pharma and Bioscience2010;1-9 Ravi kumar S, Muthu-raja M, Sivaperumal P, Gnana design M . Antibacterial activity of the mangrove leaves Excoecaria agallocha against selected fish pathogens. Asian J. Med. Sci 2010; 2(5) : 211-213. Varahala Rao, V and Chandrasekhar Naidu K . In vitro bioefficiency of marine mangrove plant activity of Rhizophora Conjugate. International Journal of PharmaceuticalTechnology2009; 1 (4) : 588-1600.

Volume VIII, Issue I, January/2019 Page No:797