INTRODUCTION:

Marine algae have been closely associated with human life and are being exhaustively used in numerous ways as a source of food, feed, fertilizer, medicine and chiefly for economically important phycocolloids.^1,2The phytochemicals from marine algae are extensively used as gelling, stabilizing and thickening agents in various industries such as food, confectionery, textile, pharmaceutical, dairy and paper. Seaweeds or marine macroalgae are the renewable living resources which are also used as food, feed and fertilizer in many parts of the world. A large number of phytochemicals belonging to several chemical classes have been shown to have inhibitory effects on all types of microbes in in vitro conditions.

The discovery of new antimicrobial compounds from the natural resources is a plausible solution to the rising emergency of antibiotic resistance and their side effects. Seaweeds are valuable sources of protein, fibre, vitamins, polyunsaturated fatty acids, macro and trace elements as well as important bioactive compounds.³They also serve as an important source of bioactive natural substances. Seaweeds are considered to produce a great variety of secondary metabolites characterized by a broad spectrum of biological activities. Red algae are mainly used as human food, feed and medicine.^4,5Compounds with cytostatic, antiviral, antihelminthic, antifungal and antibacterial activities have been detected in green, brown and red algae.^6,7 Extracts of marine algae were reported to exhibit antibacterial activity.^8-13

Today, there is a growing demand for the screening of the drugs from the natural products. Seaweeds are identified as a major source of antibiotics. The antimicrobial activity is considered to be an indicator of seaweeds to could produce the bioactive secondary metabolites.^14,15Most of the compounds of marine algae exhibit anti-bacterial activities.^16,17 Manymetabolites isolated from marine algae have been shown to possess bioactive properties.^18-20Hence, in the present study, we assessed the phytochemical and antimicrobial activity of Portieria hornemannii.

MATERIALS AND METHODS:

Collection and processing of algal sample:

The fresh species of Portieria hornemannii (Lyngbye) P. C. Silvawere collected from the coastal area of Manadapam, Ramanathapuram District, Tamil Nadu, South India. It was thoroughly washed with distilled water to remove holdfasts and epiphytes. The water was drained off from the thallus and they were spread on blotting paper to remove the excess water. The shade dried material was ground in an laboratory mixer to obtain coarse powder.

Preparation of extracts of Porteira hornemannii:

Extracts were prepared by soaking the coarse powdered materialin 100 mL of methanol with intermittent shaking. The extracts were filtered using a muslin cloth and again filtered by filter paper. The organic extracts were concentrated till solvent free by evaporation at 30°C. The residues obtained were finally dried and yield obtained was 7.5 g/100 g.

Quantitative Phytochemical Analysis:

The extracts were tested for the presence of bioactive compounds by using standard methods.^21-26

Antimicrobial activity:

The strains of Escherichia coli (ATCC 10798), Klebsiella pneumoniae (ATCC 31488), Staphylococcus aureus (ATCC 10832D-5), Pseudomonas aeruginosa (ATCC 207), were obtained from LGC Promochem India Pvt. Ltd, Bangalore, India. They were stored at 4°C in refrigerator. The different solvent extracts of the seaweeds were subjected to antimicrobial assay using disc diffusion method. The sterile Muller Hinton agar plates and potato dextrose agar were prepared and inoculated with respective bacterial cultures. A volume of 60 µL of the five different solvent extracts of P. hornemannii was introduced to respective wells and allowed for diffusion for 45 min. Solvents alone served as the control. The plates were incubated at 37°C for 24 h in upright position. A clear zone around a disc was evidence of antimicrobial activity. Discs loaded with the extracting agents were tested as controls.

Statistical analysis:

The experimental results were subjected to analysis of variance (ANOVA) using SPSS version (17.0). The results were expressed as mean ± standard error (SE).

RESULTS:

Quantitative Analysis of Phytochemicals:

The presence of various secondary metabolites in the seaweeds are a clear indication of their pharmaceutical potential. Marked variations in the quantity of many of the phytochemical contents among the seaweeds were observed. In the preliminary phytochemical analysis of crude extracts, P. hornemanniicontains phenols, alkaloids, triterpenoids, steroids, tannins, saponins, flavonoids, anthraquinones and glycosides. P. hornrmannii extract showed higher content of phenols, alkaloids, glycosides, tannins, triterpenoids and steroids (Table 1).

Table. 1 Quantitative phytochemical analysis of experimental alga (mg/g dry wt)

S. No	Phytochemicals	P. hornemannii
1	Total carbon	176.13 ± 0.80^k
2	Nitrogen	41.42 ± 0.5ⁱ
3	Total protein	126.32 ± 0.50^j
4	Total carbohydrates	39.26 ± 0.37^h
5	Total lipids	6.77 ± 0.00^e
6	Phenols	6.24 ± 0.11^e
7	Alkaloids	3.18 ± 0.1^d
8	Triterpenoids	0.08 ± 0.00^a
9	Steroids	0.97 ± 0.1^b
10	Tannins	1.12 ± 0.00^b
11	Saponins	0.09 ± 0.00^a
12	Flavonoids	0.02 ± 0.00^a
13	Antheraquinones	0.80 ± 0.57^b
14	Proteins	38.63 ± 0.1^g
15	Carbohydrates	18.96 ± 0.1^f
16	Glycosides	2.01 ± 0.1^c
F-Value		752400.0
P-Value		0.000

Values are expressed as Mean ± SEM, n=3 as Anova test p<0.05% level.

Antibacterial assays of experimental alga:

The antibacterial activity of the selected marine red alga P. hornemannii using different solvents such as water, methanol, chloroform, ethyl acetate and hexane were studied by disc diffusion method against various gram positive and gram negative bacterial pathogens.

The antibacterial activity was assessed by measuring the zone of inhibition and compared with the standard antibiotic Streptomycin. The antibacterial activity of various solvent extracts of P. hornemannii on human pathogens was presented in the Table 2.

The study showed the methanol extract of P. hornemannii showed the highest activity against S. aureus (77.77%) and followed by K. pneumonia (66.66%), P. aeruginosa (63.15%) and E. coli (58.82%). The aqueous extract showed moderate antibacterial activity against K. pneumonia (66.66%) and P. aeruginosa (63.15%) followed by other organisms. The chloroform extract was active against P. aeruginosa by showing 52.63% inhibition followed by other organisms. The ethyl acetate extract showed moderate activity against P. aeruginosa (52.63%) followed by other organisms (Figure 1). The hexane extract showed activity against the entire tested organism except E.coli with moderate inhibition (Figure 1).

Figure 1 Antibacterial activity of the silver nano particlessynthesized from P. hornemannii.

a)Escherichia coli b) Klebsiellapneumoniae c) Staphylococcus aureus d) Pseudomonas aeruginosa.

DISCUSSION:

Phytochemicals:

Seaweeds are rich in secondary metabolites such as derivatives of alkaloids, phenols, flavonoids, saponins, steroids and related active metabolites are of great medicinal value and have been extensively used in the drug and pharmaceutical industry. Phytochemical screening of the experimental alga revealed the presence of alkaloids, saponins and glycosides in methanol extract.²⁷ It has been reported that the presence of phyto-constituents such as flavonoids, tannins and polyphenols helps in preventing a number of diseases through their free radical scavenging activity.²⁸

In the present study the maximum protein content was recorded in the red alga P. hornemannii. Similarly Selvi et al., reported more protein content in red alga Hypnea valentiae.²⁹ In the present study the carbohydrate and lipid content was lesser in P. hornemannii compared to that of protein.

Steroids of plant origin are known to be important for insecticidal, antimicrobial, antiparasitic and cardiotonic properties. Steroids also play an important role in nutrition, herbal medicine and cosmetics.³⁰Regardless of their function, the presence of steroids in every organism suggests that they have a powerful role in chemosystematics.³¹

In general, the analogs of alkaloids are found to have antimicrobial properties against both Gram-positive and Gram-negative bacteria.^32,33 They are of considerable pharmaceutical importance since, they are used as drugs for the treatment of several diseases known to man.³⁴ In the present study, presence of alkaloid was confirmed in methanol extracts by quantitative analysis.

Tannins were used therapeutically as antiviral, antibacterial, antiparasitic, anti-inflammatory, antiulcer and antioxidant agents.³⁵Many tannin containing drugs are used in the treatment of piles, inflammation, burns and as astringent.³⁶Phlorotannin purified from several brown algae have been reported to possess strong antioxidant activity which may be associated with their unique molecular skeleton.³⁷ The results of the present study confirm the presence of tannin in P. hornemannii.

Saponins are considered as a key ingredient in traditional Chinese medicine and are responsible for most of the observed biological effects. Saponins are known to produce effects on inflammation and it is commercially exported as dietary supplements.³⁸Tremendous and commercially driven promotion of saponins is seen in dietary supplements and nutraceuticals.

Flavonoids, the largest group of phenolic compounds known to contain a broad spectrum of chemical and biological activities including antioxidant and free radical scavenging properties.³⁹ Several researchers have made attempts to identify the plants producing bioactive substances with success.^40-42 In the present study also, flavonoids were observed in methanol extracts of P. hornemannii.

Phenolic compounds are commonly found in plants, including seaweeds, and have been reported to have a wide range of biological activities including antioxidant properties.⁴³In general, phenolic compounds possess specific physical, chemical and biological activities that make them useful as drugs. Phenolics were also responsible for the antimicrobial, anti-inflammatory, anti-feedant, anti-viral, anticancer and vasodilatory actions.⁴⁴ The present study was promising, as alga phenolic compounds are effective antioxidants in delaying oil rancidity, and therefore, the seaweed extracts could have a potential effect on food application. So these biochemical characteristics make the seaweeds nutraceutical in nature and therefore are important as food supplements in order to give good health and resist diseases. Hence, a more detailed pharmacognostic study of these seaweeds is necessary.

It also suggested that P. hornemannii were rich sources of phytochemicals, which can be isolated and further screened for different kinds of biological activities depending on their reported therapeutic uses. Further, work will emphasize the isolation and characterization of active principles responsible for bio-efficacy and bioactivity.

Antibacterial activity of the extracts of P. hornemannii:

The production of antibacterial agents is one of capability of the seaweeds through the synthesis bioactive secondary metabolites, because the bioactive compounds or antibiotics with distinctive complex structures developed by unique metabolic and physiological mechanism. The marine macroalgae have an effective antibacterial activity against most of the human bacterial pathogens. It was reported that 151 species of macroalgae crude extracts^[46] and more information is needed on marine algae available in India. Hence, it was intended to evaluate and compare the ability of marine red alga in order to identify the bioactive potential against selected human bacterial pathogens.

Among the various organic solvents methanol, acetone, diethyl ether and ethanol extracts of eleven macroalgae were screened for their antimicrobial activity against human pathogens, the extracts of diethyl ether was found to possess bioactive compounds.⁴⁷ It was reported that methanol extracts of seven different tested seaweeds showed broad spectrum antibacterial activity against human pathogenic bacteria.⁴⁸

In the present study, the extracts of P. hornemannii exhibited broad spectrum antibacterial activity. These extracts inhibited both Gram-positive and Gram-negative strains. Methanol was a better solvent for extracting the effective antimicrobial compounds used in these experiments. This could be related to the high solubility of bioactive metabolites in the methanol. Antibacterial activity depends on both the species and the efficiency of the extraction method. It is clear that organic solvents always provide a higher efficiency in extracting compounds for antimicrobial activities compared to water-based methods.^49,50 The present investigation shows that there are remarkable differences in the activity of P. hornemannii extracts against the tested pathogenic species. It is amazing that, mostly, the antimicrobial activity toward Gram-positive species is lower than that detected against Gram-negative species. Kandhasamy and Arunachalam revealed that Gram-positive organisms were more susceptible to the crude extracts of algae used.⁴⁸ Similar kind of work was carried out by Tuney et al., Alghazeer
et al., 2012; Vinothkumar et al.^13,51,52

Some studies concerning the effectiveness of extraction methods which highlight that methanol extraction showed the higher antibacterial activity than n-hexane and ethyl acetate.⁵⁰ Whereas, other reports suggested that chloroform is better than methanol and benzene.⁵³In another study, acetone was found best among several solvents used for extracting antibacterial, substances.⁵⁴Several researchers have performed the extraction of seaweeds using chloroform and ethyl acetate also exhibited good antibacterial activity.^55,13

The present investigation has proven that marine red alga P. hornemannii possesses antimicrobial activity, which may be due to the masking of bacterial activity by the presence of some inhibitory compounds in the crude extracts. Finally, it can be concluded from the present study that the extracts of algal species used in the present investigation showed better antibacterial activity against human pathogens. They are potential sources of bioactive compounds and should be investigated for natural antibiotics. Therefore, it is suggested that further works should be performed on the isolation, identify and purify and characterization of these antibacterial compounds.

CONCLUSION:

The phytochemical screening among of methanol extracts of P. hornemannii, showed a maximum number of compounds such as tannins, flavonoids, glycosides, phenols, saponins, terpenoids. The algal extract consists of chemical constituents, that exhibits a good antimicrobial activity and hence, P. hornemannii proved to be an effective therapeutic agent.

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

ACKNOWLEDGEMENTS:

The financial support received from University Grants Commission, New Delhi (Grant No. F1-17.1/2011-12/RGNF-SC-TAM-5342/(SA-III/Website) dated 6/6/12) is gratefully acknowledged.

REFERENCES:

1. Levering T, Hoppe HA, Schmid OJ. Marine Algae. A survey of research and Utilization. Granm be Gruyter and Co., Hamburg 1969; 1-421.

2. Chapman VJ. Seaweeds and their uses. The Camelot Press Ltd., Methuen and Co Ltd., London and Southampton, II Edn1970; 63-85.

3. Ortiz J, Romero N, Robert P, Araya J, Lopez-Hernández J, Bozzo C. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulvalactuca and Durvillaeaantarctica. Food Chem 2006;99: 98–104.

4. Hoppe HA and Lerving T. Marine algae in pharmaceutical science, (Walter deGruyter, Berlin) 1982; (2): 3-48.

5. Chapman V J and Chapman D J, Seaweed and their uses, (Chapmam and Hall, London)1980; .223.

6. Lindequist U and Schweder T. Marine Biotechnology. In: Rehm, H.J., Reed,G. (Eds.) Biotechnology, vol. 10. Wiley-VCH, Weinheim2001; (2):441-484.

7. Newman DJ, Cragg GM and Snader KM. Natural products as source of new drugs over the period 1981-2002, J Nat Prod 2003; 66: 1022-1037.

8. Siddhanta AK, Mody KH, Ramavat BK, Chauhan VD, Garg VS, Goel AK, Jinandra Doss M,Srivastava MN, Patnaik GK and Kamboj VP. Bioactivity of marine organisms: Part VIII-Screening of some marine flora of Western coast of India. Indian J ExpBiol1997; 35:638-643

9. Mahasneh I, Jamal M, Kashasneh M and Ziodeh M. Antibiotic activity of marine algae against multiantibiotic resistant bacteria. Microbio1995; 83: 23-26.

10. Pandithurai M, Murugesan S and Sivamurugan V. Antibacterial activity of various solvent extracts of marine brown alga Spatoglossumasperum. International J Pharmacol Res2015; 5(6):133-138.

11. Thennarasan S andMurugesan S. Antibacterial activity of crude methanolic extract of marine brown alga Lobophoravariegata (J. V. Lamouroux). World J Pharm Res2015; 1714-1722.

12. Kotteswari M, Shanthi N, ElamvaluthiM and Murugesan S. Antibacterial activities of Caulerpascalpelliformis (R. Brown ex Turner) C. Agardh from the Gulf of MannarSouth East Coast of India. EJPMR 2015; 2(4):900-907.

13. Vinoth Kumar R,Murugesan S, Bhuvaneswari S, Thennarasan S. In vitro antibacterial effects of red alga Champiaparvula(C. Agardh) of various solvents against human pathogenic bacteria. International JAdvPharma 2015; 4(6): 111-116.

14. González del Val A, Platas G, Basilio A, Cabello A, Gorrochategui J, Suay I, et al. Screening of antimicrobial activities in red, green and brown macroalgae from Gran Canaria (Canary Islands, Spain). IntMicrobiol 2001; 4: 35-40.

15. Srivastava N, Saurav K, Mohanasrinivasan V, Kannabiran K, Singh M. Antibacterial potential of macroalgae collected from the Mandapam coast, India. Br J PharmacolToxicol 2010; 1(2): 72-76.

16. Vairappan CS, Daitoh M, Suzuki M, Abe T, Masuda M. Antibacterial halogenated metabites from the MalysianLaurencia species. Phytochemistry 2001; 58: 291-297.

17. Vlachos V, Critchley AT, Von Holy A. Differential anti-bacterial activity of extras from selected southern African macroalgalthalli. Bot Mar 1999; 42: 165-173.

18. Oh KB, Lee JH, Chung SC, Shin J, Shin HJ, Kim HK. Antimicrobial activities of the bromophenols from the red alga Odonthaliacorymbifera and some synthetic derivatives. Bioorg Med ChemLett 2008; 18: 104-108.

19. Venkateswarlu S, Panchagnula GK, Gottumukkala AL, Subbaraju GV. Synthesis, structural revision, and biological activities of 4′- chloroaurone, a metabolite of marine brown alga Spatoglossumvariabile. Tetrahedron 2007; 63: 6909-6914.

20. Yang RY, Li CY, Lin YC, Peng GT, She ZG, Zhou SN. Lactones from a brown alga endophytic fungus (No. ZZF36) from the South China Sea and their antimicrobial activities. Bioorg Med ChemLett 2006; 16: 4205-4208.

21. Van-Burden TP and Robinson WC. Formation of Complexes between Proteinand Tannin, J Agric Food Chem1981; 1: 77.

22. Trease GE and Evans. WC Pharmacognosy, 11th edn. BailliereTindall, London, 1989; 45-50.

23. Harbone, JB. Phytochemical Methods, Chapman and Hall Limited, London 1973;49-189.

24. Obdoni BO, Ochuko PO. Phytochemical studies and comparative efficacy of the crude extracts of some Homostatic plants in Edo and Delta States of Nigeria. Global J Pure ApplSci 2001; 8: 203-208.

25. Kumaran A and Karunakaran RJ. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus. Food Chemistry 2006; 97: 109–114.

26. Krishnaiah D, Devi T, BanoA and Sarbatly, R. Studies on phytochemical constituents of six Malaysian medicinal plants. J Medicinal Pl Research 2009; 3(2):67-72.

27. Lacaille-Dubois MA and Wagner H. A review of the biological and pharmacological activities of saponins. Phytomedicine 1996; 2: 363-386.

28. Vasanthi HR, Charles Dorni AI, Vidyalakshmi KS and Rajamanickam GV. Free fadical scavenging and antioxidant activity of a red alga Acanthophoraspecifera-Relation to its chemical composition. Seaweed ResUtilin 2006; 28(1):119-125

29. Selvi M, Shakila P and Selvaraj R. Studies of biochemical contents of some macro algae from the coast ofTuticorin, Tamilnadu. Seaweed ResUtiln1999; 21: 99-103.

30. Okwu DE. Evaluation of the chemical composition of Indigenous spices and flavouring agents. Glob J Pure ApplSci 2001; 7:455-9.

31. Gavidia I, Tarrio R, Rodriguez-Trelles F, Perez-Bermudez P, Seitz HU. Phytochemistry 2007; 68: 853-864.

32. Omulokoli E, Khan B and ChhabraSC.Antiplasmodial activity of four Kenyan medicinal plants. JEthnopharmacol1997; 56: 133-137.

33. Cowan MM. Plant products as antimicrobial agents.ClinMicrobiol Rev1999; 12: 564-582.

34. Okwu DE, Josiah C, Evaluation of the chemical composition of two Nigerian medicinal plants, African Journal of Biotechnology 2006; 5(4): 357-361.

35. Hemat, RAS. Fat and muscle dysfunction. In Hemat RAS, Andropathy. Dublin, Ireland: Urotext2007; 83-85.

36. Kolodziej H and Kiderlen AF. Antileishmanial activity and immune modulatory effects of tannins and related compounds on Leishmaniaparasitised RAW 264.7 cells. Phytochemistry 2005; 66(17): 2056-2071.

37. Ahn GN, Kim KN, Cha SH, Song CB, Lee J, Heo MS, Yeo, KI, Lee NH, Jee YH, Kim J.S, Heu MS and Jeon JY. Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H O -mediated DNA damage. Eur Food ResTechnol 2007; 226: 71-79.

38. ManjunathaBK.Antibacterial activity of Pterocarpussantalinus. Indian J PharmSci2006; 68(1): 115.

39. Kahkonen MP, Hopia A, Vuorela I, Rauha HJ, Pihlaja JP, Kujala K. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chem 1999; 47: 3954-3962.

40. Janakiraman N, Johnson M, SahayaSathish S/. (2012). GC-MS analysis of bioactive constituents of Peristrophebicalyculata (Retz.) Nees. (Acanthaceae). Asian Pacific J Trop Biomedicine 2012; S46-S49.

41. Sengul M, Ercislib S, Yildizb H, Gungorc N, Kavaza A, Cetina B. Antioxidant, antimicrobial activity and total phenolic content within the aerial parts of Artemisia absinthum, Artemisia santonicum and Saponariaofficinalis. Iranian J Pharm Res 2011; 10(1), 49-56

42. Sangeetha J, Vijayalakshmi K. Determination of bioactive components of ethyl acetate fraction of Punicagranatum rind extract. Int J Pharm SciDrug Res 2011; 3(2):116-122.

43. Duan XJ, Zhang WW, Li XM and Wang BG. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphoniaurceolata. Food Chem 2006;95: 37-43.

44. Aliyu AB, Musa AM, Sallau MS, Oyewale AO. Proximate composition, mineral elements and anti-nutritional factors of Anisopusmannii N.E.Br. (Asclepiadaceae). Trends ApplSciRes 2009; 4(1): 68-72.

45. Hornsey IS and Hide D. The production of antimicrobial compounds by British Marine algae and Variation of antimicrobial activity with algal generation. Br Phycol J 1985;20: 21-25.

46. Nair R, Chabhadiya R and Chanda S. Marine algae: Screening for a potent antibacterial agent. JHerbal Pharmacotherapy 2007; 7:73-86.

47. Ünci TN, Bilge Hilal A, Dilek N and Atakan S. Antimicrobial activities of extracts of marine alga from coast of Urla (Üzmir, Turkey) Turk J Biol.2006;7: 171-175.

48. Kandhasamy M and Arunachalam KD. Evaluation of in vitro antibacterial property of seaweeds of southeast coast of India. Afr J Biotechnol 2008;7: 1958-1961.

49. Ortega PC and Gonzales FRM. Antimicrobialactivities in algae extracts from the coast of Grand Canary Island (In Spanish). Benthos 1990; 6: 31-35.

50. Siddhanta AK, Mody KH, Ramavat BK, Chauhan VD, Garg HS, Goel AK, Doss MJ, Srivastava MN, Patnaik, GK and Kamboj VP. Bioactivity of marine organisms: Part VIII – Screening of some marine flora of Western coast of India. Indian J ExpBiol1997; 36:638-643.

51. TuneyI, Cadirci BH, Unal D and Sukatar A. Antimicrobial activities of the extracts of marine algae from the coast of Urla (‹zmir, Turkey). Turk J Biol 2006;30: 1-5.

52. Alghazeer R, El-Saltani H, Saleh N, Al-Najjar A and Hebail F. Antioxidant and antimicrobial properties of five medicinal Libyan plants extracts. Natural Science 2012;4: 324-335.

53. Febles CI, Arias A, Gil-Rodriguez MC, Hardisson A and Sierra Lopez A. In vitro study of antimicrobial activity in algae (Chlorophyta, Phaeophyta and Rhodophyta) collected from the coast of Tenerife (in Spanish). Anuario del Instituto de EstudiosCanarios1995; 34:181-192.

54. Jebasingh SEJ, Rosmary S, Elaiyaaja S, Sivaraman K, Lakshmikandan M and Murugan A. Potential antibacterial activity of selected green and red seaweeds. JPBMS 2011;15:1-7.

55. Rajasulochana P, Dhamotharan R and Krishnamoorthy P. Primary Phytochemical Analysis of Kappaphycus Sp. J Amer Science 2009;5(2): 91-96..

Received on 12.03.2017 Modified on 18.05.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(3): 131-136.

DOI: 10.5958/2321-5836.2017.00022.2

S.No	Name of the sample	Zone of inhibition in mm on human pathogen
S.No	Name of the sample	*E.coli*	*K.pneumoniae*	*S.aureus*	*P.aeruginosa*
1	Methanol	10 ± 0.001(58.82)	12 ± 0.005 (66.66)	14 ± 0.002 (77.77)	12 ± 0.004 (63.15)
2	Aqueous	9 ± 0.003 (52.94)	12 ± 0.002 (66.66)	9 ± 0.002 (50)	12 ± 0.001 (63.15)
3	Chloroform	7 ± 0.005 (41.17)	-	-	10 ± 0.002 (52.63)
4	Ethyl acetate	8 ± 0.004 (47.05)	7 ± 0.001(38.88)	8 ± 0.004 (44.44)	10 ± 0.003 (52.63)
5	Hexane	-	7 ± 0.003 (38.88)	8 ± 0.001 (44.44)	8 ± 0.001 (42.10)
6	Streptomycin	17 ± 0.004(100)	18(100)	18 ± 0.001(100)	19 ± 0.002(100)