1. I. Introduction

ccording to World Health Organization (WHO), the increase of resistance to antibiotics by bacterial pathogens is a growing problem in both developed and developing countries (1). The problem of microbial resistance is growing and the outlook of the use of antimicrobial drugs in future is uncertain. Therefore action must be taken to reduce this problem, for example, to control the use of antibiotics, to develop research to better understanding of the genetic mechanism of resistance and to continue study to develop new drugs either synthetic or natural (2).

For along period of time, plants have been a valuable source of natural products for maintaining human health (3). Plants are used medicinally in different countries and are a source of many potent and powerful drugs. Medicinal plants represent a rich source of antimicrobial agent (4). Different parts of plants, herbs and spices have been used for many years for the prevention of infection. The use of plants with known antimicrobial properties can be of great significance in treatment of infections (5).

A renewed interest in plant based antimicrobials has arisen during the last twenty years, but still plant based antimicrobials are poorly explored. Screening of plants extracts for antimicrobial activity has shown that higher plants represent a potential source of new antiinfective compounds (6). The antimicrobial compounds from plants may inhibit bacteria through different mechanism than the conventional antibiotics, and could therefore be of clinical value in the treatment of microbial infection (7 (8). It is well reputed in Unani System of Medicine, useful for urinary complaints and piles. Almost all parts of the plant including leaves seeds and roots are utilized in medicine. The fresh juices obtained from leaves are diuretic, laxative. Roots are used for urinary complaints and syphilitic disease; they are a reputed medicine for piles and gastrodynic pains. The seeds are expectorant, diuretic, laxative, carminative, antitussive and stomach tonic (8, 9, 10, and 11). The present study aims at assessing the antibacterial property of R. sativus seed extract, to substantiate the use of radish in Unani System of medicine in infectious diseases.

2. II. Materials and Methods

3. a) Plant Materials

The sample of seeds of Raphanus sativus

4. c) Preparation of Test Sample

A stock solution of the extracts was prepared at the concentration of 100mg/ml and store at 2 0 C till further use.

5. d) Source and Maintenance of Organisms e) Culture Media

Muellar Hinton Agar (Himedia, India) was prepared according to the manufacturer's instructions, autoclaved at 15 lbs pressure and 121ºC for required time and dispensed into petridishes more than half. Set plates were incubated overnight at 37ºC to ensure sterility before use.

6. f) Preparation of inoculums

Select & label test cultures that are to be used for (plant extract) Sensitivity Assay. Prepare nutrient agar plates. 3-4 colonies should be selected from the agar plate culture. The top of the each colony is touched with loop & transferred in to into a test tube containing4-5 ml nutrient broth. The test tubes which containing broth cultured are incubated at 37 ? C until it achieves the turbidity.

7. g) Evaluation of Antibacterial Activity

The in-vitro antibacterial activity of the extracts was determined by agar well diffusion assay (Reeves, 1989). All strains were first grown in Mueller Hinton broth (MHB) under shaking condition for 4 h 37 0 C and after the incubation period 0.1ml of the test inoculums was spread evenly with a sterile glass spreader on Mueller Hinton Agar (MHA) plates. The seeded plates were allowed to dry in the incubator at 37 0 C. Wells were made using sterile 6 mm cork borer in the inoculated MHA plates. The wells were filled with 200µl of the extracts (re-suspended in respective solvents) and negative controls 1:1 (solvent: water). The concentration of stock extracts was 200 mg /ml. The inoculated plates were incubated at 37 0 C for 24 h. The plates were observed for the presence of inhibition of bacterial growth that was indicated by a clear zone around the well. The size of zone of inhibition was measured and the bacterial activity was expressed in term of average diameter of the zone of inhibition in millimeters. The results were compared with the standard antibiotics, Chloramphenicol (25mcg) and Ciprofloxacin (25mcg). The photographs were taken in U.V-visible documentation system.

8. h) Statistical Analysis

Calculations of antibacterial activity were determined by Standard Deviation and Mean of replicates.

9. i) Screening for Secondary Metabolites

Secondary metabolites are identified in the extracts of R. sativus by using standard methods.1 mg of each extract was dissolved in 100 ml of the respective solvent and filtered through Whattman filter paper No.1. Thus, the filtrates obtained were used as test solutions for the screening. The details for the qualitative analysis (14,15,16) were described. Table1.

10. III. Results

The results are listed in Table2. Results obtained in the present study relieved that tested medicinal plant extracts posses potential antibacterial activity against all selected bacteria (agar well diffusion method). Among all the extracts Ethanolic and Methanolic extracts showed maximum antibacterial activity against all the bacterial strain used with a zone of inhibition ranges from 12-21 mm and the least activity was observed in Aqueous cold extract with zone of inhibition ranges from 7-9 mm. The test results were A total 8 strains including gram positive (Staphylococcus aureus, ATCC25923) and gram negative (E.coli-ATCC25922, Pseudomonas aeruginosa-ATCC 27853, Shigella sonnei-ATCC 25931, Salmonella typhi-ATCC 25241, Proteus vulgaris-ATCC 6380, Klebsiella pneumonie-ATCC27736, Salmonella paratyphi -ATCC 9150) bacteria were selected to assess the susceptibility test against the drug extract. The strains were obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India. They were sub cultured on nutrient agar for every 15 days and maintained on nutrient agar slants at 4 0 C. Fresh inoculums were taken for the test. compared with standard antibiotics Chloramphenicol (25µg) and Ciprofloxacin (25µg).

The plant extracts were also screened for qualitative analysis to know the relative distribution of the secondary metabolites which may be responsible for the potent antibacterial activity. Flavonoids

11. IV. Discussion

Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay (17). Crude plant extracts are generally a mixture of active and non-active compounds. A number of medicinal plants described in Unani System of Medicine still need to be testify according to the modern parameters to ensure their activity and efficacy. Many reports are available on the antibacterial, antifungal and anti-inflammatory properties of plants (18,19,20,21). Some of these observations have helped in identifying the active principle responsible for such activities and in the developing drugs for the therapeutic use in human beings.

In India, mortality rate due to infections is largely due to S. aureus, Ps. aeruginosa, K. pneumonia, E. coli, P.vulgaris, S.sonnie, S.typhi, S. paratyphi. (22). The treatment and management of infections caused by these strains has become very difficult, therefore, the challenge to discover newer and potent drugs is ever increasing. Therefore, studies were undertaken to test the extracts of R. sativus against these pathogens. The highest activity was observed in Ethanol and Methanol extracts followed by Ethyl acetate, chloroform, Benzene, aqueous hot and aqueous cold.

The highest antibacterial effect of Methanol and Ethanol extract against these organism may be due to the ability of the Ethanol and Methanol to extract some of the active properties of these plants like Flavonoids, phenolic compounds, Saponins and other secondary metabolites which are reported to antibacterial (5). Flavonoids are found to be effective antimicrobial substances against a wide range of microorganisms, probably due to their ability to complex with extra cellular and soluble proteins and to complex with bacterial cell wall; more lipophilic Flavonoids may also disrupt microbial membrane (23). Phenol and polyphenols present in the plants are known to be toxic to microorganism (24). Antibacterial activity of tannins may be related to their ability to inactivate microbial adhesins, enzymes and cell envelope transport proteins, they also complex with polysaccharides (25) .The broad spectrum antibacterial activity exhibited by R. sativus may be attributed to the various active constituents presents in it which either due to their individual or combined action.

Thus, the study ascertains the value of R. sativus used in Unani System of Medicine. This could be of considerable interest to the development of new drugs. Boil the solution for few minutes.

12. V. Acknowledgments

Figure 1.

Radish, Raphanus sativus Linn. (Brassicaceae
family) is an annual herb, consumed as vegetable.
Commonly known as Mooli. It is coarse, rough or
glabrous. Leaves are lyrate, pinnate or pinnatifid.
Flowers are large yellow, white or pale lilac, veined with
purple, in long ebracteate racemes. Seeds are
pendulous,	globose;	cotyledons	conduplicate.
Cultivated all over sub-continent up to 16,000 ft in
temperate and warm countries

Figure 2.

Figure 3. Table : 2

	Antibacterial Activity of Raphanus Sativus Linn. Seed Extract
3. 8.	Glycosides Tannins
	(a) Conc.H 2 SO 4 Ferric chloride test		1ml of conc.H2SO4 was To 1-2 ml of aq. ext. of	Formation of A bluish black	Present Present
	test		added to 1ml of test the drug add few drops of	reddish colour colour is
			solution and is allowed 5% aq. ferric chloride	produced
			to stand for 2 minutes. solution.	which
	(b) Aq NaOH test		To alc. Ext. of the drug	Formation of disappear on
			add 1ml of water and	yellow colour addition of a	Present
			adds aq.NaOH	few ml of a dil.
			solution.	Sulphuric acid
4.	Carbohydrates (a) Benedict' s test		To o.5 ml of aq. Ext. of the drug add 5 ml of Benedict's solution and	Formation of colour ppt. solu tion followed by the formation of a yellow-	Present	ear 2012 Y
			boil for 5 min.	A red -violet brown ppt.
9.	(b)Molisch's test Proteins		To 2 ml of aq. Ext. of	ring is formed	Present
A pink or reddish pink or brown colour is Honey comb like froth is formed. 9±0.5 NA 12±0.5 12±0.5 9±0.5 9.6±0.5 NA 16±0.3 at the junction of the two liquids, which addition of excess of alkali A blue or green colour is produced. A yellow ppt. is formed. A white ppt. is formed which turns red on K.pneumoniea P.valgaris Ps.aeruginosa S.aureus S.sonnie S.paratyphi Present To 5 ml of aq. ext. of the drug add drops of Sodium bicarbonate solution shake the mixture vigorously and leave for Present NA 18±5.6 NA NA 11.6±0.5 9±0.5 13.3±2.0 NA 9.3±0.5 9±0.5 9.3±0.5 7±0.5 28±0.4 20±0.5 9±0.5 14±0.4 the drug add 2 drops of freshly prepared 20% alc. ? -naphthol and H 2 SO 4 through the wall of the test tube. (a) Ferric chloride Saponins Foam test BENZENE 12.5±0.7 AQ. HOT 12±0.5 AQ. COLD NA CHLORAMPHENI 6. COL(25µG) 29±0.5 5. Phenol test (b) Aq. Lead acetate test To alc. Or aq. ext. of the drugs add 2 ml of dist. Water and add few drops of 10% aq. FeCl 3 solution. To alc. Or aq. ext. of the drugs add 5 ml of dist. water and add few drops of 1% aq. lead acetate solution. Present Present Millon's test To aq. ext. of the drug Present add 1 ml of dist. water and add 5-6 drops of E.coli ETHANOL 14.5±0.7 17±0.5 18±4.2 21.3±6.6 19±7.0 13.6±2.0 13.3±1.5 METHANOL 12.5±0.7 14.6±2.5 19.5±0.7 14.6±2.3 13.5±0.7 15.3±2.0 14.6±1.5 ETH. ACETATE 9±1.4 NA 22.5±4.9 18±0.5 18±0.5 NA NA CHLOROFORM 10±0.5 NA 19±0.5 18.3±3.5 10±0.5 NA NA mix, pour 2 ml of conc. disappears on Millon's reagent. heating. Proteus vulgaris Pseudomonas aeruginosa						16.3±1.5 NA 7±0.5 21±0.5 S.typhi 16.6±1.5 15.6±0.5 19.6±0.7 14±2.0	Global Journal of Medical Research Volume XII Issue XI Version I
Salkowski test Sterols/Steroids CIPROFLOXACIN 7. (25µG) 27±0.4		26±0.3	Add 1 ml conc. Sulphuric 3 min. 30±0.4 30±0.4	produced. Formation of A red colour is 25±0.5 27±0.4	Present Present 30±0.5	35±0.5
	1 ml of 1N NaOH acid to 2 ml of Salmonella paratyphi			yellow colour produced in Salmonella typhi
			solution w as added to chloroform ext. of the	the chloroform
			the 1ml of test solution. drug care fully through	layer or at the
			the wall of the test tube.	juncti on of the
				two liquids.

Note: EXTRACTSDiameter of Zone of inhibition(mm)

Appendix A

Appendix A.1

The authors are grateful to Dr. Mushtaq Ahmad, Director Central Research Institute of Unani Medicine (CRIUM) Hyderabad for the provision of laboratory space for the extraction process and equipments to carry out this research, as well as for his suggestions and encouragement.

Appendix B

Appendix B.1

Appendix C

Antimicrobial activity of Solanum tomentosum. A A Aliero , A J Afolayan . African Journal of Biotechnology 2006. 5 p. .
Institute of History of Medicine and Medical research, A A Sina , Al-Qanun Fi´-Tib Book , II . 1987. New Delhi. p. 300.
Physiochemical standards of Unani formulations Part-3,. Central council for research in Unani Medicine CCRUM publication No.31 p, Anonymous . 1993. p. .
Methods for testing antimicrobial effectiveness. Bailey and Scott's Diagnostic Microbiology MOS by Co, B A Forbes , O F Sahm , A S Weissfeld , E A Trevomp . 1990. Missouri, PD: St Louis. p. .
Carbohydrate polyphenol complexation. C Ya , S H Gaffney , T H Lilley , E Haslam . Chemistry and Significance of Condensed Tannins, R W Hemingway, J J Karchesy (ed.) (New York
) 1998. Plenum Press. p. .
Antibiotic assays. D S Reeves . Medical Bacteriology, A practical Approach, P M Howkey, D A Levis (ed.) (Oxford
) 1989. IRL Press. p. . (chapter)
, Evans Wc In Trease , Pharmacognosy Evans , Edn . Bailliere Tindall. London 1989. p. .
Antibacterial activity of plant extracts and phytochemicals on antibiotics-resistant bacteria. G G F Nascimento , J Locatelli , P C Freitas , G L Silva . Braz. J. Microbiol 2000. 31 p. .
, H Tsuchiya , M Sato , T Miyazaki , S Fujiwara , S Tanigaki , M Ohyama , T Tanaka , M Iinuma . J. Ethnopharmacol 1996. 50 p. .
Phytochemical Methods, A guide to modern techniques of plant analysis, 3rd Edn, J B Harborne . 1998. London: Chapman and Hall. p. .
Biodiversity: exciting prospects for drugs discovery and development. Meeting report of the Monroe Wall Symposium. J B Press . Chemtracts-Organic chemistry 1996. 9 p. .
Antibacterial activity of Indol alkaloids from Aspidosperma ramiflorum. Braz. J C A Tanaka , C C Desilva , A J B De Oliveira , C V Nakamura , B P D Filho . J. Med. boil. Res 2006. 39 p. .
Which extract should be used for the screening and isolation of antimicrobial components from plants. J N Eloff . Journal of Ethno pharmacology 1998. 60 p. .
Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. J Parekh , S Chanda . African Journal of Biology Res 2007b. 10 p. .
Medicinal Plants: An expanding role in development, J Srivastava , J Lambert , N Vietmeyer . 1996.
Antiamoebic and phytochemical screening of some Congolese medicinal plants. L Tona , K Kambu , N Ngimbi , K Cimanga , A J Vlietinck . J. Ethnopharmacol 1998. 61 p. .
, Makhzanul Kabiruddin , Mufradat . Ejaz Publishing house New Delhi. p. 558.
Plant products as antimicrobial agent. M M Cowan . Clin. Microbiol. Rev 1999. 12 p. .
, Muktanjali Arya , K Prafull , Arya . Indian J Pathol.Microbiol 2005. 48 p. .
Indian medicinal Plants. Vol. I Ed. 2 nd . Edn., India: International Book Distributors, R K Kritikar , B D Basu . 1987. 9/B, Rajpur Road, Dehradun. p. 178.
Glossary of Indian Medicinal plants (Including the supplements) Council of Scientific and Industrial Research, R N Chopra , S L Nayar , I C Chopra . 1986. New Delhi.
Antibacterial activity of some folklore medicinal plants used by tribals in Western Ghats in India. R P Samy , S Ignacimuthu . J. Ethnopharmacol 2000. 69 p. .
Indigenous phytotherapy for genito-urinary diseases used by the Kandha tribe of Orissa, India. S K Behera , M K Misra . J. Ethnopharmacol 2005. 102 p. .
, T L Mason , B Wasserman . P Phytochem 1987. 26 p. .
WHO Global Strategy for containment of antimicrobial resistance www.who.int/emedocuments/antimicrobial_resistance/docs/EGlobalst art, 2001.

Antibacterial Activity of Raphanus Sativus Linn. Seed Extract

Table of contents

Appendix A

Appendix A.1

Appendix B

Appendix B.1

Appendix C