INTRODUCTION:

Ammannia baccifera Linn , belonging to family Lythraceae Small annual herbs, growing in wet places with branches,^1,2,3 is distributed in the tropical regions of the world ,. It is a herb, often with woody troches, found in the warmer parts of India. The plant is popularly known as Dadmari (Hindi and oriya) and Agnigarva (Sanskrit)^1,3,4. The plant has been reported to used as Plant is bitter, acric, cooling appetizer, rubifacient, laxative, stomachic, diuretic, aphrodisiac and lithotriptic, it is useful in vitiated condition of kapha and pitta, burning sensation, anorexia, dyspepsia, flatulence, colic, strangury, seminal weakness, renal and vesicles calculi, rheumatism, intermittent fevers and herpetic eruptions, also reported as posses antityphoid and antitubercular activity^1,4,5,6,7. the plant is reported to contain Resin, Glucose, vitamin C, Hentriaconatane (I), dotriacontanol (II) ,1,30-triacontane diol,Betulinic acid,Lupeol,Beta-sitosterol-beta-D-glucoside,Ellagic acid ,Quercetin.^4,5.we were curious about this plant as that the traditional adhivasi(sabara) people of western Orissa were using aerial part for the treatment of fungal infection,So, The present study is intended to determine the antimicrobial activity of the plant against selected bacteria and fungus and compare it with standard drugs which are frequently used as antibiotic and antifungal agent respectively.

MATERIALS AND METHODS:

The plant material was collected in the month of July, from the village Madhyapur belongs to Balangir district of Western Orissa,and was authenticated by Botanist Dr.N.K.Dhal, department of natural product, IIMT(RRL), Bhubaneswar. The fresh aerial part(stems and leaves) was allowed to shade dried.after drying plant materials were milled into coarse powder by mechanical grinder.the crude powder was macerated for 72 hrs with water ,methanol and hexane individually and the extracts were concentrated to dry ness under vacuum(i.e cold extract) .again the damped mass of plant materials above obtained from methanol and hexane were taken in a soxhlet apparatus individually and the extracts were concentrated to dry under vacuum (i.e hot extract).the TLC analysis was been done for both cold and hot extract of hexane and methanol.the good solvent system for hexane was pet ether:ethyl acetate whereas for methanol and aqueous extract pet ether : ethyl acetate and chloroform: methanol.from the tlc spot both the cold and hot extract of hexane and methanol were found to be nearly same ,hence it was mixed and carried out for invitro screeing against the against some bacteria (pathogen) like (S.aureus, K.pneumoniae, E.coli, P.aeruginosa) and some fungi(pathogen) like (Candida albican, Kluyuviromyces mamlamus) by following a disc diffusion method. All bacterial and fungal species were used in this study, which were obtained from Post Graduate Department of Microbiology, Orissa University of Agricultural Technology(OUAT), Bhubaneswar, Orissa.

Antibacterial activity:

The extracts were screened for their antibacterial activity using disc diffusion method^8-10

The Aqueous, Methanolic, Hexane plant extracts and were dissolved in Dimethyl formamide (DMF) 6%, which was previously tested for antibacterial activity. Hexane extract was dissolved in mixture of DMF (6%) and a Surfactant SDS (Sodium Dodecyl sulfate 2%), which was previously tested for antibacterial activity against all test bacteria and found to have no activity.

The extracts were made solution of concentration 30 mg / ml and finally sterilized by filtration using 0.45 mm Millipore filters. The Sterile Discs (Hi media, Mumbai) (6mm in diameter) were impregnated with 10ml of extracts Solution of Concentration 3 mg / ml to prepare Test Discs with Concentration 300 mg/ disc and placed in inoculated area. The density of the bacterial suspension was standardized by standard Mcfarlad method¹¹

The Standard Disc (Hi-media, Mumbai) of different drugs viz. Gentamycin (10mg / disc)and indicates Amoxycillin + clavulinic acid (20+10) mg / disc. was used as standards.The controls were prepared using the same solvents employed to dissolve the extracts.The inoculated plates with the test and standard discs on them were incubated at 37°C for 24 hours.

The zones of inhibition of different extracts and standard drugs are given in the table-1. An attempt has been made to compare antibacterial activity of A.baccifera. extracts with the most potent standard effective against the selective pathogens and is illustrated in [Figure - 1]

Antibacterial studies-table-1

ORGANISM	ZONE OF INHIBITION(mm)
	EXTRACTS			STANDARDS
	HE	MT	AQ	G	AC
S.aureus	15	16	14	24.5	25.3
K.pneumoniae	16	19	15	28.3	17.1
E.coli	16	18	15	10.08	…..
P.aeuroginosa	26	27	25	26.6	12.0

Staphylococcus aureus 2. K.pneumoniae 3. Escherichia coli 4. Pseudomonas aeruginosa HE – Hexane MT- Methanolic AQ-Aqueous extract G - Indicates Gentamycin (10) mg/disc. AC indicates Amoxycillin + Clavulonic acid (20+10) mg / disc.

Fig 1-antibacterial studies

Antifungal activity:

All the three extract were made water-soluble by using tween 80 (0.2 ml for extract) followed by dilution to give test solution of desire concentration.Griseofulvin as standard was prepared in distilled water, using dimethyl formamide as co-solvent.The entire glass utensils and malt yeast agar medium were sterilized by autoclaving method (15 lb/inch²for 20 minutes).

The procedure was carried out as filter paper disc methods^12,13

15ml of malt yeast agar media was poured aseptically in each sterile petri dishes. After solidification 0.5ml of 72-hour culture of respective fungi was poured by sterile pipette. The fungi culture was spreded uniformly by specially designed glass rod with blended edges.

Whatmann filter paper no-2 disc of uniform size (6mm) dipped in various extract and isolated compound/standard drugs was placed on the inoculated medium. Plates were kept in refrigerator for 12 hour to ensure uniform diffusion of test/standard drugs in the plates. Then plates were incubated at 27⁰C. After 72 hour readings (zone of inhibition) were taken in duplicate.

Whole experiment has been carried out in aseptic condition. The zones of inhibition of different extracts and standard drug are given in the table-2. A comparative antifungal activity of A.baccifera. extracts with the most potent standard effective against the selective pathogens and is illustrated in [Figure - 2]

Antibacterial studies-table-2

Name of the fungi	Zone of inhibition in mm (mean of the reading)
	Hexane extract	Methanol extract	Aqueous extract	Griseofulvine
A	21	21	16	14
B	19	20	16	14

A- Candida albicans

B- Kluyuviromyces mamlamus

RESULT AND DISCUSSION:

In the perusal of above antimicrobial table relating to the study of zone of inhibition indicates that aqueous, methanolic. Hexane extracts showed significant zone of inhibition .the different zone of inhibition measured ranging from 14mm to 27mm in case against the bacteria (S.aureus, K.pneuminia, E.coli, P.aeuriginosa) whereas 13mm to 21 mm incase against the fungi (Candida albicans , Kluyuviromyces mamlamus).In a nutshell, it can be said that the plant extracts showed considerable inhibitory effect against the pathogen Therefore,the study justifies that different bioactive compounds may be isolated from the different solvent extract of ammannia baccifera L. And used as an antimicrobial agent. In near future , the chemical compounds responsible for the therapeutic activity may be isolated, purified, characterized, quantified and other types of activity studies may be carried out with this medicinal plant and it may be resource for scientific and clinical researches and helpful in new drug development

ACKNOWLEDGEMENT:

We are thankful to Director Dr. B.K.Mishra,IIMT(RRL)Bhubaneswar and Botanist Dr N.K. Dhal,(IIMT,Dept of Natural Product. Dr.S.Sahoo, Dr S.K Mishra Dept of pharmaceutical sciences,Utkal university , Bhubaneswar. providing the facility to carrying out the work.

REFERENCES:

1. Saxena H.O., Brahmam M.,The Flora of Orissa, 1995, vol-ii, 707

2. Gamble, Flora of British India, 1879 vol-ii, 569

3. Haines, Henry Haselfoot, The Botany of Bihar and orissa, 1922, 2:376 (396)

4. The Wealth of India (NISCAIR)vol-I, 224

5. Rastogi, Mehrotra , Compendium of Medicinal plant, vol-3, 260

6. .Indian Medicinal plant: a compendium of 500 species.

7. Purohit and Sharma ,A handbook of medicinal plant: A complete source book, sect-ii, 1st edition pg-38

8. Sahoo S. etal,Antibacterial activity of Barringtonia acutangula against selected urinary tract pathogens ,Indian journal of pharmaceutical sciences,2008,vol-70/5

9. Murray, P.R., Baron, E.J., Ptaller, M.A., Tenover, F.C. and Yolke, R.H., Manual of Clin. Microbiol ., 1995, 6, 45.

10. Bauer, A.W., Kirby, W.M.M. and Scherris, J.C., Amer. J. Clin. Pathol. , 1966, 36, 493.

11. McFarland, J., J. Amer. Med. Assoc. , 1987, 49, 1176.

12. Cowan, M.M, Plant products as antimicrobial agents, Clinical microbiological Reviews, 1999,12(4) 564-582.

13. Nistro, Germanno M.P, Extraction Methods and Bio autography for evaluation of medicinal plant antimicrobial activity, Letters in Applied Microbiology 2000, vol-30, 379-384.

The procedure was carried out as filter paper disc methods12,13

The procedure was carried out as filter paper disc methods^12,13