INTRODUCTION:

Barleria prionitis Linn (Acanthaceae) is widely distributed throughout Africa, India, Sri Lanka and tropical Asia. The crude extract of this plant is commonly used in folk medicine to treat whooping cough. The plant extract has also shown its potential applications as diaphoretic and expectorant. The plant has also shown anti-respiratory syncytial virus, anti-arthritic, anti-inflammatory and anti-fertility activities. Barleria prionitis L. is an erect bushy shrub of family Acanthacae. The height is about 1.5 metres. The leaves are narrowly to broadly elliptic lanceolate, entire, and appraised hairy beneath. The flowers are yellow, in axillary, spikate clusters. The flowering season is September to March.

In Ayurveda the leaves and the tender branches are used for treatment of toothache, strengthening of gums, whooping cough and premature ejeculation. Whole-plant extracts of porcupine flower contain iridoid glycosides, barlerin,and verbascoside, which have shown potent activity against respiratory syncytial virus in vitro and may account for the plant’s use in treating fever and several respiratory diseases in herbal medicine¹.

Plants and plant products have been used extensively throughout history to treat medical problems. Extracts of many plants are highly efficient against parasitic as well as microbial infection. It is estimated that around 70,000 plant species, from lichens to tall trees, have been used at one time to other for medicinal purposes^2-7. The medicinal plants are the plants whose parts (leaves, seeds, stem, roots, fruits, foliages etc.), extracts, infusions, decoction, powders are used in the treatment of different diseases of humans, plants and animals⁸. The medicinal plants occupy a significant place in modern medicine as a raw material for some important drugs, although synthetic drugs and antibiotics brought about a revolution in controlling different diseases. But these synthetic drugs are out of reach to million of people. Those who live in remote places depend on traditional healers, whom they know and trust. The judicious use of medicinal herbs can even cure deadly diseases that have long defied synthetic drugs⁹. Emergence of drug resistance bacteria also reduces the antimicrobial efficacy of many potential antimicrobial drugs. So, there is a increase need for new antimicrobial agents to combat this problem. Bacteria are the prokaryotes. The smallest free-living microorganism is the unicellular. The chromosome of the bacteria is located in the cytoplasm, usually attached to the cell membrane. In bacteria mitosis and meiosis nuclear divisions are absent. Nucleolus is absent in bacteria. Normally asexual reproduction is happened in bacteria. There is one chromosome is present in bacteria. In bacteria cell wall usually contains peptidoglycan; ribosomes are smaller, usually 70s; flagella structurally simple and pili are present¹⁰.

Materials and Methods:

The plant has been collected from the forest of Ichharia village, Bankura, West Bengal, India and has been authenticated by B.S.I., Shibpur, Howrah, West Bengal, India.

Preparation of Extract:

The leaves were carefully washed under running tap water followed by sterile distilled water. These were air dried at room temperature (45⁰C) for five days and pulverized to a fine powder using a sterilized mixer grinder and stored in air-tight bottles. Then the leaf powder extracted with petroleum ether (40⁰-60⁰), chloroform and maceration is done with 70% ethanol. The column fraction (70% ethanol extract) is get by column chromatography. After filtration of total extracts, the extracts were evaporated to dryness in vacuum.

Preparation of Media:

(A) Solid Media

Bacteriological Peptone (Oxoid) --1% w/v

Beef Extract (Oxoid) ----------------2% w/v

Sodium Chloride (Analar) ----------0.5% w/v

Agar (Oxoid) -------------------------1.5% w/v

Distilled Water ---------------------- Q.S. to 100ml

PH--------------------------------------7.2 - 7.4

(B) Liquid media:

Bacteriological Peptone (Oxoid) -----1% w/v

Beef extract (Oxoid) ------------------ 0.5% w/v

Sodium Chloride (Analar) ------------0.5% w/v

Distilled Water -------------------------Q.S. to 100ml

PH----------------------------------------7.2 - 7.4

(C) Peptone water:

Bacteriological peptone (Oxoid) ------1% w/v

Sodium Chloride (Analar) -------------0.5% w/v

Distilled Water -------------------------Q.S. to 100ml

PH----------------------------------------7.2 - 7.4

Bacterial strains :

The sources of bacterial strains, used for investigation of the antimicrobial properties of all the extracts are enlisted in table 1.

Determination of Minimum Inhibitory Concentration:

Gram (+ve) and gram (–ve) bacteria were grown in peptone water for 18 h; this gave an optimum growth of the test bacteria. The petroleum ether (40⁰-60⁰C) extract, chloroform extract, 70% ethanol extract and column fraction separately dissolved in dimethyl sulfoxide (DMSO) sterilized by filtration by using sintered glass filter (G-5) and stored at 4⁰C.

Petroleum ether extract was added to molten nutrient agar in the following concentration (mg/ml): 3.33; 6.66; 16.66; 33.33. Chloroform extract and column fraction was then added to molten nutrient agar in the following concentration (mg/ml): 5; 10; 25; 50. Ethanol (70%) extract added to 9 ml molten nutrient agar in the following concentration (mg/ml): 10; 20; 50; 100 and poured into sterile petridish. The pH of the media was maintained at 7.2-7.4. The inoculums consisted of an over night grown broth culture of a bacterium diluted in such a manner that a 2mm (internal diameter) loopful of that culture contain 10⁵ colony forming unit (CFU). These were then spot inoculated at the sterilized laminar air flow on nutrient agar plates containing increasing amount of a compound, inoculated at 37⁰C up to 72h for determination of the minimum inhibitory concentration (MIC)¹¹.

Results and Discussion :

Table-2: Effect of Barleria prionitis leaf extract (Petroleum ether) on different group of bacteria

Name of Strains	Concentration(mg/ml)
Name of Strains	3.33	6.66	16.66	33.33
Salmonella typhi (Ty 2-59)	+	-	-	-
Bacilus cereus (PI – 11778)	+	+	+	+
Bacilus subtilis (PI – 6633)	-	-	-	-
Vibrio cholerae (DN – 6)	+	-	-	-
Vibrio cholerae ( 793 )	+	+	+	+
Vibrio cholerae ( 813 )	+	-	-	-
Micrococus luteus ATCC– 9341	+	-	-	-
Providencia	+	+	+	+
Lactobacillus sporogenus	+	+	+	+
Citrobacter ( 8307 )	+	+	+	-

+ = Presence of growth - = Absence of growth

Discussion:

Result given in table 2 indicates that B.subtilis (PI-6633) was found to be highly sensitive to the drug as it was inhibited at 3.33mg/ml level. S.typhi (Ty 2-59), V.cholerae (DN-6), V.cholerae (813) and M.luteus (ATCC-9341) were inhibited at 6.66mg/ml level. A MIC of 33.33mg/ml was observed against Citrobacter (8307). The remaining B.cereus (PI-11778), V.cholerae (793), Providencia and L. sporogenus were resistant to the drug.

Table- 3: Effect of Barleria prionitis leaf extract (chloroform) on different group of bacteria

Name of Strains	Concentration (mg/ml)
Name of Strains	5	10	25	50
Salmonella typhi (Ty 2-59)	-	-	-	-
Bacilus cereus (PI – 11778)	+	+	+	+
Bacilus subtilis (PI – 6633)	-	-	-	-
Vibrio cholerae (DN – 6)	+	+	-	-
Vibrio cholerae ( 793 )	-	-	-	-
Vibrio cholerae ( 813 )	-	-	-	-
Micrococus luteus ATCC– 9341	-	-	-	-
Providencia	+	+	+	-
Lactobacillus sporogenus	-	-	-	-
Citrobacter ( 8307 )	-	-	-	-

+ = Presence of growth - = Absence of growth

Discussion:

Result given in table 3 indicates that S.typhi (Ty 2-59), B.subtilis (PI-6633), V.cholerae (793), V.cholerae (813), M.luteus(ATCC-9341), L.sporogenus and citrobacter (8307) were found to be highly sensitive to the drug as they were inhibited at 5mg/ml level. V.cholerae (DN-6) was inhibited at 25mg/ml level. A MIC of 50mg/ml was observed against Providencia. The remaining B.cereus (PI-11778) was resistant to the drug.

Table- 4: Effect of Barleria prionitis leaf extract (70% ethanol) on different group of bacteria:

Name of Strains	Concentration (mg/ml)
Name of Strains	10	20	50	100
Salmonella typhi (Ty 2-59)	+	-	-	-
Bacilus cereus (PI – 11778)	+	+	+	+
Bacilus subtilis (PI – 6633)	-	-	-	-
Vibrio cholerae (DN – 6)	+	+	+	-
Vibrio cholerae ( 793 )	-	-	-	-
Vibrio cholerae ( 813 )	-	-	-	-
Micrococus luteus ATCC– 9341	-	-	-	-
Providencia	+	+	+	+
Lactobacillus sporogenus	+	-	-	-
Citrobacter ( 8307 )	+	+	+	+

+= Presence of growth - = Absence of growth

Discussion:

Result given in table 4 indicates that Bacillus subtilis (PI-6633), Vibrio cholerae (793), Vibrio cholerae (813) and Micrococcus luteus (ATCC-9341) were found to be highly sensitive to the drug as they were inhibited at 10mg/ml level. Sulmonella typhi (Ty 2-59) and Lactobacillus sporogenus were inhibited at 20mg/ml level. An MIC of 100mg/ml was observed against Vibrio cholerae (DN-6). The remaining strains were resistant to the drug.

Table- 5: Effect of Barleria prionitis leaf extract (column fraction) on different group of bacteria:

Name of Strains	Concentration (mg/ml)
Name of Strains	5	10	25	50
Salmonella typhi(Ty 2-59)	-	-	-	-
Bacilus cereus (PI – 11778)	+	+	+	-
Bacilus subtilis (PI – 6633)	-	-	-	-
Vibrio cholerae (DN – 6)	-	-	-	-
Vibrio cholerae ( 793 )	+	+	+	-
Vibrio cholerae ( 813 )	-	-	-	-
Micrococus luteus ATCC– 9341	-	-	-	-
Providencia	+	+	+	-
Lactobacillus sporogenus	+	+	+	-
Citrobacter ( 8307 )	-	-	-	-

+ = Presence of growth - = Absence of growth

Discussion:

Result given in table 5 indicates that S. typhi (Ty 2-59), B. subtilis (PI-6633), V.cholerae (DN-6), V. cholerae (813), M. luteus (ATCC-9341) and Ctrobacter (8307) were found to be highly sensitive to the drug as they were inhibited at 5mg/ml level. A MIC of 50mg/ml was observed against B. cereus (PI-11778),V.cholerae (793), providencia and L. sporogenus.

Comparative study of mic:

Table- 6: Comparative study of MIC among the all extract on different group of bacteria

Name of Strains	Minimum Inhibitory Concentration (mg/ml)
Name of Strains	Pet. Ether extract	Chloroform extract	Ethanol extract	Column fraction
Salmonella typhi (Ty 2-59)	6.66mg/ml	5mg/ml	20mg/ml	5mg/ml
Bacilus cereus (PI – 11778)	N.A.	N.A.	N.A.	50mg/ml
Bacilus subtilis (PI – 6633)	3.33mg/ml	5mg/ml	10mg/ml	5mg/ml
Vibrio cholerae (DN – 6)	6.66mg/ml	25mg/ml	100mg/ml	5mg/ml
Vibrio cholerae ( 793 )	N.A	5mg/ml	10mg/ml	50mg/ml
Vibrio cholerae ( 813 )	6.66mg/ml	5mg/ml	10mg/ml	5mg/ml
M. luteus ATCC – 9341	6.66mg/ml	5mg/ml	10mg/ml	5mg/ml
Providencia	N.A.	50mg/ml	N.A.	50mg/ml
Lactobacillus sporogenus	N.A.	5mg/ml	20mg/ml	50mg/ml
Citrobacter ( 8307 )	33.33mg/ml	5mg/ml	N.A.	5mg/ml

N.A. = Not Active

NAME OF STRAINS	TYPE	SOURCE
Salmonella typhi (Ty 2-59)	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Bacilus cereus (PI – 11778)	Gram (+ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Bacilus subtilis (PI – 6633)	Gram (+ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Vibrio cholerae (DN – 6)	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Vibrio cholerae ( 793 )	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Vibrio cholerae ( 813 )	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University;Kolkata-700032
Micrococus luteus ( ATCC – 9341 )	Gram (+ve)	Dept. of Pharm. Technology; Jadavpur University; Kolkata-700032
Providencia	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Lactobacillus sporogenus	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032
Citrobacter ( 8307)	Gram (–ve)	Dept. of Pharm. Technology; Division of Microbiology; Jadavpur University; Kolkata-700032