INTRODUCTION:

In recent years food safety concerns have been focused on several pathogens. Man has been using natural products of animals, plants and microbial sources for thousands of years either in the pure forms or crude extracts .Plants have been intimately associated with human civilization since a long time. It was the human race who first learned to utilize the herbal wealth for curative purpose. Pharmacognosy- the study of plants as a sources of drugs is therefore one of the oldest disciplines. There are around 7500 species of plants used for preventive, promotive and curative application. Out of these species Allium cepa the most commonly sought after in the uses for its antibacterial properties is taken up as a research objective to determine the enzyme (protein) responsible for this activity. Onion is a good example of such plants, which has been found out to be used locally against infections, particularly in some cases of eye infections¹. Onion consists mainly the scale leaves of Allium cepa Linn. Belongs to family Lillaceae very commonly grown in North-East India. Onion extract acts as Diuretic, antibiotic, hypoglycaemiant, hypotensive, dechlorurant, anti-inflammatory, antithermic, analgesic, antineuralgic.^2,3

Allium cepa L (Onion) belongs to the family lilliceae. It is also known as 'garden onion’. Onions are effective against common cold, heart disease, diabetes, osteoporosis, coughs and sore throat⁴. The flesh part of the onion contains flavonoids. Polyphenols are the important component of onion which differentiates it from other Allium species. Anti-bacterial action of onion extracts against oral pathogenic bacteria from various region is also investigated.⁵ Antimicrobial activity of different concentrations of essential oil extracts of various type of onions (green, yellow and red) against different bacteria is evidenced.^6-7Antioxidant activity of crude onion extracts is also studied ⁸ .Sep. 2009

MATERIALS AND METHODS:

Sample Collection and isolation:

Samples were collected from Salipur of Cuttack district of state Odisha and were brought to the microbiology laboratory of Institute of Pharmacy and technology of Salipur.

Aqueous and alcoholic Extract preparation:

Fresh onion bulbs were peeled, weighed (10 g), and surface sterilized using 95% ethanol. The ethanol was allowed to evaporate in a sterile laminar flow chamber, onion was homogenized aseptically with 10 ml of sterile double distilled water using a sterile mortar and pestle. The homogenized mixture was filtered through 8 layers of muslin cloth and centrifuged at 6000 rpm for 15 min. Supernatant was collected and kept at 4ºC for further use. Edible parts of fresh Onion (Allium cepa) were rinsed thoroughly in distilled water and air dried. 10 grams were then blended and homogenized with sterile double distilled water in 1:1 ratio. The juice was then filtrated and squeezed out of it. The extract was stored at 4°C. Alcoholic extract of Allium cepa was prepared by using soxhilation apparatus and stored in an airtight container for further uses.

Quantitative chemical analysis:

Detections of alkaloid:

Few drops of extract was stirred with few drops of dilute hydrochloric acid and filtered. The filtrate when react with Mayer’s Reagent ,gives rise to crime precipitate and when react with Dragendorffs reagent give rise to orange brown precipitate and Hagers reagent gives rise to yellow ppt and with Wagner Reagent gives rise to Reddish brown ppt.

Detection of Carbohydrate and Glycoside:

Small quantity of approximately 300 mg of aqueous and alcoholic extract separately was dissolved in 4 ml of distilled water and filtered. The filtrate may be subjected to Molish test for the extraction of carbohydrates. Small quantity of extract was hydrolyzed with dil. HCl in a water bath and subjected to Libermann-Burchards. Legal and Borntragers test to detect the presence of different glycosides. A small portion of the extract was dissolved in water and treated with Fehlings, Barfodes and Benedicts reagent to detect the presence of different sugars.

Detections of Phytosterols:

Ether , acetone and alcoholic extracts were refluxed separately with solution of alcoholic potassium hydroxide till complete saponification takes place .The saponification mixture was diluted with distilled water and extracted with ether .The ethereal extract was evaporated and subjected the residue on saponifiable matter to Libermanns test and Libermanns –Burchards test.

Detection of Fixed oil and Fat:

A small quantity of petroleum ether and benzene extracts were pressed separately between two filter paper. Oil stain on the paper indicates the presence of fixed oil.

Detection of Saponine:

One ml of alcoholic and aqueous extract were diluted separately with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. A one cm layer of foam indicated the presence of saponines.

Detection of phenolic compound and Tannin:- A small quantity of alcoholic and aqueous extract were taken in separately in water and tested for the presence of phenolic compound and tannins with dil. Ferric chloride solution and aqueous bromine solution.

Detection of protein and amino acid:

A small quantity of alcoholic and aqueous extract were dissolved in a few amount of water and subject to Biuret, Millons and Ninhydrin test.

Detection of gum and mucilage:

10 ml of aqueous extract was added slowly to 25 ml of absolute alcohol with constant stirring. The precipitate was filtered and dried in air. The precipitate was examined from swelling properties.

Detection of volatile oil:

50 grams of powder Allium cepa was taken and subjected to hydro distillation for detection of volatile oil. Estimation of protein in Allium cepa Linn. by Lowris Method:

A 25% w/v egg albumin solution was prepared. To it 0.1N NaOH solution 2%NaCO₃ was added (solution A).5% CuSO₄ solution was added in 1% sodium potassium tartarate solution.(solution B). 50 ml of reagent A was mixed with 1 ml of reagent B just prior to use (solution C). Test tubes were taken and leveled with 1,2,3…7 and the reagents were added as per followings. The contents of each tube were mixed rapidly after each addition. The tubes are left as such for 30 minutes and blue color was formed.

TABLE NO:-1

Test tube no	1	2	3	4	5	6	7
Egg albumin solution (ml)	0	1	0.5	1	1	1	0.7
Sample (ml)	0	0	0	0.1	0.2	0.3	0
Water (ml)	4	3	3.5	2.9	2.8	2.7	3.3
Reagent (ml)	5.5	5.5	5.5	5.5	5.5	5.5	5.5
Folin-Ciocalteau Reagent (ml)	0.5	0.5	0.5	0.5	0.5	0.5	0.5

Table:2- Qualitative chemical examination of various extracts obtained by successive solvent extraction of plant material

Plant constitutient	Methanol extract	Water extract
Test	M	W
1.Alkaloids	+	+
2.Carbohydtates	+	+
3.Phytosterols	-	-
4.Fixed oils/fats/Lipids	+	+
5.Volitile oil/ Essential Oil	+	+
6.Saponins	-	-
7.Phenolic compounds/Tanins	+	+
8.Proteins	+	+
9.Gum/Mucilages	-	-
10.Flavonoids	+	+

[Recorded observation in +(present) or-(absent)]

Preparation of nutrient agar media:

The nutrient agar media is prepared for the growth and multiplication of micro-organism. The microorganism is generally taken Protius vulgaris and E. coli for the determination of antibacterial activity of Allium cepa.

Inoculation of bacteria:

Two bacterial species Protious vulgaris American type culture count number 3296, and E. coli were taken for determination of antibacterial activity of Allium cepa. Both are gram negative bacteria. Then after the media was transferred to the petridishes and the two bacteria species were allowed for inoculation by an incubator at 37^oc for adulation of 24 hours. Then the zone of inhibition of the bacteria is measured.

RESULTS:

All the findings are summarized in table 1,2, 3 and 4. In case of water extract of Allium cepa, it has been found that Zone radius value is less as compared to alcohol extract. And also this value is distinctly high for Proteus vulgarius in comparison to E. coli.

Table:3 Zone radius value for water extract in case of both for Proteus vulgarius as well as E. coli

Extract

Concentration

(ml)

Petridis no.

Zone radius (mm) for Proteus vulgarius (Gram negative)

Mean zone radius (mm) for Proteus vulgarius (Gram negative)

Zone radius (mm) for E. coli (Gram negative)

Mean zone radius (mm) for E. coli (Gram negative)

Water

19.5

18.0

21.3

19.6

12.2

14.7

17.5

14.8

1/10

17.1

15.4

20.0

17.5

12.8

10.2

15.7

12.9

1/100

13.3

15.4

20.0

15.4

7.5

9.0

11.1

9.2

1/1000

10.8

14.2

15.8

13.4

9.1

7.2

5.3

7.2

Table:4 Zone radius value for alcohol extract in case of both for Proteus vulgarius as well as E. coli

Extract

Concentration

(ml)

Petridis no.

Zone radius (mm) for Proteus vulgarius (Gram negative)

Mean zone radius (mm) for Proteus vulgarius (Gram negative)

Zone radius (mm) for E. coli (Gram negative)

Mean zone radius (mm) for E. coli (Gram negative)

Alcohol

21.9

20.2

18.7

20.0

14.4

14.8

15.8

15.0

1/10

17.9

15.0

21.1

18.0

14.7

12.7

14.0

13.8

1/100

16.2

14.8

16.7

15.9

8.7

10.8

12.3

10.6

1/1000

15.9

13.7

11.8

13.8

8.0

9.5

6.8

8.1