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A Comparative study of Antimicrobial activity of Datura

(Leaf, Seed), Neem (Leaf, Bark) and Orange (Peel, waste) on

Escherichia coli and Paenibacillus barcinonesis

Vivek Mishra1, Deepesh Kumar

2, V.K.Srivastava

3, Ashish shukla

4,

Aarti Gautam5, Shivani Shukla

6 , Richa Saxena

7, Abhishek Upadhyay

8,

Saksham khusharia9

1,2,3,4,5,7,8,9 Post Graduate Department of Biotechnology,

RBS Engineering Technical Campus, Bichpuri, Agra, AKTU Lucknow, (India)

6Department of Bioengineering, Integral University, Lucknow, India

ABSTRACT

In this present study was conducted to identify bacteria (Paenibacillus barcinonesis) by using biochemical test and

then to identify antimicrobial investigation against (leaf extract, bark extract) of Neem, (peel extract, waste extract)

of Orange and (leaf extract, seed extract) of Datura which is collected from the local market and Raja Balwant

singh Engineering Technical Campus Bichpuri, Agra (U.P.) India. Antimicrobial activity of crude extract

(Methanol, Isopropanol, Chloroform, Water, Butanol) were tested by using Well Diffusion Method against two

Bacterial strains (Escherichia coli and Paenibacillus barcinonesis). Neem leaves methanol crude extract produced

maximum zone of inhibition at 24 hrs, 48hrs and (2.26 ± 0.05 cm), (2.5 ± 0.17cm) and (2.6 ± 0.17cm) respectively

against Escherichia coli. Neem leaves water crude extract produced maximum zone of inhibition at 24hrs, 48hrs

and 72hrs (1.36 ± 1.01cm), (1.4 ± 0.1cm) and (1.5 ± 0.1cm) respectively against Paenibacillus barcinonesis.

Methanol crude extract Bark of Neem showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs (1.5 ± 0.43cm),

(2.06 ± 0.05cm) and (2.13 ± 0.11cm) respectively against Escherichia coli. Methanol crude extract Bark of Neem

showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs (1.3 ± 0cm), (1.4 ± 0.05cm) and (1.36 ± 0.05cm)

respectively against Paenibacillus barcinonesis. Orange peel methanol crude extract and butanol crude extract

produced maximum zone of inhibition at 24hrs and 48hrs, 72hrs (1.4 ± 0.05cm) and (1.33 ± 0.11cm), (1.46 ±

0.15cm) respectively against Escherichia coli. Orange peel methanol crude extract and water crude extract

produced maximum zone of inhibition at 24hrs and 48hrs, 72hrs (0.86 ± 0.05cm) and (0.83 ± 0.15cm), (0.86 ±

0.05cm) against Paenibacillus barcinonesis. Methanol and Butanol crude extract Waste of Orange showed

ma++++++ximum zone of inhibition at 24hrs and 48hrs, 72hrs (1.6 ± 0.51cm) and (2.7 ± 0cm), (1.03 ± 0.05cm)

against Escherichia coli. Chloroform crude extract Waste of Orange showed maximum zone of inhibition at 24hrs,

48hrs and 72hrs (0.9 ± 0.1cm), (0.9 ± 0.1cm) and (0.93 ± 0.05cm) against Paenibacillus barcinonesis. Methanol

and Water crude extract leaves of Datura showed maximum zone of inhibition at 24hrs, 72hrs and 48hrs (1.36 ±

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0.05cm), (1.56 ± 0.05cm) and (1.46 ± 0.05cm) against Escherichia coli. Methanol and Butanol crude extract leaves

of Datura showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs (3.6 ± 0cm), (3.6 ± 0.1cm) and (3.8 ±

0.05cm) against Paenibacillus barcinonesis. Datura seed methanol crude extract crude extract produced maximum

zone of inhibition at 24hrs, 48hrs and 72hrs (1.06 ± 0.28cm), (1.5 ± 0.05cm), and (1.56 ± 0.05cm) respectively

against Escherichia coli. Datura seed methanol crude extract crude extract produced maximum zone of inhibition at

24hrs, 48hrs and 72hrs (1.33 ± 0.05cm), (1.53 ± 0.15cm), and (1.6 ± 0.2cm) respectively against Paenibacillus

barcinonesis.

Keywords- Datura,Escherichia coli, Neem, Orange, Paenibacillus barcinonesis

I INTRODUCTION

Datura metel Linn (Thorn-apple, Devil trumpet, Solanaceae) is a Nigerian medicinal plant widely used in

phytomedicine to cure diseases such as asthma, cough, convulsion and insanity. The leaves and seeds are widely

used in herbal medicine as anesthetic, antispasmodic, bronchodilator and as hallucinogenic (Duke and Ayensu, 1985

and Dabur et al., 2004).). A variety of phytochemicals have been found to occur in D.metel. These phytoconstituents

comprises alkaloids, flavonoids, phenols, tannins, saponins and sterols.The solanaceous alkaloids hyoscyamine and

scopolamines have been isolated from D. metel (Chopra etal., 1986, Oliver-Bever, 1986 ).

Neem (Azadirachta indica) commonly called ‘India Lilac’ or ‘Margosa’, belongs to the family

Meliaceae, subfamily Meloideae and tribe Melieae. Neem is the most versatile, multifarious trees of tropics, with

immense potential. It possesses maximum useful non-wood products (leaves, bark, flowers, fruits, seed, gum, oil

and neem cake) than any other tree species. Various parts of the neem tree have been used as traditional Ayurvedic

medicine in India.

Neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to control leprosy, intestinal

helminthiasis, respiratory disorders, and constipation and also as a general health promoter. Neem oil finds use to

control various skin infections. Bark, leaf, root,flower and fruit together cure blood morbidity, biliary afflictions,

itching, skin ulcers, burning sensations and phthisis.

Neem tree has adaptability to a wide range of climatic, topographic and edaphic factors. It thrives well in dry, stony

shallow soils and even on soils having hard calcareous or clay pan, at a shallow depth. Neem tree requires little

water and plenty of sunlight.

Peels are generally wasted while the citrus fruits are mainly used in juice processing industries. Very large amounts

of by-product are formed as wastes during the production of citrus juices. Citrus is one of the most important

commercial fruit crops grown in all continents of the world. Oranges and Lemon are an important medicinal plant of

the family Rutaceae. It is cultivated mainly for its alkaloids, which are having anticancer activities and the

antibacterial potential in crude extracts of different parts (viz., leaves, stem, root and flower) of Lemon against

clinically significant bacterial strains has been reported. Citrus fruits are mainly used by juice processing industries

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while the peels are generally wasted. Since the juice yield of citrus is less half of the fruit weight, very large amounts

of by product wastes, such as peels are formed every year.

II MATERIAL AND METHODS

Plants Materials

The Neem leaves & Bark, Datura Leaves & Seeds were collected from RBS ETC, Bichpuri, Agra(U.P.) India and

Orange Leaves and Waste was collected from local market Bichpuri, Agra.

Microorganisms

Paenibacillus barcinonesis was isolated agriculture soil sample and E.coli was provided by RBS ETC, Bichpuri,

Agra.

Reference Drug

Reference drug used in this study was Ampicillin (10µl/disc).

Extraction- All collected materials were shade dried at room temperature for about 5-7 days. Then the samples

were cut into small pieces and powdered in a Mortar and Pestle. Extractions were carried out with Methanol,

Isopropanol, Butanol, Chloroform and Water solvent of bioactive compounds. After 24hrs the extract was filtered

with whatman filter paper then crude extract found for antimicrobial investigation.

Testing for Antimicrobial

Different concentrations of methanol, isopropanol, butanol, chloroform and water extracts of Datura,Neem and

Orange (10%, 20% and 30%). Take 10g of plant powder was added 100ml methanol, isopropanol, butanol,

chloroform and water into a conical flask for 24hrs.after 24hrs the extract were filtrate then used.(Harbone JB,

1973).

III RESULTS

We take soil sample from agricultural land from the RBS ETC, Bichpuri, Agra (U.P.) India then dilution of this

sample 1/10th

rule. Take five test tube and then mark 100-10

-4 and the last test tube diluted sample mixed well and

take 100µl in the Nutrient Agar petri plate disc for 24hrs incubated. Then I focused a single colony that plate after

then steak on a Nutrient Agar plate further incubated for 24hrs.To identified bacteria we confirmed by biochemical

tests then analyzed by Abis online which is found Paenibacillus barcinonesis.

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Serial Dilution Single colony Isolation

Biochemical Tests

Table-1

Gram-Staning

+Ve

Mannitol Test

+Ve

Starch Hydrolysis

-Ve

Catalase Activity

+Ve

Gelatin Test

+Ve

Methyl-Red Test

+Ve

Voges-Proskers Test -Ve

H2S Test

+Ve

Citrate Test

-Ve

Urease Test

-Ve

Gram’s staining Catalase Test Citrate Test

Gelatin test H2S Test Mannitol Test

Methyl red Starch Test Vp Test

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Antimicrobial activity testing by well diffusion method the antibiotic Ampicillin take as a control and then

Antimicrobial activity of crude extract (Methanol, Isopropanol, Chloroform, Water, Butanol) were tested against

two Bacterial strains (Escherichia coli and Paenibacillus barcinonesis). Neem leaves methanol crude extract

produced maximum zone of inhibition at 24 hrs, 48hrs and (2.26 ± 0.05 cm), (2.5 ± 0.17cm) and (2.6 ± 0.17cm)

respectively against Escherichia coli. Neem leaves water crude extract produced maximum zone of inhibition at

24hrs, 48hrs and 72hrs (1.36 ± 1.01cm), (1.4 ± 0.1cm) and (1.5 ± 0.1cm) respectively against Paenibacillus

barcinonesis. Methanol crude extract Bark of Neem showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs

(1.5 ± 0.43cm), (2.06 ± 0.05cm) and (2.13 ± 0.11cm) respectively against Escherichia coli. Methanol crude extract

Bark of Neem showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs (1.3 ± 0cm), (1.4 ± 0.05cm) and (1.36

± 0.05cm) respectively against Paenibacillus barcinonesis. Orange peel methanol crude extract and butanol crude

extract produced maximum zone of inhibition at 24hrs and 48hrs, 72hrs (1.4 ± 0.05cm) and (1.33 ± 0.11cm), (1.46 ±

0.15cm) respectively against Escherichia coli. Orange peel methanol crude extract and water crude extract produced

maximum zone of inhibition at 24hrs and 48hrs, 72hrs (0.86 ± 0.05cm) and (0.83 ± 0.15cm), (0.86 ± 0.05cm)

against Paenibacillus barcinonesis. Methanol and Butanol crude extract Waste of Orange showed maximum zone of

inhibition at 24hrs and 48hrs, 72hrs (1.6 ± 0.51cm) and (2.7 ± 0cm), (1.03 ± 0.05cm) against Escherichia coli.

Chloroform crude extract Waste of Orange showed maximum zone of inhibition at 24hrs, 48hrs and 72hrs (0.9 ±

0.1cm), (0.9 ± 0.1cm) and (0.93 ± 0.05cm) against Paenibacillus barcinonesis. Methanol and Water crude extract

leaves of Datura showed maximum zone of inhibition at 24hrs, 72hrs and 48hrs (1.36 ± 0.05cm), (1.56 ± 0.05cm)

and (1.46 ± 0.05cm) against Escherichia coli. Methanol and Butanol crude extract leaves of Datura showed

maximum zone of inhibition at 24hrs, 48hrs and 72hrs (3.6 ± 0cm), (3.6 ± 0.1cm) and (3.8 ± 0.05cm) against

Paenibacillus barcinonesis. Datura seed methanol crude extract crude extract produced maximum zone of inhibition

at 24hrs, 48hrs and 72hrs (1.06 ± 0.28cm), (1.5 ± 0.05cm), and (1.56 ± 0.05cm) respectively against Escherichia

coli. Datura seed methanol crude extract crude extract produced maximum zone of inhibition at 24hrs, 48hrs and

72hrs (1.33 ± 0.05cm), (1.53 ± 0.15cm), and (1.6 ± 0.2cm) respectively against Paenibacillus barcinonesis.

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Antimicrobial Pics

Datura Leaves & Datura Seed Paenibacillus barcinonesis

Neem Leaves & Bark Paenibacillus

barcinonesis

Orange Peel & Waste Paenibacillus barcinonesis

Datura Leaves & Seeds E.coli

Neem Leaves & Bark E.coli

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Orange Peel & Waste E.coli

Graphs

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IV CONCLUSION

From the above study, it concludes that the presence of constituents revealed in the solvent crude extract of leaves of

Datura, Neem, Orange could contribute for their antimicrobial activities. The various solvent extracts of the plant

showed high potential of antibacterial activities against the tested microorganisms.

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