INTRODUCTION:

Microbial assay:¹

Many therapeutic agents, which either inhibit the growth of microorganisms (antibiotics) or essential for their growth (vitamins and amino acids) can be standardised by microbiological assays.

Any change in the antibiotic molecule which may not be detected by chemical methods will be revealed by a change in the antimicrobial activity and hence microbiological assays are very useful for resolving doubts regarding possible change in potency of antibiotics. Compared with biological assay methods using animals, microbiological techniques possess the advantages of minimal requirements of space, labour, materials and time.

The minimal assay is based upon a comparison of the inhibition of growth of microorganisms by measured concentrations of the antibiotics to be examined with that produced by known concentrations of a standard preparation of the antibiotic having a known activity. Two general methods are used for microbiological assays of antibiotics.

1. Method A: Cylinder plate method or cup plate method.

2. Method B: turbidimetric method or tube assay method.

Method A:

The cylinder plate method depends upon diffusion of the antibiotic from vertical cylinder through a solidified agar layer in a Petri plate to an extent such that growth of the added microorganism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic. The turbidometry method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic.

Pharmacognosy of neem plant:²

Synonyms: Margosa

Biological Source: It consists of all aerial parts of plant known as Azadirachta indica.

Family: Meliaceae

Chemical Constituents:

Diterpenes: sugiol, nimbiol, Triterpenes: B-sitosterol, stigmasterol, Limonoids: Maliantriol, nimbidinine, Sulphurous compound: tri and tetrasulphide, Flavonol glycoside: Nimaton, quercetin, Neem leaves contain not less than 1.0% w/w of Rutin.

Uses: Insecticide, nematicide and antimicrobial properties.

Neem (Azadirachta indica A. Juss.) tree also known an “Indian Lilac” belongs to the family Meliaceae, has attracted special interest of scientists due to the presence of a variety of bioactive compounds. The tree is popularly called the "wonder tree" as it used to cure a large number of diseases, both traditional as well as modern medicine system make use of neem tree parts like leaves, bark, seed, fruit to manufacture products right from skin care to oral care, from pharmaceuticals to pesticides and manure. Neem and various other botanicals are gaining importance in crop protection in view of their selective properties, low cost and safety to ecosystem. Many botanicals have been identified to be effective in the control of plant diseases. Among the 5280 species tested, 1134, 346 and 92 plant species possessed insecticidal, fungicidal and bactericidal properties respectively³. Neem has a great potential to control various phytopathogenic bacteria and therefore, has much prospect to be used as a good bactericide. Ethanol extracts of A. indica showed bacterial toxic properties against Alternaria brassicola and F. oxysporum⁴. Therefore, the development of bio-pesticides has been focused as a viable disease control strategy. In previous years plant extracts and essential oils show antibacterial activity against a wide range of fungi^5,6,7. In order to assess the bacterial-toxic properties of neem against different bacterial pathogens.

Material:

The experiment was done in the Microbiology laboratory of Shree Sureshdada Jain Institute of Pharmaceutical Education and reseach Jamner, Natural bacterial sample viz lacto bacillus was obtained from curd. The different leaf extract forms viz., powder form, crude form, boiled form and ethanol form are isolated organised plant of neem in Department of Pharmacognosy laboratory under the supervision of head of the department Prof. Y. M. Bagad sir. Benzyl Penicillin powder obtain from pharmaceutics research laboratory of M.Pharm by the hand of Dr. S. M. Sarode sir.

Method:⁸

· Dissolve 35 mg of benzyl penicillin in 100 ml sterile water. From this, take 1ml and dilute to 100 ml of sterile water (Each ml of this contains 6-unit activity).

· By using this stock solution, prepare 1, 2, 3, 4 and 5 units / ml of standard stock solution.

· Prepare a solution Neem leaf extract in the same solvent as that of standard solution and dilute it till the level of standard preparation of the antibiotics.

· Sterilise the antibiotic assay medium by autoclave and prepare Petri plates in laminar air flow.

· Spread the test microorganism (lacto Bacillus) on the surface of Petri plate by spread plate technique.

· By using flame sterilised cork borer, prepare five to six cups (or cavity) in each plate keeping adequate distance from each other (Do not overlap each other).

· Standard and test diluted solutions (0.1 to 1ml, depending on size of cavity are added in each labelled cavity of plates.

· Transfer all the plates in refrigerator for proper diffusion of antibiotic and neem extract sample at 4°C for one to two ate hours. Incubate all the plates in incubator at 32-35°C for 24 to 48 hours.

RESULT AND OBSERVATION:

Investigations on the antibacterial activity of neem plant parts extracts of the selected plants revealed inhibitory effect of the extracts against the non-pathogen (Fig. 2) against the standard benzyl penicillin solution (Fig 1). The effect of differences in the present study the bacterial-toxicity of Azadirachta indica (neem) was evaluated in the form of ethanolic leaf extracts against bacteria i.e. lactobacillus under in-vitro condition by using cup plate method. The results prove that neem alcoholic leaf extracts significantly inhibited the growth of bacteria.

The literature review also shows that the fungitoxicity of A. indica leaf extract against Fusarium was also reported by Bansal and Gupta (2000)⁹ and Dwivedi and Shukla (2000)¹⁰. Rajput (2000)¹¹, Dubey (2001)¹² and Sharma (2004)¹³ inhibited the growth of T. penicillariae, A. tegitica and A. cyamopsides respectively by the leaf extract of neem. Alkhail (2005)¹⁴ reported strong antifungal activity of neem against F. oxysporum and R. solani and Botrytis cinerea. Neem parts extracts of some medicinal plants in our study also exhibited significant effect against some fungal pathogens. On the basis of present study, it is concluded that leaf extract of the plants which have been found effective against collar rot can be recommended against the disease after in field trials.

Fig. 1: Standard 6 zone of Benzyl Penicillin

Fig. 2: Test 6 zone of alcoholic Neem leaf extract

Observation Table:

Sr. No.	Dilution	Zone of Inhibition Standard (mm)	Zone of Inhibition Neem extract (mm)
1	1 unit/ml	8	6
2	2 unit/ml	10	7
3	3 unit/ml	11	9
4	4 unit/ml	11	10
5	5 unit/ml	12	11
6	6 unit/ml	12	11

CONCLUSION:

This research article is based on the antibacterial activity of neem. The entire study was conducted in a college laboratory to understand the concepts of microbial assay and the format of research paper writing. According to this article, we concluded that neem leaf extract exhibits efficient antibacterial activity comparable to that of standard benzylpenicillin. The benefit of using neem extract is its easy availability and low cost. However, neem is not used commercially due to its bitter taste and challenging extraction process.

REFERENCES:

1. Dr. Chandrakant Kokare. Pharmaceutical Microbiology Principle and Application. Fourth Edition. Nirali Prakashan. 2007, 19.1

2. C.K. Kokate, A.P. Purohit, S.B. Ghokhale. Pharmacognosy. 56-Edition. Nirali Prakashan, Pune. 2019 Page No.19.3, 19.4

3. Ahmed N, Grainge M. Some promising plants for pest control under small scale forming operation in developing countries working paper resource system institute, East-West Centre, Honolulu, Hawai, USA. 1982, 55.

4. Shivpuri Asha, Sharma OP, Jhamaira SL. Fungitoxic properties of plant extracts against pathogenic fungi. J Mycol. Pl. Pathol. 1997; 27: 29-31

5. Abd Alla MS, Atalla KM, Elp-Sawi MAM. Effect of some plant waste extracts on growth and aflatoxin production by Aspergillus flavus, Annals Agric Sci., Ain Shams Univ. Cairo. 2001; 46: 579-592.

6. Wilson C, Solar JM, Ghaouth-El A, Wisniewski ME. Rapid evaluation of plant extracts and essential oils for antifungal activity against Botrytis cinerea. Pl. Dis. 1997; 81: 201-210.

7. Grane M, Ahmad S. Handbook of Plants with Pest Control Properties. John Wiley and Sons, New York, 1988.

8. Chandrakant Kokare. Pharmaceutical Microbiology Experiments and Techniques. Fourth Edition, Career Publication. 2013; 222

9. Bansal RK, Gupta RK. Evaluation of plant extracts against Fusarium oxysporum, wilt pathogen of fenugreek. Indian Phytopath. 2000; 53(1): 107-108.

10. Dwivedi BP, Shukla BN. Effect of leaf extracts of some medicinal on spore germination of some Fusarium species. Karnataka J Agric. Sci. 2000; 13(1):153-154

11. Rajput SS. Management of smut in pearl millet. M.Sc. (Ag.) Thesis (JNKVV), College of Agriculture, Gwalior, 2000.

12. Dubey AK. Studies on leaf spot and flower blight of marigold M.Sc. (Ag) Thesis. JNKVV, Jabalpur (M.P.), 2001, 61.

13. Sharma RN. Studies on Alternaria blight of Guar (Cyamopsis tetragonoloba (L.) Taub.). M.Sc. (Ag) Thesis. JNKW, Jabalpur, 2004.

14. Alkhail. In-vitro evaluation of different plant extracts against some fungal pathogen. Pak. J Bol. 2005; 39(7): 2619-2624.

Received on 23.07.2024 Revised on 27.08.2024

Accepted on 21.09.2024 Published on 07.12.2024

Available online on December 30, 2024

Res.J. Pharmacology and Pharmacodynamics.2024;16(4):274-276.

DOI: 10.52711/2321-5836.2024.00047

Comparative Study of Antibacterial activity of neem leaf extract against standard benzyl penicillin by using In vitro cup plate method