Niranjan Sonkar¹, Alka Agrawal², Gopal Nath³ and C. B. Jha¹

¹Department of Rasa Shastra, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005 India. ²Department of Medicinal Chemistry, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005 India. ³Department of Microbiology, Faculty of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005 India.

Abstract

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents¹. For the present study the trial drugs taken i.e. Haritaki Churna, Vibhitaki Churna, Amalaki Churna and Triphala Churna retain their potency i.e. no deterioration is observed in terms of physical, chemical and microbiological parameters after one month, two months, four months and six months duration in the Stability chambers - Temperature 450C ± 2 & Relative Humidity 75% ± 5 and in ordinary conditions of temperature and humidity. This shelf life period may be applicable to other Churnas having similar method of preparation and constituents having similar range of phyto-chemicals, carbohydrates, cellulose etc.

Keywords

Hc. (Haritaki churna); Vc. (Vibhitaki churna); Ac. (Amalaki Churna); Tc. (Triphala churna)

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Copy the following to cite this article: Sonkar N, Agrawal A, Nath G, Jha C. B. Determination of Microbial Load in Reference to Stability Study of Churnas (powders). Biomed Pharmacol J 2010;3(1)

Copy the following to cite this URL: Sonkar N, Agrawal A, Nath G, Jha C. B. Determination of Microbial Load in Reference to Stability Study of Churnas (powders). Biomed Pharmacol J 2010;3(1). Available from: http://biomedpharmajournal.org/?p=1459

Introduction

It is well known that infectious diseases account for high proportion of health problem, especially in developing countries. Microorganisms have developed resistance to many antibiotics and this have created immense clinical problem in the treatment of infectious disease (Davis-`1994). This resistance has increased due to indiscriminate use of commercial anti microbial drugs commonly used in the treatment of infectious disease. This situation has forced scientists to search for new antimicrobial molecules from various sources, including plants (Karaman et al., 2003). Infection is considered as one of the main factors responsible for diarrhea in children. Secondary metabolites produced by plants constitute source bioactive substances and nowadays the scientific interest has increased for new drugs of plant origin.

Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 36-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16–20 hrs.) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 days.

The following tests i.e. Dilution method Diffusion method (Stoke’s method Kirby – Bauer method) were designed for the estimation of the number of viable aerobic micro-organism present and for detecting the presence of designated microbial species in pharmaceutical substance. The term “growth” is used to designate the presence and presumed proliferation of viable micro- organism. In the present paper  the antimicrobial properties of all single drugs i.e., Haritaki, Amalaki, Vibhitaki churna and all these three plant mixed as a Triphala churna were carried out on different pathogens which may help in ascertaining the mode of action as well as further specific use of the present combination in future studies.

Permissible Limits of Microbial load & Pathogens ⁵

Table 1

Microbial load	Permissible Limit As per WHO
	For in the crude plant materials (cfu per gram)	Contamination  For plant materials that have been pretreated (for topical use ) ( cfu per gram)	For other plant material for internal use (cfu per gram)
Total viable Aerobic Count	–	<107	<105
Escherichia., coli	104	102	10g
Total Yeast & mould count	105	104	103
Total Enterobacteriaceae	–	104	103
Salmonella spp.	–	None	None
Staphylococcus aureus	Absent	Absent	Absent
Pseudomonas aeruginosa	Absent	Absent	Absent
Coliforms	Absent	Absent	Absent

 

Materials and methods

The drugs specified in different groups according to the need of analysis for authentication of the data generated.

The trial drugs are divided into two batches, one batch-A samples are kept in ordinary conditions of temperature, and humidity and second batch-B is kept in stability chamber under controlled relative humidity (75 and temperature (45^oC± 2⁰C).and Batch-C  are  control group. Each batch consisting four groups and each group contains four samples. Other than this one group containing four samples of freshly prepared Haritaki Vibhitaki, Amalaki and Triphala churna are analyzed in all above stated parameters as control group for both the batches, a total of 36 samples are subjected for analysis.

Anti microbial susceptibility testing can be carried out by-

Dilution method

Diffusion method

Diffusion method is done by two methods

Stoke’s method

Kirby – Bauer method

In routine laboratory modified Kirby-Bauer method is used as suggested by NCCLS (national committee for clinical laboratory services), USA (2000).

Material required for antimicrobial study and MIC determination are:²

Mueller Hinton agar (pH 7.2 to 7.4)³

Luria Bertani broth (pH 7.2)

Sterile distilled water

Bacterial stain

Escherichia coli ATCC 25992

Staphylococcus aurens                   

Salmonella Typhi

Salmonella Paratyphi A.

Salmonella Paratyphi B

Salmonella Typhimurium

Procedures

Taken a Mullar Hinton (M.H) plate and growth on the plate with particular organism

Pure bacterial isolates were grown on nutrient agar plate.

Bacterial suspension of 106 cfu / ml was plated spread on the dry plate of M.H.agar by cotton swab

Sample preparations in the volume of 10 ml were placed on the plate.

Then incubated for 18hrs 37⁰

Observation

On the day of manufacturing of Haritaki, Vibhitaki, Amalaki and Triphala churna the bacterial load was examined and it was found that all the four preparation are free from bacterial growth.

All the preparations were divided into control group Batch-C, trial batch-A, and batch-B, and then stored at room temperature and stability chamber respectively.

Then all the samples were further tested at the end 1 month, after 2 months, after 4 months, and 6 months.

No bacterial growth is seen in any of the samples examined on the above time   duration.

Results

Showing Analytical Specifications of Samples of Control Group Batch- C and Batch – A (Samples in ordinary conditions of Temperature and humidity)

Table 2

SN.	Name of Taste	Name of Batch
		Batch-C	Batch-B
		Hc  Vc   Ac     Tc	Group – I	Group – II	Group – III		Group – IV
			H1   V1  A1    T1	H2  V2    A2     T2	H3   V3   A 3   T3		H4    V4   A4    T4
A.         B.	Microbial load Total viable aerobic count Total Enterobacteriaceae Total fungal count	Nil  Nil   Nil    Nil   Nil  Nil   Nil   Nil   Nil  Nil  Nil     Nil	Nil    Nil   Nil    Nil   Nil    Nil   Nil    Nil   Nil    Ni    Nil    Nil	Nil   Nil    Nil    Nil   Nil   Nil    Nil    Nil   Nil    Nil    Nil    Nil	Nil    Nil     Nil    Nil   Nil    Nil     Nil    Nil   Nil    Nil     Nil    Nil		Nil    Nil   Nil     Nil   Nil     Nil    Nil   Nil   Nil    Nil    Nil    Nil
	Test for specific pathogens
	Escherichia coli	Absent	Absent	Absent		Absent		Absent
	Salmonella sp.	Absent	Absent	Absent		Absent		Absent
	Staphylococcus aureus	Absent	Absent	Absent		Absent		Absent

 

Showing, Analytical Specifications of Samples of Control Group Batch – C and Batch – B (Stability chambers – Temperature 45⁰C ± 2 & Relative Humidity 75% ± 5).

Table 3

Sl.No.	Name of Taste		Name of Batch
			Batch-C		Batch-B
			Hc  Vc  Ac  Tc		Group – I	Group – II	Group – III	Group – IV
					H5  V5    A5    T5	H6  V6   A6    T6	H7   V7     A7     T7	H8    V8  A8    T8
09	Microbial load Total viable aerobic count Total Enterobacteriaceae Total fungal count		Nil  Nil  Nil  Nil   Nil  Nil  Nil  Nil   Nil  Nil  Nil  Nil		Nil   Nil    Nil    Nil   Nil    Nil   Nil     Nil   Nil     Ni     Nil   Nil	Nil   Nil     Nil    Nil   Nil   Nil     Nil    Nil   Nil   Nil    Nil    Nil	Nil    Nil     Nil     Nil   Nil    Nil     Nil     Nil   Nil   Nil     Nil      Nil	Nil    Nil  Nil     Nil   Nil   Nil     Nil   Nil   Nil   Nil     Nil   Nil
	Test for specific pathogens
	Escherichia, coli	Absent		Absent		Absent	Absent	Absent
	Salmonella sp.	Absent		Absent		Absent	Absent	Absent
	Staphylococcus aureus	Absent		Absent		Absent	Absent	Absent

 

Discussion and Conclusion

Medicinal plant materials normally have a great number of bacteria and moulds. Often of soil origin while a large number of bacteria and fungi from the naturally occurring micro flora of herbs, aerobic spore forming bacteria frequently predominate. Current practice of harvesting, handling and production often causes additional contamination and microbial growth. The determination of E. coli and mould may indicate good production and harvesting practices. Specific pathogen (E . coli, Salmonella spp., S. aureus), total viable aerobic count, total Enterobacteriaceae and total fungal count were not seen in all batches of churna up to six months.

Acknowledgement

The authors are greatly thankful to Banaras Hindu University, Varanasi for the facilities provided to carry out the research work and financial support

Reference

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