1. INTRODUCTION:

Kalmegh (Andrographis paniculata) is widely used in Indian and Chinese system of medicine¹. The plant is mainly used for liver diseases. Leaves are used in general debility, dyspepsia, stomach ailments in infants, griping. The roots are used as a tonic, stimulant and aperient. Whole plant finds its use as antispasmodic, febrifuge, stomachic, alternative, anthelmintic, anodyne, antiseptic, choleretic, cholagogue, diphoretic, expectorant, depurative, immunostimulant, laxative, astringent and antipyretic. They are used in the treatment of jaundice, diabetes, malaria, cholera, dysentery, enteritis, gastritis, pneumonia, pyelonephritis, hyperdipsia, flatulence, colic, diarrhoea, haemorrhoids and oedema.

Reports have shown that the plant possess hepatoprotective anthelmentic, antidiaarrhoeal, antimalarial, antiviral, antipyretic, antiatherosclerotic, hypotensive, immunomodulator, antifertility and other activities^2-4.

Non-standardised herbal preparation does not get accepted in the global market. It becomes wrong to assume that the biological agents are safe because they are natural.

The microbial content of herb is one of the most important parameters as they make the heel potentially dangerous for sensitive population. One of the major problems associated with herbs is its microbial contamination resulting in quality deteriorations^5-13. Inspite of substantial efforts to avoid microbial contamination, an upward trend in non-acceptance of herbals are reported. Several decontamination methods exist, but the most versatile treatment among them is the processing of herbs with gamma irradiation. Microbial decontamination of herbal products by gamma irradiation is a safe, efficient, environmentally clean and energy efficient process^14-25

Gamma irradiation at doses of 2-10 kGy has shown to be effective in eliminating pathogenic non-spore forming bacteria including other pathogens like Salmonella, Staphylococcus, Pseudomonas E.coli and others. With today’s demand for high quality herbs, gamma irradiation holds a promise for enhancing its safety and quality.

While exposure to gamma rays offers an effective alternative means of reducing microbial contamination, the dose increased to the extent of microbial kill has to be checked and also the method should be suitable to effectively reduce the microorganism and maintain this for a longer period.

Although gamma irradiation has been found as a suitable technique for microbial decontamination, there are no studies related to microbial decontamination of gamma irradiated Kalmegh. Accordingly, the objective of this study was to examine the effect on microbial contamination of irradiated samples of Kalmegh at 0, 6 and 12 months of storage.

2.MATERIALS AND METHODS:

2.1. Plant materials:

Whole plant of Kalmegh was procured from Natural remedies, Bangalore. The herb was identified and authenticated by Dr. Vasundhara, Professor. Department of horticulture GKVK, Bangalore and voucher specimen deposited. They were further ground to powder and kept at ambient temperature.

2.2 Preparation of extracts:

100 g of coarsely powdered samples were packed in 9 different sterile polythene bags at ambient temperature. Gamma irradiation was carried out at a commercial scale Cobalt 60 irradiation service facility loaded with Cobalt 60 source with strength of 398 kilocuries (KCi), owned and operated by Microtrol sterilization services Pvt.ltd. at Bangalore. The applied dose levels were 5 and 10 kGy and this absorbed dose was monitored with ceric/cerous dosimeters. Three packets were kept as such (K1- non-irradiated), three packs were gamma irradiated at a dose of 5 kGy (K2) and remaining three packets were gamma irradiated at a dose of 10 kGy (K3). 1^st set of sample (K1, K2& K3) were analysed soon after irradiation, second set was analysed after 6 months of storage and the third set was analysed after 12 months of storage.

2.3 Media for microbial studies: Total aerobic count was enumerated on nutrients agar (Hi media) and total fungal count on Sabourad’s dextrose agar. E.coli was tested on Macconkey agar and EMB agar, Selenite F broth and Brilliant green agar were the media’s for Salmonella, bismuth sulphite agar and certrimide broth were the selective medias used for determination of Pseudomonas and Staphylococcus aureus was tested on Mannitol salt agar and Vogel-Johnson medium. All the individual organisms were analysed as per the procedure given in IP²⁶.

2.4 Sampling and enumeration:

I. Total microbial load: 1 gm of each sample was suspended in 10 ml of respective medias. To determine total aerobic count the sample was incubated at 37⁰ +1⁰C for 24-48 hrs and for total fungal count – samples were incubated at 28+1ºC. Plates were examined for microbial growth, the number of colonies were counted and expressed in terms of colony forming units per gm (cfu/gm). Duplicate plates of appropriate dilutions were plated for specific organisms with there respective medias and incubated at 37º +1ºC for 24-48 hrs.

II. Identification test for Escherichia Coli:

1 gm of test substance was placed in a sterile screw-capped container with 10 ml of nutrient broth, shaken and incubated at 37⁰C for 18 to 24 hours.

a) Primary test: 0.1 ml of the enrichment culture was spread on MacConkey agar plate using sterile autoclaved spreader uniformly maintaining aseptic conditions, in laminar airflow cabinet. Plates were incubated at 37⁰C ±1⁰ C.

b) Secondary tests: 0.1 ml of the enrichment culture was spread on EMB agar plates using sterile autoclaved spreader uniformly maintaining aseptic conditions, in laminar air flow cabinet. Simultaneously 0.1 ml of enrichment culture was incorporated in tubes containing 5 ml of peptone water. Plates and tubes were incubated at 37⁰C±1⁰C, examined for the formation of metallic sheen in the agar plates and formation of indole (To test for indole add 0.5 ml of Kovac’s reagent, shake well, and allow to stand for one minute; if a red colour is produced in the reagent layer - indole is present). The formation of metallic sheen and indole in the secondary test indicates the presence of Escherichia coli.

III. Identification test for Salmonella:

1 gm of test substance was placed in a sterile screw-capped container with 10 ml of nutrient broth, shaken and incubated at 37⁰C for 1 hr (4 hours for gelatin).

a) Primary test: Added 1.0 ml of the enrichment culture to tubes containing 10 ml of Selenite F broth and incubated at 37⁰C for 24 hours. Tubes were observed for the microbial growth and for the colour of the broth. Microbial growth with no colour in tubes shows presence of salmonella.

b) Secondary test: Cultures from the preliminary test tubes were inoculated on to the brilliant green agar and bismuth sulphite agar plates. Plates were incubated at 37⁰C for 24 hr. Small, transparent and colourless, or opaque, pinkish or white (frequently surrounded by a pink or red zone) colonies on brilliant green agar and black or green colonies on bismuth sulphite agar confirms the presence of Salmonella typhi.

IV. Identification test for Pseudomonas:

a) Preliminary test: 1 gm of the sample was placed in a sterile screw capped jar containing 10 ml of Cetrimide broth

and incubated at 37⁰C for 24 hours. Subcultured on a plate containing a layer of Cetrimide agar and incubated at

37⁰C for 24-48 hours, examined for the growth by Gram’s stain.

b) Secondary test: Since the preliminary test for Pseudomonas was negative, the confirmatory secondary tests were not performed.

V. Identification test for Staphylococcus aureus:

1 gm of sample placed in a sterile screw capped jar containing 10 ml of nutrient broth and incubated at 37°C for 24 hours. Subcultured on a plate containing a layer of mannitol salt agar medium and Vogel-Johnson agar medium and incubated at 37ºC for 24 hours. Yellow colonies in mannitol salt agar medium and black colonies surrounded by yellow zones in Vogel-Johnson agar medium indicate the presence of Staphylococcus aureus.

3. RESULTS AND DISCUSSION:

Total microbial load and the effect of gamma irradiation on non-irradiated and gamma irradiated Kalmegh samples were evaluated and are shown in Graph 1.

Graph 1: Details of total microbial load in the Kalmegh samples at 0, 6 and 12 months of storage.

Before irradiation, total aerobic bacteria (1x10³cfu/gm) and total fungal count (1x10²cfu/gm) were quite high. On subjecting to specific pathogen tests as per IP, non-irradiated samples showed the presence of E.coli (3.4 x 10⁴cfu/gm) and Staphylococcus aureus (2.9x10⁵cfu/gm). Salmonella and Pseudomonas were totally absent in all the samples (Table 1). On gamma irradiating the samples at 5 kGy and 10 kGy, there was a drastic reduction in microorganism. In samples irradiated at a dose of 5 kGy initially there was reduction in total bacterial count from 1x10³cfu/gm to 1.3x10² cfu/gm and the total fungal count from 1x10²cfu/gm to acceptable levels. However, the specific microorganisms (E.coli and Staphylococcus aureus) were totally absent in samples K2 and K3. (Graph 2 and 3. Pictures of E.coli on Macconkey and EMB agar are shown in Photo 1 and 2 and pictures of Staphylococcus on Mannitol and Vogel Johnson media are shown in Photo 3 and 4.

Graph 2: E.coli in Kalmegh samples at 0, 6 and 12 months of storage.

Graph 3: Staphylococcus in Kalmegh samples at 0, 6 and 12 months of storage.

Photo 1 - E.coli on EMB agar in samples of Kalmegh.

Photo 2 - E.coli on Macconkey agar in samples of Kalmegh.

Photo 3 - Staphylococcus aureus on Mannitol

Photo 4 - Staphylococcus aureus on Vogel Johnson

Further after 6 and 12 months of storage, the total bacterial count (1.4x10³cfu/gm and 1.6x10³cfu/gm respectively) and total fungal count (1.2x10²cfu/gm and 1.3x10²cfu/gm respectively) were increased compared to 0 month analysis (Graph 1). Pathogenic organism during 6 and 12 months storage also drastically increased to - E.coli (3.4 x 10⁴cfu/gm and 3.5 x 10⁴cfu/gm respectively) and Staphylococcus aureus (2.9x10⁵cfu/gm and 3.0x10⁵cfu/gm respectively), where as gamma irradiated samples maintained their quality by showing no microbial contamination (Graph 2 and 3).

In samples irradiated at a dose of 5 kGy initially there was reduction in total bacterial count from and total fungal count to acceptable levels. However, the specific microorganisms -E.coli and Staphylococcus aureus were totally absent in samples K2and K3_.

This research clearly indicates that Kalmegh plant sample is highly contaminated. On testing for specific pathogens, Kalmegh samples were positive for presence of E.coli and Staphylococcus. Values exceeded drastically on storing it upto 12 months¹⁶. The high contamination level could be attributed to the natural microflora of the herb as well as the general conditions during cultivation, harvesting, drying, handling, processing, storage, distribution and sales²⁷. Gamma irradiation was found to be an effective technology for resolving technical trade issue (WHO 1994²⁸, FDA 2001²⁹) and thus increase the quality of Kalmegh. The killing effect of irradiation can be attributed to the ionization of water, which results in forming highly reactive radicals such as H, OH etc. These free radicals split carbon bonds of macromolecules such as DNA in living organisms, thereby killing them³⁰. They also destroy the chemical bonds by interacting with electrons of atomic constituents. Samples irradiated at 5kGy and 10kGy had significantly lower levels of microbes than the non-irradiated (control) even at 12^thmonth study³¹. 5kGy could significantly lower the microbes to acceptable levels, however complete sterility could be attained at a dose of 10kGy.

4. CONCLUSION:

A dose of 5kGy could significantly reduce the microbial load to acceptable limits. However, commercial sterility could be attained at a dose of 10kGy.

Gamma irradiated samples could maintain their stability upto 12 months from the time of irradiation, which clearly indicates that the shelf life of the products can be increased upto 12 months.

The effect of gamma irradiation on the elimination or control of microorganisms on Kalmegh powder is in agreement with that of Migdal et al³² according to whom an ionizing radiation of 10kGy could give satisfactory results pertaining to microbiological decontamination of medicinal herbs.

Thus, gamma irradiation at doses of 5kGy and 10kGy helps in extending shelf-life of Kalmegh plant samples, ensures its microbial safety and helps in overcoming quarantine barriers to international trade. Further, gamma irradiation has got the advantages of its Dosimetric release, where the products can be sterilized in its final pack and released to the market even without post sterility testing, its single exposure makes it economical and these products can compete in global market.

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Received on 07.04.2011 Modified on 10.04.2011

Research J. Pharm. and Tech. 4(6): June 2011; Page 982-986

Sl. No	Name of the sample	Pathogens tested
Sl. No	Name of the sample	*E. coli*	*Salmonella*	*Pseudomonas*	*Staphylococcus aureus*
1	K-1	+	-	-	+
2	K-2	-	-	-	-
3	K-3	-	-	-	-