Microbial Quality of Some Herbal Formulations Used in Liver Disease

Manoj Kumar Pandey¹, Rajeev Kr Sharma² and Sneh Lata³

¹Indian Pharmacopoeia Commission , Ghaziabad, India.

²Pharmacopoeia Laboratory of Indian Medicine, Ghaziabad, India.

³M.M.H.College Ghaziabad, India.

ABSTRACT:

Herbal remedies are widely used for the treatment and prevention of various diseases and often contain highly active pharmacological compounds. These products have the potential of contamination with different microorganisms. This is due to raw materials contamination and unhygienic production conditions. In this study, microbiological qualities of some herbal formulations were examined. Ten herbal products as tablet, powder and capsule were procured from local market Ghaziabad, U.P., India. The products were evaluated for microbial contamination as per method given in Indian Pharmacopoeia (2010) microbial limit test for enumeration and identification. Total aerobic count showed that all products had more than 1.5×10³cfu/g and total fungal count 2 ×10²cfu/g. Isolation and identification of microbial contamination showed that some of the samples were contaminated with Salmonella sp. There was no evidence for contamination of the samples by Staphylococcus aurous, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. In conclusion, all the samples of herbal drugs evaluated did not generally meet the standards for microbial limits as specified in official monographs. Such products can adversely affect health status of consumers as well as the stability of the products.

KEYWORDS: Microbial quality, Microbial contamination, Herbal formulation, Indian Pharmacopoeia.

INTRODUCTION:

Herbal drugs are crude preparations of various kinds of medicinal plants. In other words, herbal drug is a dried medicinal plant, or any part thereof, such as leaf, stem, root, flower or seed. Herbal medicine has a long history, probably extending over 2000 years and is quite popular with many people crude drugs and herbal medicines play an important role in home health care, health improvement, as alternative medicine and materials for medical products in many countries¹. The microbial quality of pharmaceuticals is influenced by the environment and quality of the raw materials used during formulation. Some infections outbreaks have been associated with the use of heavily contaminated raw materials of natural origin. The incidence of micro flora in non-sterile medicines generally is indicated by the nature of the ingredients (whether natural or synthetic), the quality of the vehicle, the care and attitude of persons involved in their handling among others. Most raw materials for pharmaceutical products support some form of microbial growth, depending on the nutritive properties and moisture contents. Hence, dry powder or tablets are capable of undergoing some form of microbial spoilage or degradation. The more serious problem of microbial contamination of tablets is where there are no obvious signs of spoilage.

Hence, it is contents of all drugs and medicines, whether they are required to be sterile or non-sterile , the quality control of crude drugs has been at the discretion of each pharmaceutical company; therefore, microbial contamination level varies drastically from company to company. Currently, microbial contamination on crude drugs has become an issue and certain quality assurances have been sought from the good manufacturing practices stand-point. Therefore it is necessary to estimate the microbial contamination level on crude drugs at each manufacturing stage².

In India, the unscientific methods of collection, storage, transportation and congenial climatic conditions make the raw materials of herbal drugs prone to fungal infestations. The raw materials are collected using unscientific methods and are commonly exposed to many microbial contaminants³. The raw materials are often deteriorated by microorganisms before harvesting, and during handling and storage. There are reports on aflatoxin contaminated herbal raw materials imported from India. Due to detection of aflatoxin B1 in the black pepper procured from India, some foreign pharmaceutical firms have decided to re-evaluate the suitability of Indian black pepper samples for formulation of phytomedicines⁵.In Egypt, different medicinal plant samples imported from India were reported to be contaminated by different toxigenic strains of fungi. Some of the samples have been reported to contain aflatoxin B1 by more than 20 μg/kg, beyond the tolerance level fixed by WHO⁴. Such drugs would certainly be rejected in the global market. Despite several reports on fungal contamination and aflatoxin production on foodstuff, limited researches have been carried out on microbial contamination of drug-plant samples. In the present study an attempt has been done to found out the microbial contamination of herbal formulations used as a hepatoprotective.

MATERALS AND METHODS:

Materials:

All cultures media (fluid soybean-casein digest medium, soybean casein digest agar medium, sabouraud dextrose broth, vogel-johnson agar medium, manitol-salt agar medium, cetrimide agar medium, fluid lactose medium, mac-conkey agar medium, selenite-cystine medium, bluid tetrationate medium, brilliant green agar medium, bismuth sulfite agar medium, triple sugar-iron agar medium, sabouraud dextrose agar) and chemicals (potassium tellurite,glycerin, potassium iodide, iodine, brilliant green) were obtained from Hi Media Lab Private Ltd (Mumbai).

Microorganisms:

The indicator microorganisms used in this study were all from the American Type Culture Collection (ATCC) such as S. aureus ATTC 6538, E. coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella typhi ATCC 1943 and Candida albicans ATCC 10231. These cultures were collected from Indian Pharmacopoeia Commission, Ghaziabad, U.P., India.

Preparation of samples:

Ten different herbal formulations were collected from local markets,. Ghaziabad, U,P, India. All the selected herbal formulations are used as heptoprotective drugs. Source and brands of these preparations have not been disclosed.The type of dosage form; packaging and manufacturing dates are presented in Table 1. Handling of all the selected formulations for microbiological analysis was carried out according to standard procedures. All solid dosage form samples were powdered. A portion of each sample (10 g) in solid dosage form and 10ml of liquid preparations were dispersed in fluid soybean-casein digest medium to make 100 ml in the aseptic conditions, clean rooms, areas and equipments⁶, (USP 33,2010).

Inoculation of microorganisms for recovery study:

1 ml containing not more than 100 cfu of a 24-h broth culture of the indicator micro-organisms (S. aureus, E. coli, P. aeruginosa S. typhi and C. albicans) were added to the each samples (in fluid soybean-casein digest medium or sabouraud dextrose broth), then incubated for 48 - 72 h and were evaluated for microbial growth in comparison with the colony morphology of positive blank (culture medium plus related microorganism). Doubtful results were confirmed by subculturing on selective media.

Bioburden determination:

The collected samples of herbal products were subjected to the following examinations: total yeast and mould count,total aerobic viable count (TAVC) by plate and multiple tube methods and presence or absence of S. aureus, P. aeruginosa, E. coli, Salmonella sp. and C. albicans. 10g/10ml of each sample was suspended in appropriate medium. The total volume was adjusted to 100 ml by adding soybean-casein digest medium for detection of bacteria and sabouraud dextrose broth for detection of molds and yeasts. Aerobic bacterial colony counts were made by the pour plate technique on soybean casein digest agar. Plates were incubated in duplicate at 37°C for 48 – 72h. After incubation, the number of colonies was recorded for each plate. Arithmetic mean counts were derived from each item having from 30 to 300 colonies per plate. On the other hand multiple-tube method based on Indian Pharmacopoeia 2010 for detection of total aerobic count was carried out. Following the incubation period, by examining the tubes for growth, the most probable number of microorganisms per gram/per ml of specimens was expressed by reference to related table in IP 2010.

Media and isolation of pathogenic microorganisms:

To determine the presence of S. aureus and P. aeruginosa, 10g of each sample diluted to 100 ml by adding soybean-casein digest medium and then incubated. After growth, a portion of the medium was spread on the surface of vogel-johnson agar and manitol-salt agar for detection of S. aureus and of cetrimide agar medium for detection of P. aeruginosa. Fluid lactose medium was added to 10 g of each sample to make 100 ml for detecting E. coli and Salmonella sp. Fluid lactose enrichment were streaked on to differential Mac-Conkey agar plates while 1ml aliquots of the fluid lactose cultures were transferred into 9ml fluid selenite-cystine and fluid tetrationate, respectively, to detect Salmonella sp. These cultures were incubated at 35 ± 2°C for 12 to 24 h and were further sub cultured on the surface of brilliant green agar and bismuth sulfite agar media. The butt-slant tube of triple sugar-iron agar medium was used for identification of gram-negative rods colonies. On the other hand, 10 g of each sample were added to sabouraud dextrose broth to make 100 ml for detection of C. albicans. Sabouraud dextrose broth enrichments were incubated at 20 - 25°C for 7 days. The incubated samples were examined and cultured in sabouraud dextrose agar plus chloramphenicol (SDA + C). In cases where microbial growth was observed, the colonies were identified by germ-staining test and morphological characteristics were examined microscopically.

Table -1 Product information

Formulation code	Type of formulation	Date of manufacturing	Date of expiration
Cc1	Tablet	03/2010	02/2013
Cc2	Tablet	12/2010	11/2012
Cc3	Tablet	11/2010	10/2013
Cc4	Capsule	11/2010	10/2013
Cc5	Capsule	07/2010	06/2013
Cc6	Capsule	08/2009	07/2011
Cc7	Oral Syrup	01/2010	12/2012
Cc8	Oral Syrup	07/2009	06/2012
Cc9	Oral Syrup	09/2010	08/2013
Cc10	Powder	04/2010	03/2013

Evaluation of microbial quality:

Methods prescribed in Indian Pharmacopoeia were used to test microbial quality of these herbal formulations and were compared with WHO guidelines for standards of herbal preparations^7,14. Four specific pathogens viz. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella sp. were checked for their presence along with total aerobic bacterial count and combined yeast and mould count. According to Indian Pharmacopoeia 2010(General Chapter 5.9) standards values of the microbial limits should not exceed 10³/g for total aerobic bacteria, 10²/g for yeast and moulds, 1g is free from E. coli, Salmonellae, Staphylococci and Pseudomonas.

RESULTS:

The microbial levels of herbal formulations used in this study as depicted in Table 2. All of the samples had microbial contaminants. Microbial counts by plate method ranged between 1.5 ×10³ to 6 × 10³ cfu/g (Total aerobic count), 2×10² to 4×10² (Total yeast and mould count )and by multiple tube method was >1100 cfu/g in all samples. The presence of indicator organisms in the samples is reported in Table 2. On the basis of colony appearance, Salmonella was found to be present in formulation code (Cc5 Cc7 and Cc10) examined. The suspected colonies were transferred to the specific culture media as described in IP 2010 and the plates examined and compared with the colony characteristics listed in IP 2010. The morphologic characteristics of colonies on the surface of brilliant green agar medium and bismuth sulfite agar medium confirmed the presence of salmonella species. Also, the inoculated butt-slant tube of triple sugar-iron agar medium confirmed the presence of Salmonella species. The presence of S. aureus, P. aeruginosa, E. coli and C. albicans were not observed in any of the samples.

DISCUSSION:

The microbial levels of the samples forms used in this study were not acceptable. This finding demonstrated that raw materials of natural origin in these formulations had some initial microbial levels of contaminants which related to the growing and culture conditions of medicinal plants. Similar findings have also been obtained in an earlier study on the microbiological quality of some pharmaceutical raw materials⁸ .The microbial levels associated with these herbal dosage forms could be attributed to their source of origin and their nutritive values and low standard of processing. On the other hand, high total plate counts do not have any correlation with the presence of pathogenic microorganisms. The presence of bacteria in herbal solid dosage forms constitutes a health hazard, particularly with Salmonella species which are the causative agents of harmful diseases. The presence of these harmful bacteria might be due to the application of manure to fertilize farms from which medicinal plants have been harvested.

Table 2. Identification, isolation and microbial count of some herbal formulations.

Formulation code	Number of Organism
Formulation code	Total aerobic microbial count (cfu/g)	Total yeast and moulds counts (cfu/g)	Multiple tube counts (cfu/g)	E. Coli	Salmonella sp.	Staphylococcus aureus	Pseudomonas aureginosa
Cc1	2×10⁵	4×10³	>1100	-	-	-	-
Cc2	3×10⁵	2 ×10³	>1100	-	-	-	-
Cc3	1.5×10⁵	2×10³	>1100	-	-	-	-
Cc4	6×10⁵	2×10³	>1100	-	-	-	-
Cc5	6×10⁵	2×10³	>1100	-	+	-	-
Cc6	1.5×10⁵	4×10³	>1100	-	-	-	-
Cc7	2×10⁵	4×10³	>1100	-	+	-	-
Cc8	4×10⁵	4××0³	>1100	-	-	-	-
Cc9	2×10⁵	4×10³	>1100	-	-	-	-
Cc10	3×10⁵	4×10³	>1100	-	+	-	-

Animal manure and slurries may contain a wide range of pathogenic microorganisms such as salmonella species. These organisms may survive for extended periods of time in soil and thus, increase the risk of plant contamination. Moreover, in the absence of viable cells, microbial metabolites may be toxic ^9,10. Similar results were obtained with the herbal solid dosage forms of the powder samples contaminated by E. coli and Salmonella species and herbal tablets contaminated by E. coli ¹¹. According to WHO report (2002), Salmonella food poisoning is a major problem globally and has increased in incidence in many continents in the last 25 years. Salmonella can infect plants cells and successfully evade all the defense mechanisms of plants. This shows that cleaning the surfaces of raw fruits and vegetables, for example, by washing, are not sufficient to protect against food poisoning. Previously, the only known sources of infection were plants contact with contaminated water¹². But recent studies showed that the strain of bacteria known as S. typhimurium can also invade and multiply inside plant cells. It is already known that Salmonella can survive for up to 900 days in contaminated soils, which creates a rich source of infection for plant material. However with this study, the hazard of microbial contamination of herbal solid dosage forms during manufacturing to human health has been demonstrated.

CONCLUSION:

India has a great diversity in medicinal herbal resources. More than 70% of the Indian population uses herbal drugs for the various diseases, and the manufacture of these medicines is mushrooming. Traditional herbal medicine and their preparations have been widely used in India as well as aboard for many years. But there is only little industrial organization in India who carries out quality assessment on herbal drugs¹³. WHO has set specific guidelines for the assessment for the safety efficacy and quality of the herbal medicines as a prerequisite for the global harmonization¹⁴. Still very few Ayurvedic industries follows Good Manufacturing Practice(GMP) .

In view of the above facts it is advisable to treat plant drugs with non-toxic chemicals at various stages of storage and processing. However, it would not be proper to treat herbal raw materials with synthetic chemicals because of their adverse effects on the human systems.

Recently, some of the products of higher plant origin were successfully used as botanical antimicrobials against different agricultural pests. They have merit over the synthetic antimicrobials due their biodegradable nature and non-mammalian toxicity. Among traditional plant-based antimicrobials of Indian origin, the neem tree (Azadirachta indica) represents an outstanding example. In addition, the fungitoxic components of traditionally used plants, viz. Acacia nilotica and Lawsonia inermis have been recommended to be efficacious like many synthetic antimicrobials. Hence, the traditionally used medicinal plants and their components should be tested for their efficacy in protecting herbal raw materials as botanical preservatives. The essential oil of Cinnamomum camphora has been recently recorded for protection of raw materials from fungal as well as aflatoxin B1 contamination¹⁵. Such products of higher plant origin should be screened against microorganisms of herbal raw materials and their safety profile as botanical preservative should be recorded, so that the taste and odour of the treated raw materials are not adversely affected. The mode of their practical application should also be worked out. Some of these antimicrobials of plant origin, viz. gallic acid, eugenol, carvacrol, thymol, carvone, gingerol, allicin, carnosol, rosmanol, carnosic acid and thujone are products of different spices which have been traditionally used as food items by humans for a long time. Hence there would be no or least chance of any adverse effects on human systems if such plant products are recommended in post-harvest processing of raw materials. In addition to the antimicrobial nature, these products also possess antioxidant properties. Therefore, the storage of herbal raw materials in association with the scented plant chemicals of traditionally used herbs possessing antimicrobial, antiaflatoxigenic and antioxidant efficacy may enhance the shelf-life of the raw materials by inhibiting fungal and mycotoxin contamination as well as checking their usual deterioration due to lipid peroxidation. There is a need for a large-scale screening programme so as to recommend the antifungal and antioxidants of traditionally used plants in processing and storage technologies of herbal raw materials

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Received on 01.08.2011

Accepted on 31.08.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(5): Sept.- Oct.2011, 217-220