Investigation of Microbiological Aspects of Herbal Products Used In Ayurvedic Medicines and Estimation of Measurement Uncertainty

 

Mukund Nagarnaik¹, Arun  Sarjoshi²,  D.G. Kshirsagar³,  Pranjali  Linge⁴, Shital Bhore⁵ ,

Zeba Qureshi⁶ and  Girish Pandya⁷ *

¹Managing Director, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

²QC Technical Manager, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

³Sr. Scientist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

⁴Microbiologist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

⁴Microbiologist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

5Microbiologist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

⁶Microbiologist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

⁷Sr. Scientist, Research and Development Division, Qualichem Laboratories Pvt. Ltd, 4,North Bazar Road, Gokulpeth Market, Nagpur  440010

*Corresponding Author E-mail: pandyagh@rediffmail.com

Ayurveda is a traditional medicine used worldwide, consisting principally of herbs and regulated under various legislation in each country. The Ayurvedic products are perceived as safe by consumer because of natural ingredients, but their use is not free from risks. This study aimed to assess the microbiological contamination  of some herbal products commonly used in Ayurvedic medicine, produced in India. About 20  Ayurvedic drugs bought from  different Indian shops in the form of powder, tablets, capsules, syrup, cream were analysed for total aerobic bacterial count as well as total fungi in the form of yeast and mould..Some pathogens like E. coli has also been estimated. The Ayurvedic products tested are of some concern for consumer, especially those sold in herbalist’s shops and street markets. The risk seem to be not related to the original plant, but probably to contamination of the products by bacteria and fungi and possibly by chemical substances derived from the production process. Products of industrial origin seem to be substantially safe, suggesting that complying with standard controls procedures during production and distribution process leads to better product quality and safety. In this study attempt is also made to in estimating measurement uncertainty for microbiological analysis of  drug samples. Customers are increasingly demanding  from microbiological labs to provide MU results along with routine analysis  of their products .This could serve for al those engaged in similar analysis work. Microbiological  results suggest that the products of industrial origin, bought from city drugstores, were  less contaminated than those from local production available in  street markets.

 

 

 

INTRODUCTION:

India is known for its extraordinary richness of its flora, which has more than ten thousands species of herbal medicines. They are popular because of low side-effect^1, low cost ² and high level of acceptance by patients. The plant materials used as herbal drugs are mostly organic in nature. It provides nutrition to microorganisms and facilitates the multiplication of microorganism leading to contamination; deterioration and variation in its composition. This give rise to inferior quality of herbal product with no therapeutic efficacy. The quality of herbal drug also depends on many factors like environment, collection method, cultivation, harvest, post harvest processing, transport and storage practices.

 

Herbal medicine is the oldest form of health care known to the mankind. For this reason, Herbal Medicine has been used therapeutically all around the world, being an important aspect of various traditional medicine system^3,4. Presently 90% of the world population still relies completely on raw herbs and unrefined extracts as medicines. There is a major question regarding the safety, efficacy and quality of herbal drugs. WHO has taken steps in assessment of quality, safety and efficacy of medicinal plants ⁵. Thus, the storage, packaging and handling of herbs for various ayurvedic dosages is an important aspect of investigation. As the plant material varies in its percentage of alkaloids, its therapeutic efficacy also varies according to the different places of collection, different time of collection, environmental factors, and surrounding of the cultivation of a particular medicinal plant.Vegetable drugs used as such or in formulation, should be duly identified and authenticated and be free from insects, pests, fungi, micro organism, pesticide and other animal matter including animal excreta. These may be within the permitted and specified limit for heavy metals and show no abnormal odor, color, mould or other evidence of deterioration. The quantitative tests are carried out as per Ayurvedic Pharmacopoeia of India ⁶ Safety and efficacy also depends upon storage packaging and handling of raw material, quality control during the process of product of herbal medicine, and their  self life time and preservative used.

 

Since an ad hoc regulation is not available for herbal medicines, the microbiological quality of the products was evaluated by following the indications of the Aryuvedic  Pharmacopoeia of India for microbiological examination of oral non-sterile products. These criteria establish a limit of 10⁵bacteria and 10³fungi per gram or millilitre of product and the absence of Escherichia coli. The total viable aerobic count (bacteria and fungi) was performed as an index of overall microbial contamination, and the presence of E. coli was evaluated as an indicator of possible faecal contamination.

 

MATERIALS AND METHODS:

Samples

The Herbal drug samples were collected from the local drug stores in Nagpur city.  A total of 20 different raw herbals medicinal product samples were selected .All drugs samples were examined for their bacteriological profiles.

 

 

Bacteriological incubator model BTI24, and autoclave  model BTI20  from Biotech India , were used in the studies. Petridishes, pippettes, conical flasks were from Borosil, India.

 

Media and Reagents:

Protcose and peptone broth and soyabean casein digest agar, sabouraud dextrose chloramphenicol agar, peptone, disodium hydrogen phosphate, sodium chloride, and potassium dihydrogen phosphate were procured from Hi-Media, India.

 

Preparation of sample

1.      For total microbial count: Soyabean casein digest agar was used.

2.      For fungal count Sabaurand dextrose chloramphenicol was used.

 

10 g/10 ml of sample was weighed and placed in a sterile flask. 20 ml of sterile buffered sodium chloride peptone solution was added and shaken in a flask to mix the sample. Volume was made to 100 ml with the same diluents liquid( if necessary, serial dilutions of the sample in the sterile dilution tubes is performed). For both bacterial and fungal counts, pour plates were prepared by adding 1 ml of diluted sample in duplicate in sterile petri plates. Soyabean casein digest agar was poured in bacterial count plates and sabaurand dextrose chloramphenicol agar in fungal count plates. After complete solidification of media plates, bacterial count petridishes were incubated at 30 -35 ⁰C for 5 days and for fungal count petri dishes were incubated at 20 -25 ⁰C for 5 days. After five days of incubation numbers of colonies were counted for each dilution plate for both fungal and bacterial counts. The mean count and the number of CFU per g or ml were calculated as follows:

 

CFU / gm or ml =   Number of colonies (mean) x dilution factor/ Weight of sample

 

For selective cultivation of yeast and mould, sabouraud chloramphenicol agar was used. The combination of casein enzymic hydrolysate and peptic digest provides nitrogenous compounds. Chloramphenicol inhibits a wide range of gram- positive and gram – negative bacteria .The low pH favours fungal growth and inhibits contaminating bacteria. Peptone broth is used as a growth medium.

 

Presence of Escherichia coli

10 ml of each sample solution (corresponding to 1 g of original product) was used to inoculate 100 ml of nutrient broth, homogenized and incubated at 37°C for 24 hours. 1 ml was then transferred to 5 ml of MacConkey Broth and incubated at 37°C for 48 hours. Any changes in colour or gas production in the medium were recorded: change in colour from purple to yellowish and gas production suggests growth of E. coli or coliforms. The confirmative test for these were carried out as follows:

 

1.      0.1 ml of incubated maconkey broth was added to 5 ml of fresh sterile maconkey broth.This was incubated at 44 ⁰C for 24 hours and observed for any acid and gas production.

 

2.      0.1 ml of incubated maconkey broth was added to 5ml of peptone water. This was incubated at 37 ⁰C for 24 hours. After incubation 0.5 ml of kovac’s reagent was added and formation of red color ring on the broth surface was observed.

 

RESULTS AND DISCUSSION:

Microbial analysis was carried out as per standard procedure (Ayurvedic Indian Pharmacopoeia). It included total bacterial count, total fungal count, presence of Escherichia coli. Pure culture of Escherichia coli NCIM: 2068; ATCC: 11105), were obtained from NCIM Pune ⁷. The media used for the microbial limit test were from  HiMedia Pvt. Ltd. Mumbai.

 

The Herbal Medicinal Products (HMPs) selected for this study consisted of six (6) tablets, one (1) cream, one (1) capsule, six (6) syrup, five (5) powders. All the samples were within their self life at the time of investigation. All the products had their manufacturing and expiry dates stated.

 

Standard IS method IS 5402:2002  of colony counting  was adopted to count colonies ranging between 15-300 at two consecutive dilutions. Results of colonies counted at two different dilutions of 10^-4 and 10^-5 are summarised in Table 1. Seven plates were prepared at each dilutions respectively. The number of microorganisms per ml or per gm is calculated as follows:

 

 

Number of microorganisms (N) = ∑ C /  (n₁ + 0.1 n₂ )d  

Where ∑ C is the sum of the colonies counted on all the dishes retained, n₁ is the                          number of the dishes retained in first dilution, n_{2 is} the number of the dishes retained in second  dilution, and  d  is the dilution factor corresponding to first dilution. The total microbial count obtained is 11.8 x 10 ⁴ (Table 1).

 

Similarly microbial level of marketed herbal medicines in this study were analysed and the results are depicted in Table 2. Microbial counts for various herbal medicinal products ranged between  270 to 16526. Bacterial viable count was highly observed in syrup (between 15453 to 16526) followed by powder dant manjan( 12989 to 14933). In case of other herbal medicines in the form of capsules (7368),  cream for piles (8393), and tablets for liver (690 to 11122).  All the herbal medicines used in this study were orally consumed remedies in form of alcoholic spirits, and water-extracted remedies. In addition, none of the analysed marketed herbal samples had any form of food-based tests carried out by the manufacturers. The risk of the presence of microorganisms in a pharmaceutical product depends on finality of the use, its nature and its potential damage that may be caused to the consumers. Based on pharmacopoeia, the total aerobic microbial count of herbal medicine was not more than 100,000 cfu/g. Thus, the microbial load of marketed herbal medicine which is analysed in this study was within the acceptable limit. This study demonstrates the possibility of microbial contamination of  herbal medicines which are  sold in market for  human health  problems  It also takes  into consideration the  fact that  with increase use of herbal drugs in the society and with the poor quality control measures  by the manufactures and vendors could  leave a great question   mark on the safety of consumers’ health. India is a major country and plays a lead role in the production and proliferation of standardized and therapeutically effective herbal medicines. For this the herbal products needs to be assessed using standard norms and techniques.

 

 

 

 

Table 1.  Enumeration of microorganisms – colony count at 30 ⁰C

Dilutions for plating	10-4								10-5
Number of sterile petri dishes on dilutions	1	2	3	4	5	6	7	Mean	1	2	3	4	5	6	7	Mean
Observed counts in7 sterile plated	71	69	96	63	61	77	82	74.14	18	16	17	19	17	17	16	17.14
Number of colonies per gm/ml	= ∑ C / (n1 + 0.1 n2)d =  118 x 104
Incubation temp	30 oC
Incubation time	72 hrs
Total aerobic count	11.8 x 10 4

 

 

 

Table 2  Analysis of Herbal Medicinal Products for Total Microbial count, Total Yeast and Mould and E. coli.

S.No.	Therapeutic form	Scientific contents	Use	Total microbial count, Cfu/gm	Total Yeast and mould	E. coli
1.	Powder Ayurvedic Dant manjan	Azadirachata indica, Curcna loga, Terminalia balerica, Acacia varalrica, Caryophyllus arrmaticus, Cinnamomum camphora	Helps in relieve of dental problems.	12989,14793, 14933,13137,13916		Absent
2.	Syrup		Syrup	15453,16308, 16254,16526	Absent	Absent
3	Capsules	Momordica charautia, Ocimum sanctum, Ginger, Azadrachta indica, Picorhiza kurroa, pure Commphora mukul	Karnim	7368	64	Absent
4.	Cream	Siddha Tel 21% (containing: 12.5% ​​Calatropis procera Aloe barbadensis 8%, 8% Centella asiatica, Emblica officinalis 3% 3% Belere Terminalia, Terminalia chebula 3%, Zingiber officinalis 2.5%, Piper longum 2 5%, sodium chloride 2.5%, 2.5% Embelia ribes, Plumbago zeylanica 2.5%, Adhatoda vasica 2.5%, 2.5% Baliosperomum montanum, Argemone mexicana 2.5% ferrous sulphate 2.5% sesame oil extracted - qs), 5.5% glycerol, 5% stearic acid, glycerol monostearate 3.2% cetyl alcohol, 3% oil, 0.2% cedarwood oxide 0.05% zinc, 0.05% talc, borax 0.02% Lanolin 0.25%, 0.1% butyl hydroxytoluene, methyl 4-hydroxybenzoate 0.2% Potassium hydroxide 0.1%, Propyl 4-hydroxybenzoate 0.15% demineralized water	Helps relieve hemorrhoid crisis, superficial anal fissures, anal itching	8393,	31	Absent
5	Tablets	Eclipta alba , Phyllanthus niruri ,Mandur Bhasma , Rheum emodi, Capparis spinosa , Tephrosia purpurea , Picrorrhiza kurroa , Aloe barbadensis, Cichorium intybus , Boerhaavia diffusa  , Solanum americanum , Cassia occidentalis, Tamarix articulate, Tinospora cordifolia , Berberis aristata , Plumbago zeylanica , Emblica officinalis , Embelia ribes  , Terminalia chebula, Fumaria indica	Supports the normal functioning of the liver, Supports the natural regeneration of the liver cells, Stimulates appetite, digestion and metabolism	690,10006, 9497,9666, 11122, 10468	80, 78, 79, 81, 79, 72.	Absent
6	Syrup	Embelia ribes 200 mg , Butea frondosa - 200 mg , Picrorhiza kurroa - 100 mg ,• Cyperus rotundus - 100 mg, Ocimum basillicum - 75 mg, Swertia hirayata - 75 mg,  Apium graveolens - 100 mg,  Carum carvi - 50 mg , Holarrhena antidysentrica - 50 mg, Garder gummifer - 50 mg, Punica granatum - 50 mg	Syrup	270	0	Absent

 

 

Measurement of uncertainty for microbio­logical analysis

Measurement of uncertainty has been a regular requirement in physical and chemical analyses for many years but it is only recently that the subject has been addressed by microbiologists. Whilst the accepted concept is the measurement of the “level of uncertainty” associated with a micro­biological test, the recipient really wants to know the “level of confidence” which the microbiol­ogist can put on the particular result. Laboratory accreditation procedures, and both national and in­ternational definition and standardization of labo­ratory methods seek to define the level of uncer­tainty which can be ascribed to a series of tests.

 

Well-established written quality control proce­dures, validation and procedures for measurement uncertainty (MU) determination are prerequisite for accreditation in food and  drug microbiology laboratory. It also encourages participation in proficiency testing schemes.  Reference materi­als are far more difficult to produce in a stable form for microbiological examinations than for chemical analyses. For microorganism enumerations (quantitative methods), the testing performance of laboratories is assessed through the trueness and the precision which, respectively, express the agreement. It is also noteworthy to know that “Presence or absence” data are not easily amenable to statistical analysis to express repro­ducibility or repeatability.

 

The aim of this research is to describe quality control procedures and measurement uncertainty (MU) determination in our microbiology laboratory for qualitative and quantitative type of analysis of ayurvedic drugs. Our laboratory is accredita­ted according to the standard NABL 17025:2007. General requirements for the competence of testing and calibration laboratories, which guarantees the compliance with stan­dard operating procedures and the technical competence of the staff involved.

 

A bottom - up MU estimation was adopted ⁸. 1 ml of the sample was resuspended in 99 ml dilution blank analysed for total microbial count as per standard procedures.

The steps for approach to estimation of MU are:

 

1. The formula for calculating results are : CFU observed / volume analysed (ml) = CFU / volume

2. The sources that contribute to MU are:

·        Tolerance of pipette of 1 ml capacity  : 2.5%

·        Colony counting uncertainty as standard deviation of mean of all analysts at 10% and 95% confidence level

·        Batch to batch recovery: 85 ± 15%

·        It is assumed that organisms are uniformly homogenous in samples and distribution of single plate count follows Poisson distribution

 

3. The component relative standard deviation was estimated

·        Volume w_v  = 0.025

·        Counting uncertainty w_{c = 5%} = 0.05

·        Plating medium w_{m =}  Dividing standard uncertainty (15%) by mean (85%) = 0.17

·        Distribution w_D  using relation w_D² =1/z where z is average number of colonies observed, hence  w_D² = 1/11 x 10 ⁴

·        Holding w_H =0.05

 

4.  Combining relative standard uncertainties :

 

u_c =(w_v ²+w_c ²+ w_m ² + w_D² + w_H ² ) ^1/2 = [ ( 0.025)² +            ( 0.05)²+ (0.17)²+ ( 0.09x10^-4+((0.05)2 ] ^½

= 0.185 or 18.5%

Expanding the the combined relative standard uncertainties   at 95% level of confidence

              

U = u_{c (1.96) =(0.18) (1.96) = 0.43 =  43 %}

 

Mean =  11.8 x 10 ⁴x 100 = 11.8 x 10 ⁶ CFU / 100 ml

 

Expanded uncertainty at 95% level = 11.8x 10 ⁶ x 0.43 = 5.1  x 10 ⁶ CFU /ml

 

11.8 x 10 ⁴CFU Total count /100 ml ± 0.051 x 10 ⁴x 0.43 CFU Total count/ml at 95% level.

 

CONCLUSIONS:

Credibility of measurement data has never caught the public’s attention more than today. The key principle for quality and reliability of results is comparabil­ity between laboratories on a wider.  In order to be  competent and comparable, results needs to  be re­ported with a statement of measurement uncertainty (MU). Validation measures for various types of  HMDs for  microbiological analysis are still lacking in  standardization. Measurement un­certainty determination can thus be conducted based on validation data, and is also important part of quality control in microbiology laboratory. Different ap­proaches are possible, according to literature data, in each laboratory. This research describes only one of the possible solutions, and is based on practical experience of authors in fulfilling accreditation re­quirements.

 

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3.       WHO,Regulatory Situations of Herbal Medicines: A world wide review, 1998. World health Organization, Geneva,

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Received on 10.12.2014                    Accepted on 30.12.2014  

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