Antimicrobial Activity of Herbomineral Formulations with special reference to Shweta Parpati and Sheetal Parpati
Dr. Anuradha Patil*, Dr.M.S.Nagmothi and Dr.S. S. Vaidya
LRP Ayurvedic Medical College, Maharashtra,India
December 2013
Full Text

Abstract :
Urinary Tract Infection (UTI) is one of the most common medical problems all over the world. Mutrakrichra, as mentioned in Ayurveda is a disorder of the Mutravaha Srotas. Symptoms of Mutrakrichra are similar to those of UTI, which include muhurmuhu mutra pravritti (increase frequency, micturition), mutraalpata (oliguria), and mutrendriya shool (dysuria), and kandu (pruritis) along with mutra daaha (burning micturition) in case of fungal infections.There are vast number of chemotherapeutic agents and antibiotics for its treatment in the Modern Medical Science. In spite of these advances , treatment for UTI still remains unsatisfactory particularly in sub acute, chronic and resistant cases. There are many formulations prescribed for the same in Ayurveda for treating Mutrakriccha. Shweta-parpati and Sheetal-parpati are two such formulations which are effectively used in treating chronic as well as resistant cases of UTI. These two formulations were therefore selected for evaluation of their antimicrobial activity in organisms responsible for causing UTI. In vitro antimicrobial activity was checked using Agar diffusion method and Agar dilution method. Antimicrobial activity was not seen in both the study formulations. Considering the efficacy of these two formulations in treating Mutrakruccha probably there exist a different mechanism of action other than bacteriostatic or bacteriocidal effect which needs to be explored further. The results also points towards the importance of diagnosing and treating the diseases as per fundamental principles of Ayurveda based on the doshas – Vata, Pitta and Kapha.

Keywords :
Shweta-parpati, Sheetal-parpati, Mutrakrichra, Urinary Tract Infection (UTI), In vitro Antimicrobial Activity.
Introduction :
At a very early stage of the modern medicine, man developed a concept that contagious diseases were caused by invisible living things. As microbes are invisible to the unaided eye, definitive knowledge about them had to await the development of microscopes. Medical microbiology deals with the causative agents of infectious diseases of humans, their reactions to them and the methods of protection against such diseases. Many formulations in Rasashastra too have krimighna, jantughna properties. These properties can be correlated to the antimicrobial, antibacterial drugs of the modern era which are chemically synthesized.
Urinary Tract Infection is one of the common medical problems all over the world. While considering Mutrakrichra in context with modern system, it is often correlated with dysuria or painful micturition. However, in the modern medical system, dysuria is a symptom not a disease. On careful study and analysis, Mutrakrichra does not limit itself to dysuria or painful micturition, but is a disorder of the urinary tract. There are many chemotherapeutic agents and antibiotics for its treatment in the modern medical science. In spite of the availability of a wide range of drugs and increasing facilities for laboratory investigations the treatment for urinary tract infections still remains unsatisfactory particularly in sub-acute, chronic or resistant cases.
Infection may be precipitated by urinary obstruction due to prostatic enlargement, calculi or pregnancy. About 5–7 percent of women have been reported to have urinary infection without any symptoms. While infections of the lower urinary tract seem to be ascending infection caused by fecal coliforms, pyelonephritis is probably due to hematogenous infection. Acharya Sushruta has also mentioned that when the mutravaha srotas get obstructed, they cause enlargement of the basti, mutraavarodha and medhra sthabhdata.
There are various formulations prescribed for Mutrakrichra in Ayurveda. Shweta-parpati and Sheetal-parpati are two such formulations. The evaluation of the efficacy of these formulations with respect to their anti-microbial activity is intended in the following study.

Materials :
  1. Shwetaparpati : Reference: Siddha YogaSangraha, 11th Adhyaya, Ashmari-Mutrakrichraadhikar. Shweta-parpati is a white coloured parpati devoid of parad (mercury) and gandhak (sulphur).

  2. Sheetalparpati : SiddhaBheshajManimala. Sheetal-parpati is a sagandhparpati devoid of parad, and is mentioned in the 7thshloka of MutrakrichraChikitsa in Chaturth Guccha of the text.

Organisms :
The organisms taken for the study were cultured and isolated from the Dept of Microbiology, J. N. Medical College, Belgaum. The organisms collected were:

  1. Staphylococcus aureus (S. aureus)
  2. Escherichia coli (E. coli)
  3. Proteus mirabilis (Pr. mirabilis)
  4. Candida albicans (C. albicans)
Methodology :
Samples :
Shweta Parpati: It was prepared by melting Shudha Kalamisora, Shudha Kankshi and Shudha Navasadar in proportion 16:2:1 respectively and pouring over a flat surface.(Sample A)
Sheetalparpati:It was prepared by melting and pouring Shudha Kalamisora over a flat surface and then sprinkling Shudha Gandhak over it in the proportion 48:1(Sample B).

Antimicrobial Activity :
  1. Collection of microorganisms:The organisms required for the study were identified and isolated by standard methods(Streak Culture Method31))They were then confirmed by Staining reactions, colony morphology and other biochemical reactions.The organisms were then taken for culture.
  2. Culture method: Streak Culture Method Peptone water was inoculated with the respective organisms and the turbidity adjusted to 0.5 McFarland's standard.Nichrome resistance wire(24 SWG size) was charged with the specimen and transferred onto the surface of a well-dried plate.The inoculums were spread over a small area at the periphery and then distributed thinly over the plate by streaking it with the loop in a series of parallel lines, in different segments of the plate.The loop was sterilized and cooled between different sets of streaks.
  3. Preparation of different concentrations of Sample A & Sample B 10mg of sample was added with 1gm of Tween 80 and triturated to make a paste which was further diluted with 100ml distilled water to get a concentration of 100mcg/ml,known as Stock.Further dilutions were made to obtain concentrations of 1mcg /ml, 5mcg /ml, 10mcg /ml, 25mcg /ml, and 50mcg /ml.
  4. Anti microbial activity evaluation methods:
    1. Agar diffusion method
    2. Agar dilution method
Agar Diffusion Method :
Agar plates were prepared by pouring Muller-Hinton Agar (Himedia no M-173) in disposable Petri plates (90mm) to obtain a thickness of 4mm and kept for solidification. After solidification of agar, 6 burr holes of 4mm diameter were made by using a sterile cork borer. All the plates were kept for incubation at 370C overnight to rule out the possibility of contamination.Each of the plate was inoculated with a particular organism by lawn culture using standard methods.Burr holes were filled with 10mcl (1mcg, 5mcg, 10mcg, 25mcg, 50mcg &100mcg) of test solutions, A & B.The plates were then kept for incubation at 370C overnight. The readings for the zone of inhibition were noted on the next morning.

Agar Dilution Method :
Different concentrations of the Sample A(Shweta-parpati)&Sample B(Sheetal-parpati) were prepared by mixing 1gm, 2.5gm, 5gm, 7.5gm and 10gm of the sample with 100ml of Muller-Hinton Agar (Himedia no M-173) to obtain concentrations of 1%, 2.5%, 5%, 7.5%, 10% w/v respectively and sterilized by autoclave. After cooling to approx. 500 C, the media was poured in disposable Petri plates (90mm) to obtain a thickness of 4mm and kept for solidification. All the plates were kept for incubation overnight to rule out the possibility of contamination. Each of the plate was inoculated with a particular organism by Streak cultures using standard methods.The plates were then kept for incubation at 370C overnight. The readings were noted the nextmorning.

Results :
  1. No zone of inhibition seen for any organism.
  2. Both samples do not show any antimicrobial activity with either method even at the highest given human dose, which ranges from 5 to 10 ratti(625-1250mg)
  3. Activity seen at very high concentrations in Agar Dilution method.

Discussion :
Antimicrobial study was intended as the symptoms of Mutrakrichra are similar to those of UTI, which include frequency of micturition (muhurmuhu mutra pravritti), oliguria (mutraalpata), and dysuria (mutrendriya shool), and pruritis (kandu) along with burning micturition (mutra daaha) in case of fungal infections, the severity of which are determined by the dosha vitiated, predominantly.
The two reference antimicrobial tests are the macroscopic Agar Diffusion and Agar Dilution procedures. Agar dilution method is a means of determining MIC (minimum inhibitory concentration) of AMA versus bacteria, which are not very routinely used. Occasionally, a situation may warrant a request to microbiology laboratory for a non-routine test involving AMA. The response to such requests varies with the difficulty of the test procedure and the level of staffing and sophistication of the laboratory36.
Mueller-Hinton agar was selected for testing the isolates as it closely approximates the criteria for a reproducible medium. Most pathogens grow satisfactorily, and the medium has minimal inhibitory effect on sulphonamides.
The study was done by adopting two different methods, as the first one, Agar Diffusion Method, gave negative results. It was assumed that the drugs may act at higher concentrations; hence Agar Dilution Method was done.No growth of bacteria was seen in higher concentrations.
E. coli has an optimum pH of 6.5 and a growth range between pH 4.4 & 7.8. ;Yeasts and fungi generally have an acid optimum. Candida is tolerant of acid and not sensitive to any of the antibiotics; it thrives in its normal sites in the body when broad spectrum antibiotics restrain the growth there, of the normal bacterial flora38. Bacteria show a growth range between particular ranges of pH.Microorganisms, in common with other living organisms are very susceptible to changes in the acidity or alkalinity of the surrounding medium. This is important for their growth and survival. While many bacteria show vigorous growth within a fairly wide range of acidity or alkalinity, there are others that require this to be adjusted within narrow limits before multiplication takes place. Moreover, all microorganisms have a particular alkaline or acidic or neutral reaction at which growth is optimal.
There are 2 key points while considering antimicrobial susceptibility testing as prescribed in microbiology laboratories36. First, the published standards that exist for performance of susceptibility testing apply only to aerobic and anaerobic bacteria – methods for testing fungi, parasites and viruses have not been standardized, and the reproducibility of results from the same laboratory or the comparability of results from different laboratories cannot be assured. Second, test results for bacteria are obtained in a laboratory environment, which is very different from the host environment.
Antimicrobial activity is not seen in both the samples at the given highest human doses.
Antibiotic susceptibility tests are intended to be a guide for the clinician, not a guarantee that an AMA will be effective or ineffective in therapy.We can only assume that laboratory results are predictive of clinical response, an assumption that clearly does not always hold36.
Inorganic antimicrobial agents act in one of the 3 ways– oxidation, halogenation and protein precipitation. These are the primary chemical reactions which kill the microbe or inhibit its growth. While antibiotics act at specific sites in the microbes, the inorganic antimicrobials have a nonspecific action and in high concentration, may affect microbial protein19.
Clinical and Microbiological confidence in laboratory testing is not always supportable by available scientific and clinical information.Patients may not respond to what should be the appropriate treatment, or they may recover without therapy38.
The general method to diagnose and treatment of the diseases in Ayurveda is based on the doshas – Vata, Pitta and Kapha. Thus, the Ayurvedic formulations have a different role to play.

Conclusion :
Based on the observations,it is felt that the action of the formulations may be very different in the host environment than the laboratory environment, which may be the reason for the results to be insignificant regarding their antimicrobial activity. It therefore leaves a scope for in vivo studies regarding the action of these formulations

* Asso. Prof., Dept. of Rasashastra&Bhaishajya Kalpana,Islampur,Maharashtra,India.
Acknowledgements :
Authors are grateful to the Management and staff of BMK Ayurved Mahavidyalaya,Belgaum and JN Medical College,Belgaum for providing the necessary facilities and infrastructure required for conducting the studies.

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**Sample A: Shweta Parpati
Sample B: Sheetal Parpati

Agar Diffusion Method:

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