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 5â7 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.
- Shwetaparpati : Reference: Siddha YogaSangraha, 11th Adhyaya, Ashmari-Mutrakrichraadhikar. Shweta-parpati is a white coloured parpati devoid of parad (mercury) and gandhak (sulphur).
- Sheetalparpati : SiddhaBheshajManimala. Sheetal-parpati is a sagandhparpati devoid of parad, and is mentioned in the 7thshloka of MutrakrichraChikitsa in Chaturth Guccha of the text.
The organisms taken for the study were cultured and isolated from the Dept of Microbiology, J. N. Medical College, Belgaum. The organisms collected were:
- Staphylococcus aureus (S. aureus)
- Escherichia coli (E. coli)
- Proteus mirabilis (Pr. mirabilis)
- Candida albicans (C. albicans)
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 :
Agar Diffusion Method :
- 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.
- 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.
- 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.
- Anti microbial activity evaluation methods:
- Agar diffusion method
- Agar dilution 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.
- No zone of inhibition seen for any organism.
- 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)
- Activity seen at very high concentrations in Agar Dilution method.
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 â 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 waysâ 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 â Vata, Pitta and Kapha. Thus, the Ayurvedic formulations have a different role to play.
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.
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.
**Sample A: Shweta Parpati
- TripathiK.D.,Essentials of Medical Pharmacology. 4th ed.Reprint, New Delhi:Jaypee Brothers; 2001.
- PanditDharmanandaSharma, RasaratnaSammuchchaya, 2nd ed.Varanasi:MotilalBenarasidas; 1999.
- YadavjiTrikamjiAcharya. Siddha Yoga Sangraha, 11th ed.Nasik:ShriBaidhyanath Ayurveda Bhavan ltd.; 2000.
- R Kaladhara Bhatt, LaxmiramSwami. Siddha BheshajaManimala, 2nd ed.Varanasi:Sri Krishnadas Academy; 1999.
- AmbikadattaShastry.SushrutaSamhita, 14th ed.Varanasi:Chaukhambha SanskritSansthan; 2003.
- K.R. Srikantha Murthy. AstangSangraha, Vol.III, 4th ed. Varanasi:ChaukhambhaOrientalia; 2005.
- K.R. Srikantha Murthy. AstangHridayam, Vol.II,Varanasi:Krishnadas Academy; 1995.
- KasinathaSastri, GorakhnathaChaturvedi. CharakSamhita, Vol.I, 5th ed. Varanasi:Chaukhambha Sanskrit Sansthan; 1977.
- Ghosh BN. Textbook of Pharmacology and Therapeutics, 2nded. Calcutta :Scientific Publishing Company;1969
- Nadkarni A K. Indian MateriaMedica, 3rd ed. Reprint Bombay : Popular Prakashan;2002
- KasinathaSastri. Rasatarangini, 11th ed. Reprint Varanasi : MotilalBenarasidas;2000
- Sharma P V. DravyaGunaVignan, Vol III, 12th ed. Reprint Varanasi:ChaukhambhaBharati Academy; 2002.
- Mishra Siddhinandan. Rasa Paddhati, 1st ed. Varanasi : ChaukhambhaOrientalia; 1987
- KavirajRamadarshsinh. RasendraVignanam, 1st ed. Varanasi:ChaukhambhaSanskrit Series Office; 1965.
- Subramanyam B V.Modi's Medical Jurisprudence& Toxicology, 22nded. New Delhi : Lexis NexisTripathi Publication; 2004
- UmeshwarPrasad. Economic Geology, 2nded. New Delhi : CBS Publishers Distributors; 2003
- Tuli G D, Bahi E S, Amba Prasad. Intermediate Inorganic Chemistry, 18th ed. New Delhi: S. Chand & Co. (Pvt.) Ltd.; 1974.
- Kenneth J. Anusavice. Phillip's Science of Dental Materials, 10th ed. Reprint, New Delhi: Harcourt India Private Limited.; 2002.
- Tipnis H P, Dhake A S. Inorganic Pharmaceutical Chemistry, 2nd ed.Nashik : Career Publications; 2002
- BhandariChandraraj. VanaushadhiChandrodaya, Vol.I& II, 11th ed. Varanasi : Chaukhambha Sanskrit Sansthan; 2003
- Gulrajsharma Mishra. Ayurveda Prakash, 1st ed. Reprint,Varanasi:ChaukhambhaBharati Academy; 1999.
- Gogte Vishnu Mahadev. Ayurvedic Pharmacology and Therapeutic Uses of Medicinal Plants, Mumbai:BharatiVidyaBhavan; 2000.
- Sharma Priyavrit, Sharma Guru Prasad. KaiyadevNighantu, 1st ed. Varanasi : ChaukhambhaOrientalia; 1979
- K R Kirtikar and B D Basu. Indian Medicinal Plants,Vol V & IX, 2nd ed. Uttaranchal : Oriental Enterprises; 2003
- Anne Waugh & Allison Grant. Ross &Wilson Anatomy&Physiology in Health& Illness, 9th International ed. Reprint. Edinburgh: Churchill Livingstone; 2003.
- Alphonso R Genero. Remington- The Science & Practice of Pharmacy, Vol.I, 20th ed. Reprint, B I Publications pvt. ltd.(for Indian edition); 2000
- Dennis L Kasper, Anthony S Fauci, Dan L Longo, Eugene Braunwald, Stephene L Hauser, J Larry Jameson. Harrison's Principles of Internal Medicine, Vol.I& II. 16th ed. McGraw Hill Medical Publishing Division; 2005.
- RamziCotran, Vinay Kumar, Tucker Collins. Robbins, Pathological Basis of Disease, 6th ed. Reprint. New Delhi: Harcourt India Private Limited.; 2001.
- Rajesh Bhatia, Rattan LalIchhpujani. Essentials of Medical Microbiology, 3rded. New Delhi: Jaypee Brothers; 2004.
- R. Ananthnarayan, C K JayaramPaniker. Textbook of Microbiology, 6th ed. Reprint. Chennai: Orient Longman Ltd.; 2001.
- E A Rawlins. Bentley's Textbookof Pharmaceutics, 8th ed. Reprint. Delhi: All India Traveller Book Seller; 2004.
- ParasharRadhakrishna. SharangdharSamhita, 4th ed. Nagpur:ShriBaidhyanathAyurveda Bhavan ltd.; 1994.
- BorkarDattoBallal.Saarth Rasa Chandanshu, 3rd ed. Pune:ShriGajanan Book Depot; 1983.
- J L N Shastry. IllustraredDravyagunaVijnana, Vol. II, 2nd ed. Varanasi : ChaukhambhaOrientalia; 2005
- Ronald A Sacher, Richard A McPherson. Widmann's Clinical Interpretation of Laboratory Test, 10th ed. Philadelphia: F A Davis Company; 1991.
- Elmar W Koneman, Stephen D Allen, William M Janda, Paul C Schreckenberger, Washington C Winn,Jr. DiagnosticMcrobiology, Copyright. Philadelphia: J B Lippincott Company; 1994.
- J Gerald Collee, Andrew G Fraser, Barrie P Marmion, Anthony Simmons. Mackie &McCartney Practical Medical Microbiology, 14th ed. Edinburgh: Churchill Livingstone; 1996.
- G R Chatwal. Pharmaceutical Chemistry â Inorganic, Vol. I, 2ndRevised ed. Bombay:Himalaya Publishing House; 1996.
Sample B: Sheetal Parpati
Agar Diffusion Method: