ABOUT AYURVEDA

Since the dawn of history man has been in search of ways to find cure and relief from mental and physical illness perhaps small shrubs, herbs, trees & their leaves, stems, roots or bark were the first materials to be used by the prehistoric man to get rid of his painful diseases. Ayurveda is an ancient Indian Medicine system dating back to 5000 years. Treatment in Ayurveda has 2 components preventive and curative. Preventive aspect includes personal hygiene a regular daily routine, appropriate social behavior & use of rejuvenating materials food & rasayans. Curative aspect includes 1) drugs 2) diets and exercise 3) general mode of life. Atharvaveda is one of the most ancient veda which is encyclopedia of medicinal herbs. The veda contains detailed information on approx. 2000 medical plants & their uses.

List of some veterinary and human products of Indian Herbs. Veterinary Products 1. Himalayan Batisa 2. HB Strong 3. Rumbion Bolus 4. Timpol. 5. Himax 6. Livol 7. Galog 8. Galog Strong Bolus. 9. Prajana HS 10. 11. 12. 13. 14. Coiffecu Plus. Caflon Blaze Pesto Ban Replanata

Role of Microbiology in pharmaceutical industry. Microbiology assays of herbal drugs provide information on the hygienic environment of their production and handling, procuring efficiency defects due to microbial growth and the organisms responsible for food poisoning. Herbal drugs are susceptible to spoilage by action of inherited microorganisms or those which gain entry into the product during production, procurement and packing and grow at favourable temperature during storage. The pathogenic organism or toxins producing type gives rise to drug born infection. At Indian Herbs microbiological examination of herbs & veterinary medicines are performed to check the presence of the pathogenic microorganisms because they can be hazardous to living organisms. This project work is based on the identification of microflora of crude herbs/products, which are used in medicines manufactured by Indian Herbs. A complete microbiological process involves the identification of these seprate groups of micro organs in the herbal drugs. At Indian Herbs, analysis of the presence of bacteria in the crude herbal drugs is done in accordance with WHO method.

Composition of media Reagent and culture media used during test 1. Soyabean Casein Digest Agar (SCDA) (Hi Veg MV290) Ingerdients Pancreatic digest of Casein Papaic digest of soyabean meal Sodium Chloride Agar pH gm/lit. 15.00 5.00 5.00 15.00 7.3 + 0.2

use : - A general purpose medium used for cultivation of wide variety of microorganism. 2. Eosin Methylene Blue Agar (EMB) (MO22) Ingredients Peptic digest of animal tissue Diptoassium sulphate Lactose Eosin Y Methylene blue Agar pH gm/lit. 10.00 2.00 10.00 00.40 0.065 15.00 7.1 + 0.2

use:- Recommended for the isolation of members of Enterobacteriaceae.

3. Mannitol Motility Agar (MMA) (M770) Ingredients Peptic digest of animal tissue Mannitol Potassium nitrate Phenol red Agar pH gm/lit. 20.00 2.00 1.00 0.04 3.00 7.6 + 0.2

Use:- For studying mannital fermentation and motility of bacteria. 4. Violet Red Bile Agar (VRBA) (Hig Veg MO49) Ingredients Hiveg Peptone Yeast extract Lactose Synthetic detergent NaCl Neutral red Crystal violet Agar pH gm/lit. 7.00 3.00 10.00 1.5 5.00 0.03 0.002 15.00 7.4 + 0.2

Use : - Recommended for selective isolation, detection and enumeration of aerogene bacteria in water, milk and other dairy food products. 5. Brilliant Green Agar (BGA) (MO16) Ingredients Peptone Yeast extract Lactose Sucrose NaCl Phenol Red Brilliant green Agar pH gm/lit. 10.00 3.00 10.00 10.00 5.00 00.08 0.0125 20.00 6.9 + 0.2

Use:- For selective isolation and preliminary identification of Salmonella. 6. Triple Sugar Iron Agar (TSIA) (MO21) Ingredients Peptic digest of animal tissue Casein enzymic hydroxylate Yeast extract Beef extract Lactose gm/lit. 10.00 10.00 3.00 3.00 10.00

Sucrose Dextrose NaCl Ferrous Sulphate Sodium thisulphate Phenol red Agar pH

10.00 1.00 5.00 0.20 0.30 0.024 12.00 7.4 + 0.2

Use:- For differentiating members of Enterobacteriacecae. 7. Enterobacteriaceae Enrichment Broth (EEB) (M287) Ingredients Peptic digest of animal tissue Dextrose Ox bile (Purified) Disodium phosphate Monopotassium phosphate Brilliant green pH gm/lit. 10.00 5.00 20.00 6.45 2.00 0.0135 7.2 + 0.2

use:- for selective enrichment of Enterobacteriaceae in bacteriological examination of foods. 8. Sabouraud chloramphenicol Agar (SCA) (MI067) Ingredients gm/lt.

Casein enzyme hydroxylate Peptic digest of animal tissue Dextrose Chloramphenicol Agar pH

5.00 5.00 40.00 0.05 15.00 5.6 + 0.2

Use:- Recommended for selective cultivation of yeast & mould. 9. Yeast Malt Agar (YMA) (HiVeg M424) Ingredients Hi-Veg peptone Yeast extract Malt extract Dextrose Agar pH Use:- For the preliminary test of Salmonella and E.coli. 10. Buffer solution chloride phosphate solution (BSCPS) gm/lit. 3.56 7.23 4.30 1.00 gm/lt. 5.00 3.00 3.00 10.00 20.00 6.2 + 0.2

Ingredients KH2PO4 Na2HPO4 NaCl Peptone

Tureen 80 pH

0.001 0.1 7.0 + 0.2

Use:- For the preliminary test of Salmonella and E. Coli 11. Mac Conkey Agar (HiVeg MV081) gm/lit. 1.50 1.50 17.00 10.00 1.50 5.00 0.001 0.03 15.00 7.1 + 0.2

Ingredients Hi Veg peptone Hi Veg hydroxylate Hi Veg peptone No. II Lactose Synthetic detergent Sodium chloride Crystal violet Neutral red Agar pH

12.

Tetrathionate Brilliant green bile broth (M1255) gm/lit. 8.60 8.00 6.40 20.00 20.00

Ingredients Peptone Ox bile dehydrated NaCl CaCO3 Potassium tetrathionate

Brilliant green pH

0.07 7.0 + 0.2

use:- For isolation and identification of Salmonella. 13. Simmons Citrate Agar (M099) gm/lit. 0.20 1.00 1.00 2.00 5.00 0.08 15.00 6.8 + 0.2

Ingredients MgSO4 Ammonium dehydroxylate phosphate Dipotassium phosphate Sodium citrate NaCl Bromothymol blue Agar pH

Use:- For differentiating members of Enterobacteriaceae on the basis of citrate utilization. 14. Mac Conkey Broth (HiVeg MV007) gm/lit. 23.50 10.00 1.50 5.00 0.075

Ingredients Hi Veg peptone Lactose Synthetic Detergent NaCl Neutral red

pH Use:- For enrichment & enumerative of coliforms. 15. Urea Agar Base

7.4 + 0.2

Ingredients Peptic digest of animal tissue Dextrose NaCl Monok Phosphate Phenol red Agar pH

gm/lit. 10.00 1.00 5.00 0.80 0.012 15.00 6.8+0.2

Use:- Recommended for the detection of urea production particularly by Proteus.

Determination of Micro Organisms Medicinal plant materials normally carry a great of bacteria and moulds often of soil origin. While a large range of bacteria and fungi form the naturally occurring microflora of herbs, aerobic spore forming bacteria frequently predominate. Current practices of harvesting, handling and production often cause additional contamination & microbial growth. The determination of E.coli and mould may indicate good production and harvesting practices. Methods for decontamination are restricted. For e.g:- the use of ethylene oxide has been forbidden within the EC countries treatment with ionizing irradiation requires a special registration procedure or is also forbidden in some countries. In addition the presence of aflatoxins in plant material can cause health hazards if absorbed even in very small amount. Therefore, they should be determined after using a suitable clean up procedure.

Test for specific microorganisms The condition of the test for microbial contamination are designed in such a manner as to minimize accidental contamination of the material being examined & the precautions taken must not adversely affect any microorganisms that could be revealed. Recommended procedure 1. Pretreatment of the material being examined Depending on the nature of the crude medicinal plant material, grind, dissolve, dilute, suspend or emulsify the material being examined using a suitable method & eliminate any antimicrobial properties by dilution, neutralization or filteration. Water soluble materials Dissolve or dilute 10gm or 10ml of material, unless otherwise indicated in the test procedure, in lactose broth or another suitable medium proven to have no anti microbial activity inder the conditions of the test and adjust the volume to 100ml with the same medium (some material ma

require the use of a larger volume). If necessary, adjust the pH of the suspension to about 7. Non fatty materials insoluble in water Suspend 10 gm/ 10 ml of material, unless otherwise indicated in the test procedure, in lactose broth or another suitable medium proven to have no antimicrobial activity under the condition of test & dilute to 100 ml with same medium (some material may require the use of a larger volume). If necessary, divide the material being examined & homogenize the suspension mechanically. A suitable surfactant, such as 1 mg/ml solution of polysorbate may be added. If necessary, adjust the pH of the suspension to about 7. Fatty materials Homogenize 10 gm/ 10ml of material, unless otherwise indicated in the test procedure with 5 gm of polysorbate. If necessary heat tonot more than 400 C (A temperature of not more than 410 C, for the shortest possible time, might be required). Mix carefully while maintaining the temperature in a waterbath or in an oven . add 85ml of lactose broth

or another suitable medium proven to have no antimicrobial activity in the condition of the test, heated to not more than 40 0 C. maintain this temperature for the shortest time necessary until formation of an emulsion and in any case, for not more than 30 min. if necessary, adjust the pH of the emulsion to about 7.

2. Escherichia coli Transfer a quantity of the homogenized material in Mac Conkey broth containing 1 ml of the material being examined, prepared & incubated at 43 - 450 C for 18 24 hrs. prepare a subculture on a plate with Mac Conkey agar & incubate at 43 - 450 C for 18 24 hrs. growth of red, generally non mucoid colonies of gram ve rods. Sometime surrounded by reddish zone of precipitation, indicates the possible presence of E. Coli. This may be confirm by the formation of indole at 43.5 44.50 C. The material passes the test of no such colonies are detected or if the confirmatory biochemical reactions are negative.

Procedure for examination of E. Coli in crude herbal products. Collection of sample of crude herbal products. Preparation of sample buffer solution by addition of 10 gm sample to 90ml of phosphate buffer solution. 1 ml sample inoculated in Mac Conkey broth upto 10 5 dilution using serial dilution technique. Incubate all tubes at 43 - 450C for 18 24 hrs. If growth is positive.

Streak the culture from Mac Conkey broth on Mac Cronkey Agar, Eosin Methylone blue agar. Incubate it for 18 24 hrs at 43 - 450C If growth is positive Biochemical test (Indole production test) If indole test positive, adding Kovacs reagent, cherry red color. Ring formation E. Coli positive.

Result of biochemical test for E. Coli Indole test = +ve MR test = +ve VP test = -ve

Citrate test = -ve E.coli shows the following characteristics of TSI Butt yellow Slant = yellow Gas production = absent E. coli is gram ve bacilli, rod shaped. e.coli shos pinkish, mucoid colonies on Mac Conkey Agar & metallic shine on EMB agar. 3. Enterobacteriaceae and certain other gram negative bacteria. Homogenize (buffer solution), the pretreated material appropriately & incubate at 30 - 370C for a sufficient length of time for revivification of the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 5 hrs). shake the container, transfer 1 ml of the homogenized material to 9 ml of Enterobacteriaceae enrichment broth. Serially dilute it to further I. 6. incubate at 35 - 370C for 18 24 hrs. prepare a subculture on a plate with violet red bile agar with glucose & lactose. Incubate at 35 - 370 C for 18 24 hrs. the material passes the test if no growth of colonies of gram ve bacteria are detected on the plate. Procedure for examination of Enterobacteriaceae in crude herbal products. Collection of sample of crude herbal products.

Preparation of sample buffer solution by addition of 10 gm of sample to 90 ml of phosphate buffer solution. 1 ml sample inoculated in EE broth upto 106 dilution using serial dilution technique & incubate all tubes at 370C for 24 48 hrs. After 24 hrs. incubation, gas production is positive & color also change. Now streak a loopful culture from EE broth on violet red bile agar (VRBA) & incubate the plates at 370C for 24 48 hrs. If growth is positive, do the biochemical tests (1) (2) (3) (4) (5) (6) (7) TSI MM MRVP Urease LG Indole Citrate

4. Salmonella: Incubate the solution as described above at 35 - 370 C for 24 hrs as appropriate for enrichment. Primary test: Transfer 10 ml of the enrichment culture to 90 ml of tetrathionate bile brilliant green broth & incubate at 42 - 430C for 18 24 hrs. prepare a subculture on atleast two of the following 3 agar media: - deoxycholate

citrate agar, xylose lysine deoxycholate agar or brilliant green agar incubate at 35 - 370C for 24 48 hrs carry out the secondary test if any colony are produce that confirm to the description given in the table below. Secondary Test Prepare a subculture of any colony showing the characteristics as in the table below on the surface of triple sugar iron agar & using the deep inoculation technique. This can be achieve by the first inoculating the surface of the culture medium followed by a stab culture with same inoculating needle & inoculating at 35 - 370 C for 18 24 hrs. this test is positive for the presence of Salmonella if in the deep culture, but not in the surface culture. A change of color from red to yellow is observe & usually the formation of gas, with or without production of H 2S in the agar. Confirmation is obtained by appropriate biochemical & serological tests. Medium 1 Deoxycholate agar 2 Xylose Description of Colony citrate Well colonies Lysine Well developed, red with or without black centres. 3 Brilliant green agar Small, transparent & developed

deoxycholate agar

color less or opaque, pink or white

(frequently surrounded by a pink to red zone.

Procedure for examination of Salmonella in crude herbal products:Collection of sample of crude herbal products. Preparation of sample buffer solution by adding of 10 ml of sample in 90 ml of phosphate solution. Incubate at 370C for 24 hrs. Observe for gas production in huge amount after incubation. 10 ml of sample buffer after incubation. 90 ml of Tetrathionate broth (TTB) Incubate both broths at 42 - 430 C for 24 hrs. If growth is positive. Streak the culture from TTB on DCA agar/XLD agar. Incubate at 370C for 24 hrs. after streaking If growth is positive Biochemical test.

Result of biochemical test fro Salmonella 1. TSI test Slant Butt = = red

yellow +ve

Gas production =

Presence of black spots in the media (H2S produced) 2. Urease test = 3. Citrate test = 4. Indole test = -ve +ve not produced

Test for yeast and mould in herbal drug formulation (i) Total Aerobic Count (TVAC):Collection of sample of crude herbal products From pretreated material (10 gm in 90 ml of phosphate buffer solution) Make serial dilution from 102 to 105 Transfer 1 ml from 104 and 1 ml from 105 test tubes to petriplates marked as SCDA/104 and SCDA/105. Pour SCDA medium in both plates marked as SCDA/10 4 and SCDA/105 After solidification incubate these plates at 370C for 2 days. Observe the plates after 2 days of incubation. TVAC = No. of CFU X dilution factor.

(ii)

Total Mould Count (TMC) Collection of sample of crude herbal products. From pretreated material (10 gm in 90 ml of phosphate buffer solution) Make serial dilution from 102 to 105. Transfer 1 ml from 103 and 1 ml from 104 test tubes to petriplates marked as SCA/103 and SCA/104. Pour SCA medium in both plates marked as SCA/103 and SCA/104. After solidification incubate these plates at 260C for 5 days Observe the plates after 5 days of incubation TMC = No. of CFUX dilution factor.

Bacteriology of test organisms

Enterobacteriaceae enterobacteriaceae are bacterial flora of intestine and are gram ve bacilli that are either motile with pernicious flagella or non motile facultative aerobes, oxidase ve with relatively simple nutritional requirement non acid fast fermenting sugar with a variety of end production, reduce nitrate into nitrites from catalase. Among the enteric bacteria there are many strains pathogenic to humans, animals or plants. Bacteriology of different test enteric bacteria e.Coli:- It live only in human or animal intestine detection of E.coli in drinking water is taken as evidence of recent pollution with human or animal excreta. It is given ve rod shaped often motile, non capsulated. It is aerobic & facultative anaerobic growing on simple media. It grows promptly in the presence of bile salts in EMB it grows as pink coloured convex colonies. It ferments lactose, glucose, sucrose, maltose & mannitol forming acid & gas. Indole & methyl red is +ve, VP & citrate is ve, urease is not hydrolysed, H2S is not produced.

Citrobacter member of the genus citrober are motile citrobacter freundi is a rapid fermented of lactose s it corresponds to the organism knows to water bacteriologist as knows intermediate coliform bacilli intermediate formerly it was called Escherichia

freundi it is distinguished from ecoli by being cirate +ve kcn +ve h2s +ve and lysine decarboxylase ve s usually indole ve Culture they form pale yellow colonies on mac conkey s deoxycholate citrate agar. Biochemical analysis Indole test test for indole production-tryptophan an essential amino acid is oxidized by some bacteria by enzyme tryptophanase resulting in the formation of indole, pyruvic acid & ammonia. The indole test is perfomed by inoculating bacteria in trypton broth. The indole produced during the reaction of detected by adding kovass reagent which produced a cherry red reagent ring Tryptophon tryptophanase Indole + kovass reagent Techniques Inoculate the peptone water the inoculums to be identified. Incubate at 37c for 18 hrs. After incubation add 2 to 3 drops of kovacs reagent from the side of the bube. Methyl red and voges proskauer test The methyl red (mr) & voges proskauer (vp) tests are used to differentiate two major type of facultative anaerobic enteric bacteria that produce large amount of acid & those that produse the nature product like acetone as end product. Both these are indole + pyruvic acid + nh3 rose indole +h2o

performed simultaneously because they are physiologically related medium mr-vp broth. Opposite results are usually obtained for methyl red & voges proskauer test i.e. mr (+ve) vp (-ve). In these tests if an organisms produces large amount of organic acid, acetic acid, lactic acid & succinic acid from glucose the medium will remain red (+ve test) after the addition of methyl red a ph indicator (i.e. ph remaining below 4.4.)

Technique: Incubate the MR-VP both with the inoculums to be identified. Incubate at 37c for 18 hrs. After incubation add 5 drops of mr indicater of the test tube of each set & 12 drops of vp reagent 1 & 2-3 drops of vp reagent 2 to other set test tube. Observed the change in colour of methyl red for mr test & for vp test.

3. Citrate utilization test- Citrate test is used to differentiate among enteric on the basis of the ability to utilize citrate as carbon source.

The utilization of citrate depends upon the presence of an enzyme citrase produced by the organism that breaks down citrate to oxalic acid & acetic acid. These products are later converted to ayurvedic acid and carbon dioxide ezymatically as shows below-

Citric Acid + acetic acid

Oxalo acetic acid Pyurvedic acid + CO2

Inoculating the microorganisms into an organic synthetic medium like simmons citrate Agar, where sodium citrate is the only source of carbon and energy, performs the citrate test. Bromothymol blue is used as an indicator. When the titric acid is metabolized the carbon dioxide generated combines with sodium and water to form sodium carbonate and alkaline product which change the colour of the indicator from green to blue this constitute the positive test. CO2 + 2Na + H2O Na2CO3

bromothymol blue is green when acedic (pH 6.8 and below) & blue when alkaline (pH 7.6 and higher). Test procedure 1. Inoculate the surface of the slant with the inoculums to be identified. 2. Incubate at 370C for 18-24 hrs.

3. Positive results are identified by blue colour of the medium. In the negative results the original green color of the medium stays the same.

4. Urease test:- Urea is a major organic waste product of protein digestion in most vertebrates is excreted in urine. Some

microorganisms have ability to produce the enzyme urease. The urease is a hydrolytic enzyme which attacks the carbon & nitrogen bond amide compounds with the liberation of ammonia as shown below. H2N C = H2O H2N It is a useful diagnosis test for identifyinh bacteria especially to distinguish members of the genus. Proteus from gram ve pathogens. Urease test is performed by growing the test organism om urea both of agar medium containing the pH of the medium as the pH becomes higher the phenol red changes from yellow to red or deep pink color. Failure of the development of deep pink color due to no ammonia production is evidence of a lack of urease production by microorganism. Test procedure: 2NH3 + CO2

1. Inoculate the slope of media by secondary culture os the colony to be identified. 2. Incubate the tube at 370C for 18-24 hrs. 3. Observe the reddish colour in the medium after incubation which indicates the production of Urease by test organisms.

5. TSI test: Technique 1. Using sterilizing wire gently touch colony to be identified. 2. Stab the butt of TSI agar & streak the slant. 3. Incubate the tube at 370C for 18-24 hrs. 4. Observe for colour & gas production in the media. Interpretation 1. Slant Butt 2. Slant Butt 3. Slant Butt Yellow Yellow Red Yellow Red Yellow All Sugar Fermented Only glucose is fermented None of the

sugar fermented

Break in the media indicator gas production. Black spots in the media indicate production of h2s.

CONCLUSION
Use of herbs & various plants in the formation of herbal products of medicines in corning world wide including India. But a member of micro organisms are present in these herbs & plants which plays their role in causing disease in animal and human being. So it is necessary to check the crude products before it get released into the market for control of disease and the other hand if any type of contamination is found it should be dispatched & be treated again.