msc project presentation 29 march

8/2/2019 MSc Project Presentation 29 March

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EFFECTIVE NATURAL THERAPY USINGSOME COMMON WILD HERBS AGAINST

STAPHYLOCCAL MASTITIS IN CATTLES.

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Presented by: Miss Reetibala L. BhagadkarGuided By: Prof. D. A. ChouhanDepartment of MicrobiologyD.B. Science College, Gondia.


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Drug resistance is a common phenomenon seen in pathogenicmicroorganisms. Excessive and unregulated use of antibiotics in

therapeutics is a one of the important cause.

Traditional uses of plant remedies are familiar to most peoples. The

way in which information about herbal use has been passed between

generations has been generally empirical and unquestioning.

Mastitis is considered to be the most costly disease of dairy animals

worldwide.

The control of mastitis becomes difficult in prevailing circumstances.

The major constraints include small herd size, low level of farmerseducation and lack of milk quality premium incentive etc. In the

absence of any mastitis control program and presence of high antibiotic

resistance, searching for new cost effective treatment line is essential.

BACKGROUND :


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MASTITIS

Mastitis , the inflammation of mammary glands, is a majorproduction limitingdisease of dairy animals all over the globe. It

not only reduces milk quantity but also impairs quality thus

adversely affecting milk production economics (DeGraves and

Fetrow, 1993; Sordillo et al., 1997) Infectious agents: Although, a wide variety of microorganisms

are associated with infectious mastitis, yet the bacteria are

predominantly important. Among bacteria, the most frequent

mastitogens include Staphylococcus aureus (S. aureus),Streptococcus agalactiae (Str. agalactiae), Escherichia coli (E.

coli), Corynebacterium pyogenes (C. pyogenes), Streptococcus

dysgalactiae (Str. dysgalactiae) and Streptococcus uberis (Str.

uberis) etc. OUT OF WHICH S.aureus is predominant.

INTRODUCTION:


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Mastitis

About two decades ago, worldwide losses due to mastitis have

been estimated as 35 billion dollars annually (Ratafia, 1987). In

USA, economic losses estimated to be $200 per cow per year

due to decreased production, discarded milk, treatment cost,

veterinary fees, labor costs and increased replacement costs(Smithand Hogan, 2001).

Mastitis is one of the major causes of economic loss in dairy

cattle. Staphylococcus aureus is the main pathogenic species

causing the subclinical form of mastitis. This type of udderinfection impairs alveolar function, reduces milk yield and has a

deleterious effect on milk composition, including increased milk

somatic cell count (SCC) (Gudding et al., 1984; Nickerson,

1989).

INTRODUCTION: cont


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Mastitis

Despite significant advances in mastitis control over the past 5

decades S. aureus is still an important cause of udder infection in

dairy herds (Berkema et al., 1998; Dingwell et al., 2003; Fox

and Gay, 1993; Sargeant et al., 1998; Wilson et al., 1997).

Being contagious in nature, the pathogen may be easily

transmitted within herds (Fox andGay, 1993; Lam et al., 1996).

Intrammamary infection (IMI) with S. aureus resulted inreduced

milk production and deterioration of milk quality both in termsof milkconstituents and increase somatic cell count (SCC).

INTRODUCTION Cont.


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HERBAL MEDICINES

As one would expect herbal medicine is widely used for thetreatment of animals

The use of herbal treatments for mastitis is limited by convention,regulation and lack of qualified advisers. However there appears tobe strong interest from a small but significant part of the dairyindustry for reliable advice and products.

The world wide trend continues to be one of increasing importanceof herbal medicine. Total worldwide sales are hard to measure butin 2001 were estimated at $16-20 billion (Journal of Nutrition.

2001;131:1120S-1123S) More reliable figures are available from the US where sales grew

from $2 to $4.4 billionover the period 1994 to 2005. (Ferrier et al.Nutrition Business Journal 2006).

INTRODUCTION Cont.


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There are a large number of complementary medicines

available to treat mastitis and many opinions about which is

the most effective.

Primary objective of study is to evaluate occurrence of drug resistanceamong the clinical isolates of Staphylococcus aureus from mastitic

cattles.

This study was extended for evaluation of antibacterial potential of some

commonly occurring wild herbs against these antibiotic resitistant

pathogens.

OBJECTIVE OF STUDY


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OBJECTIVE OF STUDY

Fine objectives were

Collection of clinical specimen from mastitic cattles

Isolation and identification ofStaphylococcus aureus.

Studies on occurrence of Antibiotic resistance amongClinical isolate (Staphylococcus aureus).

Screening of antibacterial potential of wild herb extracts

against clinical isolates (Staphylococcus aureus).

Phytochemical study of effective wild herb extract.

Determination of MIC


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METHODOLOGY

Collection and Identification of Plant materials Phyto-chemical analysis Isolation and identification of S. aureusfrom mastitic

cattles:

Antibiotic susceptibility testing.

Extract preparations: Aqueous and organic

Evaluation of Antibacterial activity of plant extracts: Welldiffusion method.

Determination of minimum inhibitory concentration (MIC): Determination of effect of exposure time.


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Isolation ofS. aureus from mastitic cattles: A loop full of each

clinical specimen was inoculated Baired Parker agar by four waystreaking method. The inoculated plants were incubated at 370C for24 to 48 hours acerbically. After completion of incubation,macroscopic, microscopic, examination of colonies on plant wascarried out, and Black colored colonies from BPA selected. Selected

colonies were sub-cultured on appropriate solid culture media forpurification. They were later subculture on nutrient agar slants andstored at 40C for further analysis.Identification of clinical isolates: Identification of isolates carriedout on basis of morphological, cultural and biochemicalcharacteristic. Each isolate subjected to morphological examinationlike Gram staining, motility test (hanging drop method), culturalcharacteristic on appropriate media, biochemical test like sugarfermentation test, IMViC test, Coagulase test, Nitrate reduction test,

Urease test, Catalase test etc.

METHODOLOGY cont..


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Antibiotic susceptibility testing: All isolates were subjected to analysis for

susceptibility or resistance towards available antibiotics of choice. Thechosen antibiotics were Amoxicillin, Ciprofloxacin, Gentamycin,Chloramphenicol, Ceftriazone, Cotiramaxazole, Penicillin, Tetracycline,Carbenicillin, and Kanamycin.

Antibiotic susceptibility of different isolates was tested using Kirby Bauer

disc diffusion method.

The size of the zone of inhibition was interpreted by referring zonediameter interpretive standards of the NCCLS/M 100, S12 performancestandard for antimicrobial susceptibility testing.

Measurement of multiple antibiotic resistant (MAR index)MAR index of each isolates was calculated as recommended by Krumpreman

No. of antibiotic to which test isolatesShowed resistance

MAR index of isolates = -----------------------------------------------------------No. of antibiotic used in studies

METHODOLOGY cont..


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Extract preparations

Aqueous extract: To obtain the aqueous extracts, dried and finely powdered

leaves wild herbs were weighed about 30 grams each and homogenizedusing 150ml of water. They were added to Soxhlet apparatus and the boiling

point of water was set up at 100C. The water evaporates continuously and

was recycled, thereby extracting the compounds present in the samples.They were continuously extracted until the solution looses the color.

Organic Solvent extract: To obtain the solvent extracts, dried and finely

powdered leaves of wild herbs were weighed about 30 grams each and

homogenized using 150ml of 70% organic solvent. They were added

Soxhlet apparatus set up at boiling point of solvent. The solvent wasrecycled, thereby extracting the compounds present in the samples. They

were continuously extracted until the solvent loses its color. The extract was

then transferred to a sterile Petri dish and kept for evaporation of acetone at

room temperature. Residues of extracts were collected and stored in the

refrigerator

METHODOLOGY cont..


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Phytochemical analyses

Each test sample was subjected to phytochemical analysis to detectpresence of tannins, alkalosiss and Flavanoids.

Tannins: 70 mg of pulverized plant maternal was dissolved in

one ml of distilled water and was filtered. 2ml of the filtrate was

added to 2ml of FeCl3 , blue black color indicates presence of

tannins.

Alcaloids:70 mg of plant material was dissolved in 1ml methanol

and was filtered. 2ml filtrate was added to 1% HCl and was

heated .One ml of filtrate added to 6 drops of Mayors reagent

resulted in a cream colour patch which indicates presence ofalkaloid.

Flavonoids: 70 mg of plant material dissolved in 10 ml ethanol

and was filtered. 2ml filtrate was added to Conc. HCl and add

Magnesium ribbon .Pink tomato red color indicated the presence

of Flavanoids. (Parke at al.2005)

METHODOLOGY cont..


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Antibacterial activity of plant extracts: Well diffusion method

Antibacterial activities of the aqueous and organic extracts of leavesof different wild herbs were tested using well diffusion method.100g of the sterile aqueous and organic extracts of each plant wereadded to each well.The diameter of inhibition zones were measured in mm and the

results are recorded.

Determination of minimum inhibitory concentration (MIC):The determination of minimum inhibitory concentration of the ethylacetate extracts ofTridex procumbens leafwas carried out using the

broth dilution methoddescribed by Lajubutu et al. Differentconcentrations of the extract were prepared to give a finalconcentration in the range of0.05 to 0.50. mg/ml.The lowest concentration inhibiting growth was regarded as the

minimum inhibitory concentration of the extracts.

METHODOLOGY cont..


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Effect of exposure time on (MIC) :The determination of minimum inhibitory concentration of the ethyl acetateextract ofTridex procumbens leafwas carried out using the broth dilutionmethod described by Lajubutu et al. Different concentrations of the extractwere prepared to give a final concentration in the range of 0.1 to 0.50

mg/ml.Different exposure time is allotted in study is 30 minutes, 60 minutes and120 minutes.

The lowest concentration inhibiting growth was regarded as the minimum

inhibitory concentration of the extracts for respective exposure time.

METHODOLOGY cont..


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RESULTS AND DISSCUSSION

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Isolation and identification ofS. aureus from mastiticcattles:

10 clinical specimens were tested for presence ofS aureus. All10 samples were founded to be positive for bacterial pathogens.Total 20 S. aureus strains were isolated from these clinical

specimens and identified on the basis of morphological,Biochemical and cultural characteristics.


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Antibiogram of clinical isolates:

All 20 strain tested were multiple antibiotic resistant. The antibioticsused during study were Ampicillin (10 mcg), Amikacin (10 mcg),Amoxicillin (10mcg) Co-trimaxozole (25 mcg), Ceftizidime (30 mcg),Gentamycin (10 mcg), Imipenem (10 mcg), Kanamycin (30 mcg),Nalidixic acid (30 mcg), Methicillin (5 mcg), Vancomycin (10 mcg)and Penicillin (10 mcg).All Staphylococcus aureus strain isolate found to be resistance toPenicillin, Amoxicillin, Methicillin, Ampicillin and Ceftizidime.15% strains were found to be resistant to Vancomycin & Nalidixic

acid, 20% strain resistance co-trimaxozole, 30% strain resistant toKanamycin, Gentamycin and Amikacin. Most of the strain wasshowing MDR (Multiple Drug resistance). S13, S4 strain sowingMDR index 0.75. Most of the strain showed MDR index greater than0.6.



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Sr.

No.

Antibiotics No. of isolates %

Tested Resistan

t

Sensitive Intermediat

e

R S I

1 Amoxicillin 20 20 00 00 100 00 00

2 Kanamycin 20 00 17 03 00 85 15

3 Amikacin 20 00 19 01 00 95 054 Ceftizidime 20 20 00 00 100 00 00

5 Ampicillin 20 20 00 00 100 00 00

6 Imipenem 20 05 13 02 25 65 10

7 Gentamycin 20 00 19 01 00 95 05

8 Nalidixic acid 20 10 10 00 50 50 00

9 Cotrimaxazole 20 03 16 01 15 80 05

10 Methicillin 20 17 00 03 85 00 15

11 Vancomycin 20 10 10 00 50 50 00

12 Penicillin 20 20 00 00 100 00 00


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Phytochemical analyses of wild herbs:

Sr.

No

Plant Material Alkaloids Flavanoids Tannins

1. Tridex

procumbens L

+++ ++++ ++

2 Solanum

xanthocarpum

+++ ++++ ---

3 Wild neem +++ ++++ ++


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According to world health organization report in 2000.Staphylococcus aureus is multiple drug resistance in the manyregion of the world. reported prevalence rate indicates the wildvariation exist regionally and from herd to herd. reported more

than 50% staphylococcus isolates resistance the high penicillinresistance. The wild use of intra-mammary preparationcontaining combination and browd spectrumantibiotics.(Ebrahimi A et al 2007)

Ebrahimi A et al 2007reported that 78.5% isolate were

resistance to Amikacin, 50% resistance to Penicillin, 50%resistance to tetracycline, 42.8% resistance to ampicillin, while100% susceptible to Ciprofloxacin, Carbenicillin andGentamycin.



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According to Deluyker et al 2005 reported Erythromycin andMethicillin in in-effective against Staphylococcus aureus.According to Deluyker et al 2005 broad range of antibioticssuch as Tetracycline Penicillin and Pirlimycin shows only 15

13% cure rate in mastitis cause by Staphylococcus aurous.Reason for this low success rate include1. Poor penetration of antibiotics into areas of scarring and

inflammation2. Inactivation of antibiotics by milk and serum components3. Intracellular or metabolically inactive organisms bacterial

L-forms resistance to antibiotics and improper treatmentprocedures.



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Antibacterial activity of selected herbs:

In this study different extract ( Aqueous, methanolic, Ethanolic and ethylacetate) of different wild herbs were screened for their antibacterial potential.Antibacterial activities of different extracts were summarized in Table 5.7, 5.8,5.9, 5.10. Results obtained suggested that ethyl acetate extract of Tridexpcocumbens leaf was more effective than Aqueous and methanolic extract

against all clinical isolates tested.It is also suggested that methanol extract ofMentha arvensis leaf was moreeffective than its Aqueous and ethyl acetate extract in inhibiting Staphylococcusaureus. In comparison Tridex procumbens leaf extract in ethyl acetate solventwas more effective among all wild herb tested for screening there anti-

Staphylococcus potential.Sharma B. and Kumar P., 2009 Studied antimicrobial potential of Tridexprocumbens and reported that Tridex procumbens has broad spectrum ofantimicrobial activity due to free and bound Flavanoids. Antibacterial potentialdifference extract of some wild herbs against multi drug resistanceStaphylococcus aureus from mastitis cattles was evaluated by Kirby Bauerwell diffusion method.



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Antibacterial activity of selected herbs: Result of the presence study

showed that out of 16 different extract tested ethyl acetate extract ofTridex procumbens leaf inhibiting effectively growth of Staphylococcusaureus isolates from mastitic cattles. Bioactive potential of this extract isdue to presence of Flavanoids. Several works Ilic et al 2004 alsoreported bioactive potential of Flavanoids. Samy and Ignalimupthu

1999, Taddy et al 2000 and Sanchiz et al 2005 demonstrated presenceof Flavanoids in Tridex procumbens.Tridex juices are effective against Bacillus subtilis, Staphylococcusaureus, E. coli, Pseudomonas areuginosa and is known for is woundhealing properties. (Dhar et al 2003).



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Sr. No Strain Inhibition zone (mm) Activity index

Gentamyci

n

EE ME EaE AE EE ME EaE AE

1 Sa1 16 00 12 13 00 0.00 0.75 0.81 00

2 Sa2 20 00 00 11 00 0.00 0.00 0.55 003 Sa4 16 00 12 13 00 0.00 0.76 0.81 00

4 Sa8 18 00 11 00 00 0.00 0.61 0.00 00

5 Sa10 24 00 11 00 00 0.00 0.45 0.00 00

6 Sa11 23 00 12 11 00 0.00 0.52 0.47 00

7 Sa13 20 00 00 13 00 0.00 0.00 0.61 00

8 Sa16 23 00 00 11 00 0.00 0.00 0.47 009 Sa17 20 00 15 11 00 0.00 0.75 0.55 00

10 Sa18 13 00 00 00 00 0.00 0.00 0.00 00

11 Sa19 23 00 12 15 00 0.00 0.52 0.65 00

12 Sa20 23 00 00 11 00 0.00 0.00 0.47 00

Table 5.8Antimicrobial activity ofSolanum xanthocarpum aganist S. aureus:


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Gentamyci

n


1 Sa1 16 16 16 21 00 1.00 1.00 1.31 002 Sa2 20 00 00 22 00 0.00 0.00 1.10 00

3 Sa4 16 16 16 24 00 1.00 1.00 1.50 00

4 Sa8 18 15 15 20 00 0.83 0.83 1.11 00

5 Sa10 24 13 13 22 00 0.54 0.54 0.91 00

6 Sa11 23 00 00 20 00 0.55 0.55 0.86 00

7 Sa13 20 00 00 20 00 0.00 0.00 1.00 00

8 Sa16 23 00 00 18 00 0.00 0.00 0.78 00

9 Sa17 20 16 16 23 00 0.80 0.80 1.15 00

10 Sa18 13 11 11 23 00 0.84 0.84 1.76 00

11 Sa19 23 11 11 21 00 0.47 0.47 0.91 00

12 Sa20 23 11 11 19 00 0.47 0.47 0.80 00

Table 5.7 Antimicrobial activity ofTridex procumbens against S. aureus:


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Gentamyc

in


1 Sa1 16 00 12 11 00 0.00 0.75 0.68 002 Sa2 20 00 00 14 00 0.00 0.00 0.70 00

3 Sa4 16 00 00 16 00 0.00 0.00 0.10 00

4 Sa8 18 00 11 13 00 0.00 0.61 0.72 00

5 Sa10 24 00 11 14 00 0.00 0.45 0.58 00

6 Sa11 23 00 00 16 00 0.00 0.00 0.69 00

7 Sa13 20 00 13 12 00 0.00 0.65 0.60 00

8 Sa16 23 00 00 13 00 0.00 0.00 0.56 00

9 Sa17 20 00 00 00 00 0.00 0.00 0.00 00

10 Sa18 13 00 00 00 00 0.00 0.00 0.00 00

11 Sa19 23 00 12 13 00 0.00 0.52 0.56 00

12 Sa20 23 00 00 15 00 0.00 0.00 0.60 00

Table 5.9 Antimicrobial activity of Manthaarvensis against S. aureus:


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Sr.

No

Strain Inhibition zone (mm) Activity index

Gentamyc

in


1 Sa1 16 00 00 00 00 0.00 0.00 0.00 0.00

2 Sa2 20 00 00 00 00 0.00 0.00 0.00 0.00

3 Sa4 16 00 00 00 00 0.00 0.00 0.00 0.00

4 Sa8 18 00 00 00 00 0.00 0.00 0.00 0.00

5 Sa10 24 00 00 00 00 0.00 0.00 0.00 0.00

6 Sa11 23 00 00 00 00 0.00 0.00 0.00 0.00

7 Sa13 20 00 00 13 00 0.00 0.00 0.65 0.008 Sa16 23 00 00 00 00 0.00 0.00 0.00 0.00

9 Sa17 20 00 00 00 00 0.00 0.00 0.00 0.00

10 Sa18 13 00 00 00 00 0.00 0.00 0.00 0.00

11 Sa19 23 00 00 00 00 0.00 0.00 0.00 0.00

12 Sa20 23 00 00 00 00 0.00 0.00 0.00 0.00

Table 5.10 Antimicrobial activityAndrographis peniculata (wild neem) aganist S. aureus:


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Determination of MIC of effective extract of herbs:

When ethyl acetate extract of Tridex procumbens leaf subjected todetermination of minimum inhibitory concentration. It was found thatminimum inhibition concentrate this extract against tested clinicalisolates was 0.10mg/ml against Staphylococcus aureus (S4) strain.MIC and MBC values for mast resistance strain of Staphylococcus

aureus were evaluated for ethyl acetate extract of Tridex procumbenswhich showing highest activity in agar well diffusion method. In thepresence investigation MIC value range from 0.10 0.22 mg/ml wasrecorded against multi drug resistance Staphylococcus aureus (S4)strain. Amount of extract isolated from plant parts and total activity wascalculated and recorded in table 5.12.



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Determination of MIC of effective extract of herbs:


Table 5.12 Quantity and total activity, MIC and MBC ofTridex procumbens ethyl acetate extract against S. aureus

Sr. No Strain Amount of extract

mg/gm dried plant

part

MIC

mg/ml

MBC

mg/ml

Total activity

1. Sa2 3.5 0.19 0.38 0.05

2 Sa4 3.5 0.10 0.20 0.02

3 Sa10 3.5 0.19 0.38 0.05

4 Sa13 3.5 0.22 0.44 0.06

5 Sa17 3.5 0.15 0.30 0.04

6 Sa18 3.5 0.15 0.30 0.04


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Total activity of effective extract of herbs:

Total activity indicates volume at which extract can with diluted steelhaving ability to kill microorganisms.Lean et al 1999 and Palombo and Sample 2001 reported that Tridexpcocumbens have broad spectrum of antibacterial activity. In currentstudy inhibition zone, activity index, MIC, MBC and total activity have

been evaluated.

Extract under study not only inhibit growth of selected bacterial strainbut inhibition zone develop was more or less permanent as compare toantibiotics used during study. In the light of this fact microorganismsare become resistance against antibiotic used. Present investigation is ofa great significance as far as future drugs are considered and selectedplant by used by pharmaceutical industry for preparing plant westantimicrobial drugs.



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Studies on effect of exposure time on MIC values of Ethyl acetate

extract ofTridex pcocumbens:When clinical strain S4 strain of Staphylococcus aureus subjected toaction of different concentration of ethyl acetate extract of Tridexpcocumbens leaf and also allows the exposure for different timeduration like 30min 60 min and 120 min. With increasing the exposure

time there was decrease in MIC valve was reported. For 30 minexposure time MIC of extract was found to be 0.05 mg, for 60 minexposure time MIC of extract was found to be 0.04 mg and for 120 minexposure time MIC valve is 0.02 mg/ml.




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Sr. No Exposure

time (min)

MIC

mg/ml

MBC

mg/ml

1. 30 0.05 0.10

2 60 0.04 0.07

3 120 0.02 0.04

Table 5.13 Quantity and total activity, MIC and MBC ofTridex procumbensethyl acetate extract against S. aureus

RESULTS AND DISSCUSSIONStudies on effect of exposure time on MIC values of Ethylacetate extract ofTridex pcocumbens:


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Significance of study

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The present results revealed that the extract ofTridax procumbens L. was

effective against mastitis causing S. aureus. Presence of chemicalcompounds viz. alkaloids, tannins, flavanoids ofTridax procumbens L.

may inhibit the bacterial growth.

Traditionally, Tridaxprocumbens L. was employed using/mixing with

water for treating the bacterial and other infections.

Naturally, the biologicallly active compounds whose activity can be

enhanced in the presence of ethyl acetate could have been produced

number of active compound responsible for antibacterial activity.

The present study provides the scientific information about the leaf

extract ofTridax procumbens L. and supports the usage of this plant forcuring mastitis in cattle by traditional healing and antibacterial properties.

Further, phytochemical separation studies of this plant and in VIVO

antibacterial study of Tridex extract is in under progress.

CONCLUSION


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In the absence of any mastitis control program and presence of high

antibiotic resistance, searching for new cost effective treatment line isessential.

The use of herbal treatments for mastitis is appears to be strong

interest from a small but significant part of the dairy industry for

reliable advice and products.Ethno-veterinary information about plants used in the prevention and

control of bovine mastitis.

The results of this study have shown that the leaf extract of Tridax

procumbens L. have great potential as antibacterial agents in thetreatment of mastitis caused by multidrug resistant S. aureus. Further,

detailed investigation of the active compounds of the plant for the

exact mechanism of action will contribute greatly to the development

newpharmaceuticals.


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msc project presentation 29 march

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