mirs.shalini singh yadav

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Antifungal activity of Tamarindus indica fruit extract Presented By Shalini Singh yadav Department of Microbiology RCST, Durg (CG) Under the Guidance of Prof. Yashraj yadav (Sr. Scientist) Bhopal (MP) 1

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INTRODUCTION Antifungal agent - A drug which inhibits the growth and reproduction of fungal cells and decreases the number of fungi present. such as athlete's foot, ring worm, candidiasis (thrush), serious systemic infections such as cryptococcal meningitis , and others. Some of established antifungal drugs Amphotericin B Clotrimazole Fluconazole ‘Medicinal plants represent a rich source of antimicrobial agents. Many of the plant materials used in traditional medicine are readily available in rural areas at relatively cheaper than modern medicine.’ 2

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Candida albicans is a diploid fungus (a form of yeast) and a causal agent of opportunistic oral and genital infections in humans. Systemic fungal infections (fungemias) have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). Candida albicans C. albicans Fungal Infections by C. albicans 3

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Aspergillus niger A. Niger is one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss, and, in severe cases, damage to the ear canal and tympanic membrane. if large amounts of spores are inhaled, a serious lung disease, aspergillosis can occur. Aspergillosis is, in particular, frequent among horticultural workers that inhale peat dust, which can be rich in Aspergillus spores . Fungal Infections by A. niger 4

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Extraction of plant material Thin layer chromatography (TLC) In vitro method Disc diffusion method Well diffusion method Serial dilution In vivo method (Spread pour method) OBJECIVE 5

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To search a novel antifungal agent Micro-organisms can develop resistance against antibiotics Plant medicine have less adverse effect Drugs from plant as a source can be a cheaper alternative as compared to synthetic drugs Tamarindus indica fruits are easily available plant material SIGNIFICANCE OF WORK 6

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PLANT PROFILE Kingdom Plantae Division Magnoliophyta Class Magnoliopsida Family Fabacea e Genus Tamarindus Species T. indica Tamarindus indica 7

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MATERIAL & METHOD 8 Extraction & Phytochemical Investigations Plant Selection and Collection :- Ethno botanical survey / traditional use / and literature survey Extraction :-Maceration [Ethanol: water (80: 20)] Crushed material was kept in contact with solvent mixture in closed container at room temperature with regular shaking for five days. After five days whole component was filtered and filtrate was dried at 40°C on water bath till no reduction in volume was observed (to confirm no residue of ethanol) and calculate Percent yield Characterization of extracts :- Phytochemical Investigation Thin layer Chromatography (TLC)

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I n-vitro method of antifungal activity Requirements : Media – 1. Savouraud’s Dextrose Agar Media 2. Nutrient Broth Drugs and Chemicals – 1. Standard antifungal drugs 2. Cyclophosphamide 3. Normal Saline 4. Amphotericine B Other requirements – Glassware, Laminar Air flow bench, Incubator, Oven, Autoclave, Spectrophotometer, Microscope (with camera attachment), etc. 9

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Disc Diffusion Method Principle The principle used here is that antibiotic will diffuse from a paper disc into an agar medium that contains test organisms. Inhibition is observed as a failure of the organism to grow in the region of the antibiotic. A common application of this method is the Kirby Bauer test , developed in the 1960s. Procedure Culture will be grown over night in liquid broth. O.D. will be maintained at 660 nm up to (1 x 10 6 ) Then culture will spread on savourauds agar medium Watt man filter paper(no. 6mm) will placed on disc of different conc. of plant extract 75 mg/ml dilution (25,50,75,100 %) Incubate for 8 hr at 37 ⁰C At last zone of inhibition will be measure in mm (including diameter of disc) 10

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Well diffusion method Principle The principle used here is that antibiotic will diffuse from a well into an agar medium that contains test organisms. Procedure Savourauds agar media will be prepared and autoclaved at 15 psi and 121 ⁰C Savourauds agar media will poured in Petri plat in laminar air flow Media wells where cut on the agar plate after solidification References culture will spread on media Extract of different conc. (75 mg/ml) 100,75,50,25 % will be pour in well Then plates will be incubated at 37 ⁰C for 12 – 18 hr At last zone of inhibition will be measure in mm (including diameter of disc) 11

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Serial Dilution Method (MIC) Principle The Minimum Inhibitory Concentration Assay is a technique used to determine the lowest concentration of a particular antibiotic needed to kill microorganism. This assay is typically performed on plank tonic (free floating) microbial cells. Procedure Nutrient broth will be taken in 10 test tube + blank Transfer fungal culture of known equal conc at 600 nm upto 1 x 10 6 Transfer extract 1 ml of different conc (1,2,3,……….10 mg /ml) Then plates will be incubated for 18 hr and observe fung a l growth Find out minimum inhibit concentration (MIC) at which number of fugal growth was observe 12

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In-vivo method antifungal activity Principle Immunocompromised animal will be challenged with systemic fungal infection. Capacity of test sample to protect animal from fungal infection will be investigated. Animals Required Strain : Swiss albino mice Age : 3-4 week old Gender : Either No. of animal required : 12 13 Housing Condition The animal were maintain in a controlled environment at 25 o C With a 12 hr light and dark cycle and were acclimatized for 3-5 day before use

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14 Route of administration : I.V (intravenous) , I.P (intra peritoneal) P.O (per oral) Extract - Ethanol : Acetone (1:1) Cyclophosphamide - 100, 200 mg/kg bw Grouping of animal for experiment S.No. Treatment No. of animals 1 Vehicle 4 2 Extract 4 3 Standard drug 4

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Procedure Mice weighing 25 ± 2 gram will be selected from the animal house of PBRI. In each cage not more than 5 animals will be kept and they will be provided free access to food and water through out the experiment. Each animal will be given extract p.o . throughout 15 days of experiment. (Dose will be decided on the basis of in-vitro results) On fourth day of experiment animals will be given Cyclophosphamide (200 mg/kg), i.p . On seventh day of experiment animals will be challenged with 3.0 x 10 4 CFU in 2 ml/mice. (candid albicans cell suspension for injection will be obtained from overnight culture grown on Sabouraud dextraose agar plates at 35ºC and will be suspended in sterile saline) The mortality of mice will be recorded daily and the 50% effective doses will be calculated by probit method, based on the survival rate. Animals survived at last will be sacrificed and renal burden will be assessed by culturing kidney homogenate in sabouraud dextrose agar plate by serial dilution. 15

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RESULT 1. Test For Flavonoids : Lead acetate test Negative Ferric chloride test Negative 2. Test For Glycosides : Negative 3. Test For Carbohydrates : Molisch test positive 4. Test For Alkaloids : Hagers test positive Wagners test positive 6. Test For Saponins Forth test positive 7. Test For Terpinoids : Salkowaski test positive 8. Test For Protien and Amino Acids : Biurete test Negative Result of phytoinvestigation 16 S.No . Property Extract 1. Colour dark brown 2. Consistency sticky nature (semi solid) 3. yield(w/w) % 28.26% Preliminary Physical Analysis

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17 Solvent system No. of spots Rf Acetic Acid: Chloroform (1:9) Spot A 0.2 Spot B 0.9 Thin Layer Chromatography TLC of extract was performed on different solvent system it was observed that with acetic acid and chloroform solvent system there was basically three separations occurred (one with the spot where it was marked and two spot which were observed in mid of TLC plates). Rf of two spots is mentioned In-vitro Disc Diffusion Assay Concentration Fungal strain A. Niger C. albicans 75 mg/ml 1.45 ± 0.095 1.225 ± 0.125 56.25 mg/ml 1.25 ± 0.1 1.2 ± 0.081 37.5 mg/ml 1.075 ± 0.057 1.025 ± 0.05

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18 In-vitro Disc Diffusion Assay(graph) A.niger C. albicans

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19 In-vitro Well Diffusion Assay Concentration Fungal strain A. niger C. albicans 75 mg/ml 1.9 ± 0.141 2.55 ± 0.443 56.25 mg/ml 1.4825 ± 0.023 2.37 ± 0.478 37.5 mg/ml 1.245 ± 0.33 1.2 ± 0.21 In-vitro MIC (minimum inhibitory concentration) Estimation S.No . Fungal strain MIC 1 Niger 43.75 2 C. Albicans 41.5

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20 C. albicans A.niger

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21 In-Vivo Antifungal Activity (1) Kidney Burden (on 10th fold dilution) Treatment Group Animal No Colony Vehicle treated M1 Died M2 Died M3 Died M4 Died Extract treated(500 mg/kg) M1 40 M2 Died M3 Died M4 Died Amphoterecin (0.2 mg/kg) M1 2 M2 5 M3 0 M4 Died Extract treated (twice a day dose) M1 2 M2 Died M3 6 M4 8

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22 Vehicle treated Extract treated Extract treated (twice a day dose) Standard

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23 Animal No Day of death M1 6 M2 4 M3 6 M4 5 Vehicle treated group Animal No Day of death M1 Survived M2 10 M3 9 M4 9 Extract (500 mg/kg) single dose /day treated group Animal No Day of death M1 Survived M2 14 M3 Survived M4 Survived Animal No Day of death M1 Survived M2 Survived M3 Survived M4 15 Amphoterecin (0.2 mg/kg treated group) Extract (500 g/kg) double dose/day treated group (2) Survival studies

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24 In vivo antifungal activity treatment growth Survival Vs day(time)

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25 CONCLUSION Every in-vitro result is required to be correlated with in-vivo results for ascertaining efficacy of test sample. So, this work was designed to ascertain in-vitro and in-vivo antifungal activity of T. indica against C. albicans and A. niger. This is the first investigation to establish in-vivo antifungal potential of T.indica extract. C. albicans and A.niger are cause of many fungal infections, present study gave a site for therapeutic use of T.indica. Both in-vivo and in-vitro results showed that T.indica fruit extract possess significant antifungal activity. Ethanolic extract of fruit had good yield and as method of extraction was maceration chances of degradation of thermo labile component was also absent. Phytochemical investigation revealed that extract was having Tannin, Alkaloids, Saponin and Terpens . All of these phytoconstituent are reported to possess antifungal activity.

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26 As T.indica is one of the important part of regular diet of many regions of India. From this study it can be said that this plant can be added in diet of patients suffering from fungal infection, and in this way T.indica fruit will work as an adjuvant for the fungal treatment. In future, further studies are required to isolate active component from fruit, characterize it and to assess antifungal activity of component. Detailed study is required to establish specific dose of extract that can work as antifungal agent. Thus from present investigation it can be concluded that ethanolic extract of T.indica possess significant antifungal activity against C. albicans and A. niger Cont.

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