BIOAUTOGRAPHY AND ITS APPLICATIONS.pptx

bioautography, a hyphenated chromatographic technique for the isolation of lead molecules.
it is the emerging technique and have advanced detecting possibilities.
It can be used in the field of pharmacy, chemistry, biological evaluation, herbal detection,etc.
it is broadly classified into three types ...namely planar, direct contact and diffussion chromatography.
present ppt gives idea about the bioautography in a broad sense through a thorough review.

1. BIOAUTOGRAPHY A seminar as a part of curricular for M.Pharm P resented by Tejaswini Kakade Department of Pharmaceutical Quality Assurance Under the guidance of Ms. Hiteshi Chadha Assistant Professor Anand Pharmacy College

2. C ontents Introduction to bioautography History of bioautography Types of autobiography Agar or contact bioautography Direct bioautography Immersion or agar overlay bioautography Detection of antioxidants Bioautography using beta carotene Enzyme inhibition Applications Conclusion References ANAND PHARMACY COLLEGE , ANAND 2

3. Bioautography   The identification or comparison of organic compounds separated by chromatography by means of their effect on living organisms and especially microorganisms Bioautography is a means of target directed isolation of active molecules on chromatogram. HYPHENATED TECHNIQUE---is a mix or coupling of two distinctive analytical technique with assistance of proper interface. Separation- separation Separation-identification Identification- identification ANAND PHARMACY COLLEGE , ANAND 3

4. Planar chromatographic analysis hyphenated with biological detection method is termed as bioautography . It is an effective and inexpensive technique for the phytochemical analysis of extracts to identify bioactive leads. Despite having sophisticated high performance liquid coupled bioassays , bioautography offers a simple, rapid and inexpensive method for chemical and biological screening of extracts with subsequent bioassay guided isolation. ANAND PHARMACY COLLEGE , ANAND 4

5. Fig 1. Bioautography for detection of antimicrobial compounds ANAND PHARMACY COLLEGE , ANAND 5

6. History of bioautography In 1946,Goodall and Levi introduced paper chromatography based bioautography for the first time to estimate purity of penicillin . In 1961,Fischer and Lautner introduced thin layer chromatography (TLC) based bioautography . The first review on bioautography was written by Betina in 1973. Generally , planar chromatographic ( TLC and PC) are used for bioautography , but the detection can be successfully improved by advanced chromatographic tools like HPTLC , over pressured layer chromatography (OPLC),planar electro chromatography(PEC). ANAND PHARMACY COLLEGE , ANAND 6

7. Types of bioautography Bioautographic tools are been used with TLC – and paper chromatography for detection of active components. They are: A) agar diffussion or contact bioautography B) direct TLC bioautographic detection C) immersion or agar overlay bioautography ANAND PHARMACY COLLEGE , ANAND 7

8. Fig2 : Types of bioautographic methods ANAND PHARMACY COLLEGE , ANAND 8

9. Agar diffusion or contact bioautography In contact bioautography , antimicrobial agents diffuse from a developed TLC plate or paper to an inoculated agar plate . The chromatogram is placed face down onto the inoculated agar layer for a specific period to enable diffussion . Then the chromatogram is removed and the agar layer is incubated. ANAND PHARMACY COLLEGE , ANAND 9

10. The zones of inhibition on the agar surface, corresponding to the spots in chromatographic plates, are indicative of the antimicrobial substances. An overall view of contact bioautography has been depicted in fig . Incubation time for the growth ranges between 16 and 24 h but it can be reduced to 5-6 h by spraying with 2,6 dichlorophenol-indophenol or 2,3,5-tetrazoliumchloride ANAND PHARMACY COLLEGE , ANAND 10

11. Fig 3: schematic diagram of contact / agar diffusion chromatography ANAND PHARMACY COLLEGE , ANAND 11

12. Direct TLC bioautographic detection The principle of the method is that separation and detection of biological properties of given mixture components is directly performed on TLC plates . In direct TLC bioautography , the developed TLC plate is sprayed with or dipped into a fungal or bacterial suspension. A suspension of test bacteria or fungi is used for spraying or dipping purpose The bioautogram is then incubated at 25 degrees for 48 hour under humid condition. ANAND PHARMACY COLLEGE , ANAND 12

13. For visualization of microbial growth , tetrazolium salts are used. These salts are converted by dehydrogenases or living organisms to intensely colored formazan Clear white zones against a purple background on the TLC plate indicate antimicrobial activity of the sample. The active compounds can be further identified using spectroscopic methods ANAND PHARMACY COLLEGE , ANAND 13

14. Fig 4 : schematic diagram of direct bioautography ANAND PHARMACY COLLEGE , ANAND 14

15. Immersion or agar overlay bioautography Agar overlay is a combination of contact and direct bioautography . In this method, the chromatogram is covered with a molten , seeded agar medium. After solidification, incubation and staining ( usually with tetrazolium dye), the inhibition or growth is been visualized. For gram negative bacteria, an agar solution containing the red colored bacterium serracia marcescens can be employed. ANAND PHARMACY COLLEGE , ANAND 15

16. The red colored gel is incubated overnight at room temperature and inhibition zones appear as white or pale yellow areas on a red background With other, colorless microorganisms ,zones of microbial growth inhibition are visualized with the aid of a dehydrogenase activity detecting agent ( tetrazolium salt) Metabolically active microorganisms convert the tetrazolium salts into the corresponding intensely colored formazan ANAND PHARMACY COLLEGE , ANAND 16

17. Fig 5 : schematic diagram of agar overlay bioautography ANAND PHARMACY COLLEGE , ANAND 17

18. Detection of antioxidant agents bioauto graphy Bioautography using DPPH as detection reagent The stable 2,2 diphenyl -1-picrylhydrazyl radical(DPPH) has an absorption maximum at 517 nm, which decreases upon reduction through reaction with radical scavenger. The corresponding color change can thus be observed in TLC bioassay. The developed chromatogram is sprayed with a solution of 0.2% DPPH in methanol/ ethanol. The plate is examined in daylight after 30 minutes. ANAND PHARMACY COLLEGE , ANAND 18

19. Free radical scavengers appear as cream/yellow spots against a purple background. The intensity of the yellow color can be measured with a chromameter . ANAND PHARMACY COLLEGE , ANAND 19

20. Bioautography using beta- carotene as detection reagent Completely dried TLC chromatograms are sprayed with 0.05% solution of beta carotene in chloroform. They can be left at room temperature for decolorization of the background or they can be placed under 366nm Uv light. Active compounds remain as yellow-orange spots on white background. ANAND PHARMACY COLLEGE , ANAND 20

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22. Enzyme inhibition Enzymes are important molecular targets for lead discovery in primary screening assays. The use of TLC support to screen for potential plant derived enzyme inhibitors is a rapid method which is relatively free of disturbances due to solvent. Bioautographic detection of acetyl cholinesterases inhibitor by Ellmann reaction Acetylthiocholine (ACTI) is cleaved by AchE to form thiocholine which reacts with 5,5 dithiobis ( nitrobenzoic acid) to give yellow compound. ANAND PHARMACY COLLEGE , ANAND 22

23. It could be adapted for TLC screening of AchE inhibitors In this method, a solution of DTNB and ACTI is sprayed on the chromatogram. A pale yellow background forms within about 5 minutes. AchE inhibitors appear as white spots. ANAND PHARMACY COLLEGE , ANAND 23

24. Fig 8:ELLMAN REACTION ANAND PHARMACY COLLEGE , ANAND 24

25. Xanthine oxidase inhibition The enzyme xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to uric acid and producing O 2 and H2O2 To identify XO inhibitors on TLC plates , the enzyme is suspended in agar and distributed on the TLC plate. After solidification, the plate is immersed into a solution of xanthine at 38 degree for 20 min in the dark Enzymatic oxidation of xanthine produces Oxygen which reduces the pale tetrazolium salt (NBT) to a formazan . ANAND PHARMACY COLLEGE , ANAND 25

26. Fig 9 : reaction to detect xanthine oxidase inhibitors Allopurinol an inhibitor of XO is detected as white spot on purple background. ANAND PHARMACY COLLEGE , ANAND 26

27. Applications Rapid technique for chemical and biological screening of plant extracts. Used in microbial detection Enables parallel separation of multiple samples. Used for investigating biochemical processes. In detection of antibacterial, antioxidant , enzyme inhibitory , oestrogenic activities. ANAND PHARMACY COLLEGE , ANAND 27

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29. Conclusion In spite of wide employment of sophisticated chromatographic techniques coupled with online-bioassays, bioautography is still proving its worth as simple and inexpensive tool for simultaneous chemico -biological screening of natural resources. It offers the simplest mean of bioassay guided lead discovery from natural resources. ANAND PHARMACY COLLEGE , ANAND 29

30. For the natural product the separation process is not easy, and if separated the amount is very less in maximum cases, so it is necessary to develop a process which can detect lead in a small amount and biological activity can also be measured successively. Considering these problems, we can say that bioautographic detection technique would create a new era in separation science. ANAND PHARMACY COLLEGE , ANAND 30

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32. References Saikat Dewanjee , Moumita Gangopadhyay , Niloy Bhattacharya, Ritu Khurana , Bioautography and its scope in the field of natural product chemistry, Journal of Pharmaceutical Analysis(2014) M.B. Muller, C. Dausend , C. Weins , et al., A new bioautographic screening method for the detection of estrogenic compounds, Chromatographia 60 (2004) 207–211. A. Marston, M. Maillard , K. Hostettmann , A TLC bioautographic method for the detection of α- and β- glucosidase inhibitors in plant extracts, GIT Lab. J 1 (1997) 36–39. ANAND PHARMACY COLLEGE , ANAND 32

33. References R.R. Goodall , A.A. Levi, A microchromatographic method for the detection and approximate determination of the different penicillins in a mixture, Nature 158 (1946) 675–676 K. Hostettmann , C. Terreaux , A. Marston, et al., The role of planar chromatography in the rapid screening and isolation of bioactive compounds from medicinal plants, J. Planar Chromatogr . 10 (1997) 251–258. ANAND PHARMACY COLLEGE , ANAND 33

34. Thank you ANAND PHARMACY COLLEGE , ANAND 34