1. B-Specialized screening bioassays: As described above, after having found a certain type of activity, it will be necessary to study this activity in more detail by using one or several specialized bioassays for screening and/or monitoring purposes. These in vitro or in vivo tests are more sophisticated than the primary screening bioassays and require the expertise of biochemists or pharmacologists. They can, consequently, only be performed in a multidisciplinary team. We have classified the many existing specialized screening bioassays according to the target organisms which are used. 4/2/20111Dr. Abd El Raheim Donia

2. 1- Lower organisms: Numerous research programs to detect and isolate antibacterial-, antifungal-, antiviral- and antiparasitic compounds from plants and other natural sources have been designed and are performed. Large batteries of bacteria, yeasts, fungi, viruses, insects, molluscs, protozoa, and helminthes, are thereby used in a broad range of in vitro and/or in vivo bioassays. 4/2/2011 2 Dr. Abd El Raheim Donia

3. 4/2/2011 3 Extract dilution 12345678910 1112 controlCCCCCCCCCCCCA 1/2B 1/8C 1/32D controlCCCCCCCCCCCCE 1/2F 1/8G 1/32H Different microorganisms Scheme of a plate used in the agar dilution antimicrobial test method Dr. Abd El Raheim Donia

4. 2- Isolated subcellular systems: Molecular assays look for activity using isolated systems, including enzymes and receptors. Though already known for many years in pharmacology and frequently used in industrial drug development, in studies of natural products the systematical use of these methods for screening purposes has only started. 3- Isolated cellular systems: Cellular assays utilizing intact cells of human or animal origin have been used for more than half a century to screen for cytotoxic agents from natural sources. Cytotoxicity, or toxicity to cells in culture, can be subdivided into cytostatic activity. A large number of cytotoxicity- based bioassays have been used as prescreens for antitumor and antineoplastic activity. These short-term in vitro growth inhibition assays with cultured cells include Walker carcinosarcoma 256, mouse L-120 leukemia. While these traditional cell-cytotoxicity assays are indicative for activity in leukemia, lymphoma, or a few rare tumors, their efficacy in finding products with activity in the predominantly occurring slow growing solid tumors of humans is strictly limited. 4/2/20114Dr. Abd El Raheim Donia

5. 4- Isolated organs of vertebrates: Organ-based assays are more and more replaced nowadays as the front-line primary screen, but often retain signification as secondary screens to confirm the results of, e.g. radioligand-binding bioassays and, hence, to assist in prioritization of active extracts or compounds. They represent, consequently, an essential connection between the high technology of the primary screens and the realities of the pharmacological effectiveness segments of the gastro-intestinal tract or spirally cut strips of vascular tissue are mainly used in the organ bath method for isolated organs. 4/2/20115Dr. Abd El Raheim Donia

6. 5- Whole animals: Biomedical research needs animals. This is most obvious in the case of in vivo animal experiments. However, for other scientific reasons, e.g. in vitro studies, biological material is also needed to study enzymes, membranes, receptors, cells, tissues or organs which are obtained from dead animals. Therefore, animals have to be sacrificed in biomedical laboratories, (i) at the end of in vivo experiments; (ii) during experiments where sacrifice of the animals is not part of the study but must be done when pain, distress and suffering exceed acceptable levels or if it is likely for the animal to remain in pain or distress after cessation of the experiment; and (iii) to provide biological material for in vitro studies. 4/2/20116Dr. Abd El Raheim Donia

7. In vivo testing. In vivo (Latin for "within the living") is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment.Latinorganismpartialin vitro Animal testingAnimal testing and clinical trials are two forms of in vivo research. In vivo testing is often employed over in vitro because it is better suited for observing the overall effects of an experiment on a living subject.clinical trials In vivo tests on animals often involve inducing a clinical condition in the animal to produce observable symptoms. 4/2/2011 7 DR. ABD EL RAHEIM DONIA

8. In vivo tests  There are several problems associated with in vivo testing. *It is slow and it also causes animal suffering. There are also many problems of pharmacokinetics and the result obtained may be misleading. For example, penicillin methyl ester is hydrolyzed in mice into active penicillin, while it is not hydrolyzed in humans or rabbits. Also, thalidomide is teratogenic in rabbits and humans while it is not in mice. 4/2/2011 8 Dr. Abd El Raheim Donia

9. In microbiology in vivo is often used to refer to experimentation done in live isolated cells rather than in a whole organism, for example, cultured cells derived from biopsies. In this situation, the more specific term is ex vivo. Once cells are disrupted and individual parts are tested or analyzed, this is known as in vitro.ex vivo Compounds that bind to isolated recombinant proteins are one thing; chemical tools that can perturb cell function another; and pharmacological agents that can be tolerated by a live organism and perturb its systems are yet another. If it were simple to ascertain the properties required to develop a lead discovered in vitro to one that is active in vivo, drug discovery would be as reliable as drug manufacturing. 4/2/20119Dr. Abd El Raheim Donia

10. Most in-vitro screening assays utilize a surrogate readout scheme that relies on labeled molecules or some type of coupled reaction as a readout system. The biological relevance of these assay systems to the original endogenous mechanisms can be tested with Isothermal Titration Calorimetry (ITC) to compare the results and give confidence that they mimic each other appropriately.Isothermal Titration Calorimetry (ITC) This includes all forms of ligand and receptor complexes, as well as enzyme-substrate reactions and kinetics. Both ITC and Differential Scanning Calorimetry (DSC) can be used to evaluate the quality of expressed proteins for the appropriate post-translational properties and percent activity, as well as assess the impact of assay co-factors.Differential Scanning Calorimetry (DSC) 4/2/2011 10 Dr. Abd El Raheim Donia