1. 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

2. BIOLOGICAL SCREENING 2

3. TYPES OF ASSAY Chemical Assays: Spectrophotometry, Turbidimetry Spectrofluorimetry, Gravimetry, Chromatography etc. Chemical Assays: Spectrophotometry, Turbidimetry Spectrofluorimetry, Gravimetry, Chromatography etc. Bioassays : Bioassays : In-vitro Assay In-vitro Assay In-vivo Assay In-vivo Assay Microbiological assays Microbiological assays 3

4. BIOASSAY: Estimation of the conc. / potency of a substance by measuring its biological response in living systems. Choosing the right bioassay or test system is crucial to the success of a biological screening program. Choosing the right bioassay or test system is crucial to the success of a biological screening program. The test should be simple, quick and relevant as there are usually a large number of samples to be analyzed. The test should be simple, quick and relevant as there are usually a large number of samples to be analyzed. Human testing is not possible at such early stage, so the test has to be done in vitro first. Because in vitro tests are cheaper, easier to carry out, less controversial and can be automated than in vivo one. Human testing is not possible at such early stage, so the test has to be done in vitro first. Because in vitro tests are cheaper, easier to carry out, less controversial and can be automated than in vivo one. In vivo tests needed to check the drugs interaction with specific target and to monitor their pharmacokinetics properties. In vivo tests needed to check the drugs interaction with specific target and to monitor their pharmacokinetics properties. 4

5. IN-VITRO ASSAY They do not involve live animals. Instead, specific tissues, cells, or enzymes are isolated and used. They do not involve live animals. Instead, specific tissues, cells, or enzymes are isolated and used. Enzyme inhibitors can be tested on pure enzyme in solution. Enzyme inhibitors can be tested on pure enzyme in solution. Receptor agonist and antagonists can be tested on isolated tissues or cells. Receptor agonist and antagonists can be tested on isolated tissues or cells. Antibacterial drugs are tested in vitro by measuring how effectively they inhibit or kill bacterial cells in culture Antibacterial drugs are tested in vitro by measuring how effectively they inhibit or kill bacterial cells in culture 5

6. IN-VIVO ASSAY In vivo tests on animals often involve inducing a clinical condition in the animal to produce observable symptoms. In vivo tests on animals often involve inducing a clinical condition in the animal to produce observable symptoms. The animal is then treated to see whether the drug alleviates the problem by eliminating the observable symptoms. For example, the development of non-steroidal inflammatory drugs was carried out by inducing inflammation on test animals. The animal is then treated to see whether the drug alleviates the problem by eliminating the observable symptoms. For example, the development of non-steroidal inflammatory drugs was carried out by inducing inflammation on test animals. 6

7. 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. 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. 7

8. ● After a new substance (expected to be a new drug) has been developed (extracted, isolated). -The next step is to subject it to a specified set of procedures in experimental animals. ● The aim is to determine whether or not the compound possesses the desired action. 8

9. Pharmacological screening: Requirements: 1- Animals: have a large problem such as: a- Cost b- Feeding c- Selection: e.g. : Dogs (heart/ vascular) Rats (analgesia/ inflammation) Rabbits ( CNS) Monkey ( tumor, virus) 2- Tissues: All types of muscle, heart, nerve structure. 3- Time: It take from 2-3 months and also need duplication for experiments. 4- Solubility: drug action depend on solubility so most probably in animal used: oral, i.p (Intraperitoneal). 9

10. Phytopharmacological screening: ● Antimicrobial activity Eugenia caryophyllus (Eugenol) Thymus vulgaris (Thymol) Lavendula officinalis (Linalool) ● Antineoplastic activity Catharanthus roseus (Vincrestine, vinblastin) Taxus brevifolia (Taxol) ● Antimalarial: Cinchona succirubra (Quinine) Artemisia annua (Artemisinin) 10

11. ● Hypoglycemic: Garlic (Allium sativum) (Allicin) ● Cardiotonic Digitalis purpurea (Digoxin and Digitoxin) Strophanthus kombe (K-strophanthoside) ● Antiarrythemic Cinchona succirubra (Quinidine) 11

12. Type of pharmacological screening: 1-Blind screening The aim is to see if a new compound has any useful pharmacological activity. 2- Simple screening The aim is to find a substance having a particular property. e.g. A single test for the conc. of sugar in blood may be used to screen compounds for hypoglycemic activity. 3- Programmed screening In this type of screening the most important pharmacological action of the new compound is fully investigated. 12

13. The organization of blind screening Screening for pharmacological activities proceeds from general observation on intact animals to the more specific techniques using isolated organs. It might include the following tests: A- Preliminary observations on the behaviour of conscious animals (neuropharmacological tests) B- The cardiovascular test. C- Isolated organ preparations especially the isolated guinea pig ileum. 13

14. a)Neuropharmacological tests: These tests were developed to determine the central actions of drugs. ● It can detect: sedatives, hypnotics, tranquillizers, pscychomotor stimulants, muscle relaxant, analgesics, atropine-like drugs, antipyretics, peripheral vasodilators. ● The animal used are: mice. ● The route of administration is important: - The compound is given parenterally in order to avoid the possibility of poor absorption from the intestinal tract. ● Different doses are used. - For the performance of the test 3 animals are used per dose. 14

15. The signs observed in the primary test for pharmacological activity are: 1- Consciousness or awareness. 2- Stereotype: is the frequental mechanical repetition of a movement. e.g. - Morphine: cause the mice to have circular movement. - Amphetamine: cause them to have anxiety movement. 3- Motor activity and spontaneous activity, this measures stimulation and depression of the autonomic nervous system. 4-The pain response: this measures analgesia and sedation. 15

16. 5- The touch response, may indicate the presence of anesthetic activity. 6- Central excitation, is indicated by: - Tremors. - Convulsions. - Restlessness and irritability. 7- The autonomic responses includes: - Pupil size. - Hypothermia - Secretion - Skin color - Heart rate - Respiratory rate 16

17. b) The cardiovascular tests: These tests describe the action of the new substance on blood pressure of the intact anesthetized animals. The following conclusions can be made: 1- If the test substance causes brief fall in b.p. (↓in b.p.) therefore it may have: - Muscarinic activity like ACH - Direct vasodilator activity - Cardiodepressant activity. 17

18. 3- If the test substance causes a prolonged fall in b.p. (↓↓b.p.) (more than 5 min), it may have: Ganglion blocking 4- If the test substance causes a prolonged rise in b.p. (↑↑b.p.) (5minutes or more) it may have: Monoamine oxidase inhibiting activity. 2- If the test substance causes a brief rise in blood pressure (↑ in b.p.) therefore it may have: - Sympathomimetic activity or - Direct vasopressor activity. 18

19. 5- If the test substance causes a fall in b.p. after a delay of 45 sec. it may have: Histamine-releasing activity like morphine. 7- If after administration of the test substance, the response to epinephrine injection is diminished - Sympatholytic activity. 6- If after administration of the test substance, the response to ACH is diminished. - Parasympatholytic activity like atropine. 19

20. 8- If the test substance causes the response to ACH to be augmented above the control, the test substance may have: - Anticholinesterase activity like neostigmine. 9- If the test substance causes increase in response of epinephrine above to the response obtained by giving only epinephrine (i.e. without test substance). The test substance may be monoamine oxidase inhibitor. This will confirm the observation of prolonged rise in b.p. produced by the test substance itself. 20

21. c) Isolated organ preparations especially the isolated guinea pig ileum. Isolated preparation have the following advantages: 1- Free from interference by central action (e.g. circulating hormones). 2- Relatively small amounts of the material is required. 3- Drug effect is tested directly without the factor of absorption, metabolism, etc. 4- Several preparation can be tested on a single animal. 21

22. Screening methods: Ideal methods such as: 1- Antimicrobial 2- Anticancer 3- Antiviral 4- Hepatoprotective 5- Antioxidant 22

23. Antimicrobial Screening of Natural Products Using Agar Dilution Method - The Screening methods for the detection of antimicrobial activity of natural products fall into three groups, including: - Bioautographic - Dilution methods (turbidimetric assay). - Agar diffusion (plate assay) The bioautographic is known as: Qualitative technique since this method will only give an idea of the presence or absence of substances with antimicrobial activity. 23

24. Procedure: 1- The desired concentration of natural products is dissolved in very small amount of DMSO (dimethyl sulfoxide). 2- The solution is added to 10 ml of the melted agar medium and mixed will. 3- The agar is poured into the Petri dish often desired dimensions and cooled. 4- The desired test organism are then applied a lines from the edges to the center of the plate. 5- The plate is then incubated and growth of organisms is recorded. 24

25. Results: 1- In the above figure five organisms were used to screen natural products A, B and C at 1mg/10 ml agar. 2- The used organisms are: E: Escherichia coli S: Staphylococcus aurous C: Candida albicans B: Bacillus subtillus Ps: Pseudomonas aurogenosa 3- The results revealed that: a. Natural products A and B are active against: B: Bacillus Subtillus S: Staphylococcus aurous C: Candida albicans b. Natural products C is active against: B: Bacillus Subtillus S: Staphylococcus aurous c. The fourth plate is the control showing growth of all organisms 25

26. The Dilution methods (turbidimetric assay) and agar diffusion (plate assay) are considered as: Quantitative assays once they determine the minimal inhibitory concentration (MIC).  MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.  MIC are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. 26

27. Turbidimetric assay:  The test organisms growing in test tubes that contain different concentrations of the antibiotic.  There is a direct relationship of the concentration of antibiotic to the growth of the test organism, and by measuring the growth of the organism which is indicated by the turbidity of the content of the test tube, the antibiotic titer can be determine.  Clear tubes indicate a higher antibiotic conc. than turbid tubes, and the lowest concentration of antibiotic that completely prevents the appearance of turbidity is known as MIC. 27

28. Plate assay:  Filter paper discs are impregnated with solutions of antibiotic of varying concentrations, allowed to dry, placed on agar media seeded with an appropriate test organism, and incubated.  As the concentration of the antibiotic increase, its diffusion through the agar medium increases; therefore, the size of the clear zone of growth inhibition around the filter paper disc is related to the concentration of antibiotic. 28

29. Antioxidant Screening of Natural Products Using TLC  Oxidation of a wide range of chemical compounds and the production of free radicals (contains one or more unpaired electrons) at the cellular level are believed to be involved in the cause of many cancers.  Plant derived natural products such as flavonoids, terpenoids and steroids have received considerable attention in recent years due to their diverse pharmacological properties including antioxidant and antitumor activity.  Antioxidants are compounds that help to inhibit many oxidation reactions caused by free radicals which will prevent or delay damage to the cells and tissues. 29

30. Antioxidants: classification Non-Enzymatic antioxidant: Non-Enzymatic antioxidant: Alpha tocopherol (vitamin E) Alpha tocopherol (vitamin E) Beta Carotene Beta Carotene Ascorbic acid (vitamin C) Ascorbic acid (vitamin C) Antioxidant enzymes: Superoxide dismutase (SOD) Superoxide dismutase (SOD) Glutathione peroxidase enzyme Glutathione peroxidase enzyme The catalase enzyme The catalase enzyme Free radicals and cancer Many forms of cancer are thought to be the result of reactions between free radicals and DNA, resulting in mutations that can adversely affect the cell cycle. 30

31. Natural antioxidants are presents in fruits and vegetable WHO recommendation: eat 5 fruit or vegetable per a day 31

32. EXPERIMENT Conditions: * Plant extract rich in antioxidant are: - Orange or lemon peel (dry and coarsely powder). - Green Tea leaves (coarsely powder). * Standard antioxidant (References): Gallic acid, Ellagic acid, Arbutin, Hydroquinone & Rutin (at concentration = 1-2 mg/ml MeOH). * Solvent System: -For orange peel, Lemon peel and green tea leaves is (for flavonoids): Ethylacetate : Formic acid : H 2 O (8 : 1 : 1) Or CHCl 3 : Ethylacetate (6 : 4) 32

33. * Detection : 1. DPPH solution (20 mg/100 ml in MeOH), must be freshly prepared. (DPPH= 1,1 Diphenyl-2-picrylhydrazyl) 2. Heat in Oven at 60 ο C. Procedure: 1. Spotting of the silica gel TLC plate. 2. Develop the plate. 3. Dry plates at open air for 15-20 seconds. 4. Dip the plate in DPPH which in Petri dish. 5. Dry plate in dark for 90 seconds at room temperature then for 30 seconds at 60 ο C. 6. Free radical scavenging zone were identified immediately as yellow area against violet purple background plate 33

34.  Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion (intrusion on and destruction of adjacent tissues) and sometimes metastasis (spread to other locations in the body via lymph or blood).  Causes: - Tobacco smoke - Infectious agents - Radiation - Chemical (Benzene) - Diet (coloring agent, preservative) - Pesticide - Drug Anticancer Screening of Natural Products : 34

35.  There are currently some well known structural classes of plant derived anticancer agents in the market. These are: 1.The vinca alkaloids (vinblastine, vincristine): were isolated from Catharanthus roseus and are used for the treatment of a wide variety of cancers, including leukemia, bladder cancer and testicular cancer. 2. The taxanes (paclitaxel and taxol): were isolated from Taxus brevifolia and are used for the treatment of a wide variety of cancer including Ovarian, Lung, Gastric, Cervical, Prostate & Colon cancer 3. The camptothecin derivatives (topotecan and irinotecan). Uses: Ovarian cancers, Colorectal cancer. 35

36. Cytotoxicity assays 1- Brine shrimp lethality assay:  Artemia salina is commonly known as the brine shrimp.  Larvae were hatched at 26 ºC in filtered seawater.  A lamp was placed above the tank. After 24 hrs, hatched nauplii were used for the assay.  Plant extract with different concentration was added to brine solution.  The experiment was run in five replicates of each concentration.  The number of dead and survivor brine shrimps in each tube was counted after 24 h.  To ensure that mortality observed in the bioassay is related to bioactive compounds, dead larvae in each treatment were compared to the dead larvae in the control.  The percentage of mortality was calculated as: percentage of survival in the control-percentage of survival in the treatment The brine shrimp assay is one of the best and rapid biological and toxicological assays for all the labs. 36

37.  Cirrhosis is the formation of fibrous tissue in the liver from replacing dead liver cells. The death of the liver cells can be caused by viral hepatitis, alcoholism or contact with other liver-toxic chemicals.  Diagnosis is done by checking levels of Serum glutamic oxaloacetic transaminase and Serum glutamic pyruvic transaminase Method: 1- Animals (rabbits/ rats) - Control (saline) - Standard (Silymarin) - Extracts or isolated compounds 2- Induction of cirrhosis in animals by using: - CCl 4 or CHCl 3 (reversible) - Galactosamine or α- amanitin (irreversible) 3- Administer extract and standard 24 hours measure enzymes Hepatoprotective Screening of Natural Products 37

38. Although random compound screening in animal models is still a useful approach to discover new drugs. Disadvantages: -It requires a large amount of compound. - Its sensitivity is low. - It is extremely laborious. - Since the amount of active constituents present in natural products is usually very small, it is impractical in most cases, to supply sufficient quantities of pure natural compounds for animal experimentation. 38

39. Problems with natural product screening 1. Isolation of an active component is problematic because of very small amount of active constituent along with large amount of background “rubbish”. 2.The mixtures are often very complex and contain many large macromolecules. These can often “hide” biological activity. 3.Compound isolation and structure determination difficult. 4. Structures often complex, therefore difficult to synthesize and identify the pharmacophore. 5.Plant materials are chemically and naturally variable. 39

40. 6. Herbal medicines contain a large variety of different compounds. Several of these may have biological activity, but there is a significant risk of side effects and toxicity. The active principle present in small amount, so herbals are expected to be less active than pure compound. 7.Herbal medicines may be interacting with prescribed medicines and there is no regulations or control of this matter and their uses. 8. But it is an important lead to discover and design new drugs. 9.Selective analytical methods or reference compounds may not be available commercially. 40

41. 10. The active principles are frequently unknown; 11. Standardization, stability and quality control are feasible but not easy. 12. The availability and quality of raw materials are frequently problematic. 13.Well-controlled double-blind clinical and toxicological studies to prove their efficacy and safety are rare. 14. The occurrence of undesirable side effects seems to be less frequent with herbal medicines, but well- controlled randomized clinical trials have revealed that they also exist. 41

42. High-throughput technologies: High-throughput screening (HTS) is an approach to drug discovery that has gained widespread popularity over the last three or four years. HTS is the process of assaying a large number of potential effectors of biological activity against targets. The methods of HTS are applied to the screening of stereo chemistry of compounds, genomics, protein and peptide libraries. The goal of HTS is to accelerate drug discovery by screening large libraries often composed of hundreds of thousands of compounds. 42

43. Advantage: HTS decreases the amount of testing compound required such that only microgram quantities are needed. This is advantageous for certain natural products that are difficult to isolate and purify, and permits compounds that are difficult to synthesize to be assayed. The number of compounds assayed has increased from 100 000 per year to 100 000 per day. HTS need: Highly specialized and expensive screening lab to run an HTS operation. 43

44. High-throughput screening (HTS)  Using robotics, data processing and control software, liquid handling devices, and sensitive detectors.  HTS allows a researcher to quickly conduct millions of biochemical, genetic or pharmacological tests.  The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology. 44

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46. Methodology and equipment  HTS uses automation to run an assay, or screen, of a library of candidate compounds against a target. Note: Automation: Typically, an integrated robot system consisting of one or more robots transports assay-microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. 46

47. Assay plate preparation  The key labware or testing vessel of HTS is the microtiter plate: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells.  Modern microplates for HTS generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 129mm spaced wells.  To prepare for an assay, the researcher fills each well of the plate with some biological matter such as protein or some cells, or an animal embryo.  After some incubation time has passed to allow the biological matter to absorb, bind to, or to react with the compounds in the wells, measurements are taken across all the plate's wells, either manually or by a machine. 47

48.  Manual measurements are often necessary when the researcher is using microscopy to seek changes or defects in embryonic development caused by the wells' compounds, looking for effects that a computer could not easily determine by itself. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells. In this case, the machine outputs the result of each experiment as a grid of numeric values, with each number mapping to the value obtained from a single well.  A high-capacity analysis machine can measure dozens of plates in the space of a few minutes like this, generating thousands of experimental data points very quickly. 48

49. High-Throughput Screening Up to 100,000 samples/24 hours 49

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51. Fractionation of the active extracts Fractionation of the active extracts Send fractions for biological screening Send fractions for biological screening Isolate compounds from active fractions Isolate compounds from active fractions Structural elucidation of isolated compounds Structural elucidation of isolated compounds Send pure compounds for biological screening Send pure compounds for biological screening Hopefully active molecule/s is/are found Hopefully active molecule/s is/are found BIOLOGICALLY DIRECTED PHYTOCHEMICAL STUDY The isolation of biologically active constituents Target: Pharmacological Activity 51

52. Fractionation and isolation The right chromatography techniques: CC, TLC, VC, MPLC, HPLC; solvent systems and stationary phase critical in hunting down the bioactive compound/s. 52

53. Conventional Phytochemical Evaluation 1. To study cpd. which were most easily separated (present in the largest quantities and crystallized readily) 2. To study the specific group of interest (e.g. alkaloids, terpenoids, phenols, etc.) 3. To elucidate the isolated compounds 4. To test for their biological activities (this would depend on sufficient material being available) Countless medicinally useful cpd. Have been missed in this type of approach. 53

54. The isolation of biologically active constituents  Systematic investigation 1. Every portion of the plant and every fraction of the extract is biologically tested before isolation or characterization 2. Any class of active cpd. may be isolated 3. May not be traditionally associated with a particular plant family 54