Biological and therapeutic evaluation

There are various approaches for biological and therapeutic evaluations of compounds derived from natural substances.
The classical approach is to demonstrate a pharmacological effect on animal models. For example measuring a complex response in vivo such as hypoglycaemic effect in animals with experimental alloxan induced diabetes, prevention of experimentally induced seizures, suppression of an inflammatory response, decrease in cholesterol level etc. The prior identification of a drug target is not necessary. However they are expensive and often difficult to interpret, some simpler tests may be used. These tests require less effort and better understanding of the mechanism of action of test substance is possible.
Non-animal models representing a living organism may be used. For example to investigate antihepatotoxic substances animals were subjected to toxic substances, such as carbon tetrachloride, alpha amanitine, and phalloidin. Now in vitro methods using primary cultures of rat hepatocytes subjected to prior cytotoxic effect of D-galactosamine have been developed. By measuring released transaminases or by establishing viability curves for cells the hepatoprotective power of the test compound can be presumed.

Enzyme Activators and Inhibitors
Another approach for evaluation of biological activity is to identify enzyme activators or inhibitors. As abnormality in cyclic nucleotide system occurs in many disease conditions like asthma, cancer, psoriasis, obesity and platelet aggregation, therapeutic use of activators and inhibitors of this system offers great potential. The concentration of thenucleotide 3', 5'-monophosphates responsible for the biological activity can be increased either by stimulating adenylate cyclase or by inhibiting phosphodiesterase, the enzyme responsible for the hydrolysis of 3',5'-cyclic AMP into the nucleotide 5 monophosphate.
For example Forskolin, a diterpene obtained from the roots of Coleus forskohli activates adenylate cyclase whereas papaverine, xanthines and flavonoids are the inhibitors of phosphodiesterase.

Target Selection
As majority of drugs act by targeting a functional proteins e.g. receptors, enzymes, ion-channels and carrier molecules, the prior identification of a drug target is necessary. Natural products which are mainly derived from fungal and plant sources recognise with great precision vulnerable targets in their enemies or competitors. Familiar examples include penicillin, streptomycin, vinca alkaloids, taxol, cyclosporin and rapamycin. Moreover these compounds can also serve the purpose of lead compounds. However the main disadvantages are that they are complex molecules and it is difficult to synthesise or modify by conventional synthetic chemistry and therefore commercial production is very expensive.
Another problem is that of target identification. Currently these are about 400 distinct targets and there are many more proteins which play a role in disease. However a thorough knowledge of the biochemical process involved in pathogenesis of the disease makes the job somewhat easier. For example, the knowledge that inhibition of angiotensin converting enzyme lowers blood pressure by suppressing angiotensin-II formation, a series of antihypertensive drug acting as antagonists of angiotensin-II receptors can be developed.

Tests on animal models The classical approach is to demonstrate a pharmacological effect on animal models. For example measuring a complex response in vivo such as hypoglycaemic effect in animals with experimental alloxan induced diabetes, prevention of experimentally induced seizures, suppression of an inflammatory response, decrease in cholesterol level etc. The prior identification of a drug target is not necessary. However they are expensive and often difficult to interpret, some simpler tests may be used. These tests require less effort and better understanding of the mechanism of action of test substance is possible.

Tests on Non-animal models  Non-animal models representing a living organism may be used. For example to investigate antihepatotoxic substances animals were subjected to toxic substances, such as carbon tetrachloride, alpha amanitine, and phalloidin. Now in vitro methods using primary cultures of rat hepatocytes subjected to prior cytotoxic effect of D-galactosamine have been developed. By measuring released transaminases or by establishing viability curves for cells the hepatoprotective power of the test compound can be presumed.

Some more examples of in vitro bioassays for determining useful drug effects are listed in Table:

Table  Examples of in vitro bioassays for determining useful drug effects

Assay                             Type System              Useful Effects

Angiotensin converting enzyme          In vitro                             Antihypertensive Inhibition
ATPase inhibition                                 In vitro                             Cardiotonic
HMG-CoA reductase                           In vitro                             AntihypercholesterInhibition olaemic
Platelet aggregation inhibition              In vitro                             Cardiovascular problems
COX inhibition                                      In vitro                             Antiinflammatory
Protease inhibition                               In vitro                             Anticancer
Antibacterial activity                           Bacterial culture              Antiinfective
Antimitotic activity                              Cell culture                       Anticancer
Cytotoxicity                                        Cell culture                       Anticancer
Free radicals                                      Cell culture                       Anticancer

Structure elucidation, Synthesis and Structure modification

                Structure elucidation of the isolated compound is a complicated process. It is essential that the isolated compound is pure as shown by the presence of a single spot or peak when examined by thin layer or gas or liquid chromatography.
The following characteristics of the compound should be recorded:

1. Melting point
2. Chromatographic behavior
3. UV/visible spectrum
4. IR spectrum using potassium bromide disc
5. Mass spectrum
6. Proton and C-13 Nuclear magnetic resonance(NMR)spectra

             After the determination of structure of the isolated compound it can be synthesized or its structure can be modified to improve the pharmacological activity of the compound or to produce drugs with better effects and less side effects.
Thus the plants remain a reservoir of potential for the discovery of new drugs with valuable pharmacological activities. These drugs can be used in their natural
state or can be modified to synthesize new compounds of even better therapeutic compounds of even better therapeutic value.