Random Selection Approach

❑ Pharmacological screening is performed in the plants which are selected randomly to find out any
particular activity for discovering new drug from plants are collected randomly and the extracts are
submitted for desired pharmacological activity test 

Example: Natural Cancer Institute (NIC) of USA selected 405 different plant species randomly and
evaluates the extracts for antitumor activity. Among them, 42 species showed activity. Of them, 6
species showed most promising activity. Further, isolation of active compounds from these plants had
been done.4 

Selection of plants containing specific  type of chemical compounds

Phytochemical screening: In this various parts of the plants are test for specific class of compounds  The
plants showing good positive test for the desired group of substances will be collected and subjected to
a variety of pharmacological screening 

Example: If alkaloids class compounds showed biological activity then first alkaloid test is performed for
different plants and only those showing the positive test are collected and submitted for
pharmacological screening 

Selection of plants based on a combination of criteria

Plants are selected for pharmacological screening on the basis of premade criteria such as  Traditional
medicinal uses , Chemical constituents present in plant ,Botanical aspects, etc 

Example: When all plants traditionally used for high fever was screened. Cinchona officinalis had shown
potent activity. Further, Quinine (Chloroquine) were isolated from it 

Generally this type of approach is considered expensive for profit making pharmaceutical firms .

Pharmacological Screening Techniques

Primary pharmacological screening is the preliminary investigation of pharmacological activity of crude
drugs Primary screening is always conducted on only minimum amounts of carefully authenticated
crude drugs or their extractives, If promising activity has been found during primary screening then a
sizeable quantity of authenticated plant material is acquired and secondary evaluations are organized .

Natural product screening

Natural product screening marries the search for new medicines with the search for new molecules
from natural sources. As technology has advanced, the ability to screen for new drug leads has also
increased. 

Approaches (Techniques) to Primary  Pharmacological Screening

 Single Technique- Single Goal Screening: To search for only one particular type of activity,it
involves limited observation
Example: -Plants screen for anti-diabetic activity. In this, alloxan-induce diabetic rats were used
to scree plants for only one pharmacological end point i.e. lowering of blood glucose. 
-177 plant extracts screen for anti-inflammatory  potential using only the carrageenin-induced
pedal edema assay in the rat .
-Aqueous extracts of 256 species of plants screen for  their capacity to inhibit human plasma
cholinesterase

 Screening Using a Battery of Specific Procedures: • Perform multiple test to define the
pharmacological activity.  Each test/procedure searching for a different types of information to
indicate define pharmacological activity. 
Example: Four different isolated- organ preparations to screen aqueous extracts of 55
himalayan plants of Nepal to test contraction and relaxation These preparations: guinea pig
ileum, rat uterus, rat hind limb, and rabbit heart 

 Single Technique – (Multiple Goal Screening): Using single technique designed to detect
multiple activities, take one extract to screen multiple pharmacological activities 
Examples: Intraperitoneal injection of the ethanolic  extracts of 45 crude drugs in
unanesthetized rat.  Recording of dose induced symptomatology. Based  upon dose-response
patterns, pharmacological  activities can be categorised. Such as; a) general  inactive, b) CNS
depressent, c) tranquiliz- ing activites, d) skeletal muscle relaxant, e) diuretic, f)  peripheral
vasodilation, g) sympathetic stimulant, h)  parasympathetic stimulant etc. 16 
 Combination of specific and multipurpose procedure: Some to detect specific and some to
detect multiple activities 
Example: Screening in Australia, general screening for activity (toxicity + pharmacological
activity), testing for chemotherapeutic potential and determine the effect of chronic feeding 
 Ideal requirements for a primary screen
 results must be able to be extrapolated to man either directly or by analogy with clinically
effective drugs which have also been screened by the procedure,
 potentially useful pharmacological activity must not go undetected even though the activity
may be either unexpected or unique,
 the probable nature of the activity should be indicated so that subsequent research can be
organized intelligently,
 the procedure must be unbiased and allow for the coding of all samples, including both
"known" reference materials and "unknown" test materials,
 results obtained should be reproducible if second or third "runs" are conducted,
 the screen should detect both rapid-onset and delayed-onset activity,
 the procedure should be a multilevel dose-response experiment,
 the procedures for setting up the experiment and collecting data should be standardized so as
to allow cross-comparisons with known pure and crude drugs,
 the screen should allow the use of both crude material and extractives so that the procedure
can be used to direct extraction, isolation and purification procedures for the Phyto chemist,
 completion of a single screen should not require more than 1.0 - 2.0 g of crude dry plant
material,
 with sufficient replication and the concurrent testing of a standard drug, statistically sound
bioassays should result, i.e. data from the primary screen can be incorporated into larger scale,
more definitive (publishable) research,
 potential toxic activity should be indicated by the procedure so that subsequent research does
not ignore this aspect,
 a single dosing vehicle should be used which does not affect screening results either
qualitatively or quantitatively,
 the procedure should not require expensive equipment or sophisticated laboratory
environments so that primary-level experiments might be conducted more near the sites of
collection -- especially in the case of natural products containing very labile active constituents
 the procedures should be capable of being taught easily to technicians so that highly trained
and educated scientists are not required for the day-to-day operation of the program
 the procedure should not be time-consuming.
 lastly, the screen should be relatively inexpensive to conduct over a sustained period of time --
the whole purpose of screening is lost if one selects a slightly cheaper, somewhat less efficient
screen and then proceeds to miss "useful" activity

Multidimentional Primary Screening

Multipurpose or multidimensional primary screening involves the observation of drug-induced
symptomatology in intact unanesthetized animals and deduction of the results.

Here the drug is first administered to animal and the changes occurred such as change in BP, heart beat,
CNS, eye color, motor activity, etc in the animal are observed and filled in prepared worksheet .

These were developed independently in several USA drug companies in the beginning years of the 1950
- 1960. The original impetus was to document the changes in spontaneous behavior produced by the
newly introduced psychopharmaceuticals

Multidimentional Secondary Screening

Primary screening is always conducted using only minimum amounts of carefully authenticated crude
drug. The screening of an unauthenticated sample is a waste of time and money. If promising activity
has been found in primary screening, secondary evaluations are organized Secondary testing should
confirm in another laboratory or species of animal and the activity noted in the primary screen should
consist .

Secondary evaluation should also be multidimensional and standardized so as to allow comparisons

with known drugs All the secondary testing should be conducted using either solvent free extracts or
semi pure/pure chemicals Phytochemist can make rough estimates for physiochemical characteristics
of active principles If active principles corresponds physiochemically with a known drug, all further
research is terminated If the secondary testing confirms the activity and indicates some unique
potential or if active principle also appears to be chemically new then a decision will have to be made
whether or not to launch the very expensive, very time consuming tertiary evaluation or investigation It
is believed that it neither wise nor economically sound to conduct primary, secondary, and tertiary
research using only one batch of crude drug. Taxonomists can make mistakes.

Tertiary Screening
If the secondary testing confirms the activity and indicates some unique potential and if the active
principle also appears to be chemically new then expensive and time consuming tertiary evaluations are
done  The tertiary investigation consists of  -Chemical structure determination and -Classical
pharmacological and toxicological  research. 

Tertiary investigation never truly ends as there always remains some facet left unexplained and open
for further research.

Tertiary pharmacological evaluations must be done using only chemically pure material. Large number
of authentic crude material will be needed on a regular basis until a synthetic or semi synthetic
procedure can be developed, Tertiary pharmacological investigation of a drug compound never really
ceases even after the drug is released for sale.

Bioassay
It is defined as estimation or determination of concentration or potency of physical, chemical or
biological agents by means of measuring and comparing the magnitude of the response of the test with
that of standard over a suitable biological system under standard set of conditions

Bioassays are methods used for estimation of the potency of substances by observing their
pharmacological effects on living animals (in vivo) or isolated cells/ tissues (in vitro) and comparing the
effect of these substances of unknown potency to the effect of a standard.

It is a type of scientific experiment typically conducted to measure the effects of a substance on a living
matter and  is essential in the development of new drugs and in  monitoring environmental pollutants.  

Comparative assessment of the response produced in biological system by the test compound or
mixture of  compounds compared with that of standard sample. 

It can be defined as an analytical procedure measuring a biological activity of a test substance based on
a specific, functional, biological response of a test system.

Principle of bioassay:
Types of bioassay

Direct Assay: 

• Doses of the standard and test preparations are sufficient to produce a specified response, and can  be
directly measured.  

Indirect Assay: 

• In indirect bio-assays the relationship between the  dose and response of each preparation (extract) is 
first ascertained. Then the dose corresponding to a  given response is obtained from the relation for
each  preparation separately. 

 Quantal Assay  

• This response is in the form of “all or none” means  either no response or maximum response.  • These
can be biossayed by end point method. The threshold dose producing a predetermined effect is 
measured . Quantal Responses are population response based  on an all-or-nothing (0 or 1 – presence or
absence)  response such as death  

• E.g.: Bioassay of digitalis in cats, Insulin induced  hypoglycemic convulsions in rat

Quantitative Assay
Bioassay guided fractionation

Bioassay guided fractionation

of plant extracts
linked to chromatographic
separation techniques
can leads to isolation of
biological active mole-
cules
Bioassay guided fractionation of plant extracts linked to chromatographic separation techniques can
leads to isolation of biological active molecules. As a result of the recent interest in the plant kingdom as
a potential source of new drugs, strategies for the fractionation of plant extracts based on biological
activity rath-er than on a particular class of compound have been developed. The chemical examination
follows after the isolation of the active fraction. The search for a new drug from nature is based on a
biological and ecological rationale.

Bioactivity-directed fractionation (= isolation of active compounds from biomass using a decision tree
based solely on bioactivity).

A typical protocol to isolate a pure chemical agent from natural origin is bioassay-guided fractionation,
meaning step-by-step separation of extracted components based on differences in their
physicochemical properties, and assessing the biological activity, followed by next round of separation
and assaying

Bioas-say-guided fractionation is a procedure, where-by extract is chromatographically fractionated and

refractionated until a pure biologically active compound is isolated. Each fraction produced during the
fractionation process is evaluated in a bioassay system and only active fractions are fractionated.
FIG:
Computerized program
Carrano and Truax (1968) of Atlas Chemical Industries devised the first computer evaluation method for
the superabundance of data produced by rat screen when conducted on a regular basis

The goals of computerized screen is to provide a rapid data retrieval system and a rapid and unbiased in-
depth comparison of each test substance against a library of similarly testing prototype drug with known
clinical utility

Computer evaluation programs do not replace the human evaluation of data

Different steps in computer evaluation

Out put A: consists data and technicians general comments arranged per dose levels

Out put B: presents a summary of calculation made to indicate the gross category of the drug’s activity
Out put C: represent a summary of the data for each symptom noted and is intended to assist human
evaluation of dose response and time-response relationships. The parameters which are not shown by
any dose level are not included

Out put D: lists those symptoms (important) which should be considered to be dose related and
indicated the dose at which these symptoms first appeared i.e. list of the symptoms for which there is
either marked decrease or increase in activity

Out put E: indicates the results of matching dose-related and important symptoms for the test
substance with the computer’s library of reference drugs

Out put F: the important symptoms for each dose level are ranked in order of severity

Out put G: compares the dose/effect/time matrix of the test material with corresponding matrices of all
library drugs and converts the data to a new scheme which reflects a weightage for every corresponding
match of the test drug with a library drug

The totals are then ranked by computer and gap-jump comparisons made to indicate which of the
library drug statistically match with the test drug

Phytochemical  Screening Test for Common  Secondary Metabolites

Nearly 80% of the world’s population relies on traditional medicines for primary health care, most of
which involve the use of plant extracts

• Complete phytochemical investigations of medicinal plants should be carried out, because these
secondary metabolites are responsible for medicinal activity of the plant • Secondary metabolites have
medicinal values • Preliminary qualitative phytochemical screening was carried out to have an idea of
the chemical constituents of plants

Alkaloids
Alkaloid, any of a class of naturally occurring organic nitrogen-containing bases. Alkaloids have diverse
and important physiological effects on humans and other animals. Well-known alkaloids
include morphine, strychnine,  quinine, ephedrine, and nicotine.
 Saponin
Saponins comprise a large family of structurally related compounds containing a steroid or triterpenoid
aglycone (sapogenin) linked to one or more oligosaccharide moieties. They are characterized by their
hemolytic activity and foaming properties and are responsible for imparting a bitter taste and
astringency to plant materials containing a high concentration of saponins.

Tannins

Tannin, also called tannic acid, any of a group of phenolic compounds in woody flowering plants that
are important deterrents to herbivores and have a number of industrial applications. As secondary
metabolites, tannins are sequestered in vacuoles within the plant cell, which protects the other cell
components. They occur normally in the roots, wood, bark, leaves, and fruit of many plants, particularly
in the bark of oak (Quercus) species and in sumac (Rhus) and myrobalan (Terminalia chebula). They also
occur in galls, pathological growths resulting from insect attacks.
Terpenoids
Pharmacological screening procedure for common pharmacological
activities:
Anti inflammatory
Analgesic assay
 Antioxidant screening
DPPH radical scavenging assay:

 DPPH has been widely used for measurement of free radical scavenging ability of antioxidants. This method is based on
the reduction of an alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant. Hydrogen atom or
electron-donation ability of the corresponding compounds were measured spectrophotometrically from the bleaching of
the purple-colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In this study, antioxidant activity of
tested compounds was measured using the stable radical 2, 2- diphenyl-1-picrylhydraziyl (DPPH). The free radical
scavenging capacity of betaines was determined using the DPPH according to the method of Blois with some
modifications. A solution of DPPH in methanol (0.004%) was prepared and 1mL of this solution was mixed with 1mL of
varying concentrations of betaines solution in ethanol. The reaction mixture was vortexed thoroughly and left in the dark
at room temperature. The absorbance of the mixture was spectrophotometrically measured at λ max=517nm and
compared to the standard antioxidants (BHT, BHA and ascorbic acid (vitamin C).
 Anticancer screening
Invitro
Invivo method