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Bioassay

Bioassay is a method used to estimate the potency of substances using living organisms, applicable when chemical assays are unavailable or when high sensitivity is required. It includes various types such as quantal and graded responses, and can involve different biological systems like animals, microorganisms, or plants. Bioassay-guided fractionation is crucial in drug discovery, allowing for the isolation of active compounds from natural sources, despite challenges like complexity and resource intensity.

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20 views

Bioassay

Bioassay is a method used to estimate the potency of substances using living organisms, applicable when chemical assays are unavailable or when high sensitivity is required. It includes various types such as quantal and graded responses, and can involve different biological systems like animals, microorganisms, or plants. Bioassay-guided fractionation is crucial in drug discovery, allowing for the isolation of active compounds from natural sources, despite challenges like complexity and resource intensity.

Uploaded by

sanjeev khanal
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Bioassay: Principle and Application

• Bioassay: Bio + Assay


• Bio: Living and Assay: To test
• Bioassay: produce to estimate potency of nature of substance using living organism:
which is having life.
• Example
• 1. In vivo: Bioassay of insulin using mouse.
2. In vitro: Histamine in ileum of guinea pig
• Bioassay is performed when:
a. Chemical assay method for substance is not available or possible.
b. When the quantity of sample is too small = matching bioassay is done.
c. To estimate the concentration of active principle of tissue extract.
d. To measure the pharmacological activity of new/unidentified agent.
e. To measure the drug toxicity.
f. When high sensitivity of result is required.

Note: The precision, accuracy and reproducibility depends on:


a. proper selection of tissue or method with highest sensitivity with drug
b. Bioassay is essential in development of new drugs
c. used in preclinical trial
Measurement and Types of Bioassay
• Measurement : comparing the test substance with the international standard
preparation of the same and finding out how much test substance is required to
produce the same biological effect, as produced by the standard.
• Types :
a. Quantal: all or non phenomenon (yes or no)
eg: digitalis induced cardiac arrest
b. Graded: (Gradually) response is increased with subsequent increase in dose.
• Parameters of bioassay is based on:
a. Nature of effect produced by agent. Eg: contraction of smooth muscle for the
assay of histamine.
b. Study of blood pressure response by adrenaline.

Subtypes of graded:
a. Matching
b. Bracketting
c.Interpolation
d.Multiple point
e. Demanded
f.Quantity of sample
g. Availability of exp animal
Variation of bioassays based on biological systems:

i) Animal (mouse, rat, guinea pig, rabbits etc)


ii) Micro-organisms or cell based assay using microbes like bacteria, fungi or cultured cells
for antibiotics & growth stimulants/suppressants.
iii) Plant (using plant constituents to evaluate a sample)

1. Bioassays for agonists:

a)End point method(based on quantal effect):

-The threshold dose producing the therapeutic effect is measured on the each animal.

-Then comparison is made between the average results of the two groups of animals.
-Sp./Std. are carried out simultaneously.
- Finally potency is calculated from the following formula:

Conc. of the unknown= X Conc. of Std.

b)Matching method(based on the graded response):

-The constant dose of the test is bracketed by varying doses of the standard until the exact
match is observed between the sample & the standard.

-Response of the standard is measured at 20% & 70% of the maximum response.
(For linearity : response vs. dose)

-The test will be repeated by increasing or decreasing the standard doses until the three
equal responses are obtained.
The concentration of the sample is determined by the following formula:

Concentration of the sample = X Conc. of Std

c) Graphical method(based on the graded response):

-5-6 responses of standard graded doses of the standard drug are taken & the
two doses of the sample drug are taken.

-The height of the peak is plotted against the log dose.


The dose of the sample is directly taken from the graph.

Conc. of the Sp.= [n1/t] x antilog{[(T-S1 )/(S2 -S1)] x [log n2 / n1]} x Cs

Where, n1 is the lower standard dose; n2 is the higher standard dose; t is the test dose, S1 is
response of lower standard dose; S2 is the response of the higher standard dose, T is the
response of test; Cs is the concentration of the standard.

ii) Micro-organisms or cell based assay:


-It is used to evaluate the potency of the formulations used.
(to follow purification process, its distribution within the blood & tissues when given to
animals & finally human. Monitoring antibiotics in blood & urine in a trace amount)

-Used in case of unavailability of physical or chemical method.


-Is performed using a suitable test organism.
-Most micro-biological assay procedures are highly sensitive (up to 0.1mcg) & selective.

-Sensitivity & selectivity are to measure the characteristic activity of biological products.
Assay techniques:
i)Plate diffusion
ii)Tube-dilution

i)Plate diffusion:
-The agar plates are inoculated with a test organism.
-Measured amounts of test substance are introduced into agar plates.
-It diffuses into the agar & produces zones of inhibition.
-The diameter of the zones may be plotted against log potency or conc. for a standard.
-By interpolation, potency of the test material can be determined.

-Suitable for antibiotic potency test.

ii)Tube dilution:

-Suitable dilutions of sample are introduced into an appropriate nutrient medium.

-Suppression or stimulation of growth takes place.

-Suitable for the determination of vitamin B & amino acids.

-Measurement is done by nephelometry, titration or gravimetry.


Bioassay Guided Fractionation
• Fractionation is a process used in Natural Product Chemistry to separate
a crude drug extract into its individual chemical components or fractions.

• Bioassay-guided fractionation is a process in which extract is


chromatographically fractionated and refractionated until a pure
biologically active compound is isolated.
• Each fraction produced during fractionation process is evaluated in a
bioassay system and only active fractions are fractionated.
Steps in Bioassay Guided Fractionation
1. Extraction: Involves extraction of crude mixture from natural sources.

2. Initial bioassay: The crude extract is subjected to a bioassay to evaluate it’s


biological activity. This could involve testing for antimicrobial, anti-inflammatory,
anticancer, etc.

3. Fractionation: The biologically extracted crude extract is fractionated using


various chromatographic techniques such as column chromatography, Thin Layer
Chromatography(TLC), High-Performance Liquid Chromatography(HPLC), Gas
Chromatography(GC).
4. Bioassay of Fractions: Each fraction obtained from the initial fractionation is tested in the
same bioassay to identify which fractions retain the biological activity observed in the
crude extract. This steps helps narrow down the fractions that contain the active
compounds.

5. Refractionation: Active fractions are subjected to further fractionation to isolate


individual compound. This process involves repeated cycles of fractionation and
bioassaying until pure bioactive compound are obtained.

6. Identification and Characterization: The isolatd bioactive compounds are identified and
characterized using spectroscopic techniques such as Mass Spectroscopy(MS), NMR
spectroscopy, IR spectroscopy, UV-Vis Spectroscopy.
Applications and Importance

• Drug Discovery: Bioassay-guided fractionation is fundamental in the discovery of


new drugs from natural sources, providing leads for development of pharmaceuticals.

• Quality Control: Ensures that the bioactive compounds in herbal medicines are
accurately identified and quantified, improving the quality and efficacy of these
products.

• Ethnopharmacognosy: Validates traditional medicinal uses of plants by isolating and


identifying active compounds, bridging traditional knowledge with modern science.

Challenges
• Complexity and time consuming
• Requirement of suitable bioassay
• Resource intensive
Examples of Case Study
1. Discovery of Artemisinin
• Initial Extraction: Artemisia annua (sweet wormwood) was extracted using
solvents.
• Bioassay: The crude extract showed potent antimalarial activity in bioassays.
• Fractionation: The extract was fractionated using chromatographic techniques.
• Testing Fractions: Fractions were tested for antimalarial activity, pinpointing
the active fraction.
• Isolation: Artemisinin was isolated from the active fraction.
• Identification: Structural elucidation confirmed the compound as artemisinin.
• Validation: Artemisinin was confirmed to be highly effective against malaria,
leading to its development as a drug.
2. Discovery of Paclitaxel (Taxol)
• Plant Source: Pacific yew tree (Taxus brevifolia)
• Bioactivity: Anticancer properties
• Objective: To isolate and identify the compound responsible for the anticancer activity
from Taxus brevifolia.

Steps in Bioassay-Guided Fractionation


1.Initial Testing
• Crude Extract Testing: The crude extract of Taxus brevifolia bark demonstrated
significant cytotoxic activity against various cancer cell lines in vitro.
2. Fractionation and Bioassay
• Solvent Partitioning: The crude extract was partitioned between water and chloroform.
The chloroform phase showed the highest cytotoxic activity.
• Column Chromatography: The chloroform extract was subjected to column
chromatography, yielding several fractions.
• Bioassay Screening: Each fraction was tested for cytotoxicity. Fractions with high
activity were pooled and subjected to further purification.
3. Isolation and Purification
• HPLC: Active fractions were further separated using high-performance liquid
chromatography, leading to the isolation of a pure compound.
• Structural Elucidation: Spectroscopic analysis identified the compound as
paclitaxel.

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