In vitro evaluation techniques are important for screening potential drugs before clinical trials. There are two stages of biological screening - primary and secondary assays - to identify plants with therapeutic activity. Various in vitro assays can evaluate antioxidant and anticancer properties of herbal extracts, including DPPH radical scavenging, MTT, and SRB assays. In vitro methods have advantages over animal models for initial drug screening by reducing animal use and providing reproducible, economical screening of potential drug candidates. While in vitro models cannot fully replace in vivo studies, they provide an important first step in evaluating biological activity of natural products.

1. IN- VITRO
EVALUATION TECHNIQUES
NANDINI PANDEY
http://www.free-powe

2. INTRODUCTION
Drug screening is the process by which potential drugs
are identified and optimized before selection of a
candidate drug to progress to clinical trials.
Biological screenings are carried out in stages to identify
potentially active plants against validated therapeutic
targets. There are two stages of biological screening
which are the Primary assays and the Secondary assays.

3. Need for biological screening
for herbal drugs
Over the centuries, medicinal plants have been utilized in various
cultures of the world as a natural healing tool.
These plants are mostly exercised in unrefined
or semi-processed form, often in mixtures; therefore require quality
control testing and rigorous clinical trials for scientific rationale
Galen, a Greek pharmacist and physician who showed that herbs do not
contain only medicinally beneficial constituents, but may also be const
ituted with harmful substances.
Less than 10% of herbal products in the world market are truly stand
ardized to known active components and strict quality control measures are
not always diligently adhered to .
In vitro and In vivo studies can reveal some of the risks that may b
e associated with use of herbs, therefore avoiding potential ha
rmful effects when used as medicine.

4. ANTIOXIDANTS
Oxidation is a
chemical reaction that can
produce free radicals,
thereby leading to chain
reactions that may damage
the cells of organisms.
Antioxidants terminate
these chain reactions.

5. Oxidants and their effects on the body:
• A paradox in metabolism is that,
while the vast majority of complex
life on Earth requires oxygen for
its existence, oxygen is a highly
reactive molecule that damages
living organisms by producing
reactive oxygen species.
• Antioxidant systems either prevent
these reactive species from being for
med, or remove them before they can
damage vital components of the cell.
• Some natural antioxidants are:
ASCORBIC ACID, CAROTENES,
URIC ACID

6. DPPH scavenging activity
 Hydrogen peroxide scavengin
g assay
 Nitric oxide scavenging activity
 Peroxynitrite radical scavengin
g activity
 Trolox equivalent antioxidant ca
pacity (TEAC) method
 Total radical-trapping antioxi
dant parameter (TRAP) method
 Ferric reducing-antioxidant p
ower (FRAP) assay
 Superoxide radical scavenging
activity (SOD)
 Hydroxyl radical averting capac
ity (HORAC) method
 Oxygen radical absorbance ca
pacity (ORAC) Method
 Reducing power method (RP)
 Phosphomolybdenum method
 Ferric thiocyanate (FTC)
method
 Thiobarbituric acid (TBA)
method
β-carotene linoleic acid
method/conjugated diene
assay
 Xanthine oxidase method
 Cupric ion reducing
antioxidant capacity (CUPRAC)
method
 Metal chelating activity
 DMPD (N,N-dimethyl-p-pheny
lene diamine dihydrochloride)
method

7. DPPH scavenging activity
• Change in optical density of DPPH radicals is monito
red
• Sample extract (0.2 mL) is diluted with methanol and
2 mL of DPPH solution(0.5 mM) is added.
• After 30 min, the absorbance is measured at 517 nm.
Metal chelating activity
•Ferrozine can form a complex with a red color by
forming chelates with Fe.
• This reaction is restricted in the presence of other
chelating agent.
• Measurement of the color reduction determines the
chelating activity.
Hydrogen peroxide scavenging (H2O2) assay
• A solution of hydrogen peroxide (40 mM) is prepared in
phosphate buffer (50 mM pH 7.4)
•Extract (20–60 μg/mL) in distilled water is added to
hydrogen peroxide and absorbance at 230 nm is
determined after 10 min.
• % scavenged= {(Ai-At)/Ai} * 100
Total radical-trapping antioxidant parameter (
TRAP) method
• Protection provided by antioxidants on the fluorescence decay of
R-phycoerythrin during a controlled peroxidation reaction.
• The fluorescence of R-Phycoerythrin is quenched by ABAP as a
radical generator.
• This quenching reaction is measured in the presence of
antioxidants.
Ferric reducing-antioxidant power (FRAP) a
ssay
•This method measures the ability of antioxidants to
reduce ferric iron.
•This reduction is monitored by measuring the
change in absorption at 593 nm, using a diode-array
spectrophotometer
Thiobarbituric acid (TBA) method
•TBA method was described by Ottolenghi in (1959)
• The absorbance activity of the supernatant is measur
ed at 552 nm and recorded after it has reached its max
imum.

8. Some useful antioxidants and their in vitro studies
PLANTS(FAMILY) PARTS USED MODEL FOR ASSAY
Amaranthus lividus L.
(Amaranthaceae)
stem, leaves, flower TEAC, DPPH , RP, Metal chelating,
HO
Cassia siamea Lam.
(Caesalpiniaceae)
flower TPC, RP, DPPH, H2O2, NO, Protein
oxidation, TBARS, Metal chelating
Cocos nucifera L.
(Arecaceae)
mesocarp DPPH, FRAP
Gynura procumbens(Merr.)
(Compositae)
leaves ABTS, total, RP, TPC antioxidant ac
tivity, DPPH, Xanthin oxidase,
Urtica dioica L.
(Urticaceae)
nettle Total antioxidant activity, RP, SO, D
PPH, Metal chelating, H2O2, TPC

9. Antioxidant effect of L. indica
Free radical scavenging effect of L. indica leaf extract was determined using the stable
scavenger 2, 2-diphenyl-1-picrylhydrazyl (DPPH) with slight modifications of the method
described by Brand-Williams et al. Briefly, the concentrations (100–500 μL) of extracts
were prepared in ethanol. DPPH solution (0.004%) was prepared in ethanol and 1 ml of
this solution was mixed with the same volume of methanol, ethanol, and aqueous leaf
extracts and standard ascorbic acid solution separately. The mixture was incubated for 30
minutes in the dark at room temperature and the absorbance was measured at 517 nm.
The degree of DPPH purple decolorization to DPPH yellow indicated the scavenging
efficiency of the extract. Lower absorbance of the reaction mixture indicated higher free
radical-scavenging activity.
The scavenging activity against DPPH was calculated using the equation:
DPP scavenging activity (%)= [Ac-At/Ac]*100
where Ac is the absorbance of the control reaction (1 ml of ethanol with 1 ml of DPPH
solution), and At is the absorbance of the test sample. The results were analyzed in
triplicate. The IC value is the concentration of sample required to inhibit 50% of the DPPH
free radical.

10. Key points for anti-oxidant In vitro studies
Antioxidant activity should not be concluded based on a single antioxidant
test model, in practice several in vitro test procedures are carried out for
evaluating antioxidant activities with the samples of interest.
Antioxidant test models vary in different respects.
Therefore, it is difficult to compare fully one method to other one.
Researcher has to critically verify methods of analysis before adopting that
one for his/her research purpose.
Among free radical scavenging methods, DPPH method is furthermore rapid,
simple and inexpensive in comparison to other test models. On the other
hand ABTS decolorization assay is applicable for both hydrophilic and
lipophilic antioxidants.

11. ANTICANCER
Anticancer drug, also
called anti neoplastic
drug, any drug that is
effective in the treatment of m
alignant, or cancer,
a disease in which abnormal cell
s divide uncontrollably
and destroy body tissue.

12. Causes and statistics of cancer
According to WHO:
• About 16% of people die due to cancer.(1 of 6,
globally)
• Worldwide, in 2018, 5 most common types of
cancer that kill men are: lung, liver, stomach,
colorectal and prostate cancer.
• Worldwide, in 2018, 5 most common types of
cancer that kill women are: breast, lung, stomach,
colorectal and cervical cancer.
• worldwide, only 14% of people who need
palliative care, actually receive it.

13. MODELS FOR IN VITRO
ANTICANCER DRUG STUDY
Comet
Assay
DNA
fragmentati
on assay
Thymidine
Incorporation
Assay
SRB assay (s
ulforhodamid
e-b assay)
Alamar
Blue Assay
Trypan
blue
exclusion
assay
Potato disc
tumor
Induction
assay
LDH assay
Clonogenic
Assay
MTT Assay:
(Colorimetric
Assay)

14. MTT Assay:
(Colorimetric
Assay)
• This method determines concentration of a
chemical element or chemical
compound in a solution with the aid of a col
our reagent.
• Micro culture tetrazolium salt based ass
ays (MTAs) are colorimetrically based assay, t
hey based on the reduction of a tetrazolium
salt by mitochondrial enzyme, leading to the
production of a coloured compound which is
called formazan,
quantified by spectrophotometry.
• SRB is an anionic dye and it is aminoxanth
ene, which can react with basic amino acid r
esidues of protein to forms an electrostatic c
omplex under moderately acid conditions, w
hich contribute to a susceptible and linear re
sponse.
• It is rapid, susceptible, sensitive, and inexp
ensive method for measuring the cellular pr
otein content of the cell.
SRB assay
(sulforhodamide-
b assay)

15. Clonogenic Assay
• The clonogenic assay determines cell
proliferation, it is an in-vitro type of
cell survival assay.
•The ability of a cell to proliferate indefinitel
y is said to be clonogenic, with the ability to
form a reproductive and large colony or a c
lone.
• One of the most important steps in apopt
osis is DNA fragmentation, a process whic
h result leads into degrade DNA endonucl
eases during the apoptotic program by the
activation of magnesium and calcium dep
endent nucleases.
DNA
fragmentation
assay

16. Invitro Anticancer Activity of Hydroalcoholic Extract of
Justicia tranquibariensis
Cell line and Cell Treatment
Roots of Justicia tranquibariens was collected and size reduced, dried in s
unlight, pulverized.
Soxhlet extraction method, with petroleum ether and crude hydroalcoholic extr
act devoid of solvents was obatained.
After 48hours of incubation, to each well added 15µl of MTT (5mg/ml) in phosph
ate buffered saline (PBS) and incubated at 37°C for 4 hours. The formed forma
zan crystals were solubilized in 100µl of DMSO solution. Using a micro plate
reader the absorbance was measured at 570 nm.
Percentage Cell Inhibition = [100- Abs (sample)/Abs (control)] x100

17. In vitro cell linings for different types of cancer cells
• COLO 205
• HCT-15
• KM12
COLON CANCER
• EKVX
• HOP-62
• NCI-H460
LUNG CANCER
• CCRF-CEM
• MOLT-4
• K-562
LEUKEMIA
• A498
• ACHN
• TK- 10
RENAL CANCER
• BT- 549
• HS- 578T
• MCF7
BREAST CANCER
• OVCAR- 3
• IGROV 1
• SK-OV-3
OVARIAN CANCER

18. ADVANTAGES OF IN VITRO METHODS
 In vitro methods reduce the use of mice at the antibody-production stage
 In vitro methods are usually the methods of choice for large-scale production
by the pharmaceutical industry because of the ease of culture for production
compared with use of animals, and because of economic considerations.
 In vitro methods avoid the need to submit animal protocols to IACUCs.
 In vitro methods avoid or decrease the need for laboratory personnel
experienced in animal handling.
In vitro methods using semipermeable-membrane-based systems produce mAb
in concentrations often as high as those found in ascitic fluid and are free of
mouse ascitic fluid contaminants.

19. CONCLUSION
Although, we intensively study various in vitro systems, it is often extremely difficult to repr
oduce some results in vitro. Unfortunately, we are still far from
being able to simulate complex situations such as microarchitecture and
compartmentation of organs. It may be possible to fully replace animal
experiments by in vitro systems in future. This will be the case when we :
(i) have understood all relevant mechanisms of toxicity, interactions between cell typ
es and organs including their compensatory mechanisms,
(ii) have established in vitro systems that correctly recapitulate all mechanisms. The tim
e horizon for such ambitious goals will be rather centuries than decades
(iii) In the meantime we should not neglect in vivo research. We will need more knowledge
about normally functioning and chemically compromised organs and organisms to esta
blish in vitro systems which are more than just in vitro artefacts.

20. BIBLIOGRAPHY
 Screening Methods for the Evaluation of Biological Activity in Drug Discovery by Päivi T
ammela.
 Preclinical Screening for New Anticancer Agents by Angelika M. Burger and
Heinz-Herbert Fiebig.
 In vitro models for antioxidant activity evaluation and some medicinal plants
possessing antioxidant properties: An overview, by S. Chanda and R. Dave
 Medicinal Plants Need Biological Screening: A Future Treasure as Therapeut
Agents, by Haroon Khan
 Review on in vivo and in vitro methods evaluation of antioxidant activity
by Md. Nur Alam , Nusrat Jahan Bristi, Md. Rafiquzzaman

21.  Biological screening of various medicinal plant extracts for antibacterial and
antitumor activities by Fatma Pehlivan Karakaş
 Evaluation of Anticancer, Antioxidant, and Possible Anti-inflammatory Propertis of Selec
ted Medicinal Plants Used in Indian Traditional Medication, by Rafik Shaikh,Mahesh
Pund
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803002/
 https://www.who.int/

22. Thank you