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6.1 In Vitro Cytotoxicity Studies
Drug development programme involve pre- clinical screening of a vast numbers of
chemicals for specific and non-specific cytotoxicity against many types of cells, which are
important to indicate the potential therapeutic target and safety evaluation. The screening
of plant extracts or pure compounds for exploring their antiviral properties can be more
meaningful with cytotoxicity assays (Meyer, 1982).
In a cell culture model, apparent antiviral activity of an investigational product can be the
result of host cell death after exposure to the product. Cytotoxicity tests use a series of
increasing concentrations of the antiviral product to determine what concentration results
in the death of 50 percent of the host cells. This value is referred to as the median cellular
cytotoxicity concentration (CC50 or CTC50 or CCIC50). The relative effectiveness of the
investigational product is inhibiting viral replication compared to inducing cell death is
defined as the therapeutic or selectivity index (i.e., CC50 value/EC50
value). It is desirable
to have a high therapeutic index giving maximum antiviral activity with minimal cell
toxicity. Studies determining cytotoxicity and therapeutic indexes should be conducted
before the initiation of phase 1 clinical studies (US, FDA Guidelines, 2006).
There are a number of advantages for in vitro testing using cell cultures which include
analysis of species specificity, feasibility of using only small amounts of test substances,
and facility to do mechanistic studies. Hence, twenty five extracts from four different
plants (Sida cordifolia, Sida acuta, Sida retusa and Sida spinosa) and isolated fractions
were screened to determine their cytotoxicity towards the four cell cultures (Vero, HEp-2,
A-549 and MDCK) to decide the dose which should be non toxic to the cell line used for
antiviral studies.
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6.1.1 Materials and Methods
Chemicals
3- (4,5-dimethyl thiazole-2-yl)2-5-diphemyl tetrazolium bromide (MTT) and
Sulforhodamine B (SRB) were obtained from Sigma Aldrich Co, St Louis, USA., Fetal
Bovine Serum (FBS) and New Born Calf Serum (NBCS) were obtained from PAA
laboratories, Austria, Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s
Medium (DMEM), antibiotics solution, EDTA, Glucose, Hank’s Balanced Salt Solution
(HBSS) and Phosphate Buffer Saline (PBS) from Hi-Media Laboratories Ltd., Mumbai,
Trichloro acetic acid (TCA) and tris buffer from SD fine chemicals Pvt. Ltd., Boisar, India.
Dimethyl Sulfoxide (DMSO), Glacial acetic acid and iso propranol from E. Merck Ltd.,
Mumbai, India. Ephedrine-pseudoephedrine, vasicinol-vasicinone and β-sitosterol-
stigmasterol were purchased from Sigma Aldrich Co., St Louis, USA. 25 cm2
and 75 cm2
tissue culture flasks, 6 and 96 well microtitre plates were procured from Tarson India Pvt.
Ltd. Kolkata, India. 0.22 µ filters were procured from Millipore, India.
Preparation of test solutions
For cytotoxicity studies, each extracts and fractions were weighed separately, dissolved in
distilled DMSO and volume was made up to 10 ml with MEM/DMEM, pH 7.4,
supplemented with 2 % inactivated FBS/NBCS (maintenance medium) to obtain a stock
solution of 1 mg/ml concentration, sterilized by filtration and stored at – 20 º C till use.
Serial two fold dilution was prepared from the stock solution to obtain lower
concentrations.
Cell lines and culture medium
Vero (Normal African green monkey, Kidney), HEp-2 (Human, Epithelial laryngeal
cancer), A-549 (Human, small lung carcinoma) and MDCK (Madin- Darby canine,
kidney) cell cultures were procured from National Centre for Cell Sciences (NCCS), Pune,
India. Stock cells were cultured in MEM supplemented with 10% inactivated FBS/NBCS,
Penicillin (100 IU/ml), Streptomycin (100 µg/ml) and Amphotericin B (5 µg/ml) in a
humidified atmosphere of 5 % CO2 at 37 ºC. The cells were dissociated with TPVG
solution (0.2 % trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were
grown in 25cm2 culture flasks and all experiments were carried out in either 96 or 6 well
microtitre plates.
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6.1.2 Determination of mitochondrial synthesis by MTT assay
Principle
The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity
assays. This assay is based on the assumption that dead cells or their products do not
reduce tetrazolium. The assay depends both on the number of cells present and on the
mitochondrial activity per cell. The cleavage of MTT to a blue formazan derivative by
living cells is clearly a very effective principle on which the assay is based.
The principle involved is the cleavage of tetrazolium salt MTT (3-(4,5 dimethyl thiazole-2
yl)- 2,5-diphenyl tetrazolium bromide) into a blue coloured product (formazan) by
mitochondrial enzyme succinate dehydrogenase. The numbers of cells were found to be
proportional to the extent of formazan production by the cells used (Francis and Rita,
1986).
Procedure
i. The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0x105
cells/ml using MEM/DMEM medium containing 10% FBS/NBCS.
ii. To each well of a 96 well microtitre plate, 100µl of the diluted cell suspension
(approximately 10,000 cells/well) was added.
iii. After 24 hours, when a partial monolayer was formed, the supernatant was flicked
off, the monolayer was washed once with medium and 100l of different extract
concentrations prepared in maintenance media were added per well to the partial
monolayer in microtitre plates. The plates were then incubated at 37oC for 3 days in
5% CO2 atmosphere, and microscopic examination was carried out and observations
recorded every 24 hours.
iv. After 72 hours, the extract solutions in the wells were discarded and 50l of MTT
(2mg/ml) in MEM-PR (MEM without phenol red) was added to each well.
v. The plates were gently shaken and incubated for 3 hours at 37oC in 5% CO2
atmosphere.
vi. The supernatant was removed and 50l of propanol was added and the plates were
gently shaken to solubilize the formed formazan.
vii. The absorbance was measured using a microplate reader at a wavelength of 540nm.
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The percentage growth inhibition was calculated using the following formula and
concentration of drug or test extract needed to inhibit cell growth by 50% values were
generated from the dose-response curves for each cell line.
x 100 % Growth Inhibition = 100 – Mean OD of individual test group
Mean OD of control group
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Figure 6.1 Schematic presentation of MTT assay
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6.1.3 Determination of total cell protein content by Sulforhodamine B (SRB) assay
Principle
SRB is a bright pink aminoxanthene dye with two sulfonic groups. Under mild acidic
conditions, SRB binds to protein basic amino acid residues in trichloro acetic acid (TCA)
fixed cells to provide a sensitive index of cellular protein content that is linear over a cell
density range of at least two orders of magnitude.
Colour development in SRB assay is rapid, stable and visible. The developed colour can
be measured over a broad range of visible wavelength in either a spectrophotometer or a
96 well plate reader. When TCA-fixed and SRB stained samples are air-dried, they can be
stored indefinitely without deterioration (Philip et al., 1990).
Procedure
i. The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0x105
cells/ml using MEM/DMEM medium containing 10% FBS/NBCS.
ii. To each well of a 96 well microtitre plate, 100µl of the diluted cell suspension
(approximately 10,000 cells/well) was added.
iii. After 24 hours, when a partial monolayer was formed, the supernatant was flicked
off, the monolayer was washed once with medium and 100l of different extract
concentrations prepared in maintenance media were added per well to the partial
monolayer in microtitre plates. The plates were then incubated at 37oC for 3 days in
5% CO2 atmosphere, and microscopic examination was carried out and observations
recorded every 24 hours.
iv. After 72 hours, 25l of 50% trichloro-acetic acid was added to the wells gently such
that it forms a thin layer over the extract to form a over all concentration of 10%.
v. The plates were then incubated at 4oC for 1 hr.
vi. The plates were flicked; culture was washed five times with tap water to remove
traces of medium, drug and serum, and was then air-dried.
vii. The air-dried plates were stained with SRB for 30 minutes. The unbound dye was
then removed by rapidly washing four times with 1% acetic acid. The plates were
then air-dried.
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viii. 100l of 10mM tris base was then added to the wells to solubilise the dye. The plates
were shaken vigorously for 5 minutes.
ix. The absorbance was measured using microplate reader at a wavelength of 540nm.
The percentage growth inhibition was calculated using the formula given in MTT
assay and CTC50 values were calculated.
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Figure 6.2 Schematic presentation of SRB assay
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6.2 Antiviral Studies
Viruses have caused some of the most devastating diseases that have afflicted humanity.
Smallpox is thought to have arisen more than 5000 years ago, and has killed millions of
people before it was eradicated. Similarly, poliomyelitis has been a cause for paralytic
disease since ancient times (Johnson, 1998). Fortunately, effective vaccines have been
developed for both these diseases. Vaccines led to the eradication of smallpox in 1979
(Henderson, 1987), and is likely to achieve the eradication of wild-type poliomyelitis in
the not-too-distant future (Hull et al., 1994).
However, for some of the most pressing viral pathogens of today, no vaccine is available.
Thus, despite the fact that 40 million people are currently living with human
immunodeficiency virus (HIV) infection (UNAIDS/WHO 2010 estimate), no proplylactic
vaccine is available to break the cycle of new infections. Similarly, no vaccine is available
to prevent hepatitis C virus (HCV) infection, which is estimated to infect 170,000,000
people worldwide (Lauer and Walker, 2001), leading to millions of deaths due to cirrhosis
or hepatocellular carcinoma. No vaccine is available to prevent infections with herpes
simplex virus type 2, which affect one in every five Americans (Corey and Handsfield,
2000). In fact, much effort has been expended in attempts to develop vaccines for these
diseases, which a notable lack of success. It might be argued that the esay viral candidates
for vaccine development have been exhausted. Viruses such as HCV and HIV pose a
unique challenge due to their rapid antigenic variation while other viruses such as the
herpes simplex viruses have potent immune escape mechanisms, including the
establishment of lifelong latency. Thus, the development of new vaccines for such viruses
is likely to be a slow and laborious process. Thgether with the increased awareness of
emerging viral agents such as West Nile Virus, the SARC coronavirus and Ebola, the
tortuous path to new vaccines is a sobering prospect. Fortunately, another path to the
management of viral infections has opened with the development of specific antiviral
compounds. The advent of these compounds has allowed effective management of some
viral diseases, such as herpes virus infections and HIV infection (AIDS), and has even
made possible the cure of some viral infections such as HCV.
The development of effective antiherpetic compounds was perhaps the event that set the
stage for all later efforts at antiviral therapy, and even today this stands as one of the great
success stories in medical virology. As noted above, in the 1960’s and 1970’s a large
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number of nucleoside analogs were developed and screened for antineoplastic and antiviral
activity. Some of these, such as 5’-iodo-2’-deoxyuridine (IDU) and trifluorothymidine
(TFT), came into use for topical treatment of herpes simplex virus (HSV) keratitis.
Unfortunately, these compounds were unacceptably toxic for systemic use. Another of
these so called “first generation” antiherpetics, vidarabine (Ara-A), was sufficiently well-
tolerated for systemic therapy, and found use in herpes encephalitis, neonatal herpes, and
varicella zoster infections (Whitley et al., 1977 and 1980). Vidarabine is an analog of
adenine that is activated by cellular enzymes to form a triphosphate form, which then
inhibits the HSV DNA polymerase. Despite the utility of Vidarabine, toxicity remained a
concern, as the triphosphate form would be generated in all cells, infected or not. Thus, the
search continued for compounds with true selectivity for virus-infected cells. The
culmination of these efforts resulted in the synthesis of 9-(2- hydroxyethoxymethyl)
guanine. In this guanine derivative, the 9th
position is replaced by a acyclic side chain.
Originally termed acycloguanoside, the compound which would come to be known as
acyclovir has become the standard of treatment for alphaherpesvirus infections. Ayclovir is
converted to a monophosphate by a virus encoded thymidine kinase, and subsequently
converted to di- and tri phosphates by cellular kinases (Elion et al., 1977).
Presently for the treatment of some viral diseases, mainly herpes, effective drugs such as
acyclovir, ganciclovir, valaciclovir, penciclovir, famciclovir and vidarabine, are available.
Among these acyclovir is the most commonly used drug for treatment of HSV infections,
followed by penciclovir/famciclovir (Hammer and Inouye, 1997). However, a serious
problem is the use of acyclovir is drug resistance in treated patients. Resistance to
acyclovir and related nucleoside analogues can occur following mutation in either HSV
thimidine kinase (TK) or DNA polymerase. Virus strains associated with clinical
resistance are almost always defective in TK production (Weber and Cinatl, 1996). In
relation to the involvement of different antiviral diseases and to the problems related to
drug resistance, it is very essential to explore novel antiviral molecules.
On the other hand the H1N1 influenza virus has recently spread worldwide. The
appearance of an influenza virus more virulent than pandemic H1N1 is now predicted.
Influenza viruses infect the respiratory tract in humans and causes a variety of symptoms,
including fever, nasal secretions, cough, headache, muscle pain and pneumonia. These
clinical symptoms often become severe especially in high-risk groups such as the elderly
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and infants (Nicholson et al., 2000; Thompson et al., 2003). The amantadine and
neuraminidase inhibitors zanamivir and oseltamivir have been used for the treatment and
prevention of influenza virus infection (Ison and Hayden 2001), but the appearance of
viruses resistant to them has also been reported (Wetherall et al., 2003); thus, it is
important of develop new types of anti-influenza virus agents with anti-influenza virus
actions different from those of the known agents.
Viral Infections
Viruses are obligate intracellular parasites, which contain little more than a bundle of gene
strands of either RNA or DNA, and may be surrounded by a lipid-containing envelope
(Wagner and Hewlett 1999). Yet viruses are far from simple. Unlike bacterial cells, which
are free-living entities, viruses utilize the host cell environment to propagate their progeny.
They use the reproductive machinery of cells they invade causing ailments as being as a
common wart, as irritating as a cold, or as deadly as what is known as the blood African
fever. Viruses have numerous invasion strategies. Each strain of virus has its own unique
configuration of surface molecules, which work like keys in a lock, embling viruses to
enter into hosts by precisely fitting the molecules on their surfaces to those on the
membranes of target cells. The success of viruses in evolution has been assured by four
general attributes i.e., genetic variation, variety in means of transmission, efficient
replication within host cells and the ability to persist in the host (Wagner and Hewlett,
1999). As a consequence viruses have adapted to all forms of life and have occupied
numerous ecological niches resulting in widespread diseases in humans, animals and
plants.
Virus infection control
Control of viral infections, like any other kind of infection control, can be effected either
as a prophylactic (protective) measure or therapeutically, in order to control and alleviate a
viral infection, which has already been established in the host. Unlike bacteria, fungal and
parasitic infections, viruses are not autonomous organisms and therefore, require living
cells to replicate. Consequently, most of the steps in their replication involve normal
cellular metabolic pathways, and this makes it difficult to design a treatment to attack the
virion directly or its replication, without accompanying adverse effects on the infected
cells. It is known that each class of viruses have unique features in their structure or in
their replication cycles, and these constitute potential targets. Viral enzymes play a key
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role in triggering disease and if viral enzymes could be neutralized, viral replication would
not take place and designing specific inhibitors for those enzymes is thus a desirable
objective. In fact, successful antiviral chemotherapy has been achieved against the herpes
virus with the development of acyclovir, which interferes with certain key viral enzymes
that have distinctive affinities for different nucleotide analogues (Wagner and Hewlett
1999). However, widespread usage of antiviral agents has led to drug resistant varieties of
viruses, especially in immunocompramised patients. Resistance of virus to synthetic
nucleoside analogues has been reported to develop in vitro and in vivo (Field, 2001). It is
therefore necessary to find new and effective antiviral agents to treat the viral infections
and to counter the resistant varieties of viruses.
In view of a significant number of plant extracts that have yielded positive results, it can be
concluded that a rich source of antiviral agents are still present in natural products, which
are yet to be explored. Although large number of new compounds have been isolated form
medicinal plants only some have been marketed as pharmaceutical products and have been
or are undergoing various phases of clinical trials. Based on the above information, it was
decided to screen the selected medicinal plants from the genus Sida for antiviral properties
in vitro and in vivo.
6.2.1 Materials and Methods
6.2.1.1 Preparation of virus pool
The growth medium was decanted from the bottle of monolayer cell culture and washed
with culture medium without serum. Inoculated the culture with 100µl of virus suspension
and incubated for 1 hour at 37oC for virus adsorption. Added 5ml of MEM with 2% serum
(Maintenance medium) onto the monolayer and incubated at 37oC and cytopathic effect
(CPE) was observed after 24 hours onwards. When 100% CPE was observed, the cells
were frozen at -70oC and thawed at room temperature repeatedly for 3 times and then the
cell suspension was centrifuged and the supernatant (cell free extract) collected and
distributed in vials. Virus were labeled with passage number and date, stored at -70oC in
small aliquots. This procedure was repeated to get a sufficiently good titer of virus.
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6.2.1.2 Determination of virus titer
Most of the commonly encountered human viruses produce characteristic cytopathic effect
in one or the other cell lines routinely used in virology laboratories. The infectivity titer of
these viruses can conveniently be determined by infecting a particular cell line with
increasing dilutions of the virus material and determining the highest dilution producing
cytopathic effect in 50% of the inoculated cells. The 50% end point dilution which in this
case is expressed as TCID50 /ml can be calculated using either Reed- Muench formulae
(Reed & Muench, 1938). As an example, titration of HSV-1 virus is illustrated here.
Preparation of virus dilution
The monolayer of tissue culture flask was tripsinized and 96 well plates were seeded
(10,000 cells/well). The virus stock was diluted by 10 fold (10-1
to 10-8
) serial dilution
using tissue culture medium containing 2% serum. Added 100µl of each dilution in 6 wells
each of 96 well microtitle plates. Incubated at 37 ºC with 5% CO2 atmosphere and
observed for viral CPE at every 24 h interval. Read the plate under inverted tissue culture
microscope when a confluent monolayer of Vero cells can be seen in control wells. The
50% Tissue Culture Infectivity Dose (TCID50) was calculated as per the method of Reed
and Muench (Reed and Muench, 1938).
Calculation of 50% endpoints
In any biological quantitation, the most desirable endpoint is one representing a situation
in which half of the inoculated animals or cells show the reaction (death or paralysis in the
case of animals and in CPE case of cells) and the other half do not. In other words, the
endpoint is taken as the highest dilution of the biological material, which produces desired
reaction in 50% of the animals or cells. The 50% endpoint can based on several types of
reactions. The most widely used endpoint, based on mortality, is the LD50 (50% lethal
dose). In tissue culture system TCID50 represents the dose that gives rise to Cytopathic
effect in 50% of inoculated cultures. Reed and Muench devised a simple method for
estimation of 50% endpoint based on the large total number of cells/well, which gives the
effect of using at the 2 critical dilutions between which the endpoint lies.
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Calculation of TCID50 titre by Reed- Muench method
Virus Suspention 0.2ml + 1.8ml diluent- 10-1
0.2ml + 1.8ml diluent - 10-2
0.2ml + 1.8ml diluent - 10-3
0.2ml + 1.8ml diluent - 10-4
0.2ml + 1.8ml diluent - 10-5
0.2ml + 1.8ml diluent - 10-6
0.2ml + 1.8ml diluent - 10-7
0.2ml + 1.8ml diluent - 10-8
Table 6.1 Microscopic observations
10-1
+ + + + + +
10-2
+ + + + + +
10-3
+ + + + + +
10-4
+ + + + + +
10-5
+ + + +
10-6
+
10-7
Controls
= Normal, += Died
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Table 6.2 Arrangement of data used in computation of TCID50 titer by Reed and
Muench formula
Virus
dilution
CPE
ratio
Wells
(+)
Wells
(-)
Accumulated values
CPE
(+)
CPE
(-)
CPE
ratio
Percentage
10-1
6/6 6 0 29 0 29/29 100
10-2
6/6 6 0 22 0 22/22 100
10-3
6/6 6 0 17 0 17/17 100
10-4
6/6 6 0 11 0 11/11 100
10-5
4/6 4 2 5 2 5/7 71
10-6
1/6 1 5 1 7 1/8 13
10-7
0/6 0 6 0 13 0/13 0
+= Died, - = Survived
Accumulated values for the total number of cells/well that dies or survival are obtained by
adding in the direction of lowest to the highest values. The accumulated mortality ration
and the percentage mortality for each dilution is calculated. The mortality in the 10-5
, is
higher than 50% and it the next higher dilution, 10-6
it is only 13 %. So the 50% endpoint
dilution lies between these dilutions. The proportional distance of the 50% endpoint from
these dilutions can be calculated by using following formula.
(%CPE at dilution next above 50%) - 50
TCID50 = ---------------------------------------------------------------------------------- ---------------
(%CPE at dilution next above 50%) - (% CPE at dilution next below 50%)
71- 50 21
= ------------- = ------- = 0.36 or 0.4
771-13 58
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Negative logarithm of the lowest dilution (next above-50%CPE) = -6.0 and proportionate
distance (0.4) x log dilution factor = -0.4
TCID50 titer = -5.4 /0.1ml
Log TCID50 titer = 10-6.4
/ 1 ml
1 TCID50 titer of given passage of Herpes simplex virus is approximately
= 10-6.4
/ 1 ml
10 TCID50 = 10-5
/ 1 ml
100 TCID50 = 10-4
/ 1 ml
The virus titer obtained for other viruses are as follows,
Polio Virus type I: 10 -7.5
/ 1 ml Adeno virus type VIII: 10 -6.0
/ 1 ml
HSV type I: 10 -6.4
/ 1 ml Influenza H1N1: 10 -6.2
/ 1 ml
HSV type II: 10 -6.6
/ 1 ml
Virus titration by using the plaque assay
1. Prepared confluent monolayers of cells (Vero and MDCK) in 24 well plates
(Nunc).
2. Prepared serial 10-fold dilutions (101 to 10
7 ) of virus in chilled maintenance
medium (MEM, with 2% serum).
3. Culture medium was removed and 0.2ml of virus inoculum was added, starting from
the highest dilution. Ensured that a thin film of medium completely covers the
cell sheet.
4. Incubated the plate at 37 C for 1 hour with intermittent rocking of the plate.
5. Removed the inoculum, with a pipette and then added 1.5 ml of agarose overlay
medium (growth medium with 0.3% agarose and 2.5% FCS).
6. The overlay medium was spread evenly over the monolayer, incubated at 37 C.
7. The monolayers was examined daily, starting from second day of incubation.
8. Once the plaques have developed, usually by the fourth day post inoculation,
the number of plaques were counted at each dilution, the agarose overlay was
removed and the monolayer was gently washed with PBS.
9. The plate was stained with 0.1% crystal violet solution and counted the plaques again.
10. Estimate the virus titre as a plaque forming units per ml (pfu /ml) by counting
the number of plaques at an appropriate dilution.
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The number of HSV plaques produced 10
Dilution of virus 1 x 105
Volume of inoculum 0.2 ml
Virus titre for HSV TK- = 10 x 1x105 x 1/5 pfu per ml
= 2 x 106
Virus titre for Influenza H1N1 = 1 x 106
6.2.2 In vitro antiviral studies
Figure 6.3 The in vitro antiviral testing protocol
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6.2.2.1 Cytopathic Effect (CPE) inhibition assay
Non- lytic damage that virus may do to cells are termed Cytopathic effects. These effects
vary both in terms of how the damage is manifest and how damaging the effects are to the
affect cells. If a drug is said to be antiviral, it will inhibit the CPE of virus. So for detecting
an antiviral agent, the amount of inhibition of CPE of virus can be observed
microscopically (Hu and Hsiung, 1989).
Procedure
Different nontoxic concentrations of test drugs, i.e., lower than CTC50 were tested for
antiviral property by CPE inhibition assay against different virus challenge doses 10 and
100 TCID50. The monolayer culture was trypsinized and seeded in 96 well microtitre
plates at 1×104 cells/well and incubated at 37 ºC in 5 % CO2 incubator. After 48 h, when a
complete monolayer forms the cultures were washed with fresh culture medium and
inoculated with 100 μl of 10 TCID50 and 100 TCID50 of the virus suspensions separately in
different wells, incubated for 1 h at 37 ºC in a CO2 incubator for the adsorption of the virus
on to the cells. After incubation excess virus suspension was removed by washing with
fresh culture medium. 100 μl of each selected concentration of the test compounds were
added and in quadruplicate wells and 100 μl of culture medium with 2% FBS was added
into positive (virus control), negative and solvent control wells. The culture plates were
incubated at 37 ºC with 5% CO2 atmosphere and every 24 h, the microscopic observations
were made and cytopathic effects were recorded. Antiviral activity of test samples was
determined by their inhibition of cytopathic effect compared with controls, which was
expressed as the protection offered by the test samples to the cells.
6.2.2.2 Virucidal assay
If an in vitro study of an antiviral agent shows antiviral activity, it is necessary to establish
whether the virus is inactivated in an extracellular condition (Bauer, 1972). This is done by
incubating virus suspensions with various concentrations of the antiviral agent at 37 ºC for
1 h to as long as 24 h and determining the rate of loss of infectivity by microscopical
observation. If the rate of loss of the virus infectivity exceeds that in a control preparation
incubating in the absence of the antiviral agent, it is evident that the compound is
inactivating the virus before the latter has entered the cells.
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Procedure
Different nontoxic concentrations of test drugs, i.e., lower than CTC50 were tested for
antiviral property by virucidal assay against different virus challenge doses of 10 and 100
TCID50. The virus suspensions (10 TCID50 and 100 TCID50) with various concentrations
of test compounds were incubated at 37°C for 1 hour (Test compound+ Virus suspension).
Solvent (used to dissolve test compound) alone with virus suspension were kept as virus
control. After 1 hour, 100 µl of each mixture (Test compound+ Virus suspension) were
added to the monolayer cultures grown in 96 well microtitre plates. The CPE was observed
every 24 hours for 96 hours and compared with controls, which was expressed as the
protection offered by the test samples to the cells was scored (Hu and Hsiung, 1989).
6.2.2.3 MTT antiviral assay
A rapid and sensitive procedure to evaluate antiviral compounds in vitro is based on
spectrophotometrical assessment for viability of virus- infected and mock infected cells via
in situ reduction of a tetrazolium dye MTT. Mitochondrial enzymes of viable cells convert
yellow water soluble dye MTT to a soluble, purple coloured insoluble formazan. The
quantitation of the amount of the formazan product present in each well of the microtitre
plate is then determined spectrophotometrically at 490/650 nm. While the toxicity of the
test compounds to host cells is measured concurrently in the same microtitre plate.
(Takeuchi et al., 1991)
Procedure
Cells (1×105 cells/ml) were seeded on 96-well tissue culture plates. After a 24 h period of
incubation, the medium was removed and replenished with 100 ml of medium containing
increasing concentrations of the compounds (serially diluted twofold). As cell control, 100
µl of medium only is added. After three to five days of incubation, the medium was
removed and 50 ml of MTT solution (2 mg/ml) was added to each well for 4 h at 37 ºC.
Then, 100 µl of iso-propanol was added to each well in order to dissolve the formazan
crystals. After shaking gently the plates for 10 min to dissolve the crystals, the colour
reaction was measured in an automated microplate reader at 562 nm. The untreated control
was arbitrarily set as 100%. For each compound, the percentage of cell protection/virus
inhibition can be calculated as,
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(Mean OD of control group – Mean OD of treated group) × 100
Mean OD of control group
6.2.2.4 Plaque reduction assay
Plaque assay is one of the most reliable and oldest methods of titrating infectious virus
particles in samples. The effectiveness of drug in reducing the plaque-forming units (pfu)
of virus compared with controls is an indicator of anti-viral activity (Figure 8.2)
Figure 6.3 Schematic representation of viral plaque assay.
Procedure (Shiraki et al., 1991; Shimizu et al., 2008)
1. Cells (1 × 105 cells/ml) were cultured in 60 mm tissue culture dishes.
2. When the monolayer was 80-90 % confluent it was infected with 100 plaque
forming units (PFU/0.2 ml) of virus.
3. The virus was allowed to adsorb for 1 h at 37 ºC in 5% CO2 atmosphere. The
solution was removed and the cells were washed twice with pre-warmed MEM
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medium. While virus adsorption, drug dilutions were prepared in the overlay
medium.
4. The infected cells were overlaid with 5ml 0.8% of nutrient methylcellulose (in case
of HSV) and with 0.8 % of nutrient agarose (in case of influenza virus) containing
different concentration of test compounds.
5. The plates were incubated at 37 ºC in 5% CO2 atmosphere for three to five days
before fixing with 10 % formalin solution for 30 min.
6. Cells were either stained with 1 ml per well of methylene blue or 1 % crystal violet
solution (w/v).
7. Stain were removed and rinsed gently three times with tap water and allowed to dry
inverted overnight and plaques (dark areas) were counted using low power
magnification on a binocular microscope.
Calculation of antiviral effect
The percentage of inhibition of plaque formation was calculated as follows;
(Mean number of plaques in control – Mean OD plaques in sample) × 100
Mean OD of plaques in control
The value of EC50, which is the concentration of test sample required to inhibit upto 50%
of virus growth as compared with the virus control group, were estimated from the
graphical plot of the data (Hu and Hsiung, 1989).
Determination of selective index
One of the essential requirements for a prospective antiviral agent is its high selective
index. For each virus host system, this index denotes the ration:
CTC50
Selectivity index (SI) =
EC50
Where EC50 is the minimum drug concentration which is effective to inhibit virus induced
plaque formation or cytopathic changes by 50% and CTC50 is maximum drug
concentration which causes cytotoxic effect in 50% of the cultured cells. SI of more than
three indicates potential anti-viral activity of test compound and should be further
evaluated.
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6.2.2.5 Effect on virus adsorption/penetration, or replication (Time of addition
studies)/ Mode of action of antiviral activity
Some antiviral agents may exert their activity by preventing the adsorption/penetration of
the virus to the cells. This can be determined by treating cell cultures before or during
virus adsorption. The rate of uptake of the virus by the cell in the presence or absence of
the antiviral agent(s) can be measured by assaying supernatant samples added/removed at
various time intervals during the one to tow hour adsorption period (Hu and Hsiung, 1989).
Virus adsorption assay (Penetration of virus)
The non toxic concentration of equal volumes of the extract dilutions and a virus
suspension at a concentration of 10 TCID50 and 100 TCID50 were placed in a tube and the
mixtures were incubated at 37° C for 1 h. The samples were then placed on monolayers of
cells and the virus was allowed to adsorb and penetration in the presence of the extracts.
The % cell protection/virus inhibition was calculated by formula shown in MTT assay.
Virus attachment assay (Pre treatment of cells)
Dilutions of the test extracts were added to each well of 6-well plates containing
monolayers of cells and the plates were incubated at 4°C for 1 h. Extract solutions were
then removed and virus suspensions containing concentration of 10 TCID50 and 100
TCID50 per well were added to each of the wells. Plates were incubated at 4°C for 2 h to
allow attachment, then monolayers were rinsed 3 times with cold PBS to remove the
unbound virus. Growth medium was then added to each of the wells and the plates were
incubated at 37°C for 3 days. The % cell protection/virus inhibition was calculated by
formula shown in MTT antiviral assay.
Virus replication assay
Virus suspensions containing concentration of 10 TCID50 and 100 TCID50 were prepared
on ice. Virus suspensions were added to the plates, which were incubated at 4°C for 2 h to
allow attachment and replication. Dilutions of each test extract were then added to the
appropriate wells at room temperature and plates were incubated for 10 minutes at 37°C to
allow penetration. Dilutions of extracts were then aspirated and the monolayers were
briefly washed with PBS at a pH of 3.0 to inactivate virions that had not penetrated the
cells. Growth medium was then added to each of the wells and plates were incubated at
37°C for 3 days. The % cell protection/virus inhibition was calculated by formula shown
in MTT antiviral assay (Hu and Hsiung, 1989).
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6.2.2.6 Immunofluorescence assay
1. Confluent monolayer of MDCK cells grown on cover slips in 60 mm dishes were
infected at 10 PFU/cell with A/PR/8/34 (H1N1) in the presence of test compounds.
2. The cells were washed with 3 X PBS at 6 h after infection and fixed in ice cold
acetone for 15 min.
3. The cells were directly stained with fluorescein iso-thiocyanate- conjugated goat
anti-influenza A virus antibody (ViroStat, Portland, ME, USA), which was
prepared from the purified immunoglobulin G fraction of antiserum of goat
immunized by the purified virions of USSR, H1N1 strain, diluted 1:25 in PBS at
room temperature for 40 min, washed with 3 X with PBS and mounted on glass
slides.
4. The cells were observed using an immunofluorescence microscope (Olympus,
Tokyo, Japan) at x 400 magnification (Rie et al., 2010).
6.2.3 In vivo antiviral activity against Herpes Simplex Virus type- I
On the basis of the target site of infection and disease presentation various animal models
can be used for different viruses namely mouse, guinea pigs, ferrets, rabbit, primates and
so on.
The broad host range of HSV has allowed the use of different animal models for the study
of these viruses. The most appropriate model for latency must allow virus reactivation
similar to humans. Both rabbit and the guinea pig, approximate this ideal situation,
although both suffer from limitations, and expense.
The mouse model (the most reasonable in cost), is being used extensively. As the HSV
infections in mice provide a good model for human disease, the efficacy of any extract or
compound is measured by cutaneous lesion development in mice.
The in vivo experiment is mice against HSV-I were carried out at Kyushu University of
Health and Welfare, Nobeoka, Miyazaki, Japan.
Animal
BALB/c female (6 weeks old, 18-21 g) mice were purchased from Sankyo Labs Service
Co., Ltd., Tokyo, Japan. The mice were housed in specific pathogen- free conditions, with
food and water ad libitum and under a 12 h light/12 h dark diurnal cycle (light at 7.00 am).
The temperature in the room was kept at 24 ± 2ºC. The mice were acclimated for at least
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68
five days before starting experimental procedures. Animal studies followed the animal
experimentation guidelines of Kyushu University of Health and Welfare were carried out
in an approved bio safety level.
Figure 6.4 In vivo testing protocols for anti-HSV extract/agents.
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6.2.3.1 Therapeutic efficacy in cutaneous mouse HSV infection model
Procedure
4. Plant extracts at (250 mg/kg per dose) or ACV (5 mg/kg per dose) was orally
administered in a volume of 0.2 mL/mouse by using gavages, once 4 h prior to and
twice after virus infection on day 0, and 3 times daily. 1 % DMSO solution was
used as a control. Fifteen animals (n=15) were used for each group. The
development of skin lesions and mortality was continuously observed thrice daily
(Morning, Afternoon, Eevening) and scored (Kumano et. al., 1987; Kurokawa et
al., 1993).
5. The development of skin lesions and mortality was continuously observed three
times daily and scored (figure 8.4). The infected mice were held at least for 14 days
after infection.
6. The toxicity of plant extracts was assessed in infected mice by the loss of body
weight compared with the control group. The mice were weighed everyday. The
conducted procedures were as per the National Institute of Health Guide for the
Care and Use of Laboratory Animals and the Experimentation Guidelines of the
Kyushu University of Health and Welfare, Nobeoka, Japan.
1. The right midflank of each
mouse was chipped and
depilated with a chemical
depilatory, hair remover.
2. One or two days later, the naked
skin was scratched using a
27 guage needle.
3. 5 µl of HSV-1 (7401 H strain)
suspension of 1 × 106 PFU was
applied to the scarified area
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Figure 6.5 Evaluation of Lesion Scoring
Score 0 Score 2
Score 4 Score 6
Score 8 Score 10
Score 12
• Score 0 = No lesion;
• Score 2 = Vesicles in local region;
• Score 4 = Erosion and/or ulceration in local region;
• Score 6 = Mild zoster form lesion;
• Score 8 = Moderate zoster form lesion;
• Score 10 = Severe zoster form lesion;
• Score 12 = Death.
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6.2.3.2 Determination of virus yields in skin and brain
Virus yields in the skin and brain were determined in infected mice. Mice were
cutaneously infected with wild-type HSV-1 (1 × 106 PFU/mouse), and plant extracts were
orally administered at dose of 250 mg/kg following the same schedule as described above.
The brain and skin (whole lesions that include the area (5×5 mm) encompassing the
inoculation site of infected mice) were removed under anaesthesia on day 5 after infection
and homogenized in 2 mL of phosphate- buffered saline as described previously
(Kurokawa et al., 1995). The homogenate was centrifuged at 3,000 rpm for 15 min, and
the virus yield in the supernatant was determined by the plaque assay on Vero cells
(Kurokawa et al., 1993).
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6.3 Fractionation on active extracts
An attempt was made to fractionate the extracts for its active component which could be
responsible for its antiviral activity. Among the extracts from different plants tested, three
extracts (hydroalcohol (HA), toluene(Tol) and methanol(MeOH)) of Sida cordifolia
showed potent antiviral activity and were taken for fractionation. The high performance
thin layer chromatography (HPTLC) was performed for HA, Tol and MeOH extracts of
Sida cordifolia for developing fingerprints and to find constituents of biological
importance.
6.3.1 Materials and Methods
6.3.1.1 High Performance Thin Layer Chromatography (HPTLC) fingerprint profile
of Sida cordifolia
Procedure
The solutions of FT, FHA and FMeOH (10 mg/ml, 10 μl) were used for the HPTLC
fingerprint analysis. The mobile phase used for fingerprint and screening was Ethyl
acetate: Acetic acid: Formic acid (16:0.4:0.4) for FHA and FMeOH extracts and Toluene:
Ethyl acetate: Acetic acid (15:3.5:0.5) for Tol extract. The samples were analysed as per
the HPTLC method described below (Wagner and Baldt, 1996).
In brief, precoated TLC Silica gel 60 F254 Plates (Merck) were used as stationary phase.
Samples were applied as 8 mm band using Camag Linomat IV applicator. Application was
done on the plate at a distance of 15 mm from bottom and 12 mm from the left margin
with a 6 mm distance between the tracks at a constant application rate of 10 s/µl using
nitrogen aspirator. Development was carried out in Camag twin trough development
chamber which has been previously saturated with the specified mobile phase. The length
of chromatogram run was maintained to 6 cm from applied position. After the
development, TLC plates were dried in an air current with the help of a hair-dryer. The slit
dimension setting of length 4 mm and width 0.30 mm, and a scanning rate of 20 mm/s and
data resolution of 100 µm/step were used. Deuterium lamp, mercury lamp and tungsten
lamp were used for scanning at 256 nm, 366 nm and 400-800 nm respectively.
6.3.1.2 Isolation of total alkaloids and phytosterols from HA, Tol and MeOH extracts
of Sida cordifolia L.
Among the extracts from different plants tested HA, Tol and MeOH extracts of Sida
cordifolia showed potent antiviral activity. The phytochemical studies indicated the
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presence of alkaloids in HA and MeOH extracts and phytosterols in Tol extract. Hence, it
was chosen for isolating phytoconstituents, which might be responsible for its biological
activities.
The alkaloids from HA and MeOH were fractionized by solvent partitioning with acid base
treatment. In brief, crude HA and MeOH extract (1g) was dissolved in methanol and
filtered through a Whatman filter paper. The filtrate was treated with 10 % hydrochloric
acid for half an hour and 10 % ammonium hydroxide solution was added to it, after an
hour equal volume of chloroform was added and the mixture was kept for an hr. The
aqueous and acidic layers were separated and dried under pressure and reduced
temperature (Wagner and Baldt, 1996). The phytosterols from Tol extract were
fractionated by solvent crystallization method (Poulos et. al., 1961). The white crystals
thus obtained were stored at 4 º C until further use. The fractions were named as FHA,
FMeOH and FT.
The UV- visible spectroscopy, HPLC, IR and LC-MS/MS were carried out for the isolated
fractions and were compared with standard compounds i.e., Ephedrine-pseudo ephedrine,
vasicinol-vasicinone, and β-sitosterol-stigmasterol which are said to be present in those
extracts (Ghosh and Dutt, 1930; Sutradhar et al., 2007, 2008).
6.3.1.3 UV – Visible Spectroscopy analysis
The isolated fraction (FHA, FT and FMeOH) and standard mixture of compounds namely,
Ephedrine-pseudo ephedrine, vasicinol-vasicinone, and β-sitosterol-stigmasterol were
analyzed with UV – Visible Spectroscopy using methanol as a solvent.
The absorption spectra of plant constituents can be measured in very dilute solution against
a solvent blank using an automatic recording spectrophotometer. For colorless compounds,
measurements were made in the range of 200 – 400 nm and for colored compounds, the
range was 400 – 800 nm. The wavelengths of the maxima and minima of the absorption
spectrum so obtained were recorded (in nm) and also the intensity of the absorbance (or
optical density) at the particular maxima and minima. Only traces of material are required,
since the standard spectrophotometer cell (1 × 1cm) holds only 3ml of solution and, using
special cells, only one tenth of this volume is required for spectrophotometer. Such
spectral measurements are important in the identification of many plant constituents, for
monitoring the eluates of chromatographic columns during purification of plant products
and for screening crude plant extracts for the presence of particular classes of compounds.
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6.3.1.4 HPLC isolated fractions of Sida cordifolia L.
For HPLC analysis, the Shimadzu prominence HPLC system was equipped with model
series LC- 20 AT pump, Rheodyne 7752i injector with 20μl loop and SPD- 20A UV/VIS
detector, reverse phase phenomenex C18 column (250 X 4.60 mm id, 5 μm) and
spinchrom chromatography station software performed the data acquisition. The mobile
phase consist of mixture of Methanol: Water (70:30) were filtered through 0.2 micron
membrane filter before use, and pumped from the solvent reservoir at a flow rate of 0.5
ml/min, which yielded column backup, pressure of 160-170 kgf/cm2. The column was
maintained at 25º C. Syringe (Bonaduz schweiz, Hamilton) was used for injection of 20 μl
of respective samples.
HPLC Purification of extracts
Isolated fraction and Standard drugs 10 mg/100 ml dissolved in Mobile phase
Injection volume 20 μl
Flow rate 0.5 ml/min
Wavelength (λ) 270 nm
Mobile phase Methanol: Water (70:30)
Detector UV/VIS
Column Phenomenex C18 column (250 X 4.60 mm
id, 5 μm)
Sample preparation
The isolated fractions of Sida cordifolia L. were concentrated to obtain the respective
residues. Each residue (10mg) was dissolved in mobile phase Methanol: Water (70:30) in
10 ml standard flask and filtered through 0.2 μ filter paper. These solutions were used for
HPLC analysis. The 20 μl of the sample was injected using Hamilton syringe. The
programme was run up to 10-15 minutes.
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6.3.1.5 LC-MS/MS
Shimadzu LC-MS/MS equipped with double quadruple with following configuration LC-
10 AD-Vp solvent delivery system (pump), SIL 10 AD-Vp Auto injector, CTO 10Vp
column oven, DGU 14 AM de gasser and LC-MS/MS solution data station was used. The
separation was performed on a POLARIS C-18-A-50X2.00mm column using a mixture of
Methanol: Water (70:30) at a flow rate of 0.5 ml/min, and eluent was introduced into
positive ESI-MS/MS. The ion source and desolvation temperature were set at 200º C and
200º C respectively, capillary voltage was set to 1.3 KV and peak areas of analyses were
automatically integrated using LC-MS/MS solution data station.
6.4 Antiviral studies on isolated fractions
The antiviral activity of the isolated fraction (FHA, FTol and FMeOH) and standard drugs
(ephedrine and pseudoephedrine, vasicinol and vasicinone and β-sitosterol and
stigmasterol) were determined by CPE inhibition assay, virucidal assay and MTT antiviral
assay against HSV-I & II, Adenovirus type VIII, Poliovirus type I and Influenza virus type
A H1N1.