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45

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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46

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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48

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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50

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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51

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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53

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

Antiviral studies

54

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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55

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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56

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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60

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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61

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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62

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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64

(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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65

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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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.