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In Vitro Anti-HSV-1 Activities of d-lenolate at different concentrations

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Source – The Science Behind d-Lanelate® Studies 1999 – 2014. East Park™ Research

Objective

To determine the in vitro anti-HSV-1 activities of East Park Research’s d-lenolate at different concentrations.

Methods

Both 5% and 10% d-lenolate stock solutions were prepared by resuspending 2 grams and 4 grams of the powdered olive leaf extract in 40mL of PBS. The extract was vigorously vortexed and centrifuged to sediment under sterile conditions. The supernatants were used to test the antiviral activity of the desired compound. Ten-fold dilutions of each d-lenolate extract stock solution were prepared in PBS. Varying dilutions of HSV-1 were added to each of the dilutions of d-lenolate.

For the adsorption only tests 200uL of each mixture was adsorbed onto Vero cell monolayers for one hour. The mixture was aspirated prior to the addition of DMEM with 1% carboxymethylcellulose. Plates were incubated at 37C and 5% CO2 for 72 hours. Virus controls were maintained.

For the adsorption + infection tests, 200uL of the mixture containing DMEM with 1% carboxymethylcellulose was incubated at 37C and 5% CO2 for 72 hours. Virus controls were maintained.

The cells were fixed with for 1 hour; washed with PBS; histochemically stained with Vecta Stain (Vector Labs) according to manufacturer’s protocol.

The ability of different concentrations of d-lenolate extract to inhibit fusion of Vero cell monolayers caused by syncytial mutants HSV-1(oncsyn) and HSV-1(gK) were assessed by phase contrast microscopy. Confluent monolayers of cells were infected with an MOI of 10 PFU/cell, various concentrations of the d-lenolate was added to media immediately after adsorption, and fusion was assessed at 12-18 hours after infection.

Observations

For the adsorption experiments, using the 1% and 0.5% d-lenolate extract treatments, no viral Plaques were observed. A greater than 4 log reduction in the number of viral plaques was observed in the 0.25% treatment group. Approximately a 1.5 log reduction in plaques was seen in the 0.125% treatment group.

For the adsorption + infection experiments, d-lenolate extract was found to be toxic in the 1% and 0.5% treatment groups. At 0.25% an approximately 4 log reduction in the number of viral plaques was observed. Approximately a 1.5 log reduction in plaques was seen in the 0.125% treatment group.

For the fusion inhibition assay, d-lenolate inhibited the fusion activity of both the onccsyn and gK fusing viruses.

Conclusion

The d-lenolate solution demonstrated antiviral activity at higher concentrations. The effect decreased with increasing dilutions. The fusion activities of two fusing viruses were inhibited.

HSV-1

Adsorption only

Concentration Plaque#(dilution) Plaque#/mL Log difference from control
1% d-lenolate extract (10% stock) 0(10-3) 0 >5 logs
0.5% d-lenolate extract (10% stock) 0(10-3) 0 >5 logs
0.25% d-lenolate extract(10% stock) 11(10-4) 4.4 x 105 ~4 logs
0.5% d-lenolate extract(5% stock) 0(10-3) 0 > 5 logs
0.25% d-lenolate extract(5% stock) 6(10-4) 2.4 x 105 > 4 logs
0.125% d-lenolate extract(5% stock) 4(10-6) 1.6 x 107 ~1.5 logs
Control 14(10-8) 5.6 x 109 —–

 

HSV-1

Adsorption + Infection

Concentration Plaque#(dilution) Plaque#/mL Log difference from control
1% d-lenolate extract (10% stock) toxic 0 —–
0.5% d-lenolate extract (10% stock) toxic 0 >5 logs
0.25% d-lenolate extract(10% stock) 8(10-4) 3.2 x 105 ~4 logs
0.5% d-lenolate extract(5% stock) toxic 0 —-
0.25% d-lenolate extract(5% stock) 9(10-4) 3.6 x 105 ~4 logs
0.125% d-lenolate extract(5% stock) 7(10-6) 2.8 x 107 ~1.5 logs
Control 12(10-8) 4.8 x 109 —-

 
 

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