The Effect of d-Lenolate® on the Immune Paramaters in Humans

BACKGROUND AND INTRODUCTION

The purpose of this study was to test the immune effects of d-Lenolate in healthy volunteers at the Department of Cytogenetics and Immunology of NICS. d-Lenolate is a dietary supplement patented by East Park Research. d-Lenolate formulation is prepared on a patented extraction process of selected olive leaves that contain Oleuropein. This study will examine the effect of 21 days of d-Lenolate treatment on the immune parameters of healthy volunteers.

Immune-toxicology examines the damaging/modifying effects caused by exposure at the workplace, environment or therapy on the immune system. Its task is to detect and assess the modifying factors affecting the immune system especially from the aspect of their effect on human health. An immune response may be elicited when the immune system is the passive target of a chemical agent or when the chemical, as antigen, triggers a specific response. In consequence of the complexity of the immune system the chemical agents have a broad target of attack. They can affect the development, maturation, division, differentiation and function of cells, or modify the regulation of the immune system.

The immunology tests were carried out on White blood cells (WBC). WBC is involved in all aspects of the immune reaction and is an important role in the defense mechanisms of the body. WBC can be broken down into 3 main types: lymphocytes, monocytes and granulocytes. Monocytes make up about 2-9% of WBC and are activated by lymphokines that are secreted. As a result they become able to phagocytose foreign matter such as bacteria, and can release a number of inflammatory mediators. Lymphocytes become ‘activated’ when they encounter the foreign object/molecule for which they are designed. We can respond to at least two million different foreign molecules because our lymphocytes are pre-programmed to recognize them over the course of our lifetimes, but only when they encounter those specific molecules. Granulocytes has a very important role in the development if inflammatory and allergic reactions. Most of the granulocytes are made up of neutrophils. Neutrophils are the basis of cellular protection against infection, and can enter the tissues in large quantities. In the course of bacterial or fungal infection, the neutrophil granulocytes, phagocytose and destroy the pathogens. Oxygen-independent enzymes and oxygen-dependent enzymatic systems achieve this intracellular killing of pathogens.

The activated phagocytic cells produce antimicrobial relative radicals, so called Reactive Oxygen Intermediates (ROI). ROI is the ability of the neutrophils described above to kill foreign cells or other unrecognized materials in the blood or tissues. The ROI molecules are very toxic and therefore kept within the neutrophils and only exposed to the target once it is engulfed by the neutrophil. Therefore, the killing capacity of WBC was determined by measuring the production of ROI.

METHODS

30 healthy volunteers, 15 men and 15 women, participated in a 21 day d-Lenolate treatment which consisted of taking 2 capsules 3 times a day. The measurements were done on the 1 ^st, 8^th, 15^th, and 21^st day. Blood samples were taken for determining the qualitative and quantitative blood count for the immunology tests and were analyzed by the lab.

RESULTS AND DISCUSSION

In this study, we see that a greater number of lymphocytes are ‘activated’ as seen by the development of certain surface markers. It is not clear why these cells are ‘activated’. One idea is that they were sluggish before and now recognizes the foreign molecules they were supposed to recognize before. However, the ROI production of neutrophils increased significantly in both the control and the stimulated samples from the first week of the treatment with continual growth throughout the study. The neutrophils responded to several foreign molecules that were presented to them in blood samples. A (weak) stimulus, fMLP chemotatic peptide, was able to stimulate increased ROI response. A particulate (solid) stimulus using, E. coli coated with antibodies, also increased the ROI response, and PMA (a strong signal) increased the amount of ROI. The slight increase in WBC is entirely due to an increase in the number of neutrophils. In conclusion d-Lenolate showed a potential immune building response along with the ability to fight off weak, solid, and strong bacterial stimuli.

 

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

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% CO ₂ 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% CO ₂ 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	——

 

HERPES SIMPLEX VIRUS (HSV)

d-Lenolate®, Aloe and Neem Tissue Culture Experiments.

Purpose: To test d-Lenolate®, d-Lenolate® plus aloe, and d-Lenolate® plus neem for anti Herpes simplex type I activity. Once the optimal concentration of each compound or combination was determined, a final concoction yielding the greatest activity with the least (or acceptable) toxicity could be defined.

Plaque Formation: The mechanism of plaque formation is that ideally one virus particle lands on one tissue culture cell and infects it. The viruses take over the machinery of the tissue culture cell and multiply sometimes hundreds of times. That original cell either leaks out new virus particles or it bursts, releasing viruses to the surrounding tissue cells. Each of the surrounding cells becomes infected with new virus particles and the cycle repeats. As the cells are killed by viruses, a hole is created in the smooth surface of the tissue culture. Each ‘hole’ is equal to a plaque. If the plaques are large, this indicates that the viruses spread easily. If the plaques are small, or become smaller with a treatment, this indicates a lack of spreading. The LSU team found that not only did plaque formation decrease but also the plaque size decreased with some compounds. Therefore, both inhibition of infection andinhibition of spreading occurred. This is understated and very important to minimizing the extent of the disease and presumably promoting healing faster.

The Array of Compounds was tested:

The optimal concentrations of ‘inactive’ ingredients and gel was found through testing.

d-Lenolate® 0.2% 51.9% reduction in plaques/effectiveness

Aloe 0.05% 0%

Neem 0.2% 0%

d-Lenolate® + aloe 0.2/0.05% 76.7%

d-Lenolate® + aloe + neem 0.2/0.05/0.2% 80.6%

Optimum Gel Concentration:

The gel itself had some anti-viral activity = 67.6% particularly in preventing spreading (movement of viruses from the first infected cell to the surrounding cells).

Testing of Active Ingredients:

Menthol Possible 0.05% concentration

Benzocaine Possible 0.125% concentration

Benzyl alcohol Incompatible with the gel

Menthol was chosen as the best ‘active’ ingredient at 0.05%. The final mix contained 0.2% d-Lenolate®, 0.2% neem, 0.05% aloe, 0.05% menthol and 16% F-127 (gel). This combination completely eliminated a viral concentration of 10 ³ or 1000 viral particles. Although this combination was very slightly toxic to tissue culture cells, it will probably have no toxic effect on epidermis (outer skin) of the animals.

It is an unexpected result that the gel had a significant anti-spreading capability. Along with d-Lenolate® and the other natural ingredients, the degree of inhibition of viral infection and spread is really promising .

We do not know what the anti-viral effect of menthol, benzocaine and benyl alcohol are, however, the experiments were designed to measure the effects of d-Lenolate® primarily, and neem and aloe secondarily. The ‘active’ ingredients were pretty much tested for ability to be compatible with the gel and the other ingredients, not how effective they were against the viruses.

Conclusion:

The findings using tissue cultures and Herpes simplex type I were very positive and are encouraging for the development of a safe and effective topical product. One of the important findings is the best concentrations of each ingredient to make the most effective mixture. This mixture should be set (unless for some reason it causes problems on the skin) because it is almost assured that anything placed on open tissue which results in very low toxicity would be very well tolerated on the skin. The anti-viral topical has been created!

 

Safety Report

Analysis of the LSU Safety Letter dated May 11, 2009

This is a safety report on experiments done at LSU using hairless mice and a mixture of d-Lenolate®, Neem, Aloe, and Menthol. Two concentrations of these ingredients were tested.

Concentration A contained 0.2% d-Lenolate®, 0.2% Neem, 0.05% Aloe and 0.05% Menthol.

Concentration C contained a tenfold higher concentration of each ingredient: 2% d-Lenolate®, 0.2% Neem, 0.5% Aloe and 0.2% Menthol.

***Control contained no d-Lenolate® or the mice were untreated

Three groups of hairless mice were used – 10 mice each. The skin of the mice was scratched and after 12 hours, two gel formulations, Concentrations A and C, were applied to the scratched area of 10 mice each (3 times/day for 10 days). The remaining 10 mice made up the Control group.

The results showed that no detectable effects of the application of Concentration A or C were seen compared to the control . Since this was only a safety test and not a test of the efficacy of the ingredients, the test results are exactly what we expected and wanted to see. There are no adverse reactions to the gel mixture during testing up to 2% d-Lenolate®.

General Conclusions: d-Lenolate® was well tolerated and no toxic effects were seen. In this short-term test with hairless mice, the application of d-Lenolate® in two concentrations using a gel vehicle resulted in no difference from the control.