THE SCIENCE BEHIND

d-Lenolate®

Studies

1999 - 2014

Baton Rouge, Louisiana 70803 (225) 578-9683 Fax (225) 578-9655

L O U I S I A N A S T A T E U N I V E R S I T Y A N D A G R I C U L T U R A L A N D M E C H A N I C A L C O L L E G E

Division of Biotechnology and Molecular Medicine (BIOMMED) LSU School of Veterinary Medicine

April 6, 2009

Mr. Gordon Melcher, CEO East Park Research, Inc. 2709 Horseshoe Drive Las Vegas, NV 89120 Dear. Mr. Melcher: As you know, I have extensive experience in infectious diseases and the development of therapeutic modalities to combat serious viral and bacterial infections of humans. In my positions as the Director of the LSU Division of Biotechnology and Molecular Medicine and Director of the LSU-Tulane Center for Experimental Infectious Diseases (CEIDR), I have been involved in a number of pre-clinical and clinical trials to determine the safety, efficacy and potency of specific antiviral and anti-bacterial drugs and other therapeutics. I am particularly impressed with the potent antiviral activity of d-Lenolate against herpes simplex virus type-1 (HSV-1), the causative agent of human cold sores as well as genital infections. I am not aware of any other “natural” compound that exhibits these properties. We have just started the treatment of infected mice, and I believe that we will find similar results. I recommend the further testing of d-Lenolate in a limited clinical trial in human patients to determine the efficacy of the compound against HSV-1 and HSV-2 (genital infections). I hold adjunct appointments at the LSU Health Sciences Center in New Orleans and I can organize a human trial in collaboration with Dr. John Estrada, Director of the Clinical/Translational Center in New Orleans. Also, I highly recommend testing of d-Lenolate against human immunodeficiency virus (HIV). We have worked closely with the Tulane National Primate Research Center (TNPRC) in HIV studies using the simian immunodeficiency virus (SIV) rhesus macaque animal model. Cell culture studies can be performed in my laboratory, while animal studies can be performed at the TNPRC by my colleague, Dr. Preston Marx, an international authority on HIV/SIV infections.

I believe that these are initial “threshold tests” that will validate d-Lenolate’s efficacy on two important viral pathogens, herpes simplex virus and human immunodeficiency virus. Additional experiments should be performed against other viral and bacterial pathogens in the future.

Again, I am very impressed by the positive results to date and as we move forward, I am confident that we will be discussing additional research paths to explore.

Best regards,

Gus Kousoulas, Ph.D. The Mary Lou Martin Professor of Virology & Biotechnology Director, LSU-Tulane NIH COBRE Center in Experimental Infectious Disease Research

COMPANY/PRODUCT OVERVIEW Headquartered in Las Vegas, Nevada, East Park Research Inc. (EPR) was founded in 1990 by Gordon Melcher, CEO, and incorporated in 1995. For the last 17 years EPR has manufactured and distributed a patented, special dietary food supplement which is obtained from olive leaf extract containing oleuropein. d-lenolate® possesses broad biological properties. Early evidence suggested increased activity against numerous pathogens (virus, bacteria, yeast, fungus and spectrum antibiotic parasites).1, 2Anecdotal evidence also exists for d-lenolate activity against amoebae, rabies virus, Rubella virus, Chlamydia and bacteria which cause botulism poisoning. Initial clinical tests have also shown it to be effective against anthrax bacteria, influenza virus, Candida and E.Coli and herpes. Although most scientific research was undertaken prior to 1995, it was not until EPR began investigating the benefits of d-lenolate, that a clinical application of the natural olive leaf extract was developed. It was at this time that EPR’s Olive Leaf Extract, which was named d-lenolate, was born. EPR, Inc. continued to research the product, as additional benefits were identified. The active ingredient in EPR’s products is d-lenolate®, also known as olive leaf (Olea europaea) extract. d-lenolate®’s formulation is based on a patented extraction process of selected olive leaves that contain Oleuropein, the miracle element found in the leaves of certain olive leaf trees. EPR believes that the molecules (isomers) are delicate and that the extraction process usually destroys some of the effectiveness of olive leave extract. By using EPR’s patented method of extraction, the resultant extract (d-lenolate) is produced, which minimizes blood serum protein binding. (Ongoing research at UNLV is determining the exact level of binding permitted. This minimal binding, provides an extremely effective supplement which significantly reduces the level of pathogens thereby strengthening the immune system and boosting its pathogenic removal ability to a much higher levels of elimination than ever before. Additionally, there was research performed (or data collected) that showed a direct correlation of d-lenolate against Candida albicans. Other third party studies of Olea europaea have also shown that olive leaf extract (or d-lenolate) contributes to increased cardio-vascular health with normal usage.

Although d-lenolate is the core sales product, EPR also manufactures and distributes a line of products that utilizes the d-lenolate formulation as its key ingredient. The product mix contains a topical cream, a cleansing bar, a shampoo, a conditioner, and a new combination product which features the d-lenolate formulation and another very effective immune system enhancer, Manapol, produced by Carrington Labs in Dallas, Texas. Use of these products has generated a mass of anecdotal, testimonial evidence, all attesting to their effectiveness and safety (See Appendix H for a full catalogue). It is important to note that d-lenolate has been in public use for over ten years and taken by tens of thousands of customers without one reported instance of serious side effects or hospitalization. Significant reports, produced by some of the most prominent research scientists, associated with major domestic and international universities, further indicate d-Lenolate and its effective components have a direct causal, and documentable, effect on some of the worst diseases afflicting the world such as Anthrax, Influenza, e-Coli, HIV and Leukemia. Additional and expanded testing have been initiated and planned. References: 1. Robinson TWE, Cureton RJR, Heather RB. 1969. The effect of cyclophosphamide on sendai virus infection of mice. J Med Microbial. 2:137-145. 2. Fujisawa H, Tsuru S, Taniguchi M, Zinnaka Y, Nomoto K. 1987. Protective mechanisms against pulmonary infection with influenza virus. I. Relative contribution of polymorphonuclear leukocytes and of alveolar macrophages to protection during the early phase of intranasal infection. J Gen Virol. 68:425-432. 3. Sumiaki Tsuru, Machio Ibusuki, Sakae Ohtake, Yoshimi Umezawa, Masatoshi Kaneko 2000. Enhanced Resistance against Influenza Virus by Treatment with Dietary Supplement d-Lenolate in Neutropenic Mice Induced by Cyclphosphamide 4. Sumiaki Tsuru, Akihito Nagae, Takuya Ohta, Sakae Ohtake, Machio Ibusuki, Masatosi Kaneko 2001 Therapeutic Effect of d-Lenolate Against Experimental Infections in Immuno-compromised Mice.

TABLE OF CONTENTS

EVALUATION LETTERS

Dr. Gus Kousoulas, Ph.D. Director, LSU-Tulane NIH COBRE Center In Experimental Infectous Disease Research

Dr. Penny Amy, Ph.D. UNLV Proffessor of Microbiology, Nevada System of Higher Education Health Sciences Systems

INTRODUCTION & OVERVIEW

Company Overview Table of Contents Therapeutic Components in d-Lenolate

STUDIES & RESEARCH

The Effect of d-lenolate® on the Immune Parameters of Humans Preliminary Testing for Antiviral Properties of d-Lenolate in Gel Formulation In-vitro anti-HSV-1 Activities of d-Lenolate at Different Concentrations Preliminary testing of anti-viral properties of d-Lenolate in gel formulation Preliminary testing of anti-viral properties of d-Lenolate, Aloe & Neem Tissue

Culture Experiments Analysis of the LSU Saftey Letter dated May 11, 2009 Final Report from Animal Studies with East Park Research Formultaion for an

Effective Herpes Simplex Type 1 Treatment Evaluation of East Park Research Formulations Containing d-Lenolate as Antiviral

for Effectiveness in Treating Topical Herpes Infections In Vitro Anti-West Nile Virus (WNV) Activities of d-Lenolate Antimicrobial Activities of d-Lenolate Therapeutic Effect of d-Lenolate Against Experimental Infections in

Immunocompromised Mice. (E.coli, Pseudomonas aeruginosa and Candida albicans) Enhanced Resistance against Influenza Virus by Treatment with Dietary Supplement

d-Lenolate in Neutropenic Mice Induced by Cyclophosphamide In-Vitro Antibacterial Effect of d-Lenolate Against B. Anthracis Evaluation of the Efficacy of d-Lenolate in the Control of Symptoms of Candida

Hypersensitivity Syndrome Effects of d-Lenolate in Modifying Symptoms of Arthritis

APPENDIX Professional Testimonials/Evaluation Letters

THE EFFECT OF d-Lenolate® ON THE IMMUNE PARAMETERS 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 1st, 8th, 15th, and 21st 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.

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“Antimicrobial activity of d-Lenolate”

A. Background

Resistance to antimicrobials increases the fatality rate of infections, and the prolonged disease state allows greater chance for pathogenic organism to spread across the population. Furthermore, antimicrobial resistance increases care cost by requiring the use of more expensive drugs.

Of the resistant organisms now proliferating around the world, none carries more potential for destruction than the emergence of hospital-acquired "super-infections". In the

United States alone, some 14,000 individuals die each year from drug-resistant microbes picked up in hospitals. The resurgence of these “super-bugs” has created the need for the discovery of novel anti-infective compounds.

Products obtained from natural sources, such as plants, marine habitats, and microbial communities have been used extensively as the source of novel drugs. In particular, olive leaf extracts have been shown to possess broad biological activity, including antimicrobial properties. Although it is believed that phenolic compounds in these extract are

responsible for their effects, a direct separation, purification, and individual compound analysis has not been attempted. d-Lenolate® is an olive extract produced by East Park Research, a company based in Las Vegas, using a patented extraction process. Evidence suggests that d-Lenolate possesses increased activity against numerous pathogens (virus, bacteria, and yeast, fungus and spectrum antibiotic parasites). Anecdotal evidence also exists for d-Lenolate activity against Amoeba, Botulism, Influenza, Rabies, Rubella, and Chlamydia. Initial clinical tests have also shown it to be effective against Anthrax, Influenza and E. coli.

In this project, separated and purified individual compounds from d-Lenolate extracts. We tested these compounds for their stability against serum binding and we determined their antibiotic properties.

Fig. 1. Vancomycin resistance has shown a steady increase 

in hospitalized patients. Source: CDC 

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B. Results and discussion B1. Solubility of d-Lenolate Experimental: Samples of d-Lenolate (1 g) were weighted and placed in 10 individual vials. Each vial was independently treated with 1 ml of hexane, dichloromethane, methanol, tetrahydrofuran, ethyl acetate, acetonitrile, DMSO, water, 5% HCl, and 5% NaOH. The samples were vortexed for one minute. Vials were then centrifuged for five minutes. Solvents were decanted and vials dried at 50 ºC under vacuum. The weight of the remaining d-Lenolate was determined. The percentage of dissolved d-Lenolate was calculated by dividing the weight of the remaining d-Lenolate by the weight of the original d-Lenolate sample. Results showed that methanol was the best solvent to dissolve d-Lenolate, followed by DMSO, and double distilled water. Furthermore, it seems that d-Lenolate is composed mostly of polar, basic compounds. Results: As seen in table 1, oleuropein was most soluble in MeOH, DMSO and water. Oleuropein was not very soluble in non-polar organic solvents or in basic solutions. B2. Qualitative determination of phenolic compounds in d-Lenolate Experimental: d-Lenolate was dissolved in methanol and the solution applied to thin layer chromatography (TLC) plate. The plates were individually developed in 0, 1%, 2%, 3%, 5%, 10%, 12.5%, 15% and 20% methanol/dichloromethane solutions. TLC plates were observed under UV light to find spots corresponding to phenolic compounds. Results: Oleuropein was identified as the major active compound by comparison with a known commercial sample. Three other phenolic compounds were also observed. These compounds will be purified and their physical and biological activities determined.

B3. Purification of oleuropein from d-Lenolate: The project began with the chemical and physical characterization of purified oleuropein from Indofine Chemical Company, Inc. This commercial product was used to determine that oleuropein is soluble in methanol. Methanol was then used to extract soluble compounds from d-Lenolate. Thin Layer Chromatography (TLC) technique was performed with the extracted d-Lenolate sample and pure oleuropein as a control to help determine the presence of oleuropein in the D-Lenolate extracted sample. The TLC

Table 1. Solubility of d-Lenolate Solvent Percentage soluble material Methanol 97.4% DMSO 91.6% Water 80.9% 5% HCl 74.4% tetrahydrofuran 70.1% Acetonitrile 26.8% ethyl acetate 21.4% dichloromethane 16.9% Hexane 4.6% 5% NaOH 0.0%

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procedure also helped conclude that 15% MeOH/CH2Cl2 will allow purifying oleuropein from d-Lenolate when performing a column chromatography. The sample is then dried in preparation for column chromatography. Column chromatography is used to purify compounds from a crude extract. The column chromatography experiment was performed using a solvent elution gradient of 100% CH2Cl2, 5 % MeOH/CH2Cl2, 10 % MeOH/CH2Cl2, 15 % MeOH/CH2Cl2, and 20 % MeOH/CH2Cl2. The fractions collected were then analyzed by TLC to determine the presence of pure oleuropein. As expected, pure oleuropein was eluted in the 15 % MeOH/CH2Cl2 fraction. The column was repeated twice to ensure pure oleuropein was extracted. Results:

This above protocol was used to extract and purify oleuropein from d-Lenolate and the olive leave extract from a competitor company (labeled “C”). Oleuropein was also attempted to be purified from olive leaf extract from a second competitor (labeled “B”). Even though, there was oleuropein present in the extract B, complete purification was not achieved.

B4. Binding of commercial and d-Lenolate purified oleuropein to serum Experimental: The UV-visible spectrum of a 1 mM solution of oleuropein in water was obtained. The oleuropein spectrum shows strong absorption at 235, 275 nm and a weaker peak at 330 nm. A serum sample, on the other hand, showed absorbance peaks at 220 and 280 nm. Due to overlap of the absorbance spectra, serum-oleuropein binding could only be determined using the oleuropein peak at 330 nm. a 1 mM solution of oleuropein in water was mixed with an equal volume of dialyzed serum. The mixture was partitioned using a centrifuge size-exclusion filter. Flow-through and retentive samples were analyzed by UV-visible spectroscopy to determine the amount of free and serum-bound oleuropein, respectively. Each sample was normalized by dividing each spectrum by the corresponding spectrum of a serum-free oleuropein sample. If oleuropein do not interact with serum, it is expected that the both the flow-through spectra will have maximum values of 1. On the other hand, if serum proteins can bind oleuropein, it is expected that the retentate will have a positive peak with a maximum >1. Correspondingly, the flow-through spectra will show a negative peak with a minimum value <1. Results:

As seen on Fig. 2, oleuropein in the retentate shows a positive peak of with a maximum of 1.2. Oleuropein on the follow-through, on the other hand, shows a negative peak with a minimum of approximately 0.65. Thus, 20-35% of oleuropein seems to be bound to serum proteins. Preliminary data for oleuropein purified from d-Lenolate shows a similar trend.

Fig. 2. Analysis of oleuropein-serum binding. Retentate represents the normalized absorbance of serum-bound oleuropein. Flow-trough represents the normalized absorbance of free oleuropein.

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B5. Testing of Oleuropein Binding to BSA Experimental:

The binding of oleuropein to serum was performed with oleuropein from d-Lenolate and oleuropein from extract C. However, complete serum could not be used due to the high background in the absorbance spectra. Instead, bovine serum albumin (BSA) was used as surrogate for serum. BSA is the most abundant protein in serum and has a tendency for binding exogenous compounds. To determine binding of oleuropein to BSA, equilibrium dialysis procedure was used. The apparatus used in this experiment was the Spectrum Equilibrium Dialyzer from Spectrum Labs. Different millimolar concentrations of both the extracted oleuropein and BSA were tested during this part of the experiment. The procedure included testing 0.5mM EPR extracted oleuropein against 0.1 mM BSA, 0.125 mM EPR extracted oleuropein against 0.1 mM BSA and 0.25mM EPR extracted oleuropein against 0.25 mM BSA. The same protocol was applied with sample C extracted oleuropein.

The basis of this test is in each cell of the apparatus, there are two chambers. One cell is loaded with oleuropein along with BSA and in the second cell, only oleuropein. A membrane disc that only allows the diffusion of oleuropein is placed in between the two chambers. The equilibrium dialysis will run for 24 hours. In that 24-hour span, free (nonbinding) oleuropein will reach equilibrium between the two chambers. Absorbance readings of each chamber are then taken using the InfiniteM200 spectrophotometer from Tecan. The wavelength used to measure the absorbance of oleuropein is 330 nm, which is unique to oleuropein and is not interfered by BSA.

Results:

During the 24 hours oleuropein diffuses back and forth until there is the same amount of free oleuropein on each side of the membrane. If oleuropein binds to BSA in one chamber, then the free oleuropein from the second chamber will have to diffuse through the membrane until it reaches equilibrium again. So if binding occurs, one chamber will contain only free oleuropein, the other chamber would contain the same amount of free oleuropein in addition to bound oleuropein. Hence, the chamber with the BSA and oleuropein will now have a higher absorbance reading at 330 nm than the other chamber because it now contains more oleuropein than when it started. If binding does not occur, then both chamber readings should be the same.

According to the data obtained, it could be assumed that neither East Park Research extracted neither oleuropein nor sample C extracted oleuropein bind to BSA in any of the conditions tested. B6. Fractionation of d-Lenolate Experimental:

An extraction method was used to fractionate desired products from the olive leaf extract. The extraction method was carried out using the soxhlet apparatus. d-Lenolate powder (200 g) was extracted for 8-10 hours with methanol to ensure that all alcohol soluble compounds are collected. The resulting extract is then dried using the rotary evaporator and grinded with a pestle and mortar to ensure that there is no clumping that would interfere with uniform separation during column chromatography. The dried extract was then further purified by running the dry column vacuum chromatography (Pedersen & Rosenbohm). The extract was sequentially extracted with solvents

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with increased polarity. This ensures that compounds will be fractionated according to their polarity. d-Lenolate methanol residue was thus sequentially extracted starting with 100% hexane and ending at 100% ethyl acetate increasing in 10% increments. This was followed by 10% MeOH/Ethyl acetate, ending at 100% MeOH, also increasing in 10% increments. Results:

Twenty-one fractions from the dry column vacuum chromatography were collected and then dried. These 21 samples (labeled DCVC 1-21) were then tested for antimicrobial activity. B7. Testing of d-Lenolate Fractions for Antimicrobial Activity Experimental:

To test the 21 samples previously obtained for the presence of antimicrobial activity, the extracts were tested against Bacillus anthracis, Sterne strain (non-infectious) and Escherichia coli, DH5α. Determining antimicrobial properties were achieved by applying the disk diffusion test and the growth inhibition test. Both procedures test for the growth of the bacteria, but the difference is disk diffusion is done on solid media as opposed to liquid media for growth inhibition.

Disk diffusion is a process where a disk shaped filter paper is saturated with the sample. The disk is then placed onto a lawn of bacteria on an agar plate. The plate is incubated for 24 hours at 37°C. A clear zone around the disk is will be observed after a day of incubation if antimicrobial activity is present.

Growth inhibition test differs from disk diffusion in that growth is determined in liquid media instead of solid media. Samples along with bacteria is diluted in liquid broth and incubated for four hours. Optical density is taken at 30 minute intervals to measure bacterial growth. Optical density is a measure of transmittance. Higher OD readings equal lower transmittance, which would indicate bacterial growth. Relative OD is the readings of all the ODs relative to the optical density at time 0 hr. Optical density readings were measured using the Lab systems iEMS Reader MF. Results:

When testing for antimicrobial activity using the disk diffusion test, none of the 21 samples (DCVC 1-21) nor d-Lenolate, exhibited antimicrobial activity. No clear zone was present, which would have indicated antimicrobial activity.

However, growth inhibition tests at saturated concentrations of DCVC 1-21determined some of the fractionated samples of d-Lenolate as well as d-Lenolate itself, presented antimicrobial properties when tested against B. anthracis and E. coli (Table 2). Results from the growth inhibition assay suggest

Table 2. Bacteria growth in the presence of d-Lenolate extracts after three hours

Relative growth

Conditions B. anthracis E. coli

No treatment 100 100 Oleuropein 78 76 Chloramphenicol 3 0 d-D-Lenolate 26 31 20% MeOH/EtOAc 40 57 30% MeOH/EtOAc 50 45 40% MeOH/EtOAc 70 76 50% MeOH/EtOAc 33 64 60% MeOH/EtOAc 76 75

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that the fractionated d-Lenolate samples slowed the growth rate of both B. anthracis and E. coli. Samples DCVC10, DCVC11, and DCVC12 showed the greatest activity against both

gram positive and gram-negative bacteria. To assess compounds present in these samples, thin layer chromatography was performed to see if there was any unique characteristics of those three samples. The analysis of the TLC of the 21 samples showed the presence of three compounds that were at higher concentrations in DCVC10, DCVC11, and DCVC12 than in other samples. Because DCVC10, DCVC11, and DCVC12 showed greater activity than the other samples tested, it can be assumed that these three compounds might have a role with their ability to slow down bacterial growth.

The next step is to separate and purify the three compounds of samples DCVC10, DCVC11, and DCVC12. Since DCVC10, DCVC11 show similar compounds by TLC, these two extracts were combined for further purification. When the separation and purification process is done, the separate compounds will then be tested for antimicrobial activity. B8. Purification of d-Lenolate Fractions Experimental:

Further separation and purification of the DCVC10, DCVC11, and DCVC12 samples was achieved by column chromatography. This is procedure starts out with dissolving 3.0g of the combination of samples DCVC10 and DCVC11 in about 25mL of 50% MeOH/CHCl3. The sample was loaded into a column that has been packed with a slurry of 222.0 grams of 100-200 mesh silica gel in 50% MeOH/CHCl3. A layer of glass wool was added to the top of the column to protect the column. The column was then flushed out with 100% CH2Cl2 to get rid of all the methanol and chloroform creating a less polar column. This will allow the column to start at a nonpolar environment and work its way up to a higher polar solvent. The procedure was continued with 2 liters 2.5% MeOH/CH2Cl2. 10mL fractions were collected and each fraction was analyzed by TLC to determine if compounds have eluted and whether these compounds were pure. The fractions that contained the same compounds were combined and further separation and purification was followed. From these procedures, a fraction was selected for further purification. This sample was labeled A1.

1.5g of sample A1 (from separation of DCVC10 and DCVC11) was dissolved in about 15mL of 50% MeOH/CHCl3. The sample was loaded into a column that was vacuumed packed with 270.0 grams of 100-200 mesh silica gel creating a dry column instead of slurry (wet column). The column was then flushed out with 100% dichloromethane and continued with 2.5 liters of 5% MeOH/CH2Cl2. The fractions were collected and analyzed by TLC. Fractions A1.3, A1.4, and A1.5 were combined and analyzed by further separation.

A1 (from separation of DCVC10 and DCVC11) was also separated by medium pressure liquid chromatography (MPLC). MPLC does not use gravity or a vacuum for the separation, instead, a pump is used. It uses more pressure than gravity, but not as much pressure as the vacuum. 1.5 grams of the 2.5g have been previously separated during the first set of purification leaving 1.0g for this procedure. The 1.0g of sample was dissolved in 9mL of 50% ACN/CH2Cl2 and loaded into a Michael Miller column packed with about 110 grams of 5-40um silica gel slurry. 100% dichloromethane was used to flush out the 50% ACN/CH2Cl2. 300mL of 8% ACN/CH2Cl2 was used to run the column. 2mL fractions were collected and analyzed by TLC.

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600 mg of samples A1.3, A1.4, and A1.5 (from separation of sample A1 above) were combined and dissolved in about 4mL of 2.5% ACN/CH2Cl2 and loaded into the column packed with 110g of 15-40um silica gel. The column was vacuumed to form a tight packed column. 500mL of 2.5% ACN/CH2Cl2 and 500mL of 5% ACN/CH2Cl2 were used to run the column. 3mL fractions were collected and analyzed by TLC.

A different set of column chromatography was also performed but with sample DCVC12 from the dry vacuum column chromatography. There were about 8.5 grams of fraction DCVC12 that could be separated and purified. This column was performed with the flash chromatography technique. Vacuumed is used as opposed to gravity as described during the previous separations. 400 grams of 100-200 mesh silica gel was used to pack the column very tightly with the vacuum. The 8.5g of fraction DCVC12 was dissolved in about 11.4mL of methanol and loaded into the column. 300mL fractions were collected with the use of the vacuum and analyzed by TLC. Three fractions were obtained and labeled as B7, B8, and B10 from these procedures, fraction B7 was selected for further purification. Samples from the previous flash chromatography were further separated and purified. 50.0mg of B7 was dissolved in about 3mL of 50% ACN/CH2Cl2 and loaded into a column that was vacuumed packed with about 30.0g of 15-40um silica gel. The column was first flushed with 100% dichloromethane and followed with about 300mL of 12% ACN/CH2Cl2and proceeded with 300mL of 25% ACN/CH2Cl2. Fractions of 1mL were collected and analyzed by thin layer chromatography.

Results: The separation and purification of sample DCVC10 and DCVC11 (from separation of dry

column vacuum chromatography (DCVC)) lead to the separation and purification of sample A1 (from separation DCVC10 and DCVC11 ), which was followed by the separation and purification of samples A1.3, A1.4, and A1.5 (from separation of sample A1). The samples collected from the last purification were analyzed by thin layer chromatography. After looking at all the plates it was determined that only fraction A1.3.12 (from separation of A1.3, A1.4, and A1.5) was completely pure and that will be used to test for antimicrobial activity. Fraction A1.3.4 (from separation of A1.3, A1.4, and A1.5) was not completely pure, but pure enough for further testing. An antimicrobial test was done with fractions A1.3.4 and A1.3.12 to test for activity against B. anthracis and E. coli.

The separation and purification of DCVC12 (from separation of DCVC) lead to the separation and purification of B7 (from separation of DCVC12). The samples collected from the purification of B7 (from separation of DCVC12) were then analyzed by TLC. Samples B7.5 and B7.6 (from separation of B7) were the only samples determined to be pure. Two other samples (B7.10 and B7.11) were partially purified. The growth inhibition test was done with all of the samples collected from the separation of samples B7.5, B7.6, B7.10 and B7.11.

The separation and purification of 1.0g of sample 10 and 11(from separation of dry column vacuum chromatography (DCVC)) lead to the separation and purification of sample MPLC1 (from separation DCVC10 and DCVC11) by way of medium pressure liquid chromatography. The samples from the purification by MPLC were collected and analyzed by TLC. Samples 7M, 8M, and 11M from the purification look to be very pure. All of the samples were tested for antimicrobial properties.

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There are no IR and NMR data for the samples that are pure because there were not enough samples to run all of the tests. Nevertheless, samples were submitted for mass spectrometry. Samples are being currently analyzed at the University of Arizona. . B9. Testing of Fractions for Antimicrobial Activity Experimental: All of the samples obtained from the previous separations and purifications were tested for antimicrobial activity using the growth inhibition test. Results:

According to the data of the growth inhibition test performed with 0.2mg of samples A1.3.4 and A1.3.12 from the first set of separations; these two compounds do not possess any antimicrobial activity. The procedure was repeated to ensure that the data was accurate. Sample A1.3.4 and A1.3.12 were combined to determine whether the tow compounds together would have increased antimicrobial activity. According to the data, there were no antimicrobial properties of these two samples combined together. .

The growth inhibition results for the samples from the flash chromatography determined that samples B7, B8 and B10 at 0.2mg presented antimicrobial activity. Sample B10 contained a large amount (based on intensity of the spot) oleuropein, which may have played a big factor in its activity. We are only concerned with the samples that do not contain oleuropein. Since sample B7 had greater activity than B8, B7 was further separated and purified.

Fractions from the separation and purification of B7 were obtained and tested for antimicrobial activity. Based on the results obtained from the growth inhibition test, sample B7.5, B7.6, and B7.11 (from separation of B7) presented antimicrobial activity. According to TLC analysis, samples B7.5 and B7.6 are the same compound, but the test shows that sample 5 does not have activity. Sample B7.6 was tested at 0.08mg because that was all that was obtained and sample B7.5 was tested at 0.2mg. The test was repeated and again indicated that only samples B7.6 (0.08mg), B7.10 (0.2mg), and B7.11 (0.2mg) had activity. Sample B7.5 was then tested again at 0.08mg instead of 0.2mg to try to duplicate the results of sample B7.6. The results indicated that sample B7.5 does not possess any antimicrobial activity. The test was repeated and the same results were obtained. Further separation and purification will now be focused on samples B7.10 and B7.11 (from separation of B7)

Samples from the separation and purification of MPLC1 were tested against B. anthracis and E. coli for activity. The data collected indicated no antimicrobial properties of samples 1M through 11M. The test was repeated to confirm the previous results. Samples 7M, 8M, and 11M are pure and might even be the same compound. These three samples were prepared and sent to Arizona for mass spectrometry. B10. Comparing antimicrobial activity of d-Lenolate and Spanish olive leaf extract Experimental:

The antimicrobial activity of East Park Research’s d-Lenolate and a comparable olive leaf extract from Spain were tested simultaneously against Bacillus anthracis and Escherichia coli during the growth inhibition test. The optical density readings were recorded and analyzed. The procedure was repeated to ensure the results were accurate.

  9

Results: The results indicated that both d-Lenolate and the Spanish olive leaf extract presented

similar antimicrobial activity. TLC data also showed that the two extracts have similar composition. B11. Cell Killing with Macrophages Experimental:

The mammalian cell line used for this part of the experiment is the J774a.1 murine macrophages. These cells were generously provided by Dr. Brojatsch at Albert Einstein College of Medicine, NY. There are certain conditions required for the medium and growth of these macrophages. One of the very important conditions needed is an incubator set at 37°C and humidified with 5% CO2.

Sample B7.5 (from separation of B7) was used to determine the effects it would have on macrophages. After a few subculture procedures were performed, the cells were ready for testing. The procedure begins with the loading 80uL of macrophages into each well of the 96- well plate and incubated for 30mins to allow cells to attach. 10uL of propidium iodine (PI) is then added to each well and allowed to sit in the incubator for 10 minutes. PI is a fluorescent dye that stains the nucleus of the cell. PI will stain cells red only if the cells are dead. Living cells will not fluoresce. After the 10 minutes of incubation, a fluorescent reading was done using the InfiniteM200 from Tecan. Sample B7.5 (separated from B7) was then loaded into its respective wells and the 96-well plate was placed back into the incubator. Fluorescent readings were taken at 0hr, .5hr, 1.0hr, 2.0hr, 3.0hr, and 4.0hr.

Results:

The results indicated that sample B7.5 (separated from B7) does not harm mammalian macrophages. Increasing numbers during the readings would indicate the killing of the cells. The numbers of the readings with the samples do not deviate too much from the readings of the wells with just methanol (control). The readings of the different time intervals of the wells with the sample were consistent with each other. B12. Artificial Gastric Juice Stability of Oleuropein Experimental:

It is an interest to determine how stable oleuropein is in the gut of a human being to ensure that oleuropein can indeed be administered orally. This is a test that will help indicate if gastric juice from the stomach will or will not degrade the compound before it can reach the blood stream.

Oleuropein (Indofine Chemical Company, Inc.) was tested against artificial gastric juice (VWR) to determine

Fig. 3. Stability of oleuropein to gastric juisce. Oleuropein was incubated with artificial gastric juice and spotted for TLC 0, 1, 2, 3, 4, and 18 hours post-exposure. Degradation of oleuropein can be seen as new spots on the TLC plate

  10

its stability in this acidic environment. It was determined that each adult size capsule from EPR contains 500mg of d-Lenolate. Because there is only at most 20% of oleuropein in each capsule, 100mg of commercial oleuropein was used for this test. The 100mg of oleuropein was dissolved in 500uL of MeOH. The 500uL of samples was then added to a culture tube with 4.5mL of artificial gastric juice making the ratio of oleuropein to gastric juice 1:9. The tube was then incubated at 37°C. A TLC was spotted with the sample at 0hr, 1hr, 2hr, 3hr, 4hr, and overnight incubation. The plate was then be analyzed to see if there are any changes to the oleuropein compound. If all the spots at different hours look the same, then the compound was not destroyed by the gastric juice and can be taken orally and vice versa. Results: The six spots (0hr far left and overnight far right) on the TLC plate were analyzed. It can be seen at 0hr; oleuropein is pure and only has one spot. After one hour of incubation, an additional compound along with oleuropein can be seen. As the time of incubation increased, the addition compounds became more distinct and the intensity of the oleuropein decreased. After incubation throughout the evening, two addition compounds along with oleuropein (total of three) can clearly be seen. It could be assumed that oleuropein is not very stable in artificial gastric juice. Based on the results of this test, gastric juice degrades oleuropein and in the process produces two unknown additional compounds. C. Conclusions

Olive leaf extract has a long tradition in traditional medicine. In the present study, we show that oleuropein is soluble in polar solvents, but not in non-polar or basic solvents. Oleuropein was shown to not bind to serum or BSA. However, the acidic environment of the stomach can degrade oleuropein. Whether these degradation products are harmful to humans, or are active against bacteria is yet to be determined. Thus, olive leaf extracts should be formulated to protect oleuropein from degradation by the GI tract. Oleuropein has been shown to have antimicrobial activity. In our hand, olive leaf extracts and purified oleuropein can reduce bacterial growth but not eliminate it. In contrast, olive leaf components did not cause harm mammalian cells. This is consistent with previous report that show oleuropein to be a modulator of the immune system. By retarding bacterial growth, olive leaf extracts could allow the immune system to clear up bacterial infections. Interestingly, olive leaf extracts were more potent in antimicrobial tests than purified oleuropein. In fact, fractions of olive leaf extracts devoid of oleuropein were significantly more active than purified oleuropein. This shows that oleuropein is not the only active compound in olive leaf extract. However, when the oleuropein-less fractions were further purified, their antimicrobial activity was reduced or eliminated. This argues that the mixture of compounds in olive leaf extract is required for maximal anti-microbial activity and that no single component is the major antimicrobial compound.

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% 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  ‐‐‐‐‐‐ 

 

 

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 and inhibition 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 103 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.

Evaluation of East Park Research Formulations Containing d-Lenolate as an Antiviral for Effectiveness in Treating Topical (Skin) Herpes

Infections

Purpose: To evaluate the efficacy of a topical application of East Park Research formulations containing d-Lenolate in treating dermal herpes infections in mice. Procedure: Human HSV-1 clinical strain ABGK1 was used to inoculate SKH-1 hairless mice at scarification sites located on the dermis of the lower left dorsal flank (approximately 2.5 x 104 PFU/mouse). Beginning 12 hours post infection, 20 µL of vehicle plus EPR formulation at 3 different concentrations were applied to the sites of infection 3 times a day at 8 am, 2 pm and 8 pm, with 8 animals per treatment group. Treatments were also included 20 µL of PBS as a negative control and 25 µL of Abreva as a positive control Treatments were continued for 14 days followed by euthanasia on day 16. The resulting herpes lesions were scored daily on a 5 point scale of increasing pathology. Results: Mice began to develop detectable lesions at sites of infection on day 2 post-infection with a majority showing signs of infection by day 4. Early/low scoring lesions were typically characterized by discreet vesicular eruptions associated would then merge to form scabbed over bands (Score 2). Lateral lesion spread and/or banding occurred when secondary eruptions appeared outside the area defined by the initial site of infection. In more advanced cases, lesion banding spread to define a dermatome extending from the site of infection down the outside of the left leg. Subsequent dermatome expansion onto the paw was often accompanied by leg retraction and paw curling, along with secondary lesion development on the left ventral posterior abdomen proximal to the vagina and anus (sore 3). Extreme cases (score 4) involved distal lesion development across the anterior-posterior midline from the SOI. Cross-midline lesions could appear on either dorsal or ventral surfaces. Cross midline lesions were frequently a prelude to more severe complications leading to euthanasia. Animals that presented with hind limb paralysis, severe weight loss, or signs of dehydration were given scores of 5 and euthanized. By day 15 all surviving mice showed visible signs of improvement reflected in declining lesion scores. Analysis: Lesion scores for individual mice were plotted vs. Days post-infection and the area under the curve (AUC) for each animal was determined. The Mean AUC for each treatment group was calculated from days 1-13 and plotted . One-way ANOVA with Bonferroni’s correction for multiple comparisons showed that the experimental treatments had a significant effect on the mean AUC. Individual unpaired two tailed t-tests using Welch’s correction for unequal group variances showed that treatment with the highest d-Lenolate concentration (dose C), significantly reduced the mean AUC as

compared with all other treatments excluding Abreva. The difference between the mean AUC of the PBS control-treatment group and the mean AUC of the Dose C group was highly significant (two-tailed t test p value=0.0031). These results were also supported by analysis of animal survival data. Mean survival curves were established for each group and are shown in Figure 3, plotted as % survival over time (days post infection). The survival curve for each experimental group is shown alongside the survival curve derived from the PNS-treated control group for comparison. Survival curves of the experimental treatments were compared with that derived from PBS treatment using Log rank (Mantel-Cox) and Gehan Breslow-Wilcoon tests. The results from these tests agree that mortality was significantly reduced in groups receiving treatment with Dose C (p values=0.0088 and 0.0069 respectively) while other treatments displayed no significant differences in mortality. Controls in this experiment behaved as expected. Herpes lesion and symptom development in mice treated with PBS were the most rapid and the most severe. Surprisingly however, treatment with Abreva had, at best, a minimal effect on herpes symptom development and duration. Treatments with the lower doses of d-lenolate (doses A and B) did not display any measurable therapeutic effect under the conditions of this study. In contrast, treatment with dose C was clearly effective in mitigating and ameliorating herpes disease symptoms. Conclusions: There were no significant deference between any of the mean AUCs for groups treated with PBS, vehicle, dose A and dose B. In other words this study was unable to detect any effect of these treatments on herpes infection in the SKH1 murine model. In contrast, the dose C experimental treatment group presented with significantly reduced symptoms, characterized not only by a decrease in the number of animals with herpes symptoms, but also a decrease in mortality and in the severity of disease in symptomatic animals. Statistical analyses support this conclusion. p values calculated for the comparison of herpes lesion scores and mortality between dose C and PBS treatments suggest that the observed differences are highly significant. We conclude that treatment with dose C inhibits human herpes simplex virus pathogenesis in the SKH1 model.

Final Report from Animal Studies with East Park Research Formulation for an Effective Herpes Simplex Type 1 Treatment

Prior to this series of experiments, safety testing was performed at a leading university on nude mice using the East Park Research formulation. The results showed that no detectable effects of the application of Concentration A or C (1X and 10X of the active ingredient) were seen compared to the control. There were no adverse reactions.

In the current testing regime, a strain of human HSV-1 (Herpes simplex type 1) was used to treat hairless mice by means of scraping a small area on the left hind flank of the mouse and then applying various treatments to the area following introduction of the virus to the scraped skin. The number of virus particles was approximately 25,000 per mouse.

The treatments were as follows:

Negative control: 20 microliters of saline water (PBS)

Control without additives: 20 microliters of ‘vehicle’ which is the gel alone

Positive Control: 25 microliters of Abreva

Treatments: 20 microliters of the gel vehicle plus one of three concentrations of the East Park Research formulation.

Concentration A: 1X Concentration B: 3X Concentration C: 10X

All treatments were applied 3 times per day for 14 days.

Results:

Between 2-4 days after treatment with the virus, the mice developed symptoms of virus infection on the skin. Some mice developed further infection with the severity of the infection scored 1-5 from mildest to most severe. A value of 5 required early euthanasia. All remaining mice were euthanized at day 16.

Mice were scored for severity of infection daily. The scores per treatment were added and divided by the number of living mice scored. For example, on day 7, all mice in the concentration C treatment group were scored, the scores added and divided by 8, which was the number of test animals alive on day 7. The resulting average or mean score was plotted in Figure 2. The higher the line on the graph, the less effective the treatment. The order from least effective to most effective was:

PBS -> Concentration B-> vehicle gel alone, Abeva and Concentration A (all three had equal results) -> Concentration C

Not only did the Concentration C of the EPR formulation work best, it held the score, or severity, of the infection from approximately day 7-13. This means that it kept the virus from spreading and held the score to just over 1, which was a mild infection.

The difference between Concentration C treatment group and all the others was statistically highly significant (p value = 0.0031). In statistical tests, the smaller the p value, the more confidence there is in a difference between scores.

In Figure 3, the survival of the mice is compared to the PBS negative control in treatment pairs. The higher the line, the more mice survived. In each case there was significant death at days 7 and 12 with the exception of Concentration C.

A description of each pair and overall comparison is as follows:

PBS and Abreva – death at days 7 and 12. Abreva was mildly protective.

PBS and Vehicle (gel) alone – death of mice at days 7 and 12. The gel is mildly protective.

PBS and Concentration A – death of mice at days 7 and 12 in both cases. The EPR Concentration A was mildly protective.

PBS and Concentration B – death of mice at days 7 and 12. Concentration B was mildly protective but not as much as Concentration A, Abreva and the gel vehicle.

PBS and Concentration C – this treatment provided for the best survival of all the treatments. Although the survival with the negative control (PBS) was below 50%, Concentration C allowed 100% survival until day 12 when the survival dropped to approximately 90%. This treatment was highly effective.

Conclusions:

PBS treatment allowed the maximum infection and mouse mortality. Concentration B was not protective in either the infection score (severity) or the survival of the mice. Abreva, Concentration A and the gel vehicle provided mild protection from severe virus infection and in survival of the mice. Note that Abreva was only as effective as the gel alone. Concentration C was highly effective, reducing the infectivity score to less than 1.5 and allowing nearly all the mice to survive. A score of 1 was defined as a discrete vesicular lesion that crusted over by day 5 and healed. A score of 2 was defined as more than one discrete vesicle that merged to form a band and followed by scabbed over bands. A score of 1 or 2 resulted in scabbing and healing of the mouse lesions, therefore a score of less than 1.5, as seen with Concentration C, resulted in healing and survival. All other scores were from much more severe infections with a score of 5 resulting in early death.

In Vitro Anti‐West Nile Virus (WNV) Activities of d‐Lenolate 

 

Purpose  To determine the in vitro anti‐West Nile Virus (WNV) activities of East Park Research’s  d‐Lenolate at different concentrations.  

Methods  A 10% stock solution was prepared by resuspending 4 grams of the powdered d‐lenolate extract in 40mL  of PBS. The extract was vigorously vortexed and filtered through 0.45um Acrodisc syringe filters under  sterile conditions. This filtrate was used to test the antiviral activity of d‐lenolate.    Ten‐ fold dilutions of the 10% d‐lenolate extract stock solution were prepared in serum‐free DMEM. 100  PFU of WNV was added to each of the dilutions of d‐lenolate extract. 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  and cell controls were maintained. All samples were done in triplicate. The cells were fixed with 10%  neutral buffered formalin for 1 hour; washed with PBS; stained with 0.1% crystal violet and the plaques  were counted.  

Observations In the 5% d‐lenolate extract treatment, no viral plaques were observed. An 80% reduction in viral  plaques was observed in 0.5% treatment. No decrease in the number of viral plaques was observed in  the 0.05% and 0.005% treatments. No toxic effects were observed.  

Conclusion     The d‐lenolate solution demonstrated antiviral activity at higher concentrations. The effect decreased with increasing dilutions.   

WNV  Adsorption Only 

Concentration # Plaques 

(Mean of 3 replicates) % Plaque Reduction 

5% d‐lenolate extract (10% stock) 

0  100% 

0.5% d‐lenolate extract (10% stock) 

20  80% 

0.05% d‐lenolate  extract (10% stock) 

100  0% 

0.005% d‐lenolate extract (10% stock) 

100  0% 

Virus control  100  ‐‐‐‐‐‐ 

Cell Control  0  ‐‐‐‐‐‐ 

 

 

   

Enhanced Resistance against Influenza Virus by Treatment with Dietary Supplement d-Lenolate in Neutropenic Mice Induced by Cyclophosphamide. 2001. Sumiaki Tsuru, Machio Ibusuki, Sakae Ohtake, Yoshimi Umezawa, and Masatoshi Kaneko. Journal of Orthomolecular Medicine, 16:102-114.

d-Lenolate, a natural neutraceutical extracted from olive leaves, was evaluated for

its effectiveness against infection by influenza virus in tissue culture and mice. Four basic treatments were made with tissue culture or whole mice prior to exposure with the influenza virus: 1) untreated control; treatment with CY (cyclophosamide); treatment with d-Lenolate; and treatment with d-Lenolate followed by treatment with CY. CY depresses the immune system, creating an immunocompromised state, and generally accelerates the infectious process.

d-Lenolate had previously been found to inhibit bacteria that grow outside of animal cells in the early stages of infection. These included: Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pneumoniae. (2,3) A recent study by the authors indicated that d-Lenolate helped restore white blood cell count which augmented protection against several bacterial infections in immunocompromised mice. (13) Potential for protection or immune system augmentation of HIV patients might be considered. There is some evidence that Polymorphonuclear leukocytes (PMN) were also protective against influenza virus infection. (5) This study was designed to assess in more detail, the role of PMN, macrophages and d-Lenolate in protecting tissue cultures and mice against infection and death due to influenza virus. Findings: 1. When mice were exposed to influenza virus with no pretreatment (contol) or treatment with d-Lenolate, no mice died, however, when exposed to CY prior to influenza exposure, all the mice died. Mice treated with CY could be ‘rescued’ from death in 30% of the cases when d-Lenolate was given after CY treatment. 2. Correlated with mouse death was the number of virus particles found in the lungs of virus-inflected mice. The number was similar in the control (untreated mice) and the d-Lenolate-treated mice, however, it was lower with d-Lenolate treatment. When treated with CY, the number of viruses increased to the highest level found in any treatment. Since CY treated mice could be ‘rescued’ in about 30% of the mice as described in #1. The number of viruses in ‘rescued’ mice fell between the high level with CY treatment and the lowest level with d-Lenolate treatment (treatment started 7 days prior to influenza exposure). There appears to be a direct correlation between virus number in the lungs and death due to influenza infection.

3. The number of PMN in the blood of mice treated with CY fell rapidly. If mice were given d-Lenolate post exposure to CY, the PMN numbers were restored faster than in CY treated mice without d-Lenolate. 4. Tissue culture cells were exposed to influenza virus then the number of PMN which could not clear, or destroy, the virus were measured. These PMN were infected and not able to perform their function of engulfing foreign particles and destroying them. In every case with d-Lenolate treatment, PMN unable to clear viruses decreased significantly from the untreated control. It was also shown that lung macrophages, another type of white blood cell that engulfs viruses, did not change dramatically although there was some ability for d-Lenolate to enhance clearing of viruses from macrophages as well. 5. d-Lenolate treatment resulted in the production of antibodies to influenza virus at a comparable level to control mice. CY-treated mice had a much lower level of antibody production and once again, d-Lenolate was able to restore some of the antibody production capability if given after CY treatment.

This article demonstrates that early protection against influenza virus infection is related to the number and function of PMN in the blood and, to a lesser extent, macrophages in the lung. d-Lenolate prevented the extreme loss of PMN found when mice were treated with CY, lowered the number of viruses found in infected lungs and assisted the PMN in destroying viruses. It further helped restore PMN numbers in mice previously treated with CY.

In a study conducted by the authors, but not described in this article, d-Lenolate was able to restore antibody production after chemotherapy. Therefore, it appears that d-Lenolate functions in the early stages of infection by enhancing PMN number and function and, antibody production capability of B cells as well. All of these d-Lenolate induced functions make the efficacy of cellular mechanisms in late stage influenza virus infection more successful when the immune system is depressed and at least equivalent to healthy mice responses.

It appears that d-Lenolate is a successful treatment for influenza and extracellular bacterial infection. It is particularly useful as a preventative treatment prior to exposure and as a treatment to enhance healing in exposed and immunocompromised mice. References: 1. Mitsuyama M, Takeya M, Takeya K, Nomoto K. 1978. Differing contribution of polymorphonuclear cells and macrophages to protection of mice against Listeria monocytogenses and Pseudomonas aeruginosa. J Gen Microbiol. 115:161-166. 2. Tsuru S, Nomoto K, Mitsuyama M, Zinnaka Y, Takeya K. 1981. Importance of polymorphonuclear leukocytes in protection of mice against Escherichia coli. J Gen Microbiol. 122:335-338. 3. Robinson TWE, Cureton RJR, Heather RB. 1969. The effect of cyclophosphamide on sendai virus infection of mice. J Med Microbiol. 2:137-145. 4. Fujisawa H, Tsuru S, Taniguchi M, Zinnaka Y, Nomoto K. 1987. Protective mechanisms against pulmonary infection with influenza virus. I. Relative contribution of polymorphonuclear leukocytes and of alveolar macrophages to protection during the early phase of intrasnasal infection. J Gen Virol. 68:425-432.

Therapeutic Effect of d-Lenolate Against Experimental Infections in Immuno-compromised Mice. 2000. Sumiaki Tsuru, Akihito Nagae, Takuya Ohta, Sakae Ohtake, Machio Ibusuki, Masatosi Kaneko. Journal of Orthomolecular Medicine. 15:127-138.

d-Lenolate, a natural neutraceutical extracted from olive leaves, was evaluated as a protective agent against two bacterial species in mice: E. coli and Pseudomonas aeruginosa; and the yeast, Candida albicans. A number of compounds were used to stimulate the mouse immune system; these are called adjuvants. The adjuvants included: Lithium carbonate (LiCO3); A group A streptococcal preparation (OK-432); and a lipopolysaccaride preparation from E. coli. Two compounds were used to depress the immune system: Cyclophosphamide (CY) and prednisone (steroid immune depressant). Additionally, X-irradiation was used to kill white blood cells, thus making the mice immuncompromised.

The present study focused on the effect of d-Lenolate on enhancement or restoration of immune system function. d-Lenolate was given either orally (po), by intraperitoneal injection ip (injected in the cavity in which the stomach and intestines reside), or by intravenous administration (iv). The dosage was either 250 or 500 mg/kg. This is equivalent to 18.75 or 37.5 gm, respectively, in a 75 kg/165lb human

Findings:

d-Lenolate was given po (oral) and ip (intraperitoneal) to mice before CY and microbial injection. Six days later the mice were injected ip with CY. The number of white blood cells present in peripheral blood with CY-treated mice fell dramatically immediately after treatment and reached its lowest level at day 2. Thereafter, the number began to be naturally restored at days 4, 6, and 8. The high dose of d-Lenolate one day before exposure to microbes, showed the same general pattern of white blood cell loss but to much lesser extent. For example, at day 2, CY-treated mice had white blood cell counts of less than 2,000 per square mm2 whereas those treated with both CY and d-Lenolate had counts of 6,000 per mm2. The pretreated number was 9,000 per mm2 for both untreated controls and prior to CY treatment. By 8 days, the d-Lenolate-treated mice showed counts higher than the initial value (11,000) while the CY-treated or immunodepressed mice were unable to restore the white blood cell counts to the original value.

1. In a similar experiment, the white blood cell count of X-irradiated mice was measured, as well as mice X-irradiated but treated with oral d-Lenolate 7 days before the X-ray treatment. Once again, the d-Lenolate, while not totally eliminating a decrease in white blood cells, maintained a higher count than the mice only receiving the X-ray treatment throughout the experiment.

2. To create a depressed immune state, CY was given on the day of microbial injection, or X-irradiation was performed one day prior to injection. Groups of 10 X-irradiated mice were given a lethal dose of microbes either in muscle tissue

(im) or in the veins (iv). Seven consecutive days prior to exposure in some groups, either the high dose (500 mg/kg) or the low dose (250 mg/kg) of d-Lenolate, or OK-432, was given orally. Control mice were given the same volume of a saline solution by the same route.

Treatment - all with E. coli by iv exposure Survival at 14 days

X-ray irradiated and infected mice 0% (7days)

+ 250mg/kg d-Lenolate 90%

+500 mg/kg d-Lenolate 100%

+1 KE/kg of OK-432 60%

Treatment with E.coli by im exposure Survival at 14 days

X-ray irradiated and infected mice 0% (8days)

+ 250mg/kg d-Lenolate 50%

+ 500 mg/kg d-Lenolate 70%

+ 1 KE/kg of OK-432 20%

d-Lenolate was clearly protective for mice inoculated with a lethal dose of E. coli. A similar result was found with X-irradiated mice exposed to P. aeruginosa. Mice were X-irradiated and one day later injected with bacteria by iv. D-Lenolate and OK-432 were administered orally each of the 7 days prior to injection. All X-ray treated control mice died within 14 days; 80% of mice also treated with 250 mg/kg d-Lenolate survived; 100% of the mice treated with 500 mg/kg d-Lenolate survived; and 60% of the mice treated with OK-432 survived.

A third experiment, set up in the same manner but using C. albicans, was conducted. This microbe is a yeast and not bacterial in nature. Control mice were exposed one day after X-irradiation with C. albicans; all mice were dead by 12 days. Additional treatment with 250 mg/kg d-Lenolate resulted in 30% survival at 14 days, 500 mg/kg d-Lenolate treatment resulted in 70% survival, and OK-432 treatment resulted in death of all mice by 14 days.

d-Lenolate was the most effective treatment in restoring health to microbially-infected X-irradiated mice. The adjuvant, OK-432, was also somewhat protective but to a lesser

extent. This adjuvant is known to stimulate the immune system, however, it is not as effective as d-Lenolate in immune system stimulation.

4. After mice were X-irradiated, their depressed immune systems showed difficulty in clearing bacteria from their blood. When d-Lenolate or OK-432 were given 6 days prior to X-irradiation followed by bacterial injection, the higher dose of d-Lenolate was able to assist in recovery of immune system function to the level of the non-irradiated controls. The lower dose was not as effective but still showed blood bacterial counts only half those in the irradiated controls. OK-432 was effective at a level between the low and high doses of d-Lenolate.

5. A similar experiment was performed without X-irradiation, testing the ability of several compounds to effect phagocytosis (uptake of a killed yeast suspension by PMN). The yeast suspension was injected ip into the mouse immediately after treatment with orally administered d-Lenolate, both high and low doses; d-Lenolate given ip, both high and low doses; LiCO3 given ip at both high and low doses; and OK-432 given ip at high and low doses. The results showed that low dose d-Lenolate or OK-432 given orally or ip enhanced phagocytosis of the killed yeast cells a little more than 150% of the untreated control. The LiCO3 was not as effective. The high dose d-Lenolate, given either orally or ip, or high dose LiCO3 enhanced the uptake of the yeast cells by approximately 200%. High dose OK-432 was not more effective than the lower dose.

6. Mice were given prednisolone to determine the effect on peritoneal macrophages. These are a second cell type capable of phagocytosis (ability to take up killed yeast cells). Prednisolone depressed macrophage activity by 30%, however the addition of oral d-Lenolate at either low or high dose restored the macrophage phagocytic activity to 100%.

7. Reduction of a compound (NBT), used to measure respiratory activity in PMN, was tested in rats and Guinea pigs. Reduction of NBT represents the cell’s metabolic ability or activity. PMN were harvested from rats and Guinea pigs 2 hours after administration of treatment with d-Lenolate or OK-432 to stimulate the immune response. In rats, orally administered d-Lenolate resulted in nearly 200% enhancement of PMN activity, measured as NBT reduction. OK-432 provided approximately 150% enhancement over the control rat PMN level. In Guinea pigs, high dose, orally administered d-Lenolate enhanced activity approximately 200% and the same dose given ip resulted in nearly 300% enhancement in macrophage activity. Low dose, d-Lenolate given ip enhanced macrophage activity closer to 150%.

8. Interleukin-1 production is a cellular defense mechanism against infection. It is often seen with virus exposure where it helps prevent viral spread from cell to cell. Interleukin-1 was measured from peritoneal macrophages two hours after exposure to low and high doses of d-Lenolate, low and high doses of LPS (lipopolysaccharide mimics bacterial cell infection), and OK-432. The amount of IL-1 produced was found to be in the following sequence: high dose LPS > high dose d-Lenolate > low dose LPS > d-Lenolate and OK-432 > control. With yet another parameter of immune response, d-Lenolate was a highly effective stimulant.

d-Lenolate enhanced the restoration of white blood cells to the normal level and protected against infection with bacteria and yeast. Protection in early stage infection has been primarily attributed to PMN production and phagocytic activity. A reduction in PMN and risk from infection has been shown in humans (Peters et al., 1972; Catane et al., 1977). In this study, d-Lenolate restored PMN levels in immunocompromised mice and also enhanced clearance of E. coli from blood., both restorative effects after infection with bacteria. These two factors make d-Lenolate a powerful protectant from infection as well as an enhancer to recovery post infection. Compounds tested in comparison to d-Lenolate, such as OK-432, LPS, LiCO3 are known to result in immune stimulation. Since d-Lenolate out performed these treatments in nearly all cases, it appears to function through immune system stimulation. LiCO3 has also been shown to alleviate reduction in white blood cells in chemotherapy patients (Stein et al., 1977; Bandini et al., 1980; Lyman et al., 1980). D-Lenolate may potentially be highly effective in stimulating white blood cells production and activity in chemotherapy patients as well.

X-irradiation, prednisolone, and CY all depress the immune system by rapid reduction in white blood cell counts. D-Lenolate was able to restore immune function after treatment with all of these agents.

Production of IL-1 activates both specific cellular immunity (T cells) and antibody production (B cells). D-Lenolate effectively stimulated production of IL-1 in macrophages within two hours of exposure. Thus, not only does d-Lenolate stimulate non-specific phagocytosis by PMN and macrophages, but it also stimulates the specific immune response to particular bacteria, fungi and, presumably, viruses.

References:

Peters WP, Holland JF, Senn H, et al. 1972. Corticosteroid administration and localized leukocyte mobilization in man. New England Journal of Medicine. 282:342-345.

Catane R, Kaufman J, Mittelman A, Murphy GP. 1977. Attenuation of immunosuppression with lithium. New England Journal of Medicine. 297:452-453.

Stein RS, Breaman RN, Ali MY et al. 1977. Lithium carbonate attenuation of chemotherapy induced neutropenia. New England Journal of Medicine. 297:430-431.

Bandini G, Ricci P, Visani G, Tura S. 1980. Lithium carbonate hematology. Lancet II. 926.

Lyman GH, Charles MD, Williams MD, Preston D. 1980. The use of lithium carbonate to reduce infection and leucopenia during systemic chemotherapy. New England Journal of Medicine. 302:257-259.

See also: Review of Enhanced Resistance against Influenza Virus by Treatment with Dietary Supplement d-Lenolate in Neutropenic Mice Induced by Cyclophosphamide. 2001. Sumiaki Tsuru, Machio Ibusuki, Sakae Ohtake, Yoshimi Umezawa, and Masatoshi Kaneko. Journal of Orthomolecular Medicine, 16:102-114.

 

© Copyright Envision Corporation. 2002. All rights reserved. Protected by the copyright laws of the United States & Canada and by international treaties. IT IS ILLEGAL AND STRICTLY PROHIBITED TO DISTRIBUTE, PUBLISH, OFFER FOR SALE, LICENSE OR SUBLICENSE, GIVE OR DISCLOSE TO ANY OTHER PARTY, THIS PRODUCT IN HARD COPY OR DIGITAL FORM. ALL OFFENDERS WILL BE SUED IN A COURT OF LAW.

IN-VITRO ANTIBACTERIAL EFFECT OF D-LENOLATE AGAINST B. ANTHRACIS

INTRODUCTION AND DESIGN The purpose of this study was to test the ant-B. anthracis activities of olive leaf extract, contained in the d-lenolate product, against B. anthracis spores and vegetative cells, including inhibition of spore germination in an in vitro system. Extraction of d-lenolate was vortexed (vigorous shaking) for 30 seconds and then rocked in solution for 1 hour. Three solutions were made and are as follows; pure water 1:1, methanol/acetone 1:1, methanol/water, and direct OLE powder and placed onto an agar. Extracted d-Lenolate mixture was added to paper disks and dried. These were applied to the surface of Petri dishes containing Luria Agar (SA) and covered with B. anthracis to observe inhibition zones. RESULTS AND DISCUSSION The presence of olive leaf extract at the concentrations of 2.5 mg/ml or higher completely inhibited spore growth in LB medium. The East Park Research d-Lenolate powder placed directly onto the agar surface showed somewhat more inhibition - 37 mm inhibition zone with 20 mm powder diameter. The olive leaf extract strongly inhibits the B. anthracis spore growth in the in vitro LB medium culture. However the activity of olive leaf extract is lower than a mixture of penicillin and streptomycin. This is a good result taking into account that penicillin is one of the most potent antibiotics against anthrax and that the antibacterial compound in olive leaf extract represents only a fraction of its total weight. Another consideration is that olive leaf extract can be administered at higher dose to compensate for its lower activity, or it could be taken prophylactic ally. Olive leaf extract effect seems to be not bactericidal but bacteriostatic. In general, this effect for a natural compound like olive leaf extract has been expected. It should not be considered as a disadvantage, as many antibacterial drugs are bacteriostatic. In the simplest case this means that olive leaf extract may need to be taken for a prolonged time or in combination with other drugs, or as a prophylaxis. The exact medical recommendation may be developed later after we learn more about the olive leaf extract

© Copyright Envision Corporation. 2002. All rights reserved. Protected by the copyright laws of the United States & Canada and by international treaties. IT IS ILLEGAL AND STRICTLY PROHIBITED TO DISTRIBUTE, PUBLISH, OFFER FOR SALE, LICENSE OR SUBLICENSE, GIVE OR DISCLOSE TO ANY OTHER PARTY, THIS PRODUCT IN HARD COPY OR DIGITAL FORM. ALL OFFENDERS WILL BE SUED IN A COURT OF LAW.

ANTI-B ANTHRACIS ACTIVITIES OF OLIVELEAF EXTRACT: IN VITRO STUDY

INTRODUCTION AND DESIGN Olive leaf extract has previously demonstrated antimicrobial potential against a number of organisms. Of particular interest is its established antibacterial activity against Bacillus cereus. Thus, the goal of this study was to determine the potential antibacterial effect of olive leaf extract via D-lenolate against B. anthracis. The d-Lenolate solution was dissolved in dimethyl Sulfoxide (DMSO) at 100mg/ml in water and was added to Luria Broth (LB) to observe growth in liquid culture using a spectrophotometer to measure cloudiness (turbidity) at a wavelength of 600 nanometers. RESULTS AND DISCUSSION

Uninhibited spore growth without d-Lenolate, delayed growth for 6 hrs with 1 mg/ml d-

Lenolate, total inhibition with 5 mg/ml to 20 mg/ml d-Lenolate. At 24 hrs the 1 or 1.25 mg/ml growth level equaled the control without d-Lenolate but the 2.5-10 mg/ml cultures continued to show little or no growth. Effect on vegetative cells showed the same results as with spores: 1 mg/ml d-Lenolate retarded growth for 6 hrs with growth reaching the same level as the control (no d-Lenolate) within 24 hrs. 5-20 mg/ml totally prevented growth for at least 24 hrs. Cells having grown in LB with d-Lenolate concentrations that totally inhibited growth were washed with fresh medium without d-Lenolate – cells grew fine on LA plates. Therefore d-Lenolate is bacteriostatic or, inhibits cell growth as long as it is present, and not bactericidal. Fetal bovine serum (FBS) at 25% in growth medium (LB) reduced the effectiveness of d-Lenolate so that it required 5 mg/ml d-Lenolate to inhibit growth at 6 hrs. Whereas no growth was seen with d-Lenolate in LB at 2.5 and 5.0 mg/ml at 24 hrs. The disk diffusion study demonstrated that antibiotic activity against B. anthracis is associated with extracts from D-lenolate. When compared to clearance zones produced by pen-strep disks, d-lenolate inhibited growth in a much smaller are containing a 100-fold dilution of pen-strep.

Evaluation of the Efficacy of d-lenolate in the control of symptoms of Candida Hypersensitivity Syndrome

Bernard J. Mizock, MD, MS, FACS, 1436 West Wrightwood Avenue,Chicago, IL 60614

Purpose To evaluate the efficacy of East Park d-Lenolate in the control of symptoms of Candida Hypersensitivity Syndrome. Method Candidates for the study were obtained through advertising. Subjects identified themselves as having been treated for several years for yeast infections without success. A double-blind, randomized placebo control study was conducted in 30 subjects for 60 days. Basal metabolic panels, complete blood counts, and Somatomedin-C levels of each subject were recorded at the beginning and completion of the study. A yeast infection questionnaire was filled out by each participant at the beginning and conclusion of the study. Subjects took 6 capsules per day of either the d-Lenolate or the placebo. There were no modifications to the diet or exercise programs of the participants during the study period, and no additional nutritional supplements were prescribed. Our study was conducted from January through June of 1999. Results Virtually all of the subjects had normal blood count and basal metabolic panels. The mean score at baseline was 250. Within 60 days of taking the d-Lenolate, symptoms were reduced by greater than 50%. The reduction of symptoms in the placebo group was significantly less than in the group taking the d-Lenolate. Conclusion East Park d-Lenolate will reduce the symptoms of chronic yeast infections by greater than 50% within 60 days of its use with no apparent side effects. Continued use may be warranted to enhance an ongoing treatment protocol.

Bernard Mizock, MS, MD, FACS

REPRODUCTION- ORIGINAL ON FILE COPYRIGHT 1999 EAST PARK RESEARCH, INC.

Effects of d-Lenolate in modifying symptoms of Arthritis

Purpose To study the efficacy of East Park d-Lenolate in modifying symptoms of arthritis. Method Thirty subjects were enrolled into the study who had a history of arthritis. They were asked to take two capsules of d-Lenolate three times daily for sixty days. Arthritis symptoms were assessed using the Western Ontario and McMaster Universities (WOMAC) arthritis index. Scores were obtained at baseline, 20, 40, and 60 days. The study was carried out by mail, from September 2000 to January 2001. No changes were made in current diet or medications. Results Time Course (Days) WOMAC Composite Score % Improvement Baseline 50 20 40 20 40 32 36 60 32 36 Conclusion East Park d-Lenolate improves the symptoms of arthritis. The effects are first noted within 20 days, and reach a maximum effect of 32% improvement of 40 days.

BERNARD J. MIZOCK, MD, FACS 1100 PACIFIC MARINA #712 ALAMEDA, CA 94501

 

Professional Testimonials 

and Evaluation Letters 

Dear Mr. Melcher: In response to your inquiry, please be informed of the following facts: During mid-1996, my interest in olive leaf extract was heightened after talking to Gordon G. Melcher, President of East Park Research of Henderson, Nevada. Mr. Melcher’s description to me of people eliminating the effects of chronic fatigue syndrome, curing psoriasis, and experiencing immense increases in energy merely by taking the olive leaf extract seemed unbelievable. Mr. Melcher insisted that I come to Las Vegas to view the evidence and clear the validity of his claims, which I did. Upon my arrival, I telephoned a dozen or more doctors as well as many people taking the olive leaf extract as a food supplement. After receiving an amazing response from these people, I knew I was on the threshold of a best-selling book. Mr. Melcher opened his research files to me and continued to feed me testimonials he received from doctors and patients from around the world. I did write the book and did dedicate it to Les Nachman, Mr. Melcher’s partner. Mr. Nachman is the individual responsible for most of the preliminary research on olive leaf extract that I reviewed. I also had mentioned East Park Research many times in my manuscript. Mr. Melcher submitted this manuscript to the company’s attorney prior to my sending it for publication. His attorney instructed Mr. Melcher to remove the East Park Research name from the book as it would be in violation of U.S. Federal Trade Regulations; otherwise, I would have identified East Park Research as the inspiration for my book. Regardless of what others may claim, my book was written around East Park Research’s Olive Leaf Extract. Furthermore, it is the olive leaf extract of my choice inasmuch as my wife and I take it every day. This letter is being sent to you, Gordon Melcher, with the understanding that it will not be disseminated in conjunction with the sale of any product. I require this promise from you in order to avoid my letter’s becoming labeling for that product, which might be in violation of the U.S. Food and Drug Administration’s rulings, something which I do not want to happen. Sincerely,

Dr. Morton Walker, Editorial Director FREELANCE COMMUNICATIONS

REPRODUCTION – ORIGINAL ON FILE COPYRIGHT EAST PARK RESEARCH, INC 2000

May 13, 1998 Lorraine Rosenthal, Director Cancer Control Society 2043 N Berendo Street Los Angeles, CA 90027 Dear Lorraine: I returned this week from visiting Budapest, Hungary, where I interviewed three hospital- based physicians on a dramatic medical breakthrough. With your permission, I will introduce this breakthrough for the first time into the United States at the 1998 Labor Day weekend conference of the Cancer Control Society. As we discussed at the Ft. Lauderdale ACAM convention, my exposure in Budapest has been to a fungus that causes hormonal cancer in women. I interviewed oncological gynecologist Eric Bottyan, M.D, who successfully reduces Pap smear readings for women at cancer risk from Phase 5 to Phase 2. Dr. Bottyan has eliminated cervical, uterine, ovarian and breast cancers not by treating the cancer, but by killing the patient’s fungus, Candida Ghia Mundie, which promotes flourishment of the human papilloma virus (HPV) and the development of hormonal cancers. Shown by the Hungarian family practice specialist, Dr. Robert Lyons, this same fungus promotes the breakout of psoriasis. But psoriatic lesions disappear permanently within two months when the patient’s fungal infection is removed using olive leaf extract capsules and/or gel. Thus, audience education about psoriasis healing will also be part of my Cancer Control Society presentation. I will need a projector for showing slides of psoriatic lesions before and after fungal treatment. This, too, will be the first time ever that Candida Ghia Mundie has been cited as the source of psoriasis. I request that you allow me the lecture period just preceding lunch on Saturday, Sept. 5, 1998. Please advise that this arrangement conforms to your needs. You’ll likely want to sell my mass-market paperback, Olive Leaf Extract, retailing at $5.99. Thank you. Yours Fondly, Dr Morton Walker, Editorial Director FREELANCE COMMUNICATIONS

REPRODUCTION- ORIGINAL ON FILE COPYRIGHT 1999 EAST PARK RESEARCH

Bernard J. Mizock, MD, MS, FACS

1436 West Wrightwood Avenue Chicago, IL 60614

EAST PARK OLIVE LEAF EXTRACT STUDY RESULTS

“d-LENOLATE”

Purpose To evaluate the efficacy of East Park d-LENOLATE in the control of symptoms of Candida Hypersensitivity Syndrome.

Method Candidates for the study were obtained through advertising. Subjects identified themselves as having been treated for several years for yeast infections without success. A double-blind, randomized placebo control study was conducted in 30 subjects for 60 days. Basal metabolic panels, complete blood counts, and Somatomedin-C levels of each subject were recorded at the beginning and completion of the study. A yeast infection questionnaire was filled out by each participant at the beginning and conclusion of the study. Subjects took 6 capsules per day of either the d-LENOLATE or the placebo. There were no modifications to the diet or exercise programs of the participants during the study period, and no additional nutritional supplements were prescribed. Our study was conducted from January through June of 1999.

Results Virtually all of the subjects had normal blood count and basal metabolic panels. The mean score at baseline was 250. Within 60 days of taking the d-LENOLATE, symptoms were reduced by greater than 50%. The reduction of symptoms in the placebo group was significantly less than in the group taking the d-LENOLATE. Conclusion East Park d-LENOLATE will reduce the symptoms of chronic yeast infections by greater than 50% within 60 days of its use with no apparent side effects. Continued use may be warranted to enhance an ongoing treatment protocol.

Bernard Mizock, MS, MD, FACS

Practitioner Report: Michael Coyle Dear Mr. Melcher: This letter is to inform you of some research findings. Following are some applications that we have determined to be efficacious for the d-Lenolate, Olive Leaf Extract.

We have experienced a complete reversal of Hepatitis C based on testing for viral titers and hepatic enzyme levels. Dosage was one capsule per hour at approximately 15 hours per day. We also asked the subject to administer one capsule anytime they may awaken at night.

Complete reversal of HIV viral titers. We will be reporting more later regarding the ongoing progress of this subject.

Immunity from amoebic dysentery while all others associated with the subject were required to use repeated doses of antibiotics.

Reversal of severe poison oak symptoms with topical application of the Topical Gel. This was only effective with dietary changes that balanced the subjects’ pH. Without pH balancing, there was only palliative relief, with pH balancing, there was reversal.

Dissipation of warts. Reversal of 50% of tumors in a skin cancer study. The subject is erratic in

application and this will be remedied. Results report will follow. Reversal of flu symptoms. This was most effective with high dosage applications

of lactobacillus-bifidus cultures and pH balancing. Reversal of Candidiasis symptoms where dietary restraint of sugar and alcohol

was observed. The main consideration here was to administer at a rate that doesn’t create so much die-off that the person starts to binge on sugars. The fungi put out chemicals that mimic hormones and trigger the brain to crave dietary glucose input.

We are working with a Hepatitis C/Herpes Simplex II subject with the intention of completely reversing viral load. So far, although the viral load is still considerable and the hepatic enzymes disregulated, blood panels indicate that the basic immunity is very strong since the commencement of the study. This is great improvement and there has been abatement of symptoms. We will keep you informed.

REPRODUCTION – ORIGINAL ON FILE COPYRIGHT EAST PARK RESEARCH, INC 2000

Please note that with some of these subjects, they were hesitant to participate in the study because they had used other versions of Olive Leaf Extract to no avail. We had to educate them as to the difference in the functional efficacy of the d-Lenolate formula before we could proceed. Our observation is that the d-Lenolate is invaluable for those who are immuno-compromised. For instance, it reversed strong tendency towards cold and flu in some who had mercury amalgrams suppressing their immunity and contributing to systemic fungal infections, thereby leaving them vulnerable through lack of sufficient immune response. The d-Lenolate very much changed the quality of their life. It should be noted that the numerous practitioners (500 or so) that we have informed of the d-Lenolate, many of whom we have trained in the application of same in conjunction with blood studies have been very happy with their results. Many now use it as a staple. I would say that it is utilized by ourselves and many of our students as the #1 resort in the case of bacterial, viral and fungal profiles. We also find that it deals with many parasitic infections. Thank you for your cooperation and support in our studies, especially for developing such a fine product and supplying it for our research purposes. Please look forward to further reports as matters develop. Best wishes in good health, Michael Coyle, NuLife Sciences

REPRODUCTION – ORIGINAL ON FILE COPYRIGHT EAST PARK RESEARCH, INC 2000

d-LENOLATE CASE Study #1

BAZUCAR AND d-LENOLATE IN OVERWHELMING INFECTION IN DIABETIC PATIENT

A 59 year old black male, auto salesman was admitted to Doctors Hospital January, 2003. He was a known diabetic and hypertensive. He presented to hospital with intense abdominal pain and vomiting and was toxic from overwhelming infection in the abdomen and blood. The sigmoid colon had ruptured at the site of a diverticulum, the blood pressure was low and he was disoriented and confused from the infection. After antibiotics were administered intravenously and fluids replenished for several hours to restore blood pressure to normal he was taken to the operating room for emergency surgery to remove the ruptured portion of the colon and a temporary colostomy was performed. Despite the most potent antibiotics, he developed an abdominal abscess requiring a second abdominal surgery for drainage. The infection in the blood resulted in pneumonia in all lobes of the lung. His incision also became infected and the abdomen eventually ruptured through the incision requiring yet another surgery for re-closure. The blood sugar which was precarious required filtrated doses of insulin to attempt control. Candida was seen in lung washing specimens and staphylococcus was cultured from the blood. After 27 days of optimal intravenous potent antibiotic therapy with no resolution and fluctuating blood sugar levels, despite varying intravenous insulin doses, d-Lenolate (two capsules every four (4) hours) and Bazucar (one capsule every eight (8) hours) were commenced. Significant subjective and physiological improvement was noted after only twenty-four (24) hours of therapy. All conventional antibiotics were discontinued the following day. He was discharged from the Intensive Car Unit, to the Step-Down Unit three (3) days later and left the hospital eleven (11) days later.

d-LENOLATE CASE STUDY #2

EFFICACY OF d-LENOLATE IN OVERWHELMING INFECTION

A 63 year old truck driver/mechanic, known heavy smoker and alcoholic was involved in a road

traffic accident February 2003. He sustained a skull fracture, fracture of the right femur and crush injury to the left ankle.

His ankle injury became infected and non-viable necessitating amputation three days later. The infection had become blood-borne and was complicated by pneumonia in both lungs. Despite optimal combinations of potent antibiotics the pneumonia was not responding.

After three (3) weeks without resolution, two d-Lenolate every four (4) hours was prescribed. Within three (3) days his temperature was normal and a day later he did not require oxygen any longer.

Barrett A. McCartney, M.D. Director of Critical Care Services

Doctors Hospital Nassau, Bahamas

Dear Mr. Melcher, By way of introduction, let me begin by telling you that I have been practicing medicine for more then 20 years, and have a private clinic that has been established for 10 years. My focus is to combine the best of the traditional school of medicine with the best of alternative, complimentary medicine on a holistic basis. Having encountered the problems in the traditional school of medicine, that usually we are not about to ‘cure’ anything, but rather as a doctor, you are drilled to prescribe pharmaceuticals which at best can only alleviate or reduce the symptoms. As a natural consequence of this, my motivation for a combined effort grew stronger and has since been the basis for my practice as a doctor and specialist of internal medicines. I have seen in my practice within the last 5 years more and more patients with the digestive system, stomach and intestinal problem developing food allergies and food intolerances. I have seen first hand and documented the relations to these problems as overgrowth of fungus, often of the Candida species. As a consequence, I have been using different pharmaceutical products and learned that they are not able to cure the patient or give permanent relief. I was fortunate to read some scientific reports and clinical studies about East Park Research’s d-Lenolate® and as a result, I have been testing this new preparation. I can say with confidence that this has proven to be a superior treatment modality over and above any pharmaceutical product available. In addition, we have the great advantage of using a potent fungi killer that has no side effects, whatsoever. This makes this new treatment extremely fascinating. Now you can combine treatment potency and safety. This preparation has broad-spectrum abilities. It kills off not only candida albicans, but also several other pathogens, bacteria, viruses, and parasites. I have had the opportunity to test East Park Research’s d-Lenolate® on a large variety of different medical problems and diseases. One of the most outstanding patient cases is that of a 5-year-old girl with psoriasis. She had never succeeded with any of the many pharmaceutical treatments tried. However, we experienced dramatic clinical effects within just two weeks with the combination of applying East Park Research’s topical gel and also taking East Park Research’s d-Lenolate® orally. Thrilled about the initial success, treatment continued and for the first time ever, she is now rid of any symptoms of this otherwise chronic disease. Another astounding success was the treatment of an 18-year-old girl with a chronic debilitating inflammatory bowel disease called Crohn’s Disease. When I first saw her, she was in extremely poor condition. She weighed about 80 pounds and was extremely weak. She had been admitted to the hospital for extended periods of time and they wanted to perform an intestinal amputation of a large part of the small intestine, which, of course,

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would have serious consequences for the rest of her life. Having now the experience of the powerful killing effects on pathogenic microorganisms, I proceeded with treatment of East Park Research’s d-Lenolate® and it was a matter of a few days before she started feeling better and was able to eat and drink properly and started to regain her strength and the weight she had lost. The specialist assigned to her in the hospital did not understand what was happening, but was of the opinion that it was a miracle. I later was able to read the results of my blood screening prior to my treatment of her with East Park d-Lenolate®, whereby the functional lab testing documents extreme high Candida Albicans IgG-titers and later after treatment had begun, when the ‘miracle’ occurred, showed that these high titer counts were diminished telling us that the prime cause of her chronic intestinal inflammation was a result of extreme overgrowth of these candida pathogens. The East Park Research d-Lenolate® showed to be highly effective in killing off this fungus and eventually saving this young girl from an intestinal amputation. (She has continued to use East Park Research’s d-Lenolate® as a maintenance using one to three capsules daily. This is a cheaper insurance to good health and the cure has shown to be effective, in my opinion, on a permanent basis.) With the modern lifestyle and common Western diet, it is necessary to realize now that more and more people are having or will have problems with overgrowth of pathogens and candida albicans will be a prominent marker for this problem. As it is, in my opinion, the potential course of many of the health problems in the Western world, I am quite confident that this treatment modality (East Park Research’s d-Lenolate®) will become a major contributor to the health and high quality of life for mankind into the new millennium. It is a shifting paradigm. For the last 3 to 4 generations, we have focused in the potential possibilities of pharmaceuticals and relying on the development of synthetic drugs. We are now realizing that this is false; we cannot base our well being on synthetic drugs. We have taken a step backwards (and now need to go back to) using natural remedies. Fortunately, we now have the technologies whereby we can extract the active ingredients, test them out, and provide new, extremely potent natural medicines. This new type of herbal medicines can be more potent than pharmaceuticals and in addition, bear the great advantage of having no damaging side effects, such as East Park Research d-Lenolate® illustrating a true current example.

Allan Fjelstrup, M.D. Specialist Internal Medicine Bayline Clinics NO-4002 Stavanger, Norway

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Practitioner Report: Grovit

Les Nachman 2709 Horseshoe Drive Las Vegas, Nevada 89120

December 7, 1999

Dear Mr. Nachman:

Several months ago, I spoke with you with particular regard to the potential benefit of d-Lenolate controlling bacterial overgrowth in the short bowel syndrome. I have had much success in the nutritional management of the short bowel syndrome, but the control of bacterial overgrowth has been recalcitrant.

The chronic use of broad spectrum antibiotics for several days each month has resulted in resistant organisms and unfortunately tendinopathy associated with one of the quinolones; hence, my concern to pursue another avenue for controlling bacteria overgrowth.

I am quite happy to report to report that one capsule of d-Lenolate in the morning and two capsules in the evening mixed into the food bolus has resulted in considerable benefits by significantly extending the symptom-free period. I am at a loss to explain the very perceptible response to a seemingly low dose of d-Lenolate.

I am most grateful that you have made d-Lenolate available for use and will surely keep you informed as to the outcome of my clinical experience.

Sincerely Yours, Melvyn Grovit, D.P.M, M.S., C.N.S Podiatric Medicine Consulting Nutritionist Certified Diabetes Educator

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Adrian Hohenwarter 326 W Chocolate Ave Hershey, PA 17033 Dear Mr. Melcher, This letter is to document my recent success with your product ‘Olive Leaf Gel’. The case involved a 59-year-old white male who complained of a skin lesion under his right eye immediately adjacent to the lower eyelid. It was first noticed over two months prior. An exam revealed a classic basal cell carcinoma characterized by a firm, approximately 7-8 mm papule with a rolled translucent border with obvious telangectasia. Biopsy and/or referral were deferred so that a trial of topical olive leaf gel could be implemented. The patient was next seen two weeks later. At this time, it was evident that the lesion was markedly improved, no longer demonstrating any of the diagnostic stigmata of a basal cell. The patient admitted to only applying the gel ten times in the two weeks. Now, at four weeks since beginning treatment, there is only a barely noticeable soft papule at the site. The patient continues to apply the Olive Leaf Gel at similar frequency. This dramatic therapeutic response exemplifies the case histories that you have discussed with me from your other clients. Apparently, the gel increases immunity at the local site of application and/or has a anti-cancer property. I am excited to witness future clinical use of this product and I encourage you to promote its use. Thank you. Sincerely,

Adrian Hohenwarter, M.D.

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Practitioner Report: Dr. Hugh Smith June 12, 2000 Mr. Nachman: It is rare in the extreme that I write letters praising a product, but d-Lenolate is one unique supplement that I find I must do so. In our clinic, we have observed (using DIC and phase contrast microscopy) on an ever-increasing level, the markers for mycroplasmas in the living blood of hundreds of clients. As you know, mycroplasmas are implicated in a myriad of chronic illnesses from lupus to chronic fatigue and arthritis. One of the problems with mycroplasmas is that they invade the DNA strand of the T cells, making eradication by the immune system nearly impossible. Another problem is the ability of mycroplasmas to be transmitted through casual contact. While many products enhance the immune system, that is simply not effective against stealth pathogens and cell wall deficient forms such as mycroplasmas because they are effectively ‘invisible’ to T cells. Thus, one must find something that will kill them rather then simply enhance immunity, and one must find something that will do so at the DNA level. ‘Standard’ treatment by most physicians is a 90-day course of either tetracycline or doxycycline. While these treatments may indeed work, at least symptomatically, the side effects and long-term problems they create are not encouraging. Most antibiotics, like penicillin, work by attacking the protein layer of pathogens. Unfortunately, mycroplasmas have no such layer. Enter d-Lenolate. We have been conducting our own research regarding this supplement. While not a ‘standard’ double blind study and therefore considered anecdotal, the proof is in the health of the individuals to whom we have recommended d-Lenolate. I have included a copy of a client with mycroplasmas markers and a photograph 90 days later- please note the difference in the blood cells. No markers! Our recommendation is (depending upon age, weight and severity of symptoms) 2 capsules three times daily for the first 30 days, 1 capsule three times daily for 60 days

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thereafter. In every case, where the regimen is faithfully followed, symptoms and markers have either disappeared or been dramatically reduced. It is important to note there are many products claiming to be olive leaf extract, but in our office, we insist that our clients take only d-Lenolate from East Park Research. On the rare occasions where a client has found a ‘cheaper substitute’ the results have been discouraging… none whatsoever! One’s health is the issue, not bargain basement supplements! We have recommended this product to many of our colleagues (some of whom are MD’s) who suffer from such chronic conditions as Epstein Barr with similar results. While the pursuit of Ehrlich’s ‘magic bullet’ may be futile, d-Lenolate come as close to it as any we have recommended (and believe me- we’ve tried it all). Permit me, if you will, to wax philosophic for a moment. As you mention in your literature, Revelation 2:7 mentions the ‘tree of life’ possibly being the olive tree. If one examines the Tree of Life from the Kaballah, one must note that it is a representation of the double helix of DNA. I find that extremely interesting since it is our contention that d-Lenolate is effective in mercilessly eradicating stealth pathogens at the DNA level, thus confirming (in our minds) the Biblical promise of ‘healing for the nations’, Revelations 22:2. You have a product that simply must be integrated into western medicine if we are to survive this epidemic of mycroplasmas. Thank you for your patience with this long ‘note’ and feel free to use my name/and or clinic to help pass the word. Sincerely,

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REPORT FROM HUNGARY (East Park d-LENOLATE® is sold under the brand name “EDEN” in Hungary)

Eden’s new aspects in human therapy Eric Bottyan M.D. Gynecologist and Clinical Oncologist, Budapest, Hungary. Keywords: Eden, vulvovaginitis, immunostimulation, Author, at first in Hungary, applied Eden in his gynecologic private practice and observed unexpectedly high and good response as regards recurrent and persistent vulvovaginitis with different portion alterations. Material, method and results Since 01/01/1996. thanks to Dr. Robert Lyons — had the chance to treat 216 patients with the following diseases: -mainly (at first) persistent or recurrent vulvovaginitis patients, whom I sent to the Hungarian-STD (sexually transmitted disease) Center, and after their “failure” I introduced Eden: food supplement as therapy!!! 142 of 163 -87%!!! reacted positively — My Goodness!!! (Naturally they needed at least 2 or 3 of the 60 capsule bottles, and there is no official support on this preparation!) -Herpes labialis. Herpes genitalis: 16 patients of 16 - 100%!!! -Portio erosion with Pap 2 and inflammation: 121 of 128 have shown healing process - 95%! ! ! -hair loose/looking for focus: 11 cases/IUD, oophoritis, cholecystitis etc. It merits extra attention — another study! -Clearing effect: 22 cases: 16 patients with different Cancers and depression 5 patients with postmenopausal depression

1 patient/writer with schizophrenia Absolutely interesting!!!!

-Helicobacter pylod: 1 case after 2 x 60 capsules laboratorial negative-HP V-cervical cancer: I have the opportunity to identify the Human Papillom Virus tribes with high malignant potential, and I have a patient of 25 years, no pregnancies, with colposcopic leukoplaky, Pap 3 with a type — I have made fro.abrasioconisation and the histology was: CIN II. “Naturally” Eden was introduced, and after the first 3 months HPV is not detectable. What can come??-dermatomycosis: 2 patients, still, who are taking the capsules, showing a great remission after being treated in several clinics. Here are “some” aspects of Eden, no trial about hypertension, cerebralvascular, or even vascular effects, or cod etc. Summary As the Vice Medical Director of Budapest’s third greatest hospital, I was deeply impressed by Dr. Robert Lyons’ performance with Eden. My personal experiences and my patients have convinced me that you/we have found something to follow up on.

Practitioner Report: Life Sources Inc.

East Park Research 2709 Horseshoe Dr. Las Vegas, NV 89120 Attention: Gordon Melcher/President Dear Gordon: As you know, Life Sources has developed an unique enzyme/energy formula based on our years of Vital Hematology (the study of living blood). ReGenesis was designed to address a number of conditions that MS, CFS, and FMS sufferers have in common: stealth pathogens and the damage done to the human body by these cell wall deficient forms. Standard treatment for MS usually falls into two categories: the control of the symptoms, or the slowing of progression or exacerbations. Unfortunately, none of these allopathic methodologies address the causative issues of MS. Life Sources has developed a protocol using our exclusive product, ReGenesis, and East Park Research’s d-Lenolate that has proved to be exceptionally effective. Together, these two products address the basic issues of controlling the symptoms and reversing MS. In a nutshell, East Park’s d-Lenolate addresses the issue(s) of stealth pathogens (and several other causal factors such as free radical damage) in MS. ReGenesis is a synergetic enzyme based supplement whose individual components have proved to: detoxify the system of harmful metals and contaminants, reduce inflammation, reduce spasms, enhance cardiovascular health, increase energy, increase nerve function, synthesize uric acid and cholesterol, relieve pain and increase oxygenation of cells. Together, East Park Research and Life Sources have products that address a myriad of chronic conditions with vague and inconsistent symptoms that have eluded standard diagnosis and treatment. While we cannot claim that either product cures anything, we have hundreds of anecdotal reports that are extremely encouraging. Our many thanks to East Park Research who has put aside any personal agenda for the sake of helping those in need of relief from this and other devastating diseases. P. Andrea McCreery, Ph.D/President-Life Sources Inc.

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BERNARD J. MIZOCK, MD, FACS

1100 PACIFIC MARINA #712 ALAMEDA, CA 94501

EAST PARK OLIVE LEAF EXTRACT STUDY RESULTS

“d-Lenolate” Purpose To study the efficacy of East Park d-Lenolate in modifying symptoms of arthritis. Method Thirty subjects were enrolled into the study who had a history of arthritis. They were asked to take two capsules of d-Lenolate three times daily for sixty days. Arthritis symptoms were assessed using the Western Ontario and McMaster Universities (WOMAC) arthritis index. Scores were obtained at baseline, 20, 40, and 60 days. The study was carried out by mail, from September 2000 to January 2001. No changes were made in current diet or medications. Results Time Course (Days) WOMAC Composite Score % Improvement Baseline 50 20 40 20 40 32 36 60 32 36 Conclusion East Park d-Lenolate improves the symptoms of arthritis. The effects are first noted within 20 days, and reach a maximum effect of 32% improvement of 40 days.

Bernard Mizock, MS, MD, FACS

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