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DV Biologics Reports: Earth Science Tech Hemp CBD Oil Deters the Formation of Reactive Oxygen Species (ROS) on Human Brain Cells

DV Biologics, a Leading Biological company, Reports Earth Science Tech (ETST) High Grade Hemp CBD (Cannabidiol) Oil Deters the Formation of Reactive Oxygen Species (ROS) on Human Brain Cells: May serve as a Neuroprotectant

BOCA RATON, FL / ACCESSWIRE / August 18, 2015 / Earth Science Tech, Inc. ("ETST" or "the Company"), a unique biotechnology company focused on nutraceuticals, bioceuticals and dietary supplements, announced today that DV Biologics, a leading biological company, has confirmed ETST High Grade Hemp CBD (Cannabidiol) Oil deterred the formation of reactive oxygen species and cell death in human brain cells in vitro. The goal of these studies is to test if ETST High Grade Hemp CBD (Cannabidiol) Oil, as a dietary supplement, serves as a neuroprotectant which may help with neurodegenerative disorders such as Alzheimer's and Parkinson's. The scientific study which concludes the in vitro experiments done independently on human brain cells by DV Biologics, a company that provides biological tools and research for the advancement of medicine, illustrates that using ETST High Grade Hemp CBD (Cannabidiol) Oil can rescue human brain cells from the detrimental effects of reactive oxygen species and free radical formation which cause damage to brain cells (see study below). Recently, independent studies demonstrated that ETST High Grade Hemp CBD (Cannabidiol) Oil can reduce lipid peroxidation (oxidative stress) on human brain cells following the addition of hydrogen peroxide. This new data supports and extends the previous data that ETST High Grade Hemp CBD (Cannabidiol) Oil may help serve as a neuroprotectant. This study looks at another pathway of free radical formation, oxidative stress and cell death that occurs in the brain during neurodegenerative disorders such as Alzheimers and Parkinsons. This data suggest that ETST High Grade Hemp CBD (Cannabidiol) Oil may have neuroprotective properties.

"Here at ETST we will continue focusing on providing the best All-Natural High Grade Hemp CBD (Cannabidiol) Oil as a dietary supplement. We believe the only way to do this is by studying the effects of ETST High Grade Hemp CBD Oils in various experiments. Science is what will show the world that ETST provides high quality products as well as the best All-Natural High Grade Hemp CBD (Cannabidiol) Oil designed to give consumers maximum overall wellness benefits. We have relatively exciting studies which we will be starting soon in our continual goal of providing the best products for consumers worldwide and great value to our investors," stated Matthew Cohen, CEO of ETST.

According to Dr Rafael Gonzalez, VP of Research & Development at DV Biologics, "The data speaks for itself. This in vitro study is very encouraging as it demonstrates that ETST High Grade Hemp CBD (Cannabidiol) Oil may be beneficial for the brain. The great thing is that the data is supported by 3 different experiments, all showing the same result that it works. With this data if I had a neurodegenerative disorder such as Parkinsons, I would seriously consider taking this as there isnt much available that can deter the progression of the disorder. We hope that ETST will continue to give us the opportunity to work with their High Grade Hemp CBD (Cannabidiol) Oil."

*Complete DV Biologics study shown below*

Does Earth Science Tech High Grade Cannabidiol (CBD) Rich Hemp Oil Have a Neuroprotective Effect? The DV Biologics Report and data below is encouraging and clearly demonstrates that Earth Science Technology (ETST) High Grade HEMP CBD (Cannabidiol) Oil is protective against lipid peroxidation in human brain cells in vitro.

The Brain

The brain contains trillions of cells. From neurons with its long axons (nerve fibers) which can extend greater than 2 feet and serve to communicate to glial cells which support the brain in all its complex task. Two-thirds of the brain is composed of lipids. The vast majority of the different cell types in the brain require lipids for correct transmission of signals, providing energy and structural support to our cell membranes. Cells in the body and particularly in the brain are under constant attack by free radicals and oxidative stress. There are protective mechanisms that help deter this normal damage that occurs; however, with aging the protective mechanism declines substantially and degeneration of the brain occurs.

Lipid Peroxidation

Lipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the production of end products such as malondialdehyde (MDA).

Reactive oxygen species (ROS)

Reactive oxygen species are molecules that contain oxygen that are chemically active (i.e. oxygen ions and peroxides). They are formed as a natural byproduct of metabolism in our bodies. However, during stress and disease or dysfunction, these byproducts are not taken removed correctly and damage occurs in our body. The harmful effect of ROS occurs in cells in the form of DNA damage and oxidation of amino acids in proteins. ROS come in various forms such as superoxides and hydroxyl radicals and it is well documented that ROS play a crucial role in the formation of neurodegenerative disorders, cancer and aging.

Experiment:

In this experiment, we look at three (3) different parameters:

1. Fluorometric intracellular ROS with a Kit that provides a sensitive, one-step fluorometric assay to detect intracellular ROS (especially superoxide and hydroxyl radicals) after induction of peroxidation with hydrogen peroxide (with and without ETST High Grade Hemp CBD (Cannabidiol) Oil).

2. Lipid peroxidation is determined by the reaction of MDA with thiobarbituric acid (TBA) to form a fluorometric complex, proportional to the amount of MDA present, after induction of peroxidation with hydrogen peroxide (with and without ETST High Grade Hemp CBD (Cannabidiol) Oil).

3. Neutral Red Assay to determine viability of cells after induction of peroxidation with hydrogen peroxide (with and without ETST High Grade Hemp CBD (Cannabidiol) Oil).

Experimental procedure ROS:

*Cells were plated in 96 well until it reach 90%. Neural cells were at a density of 2 X 104 cells/cm2. The cells were grown in neural media supplemented with 20 ng/ml of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)
*Media was changed every 2-3 days.
*ETST Hemp CBD/Neural-Growth media was prepared by diluting the ETST Hemp CBD in dimethyl sulfoxide (DMSO). The suspension was filtered with a 0.22 micron filter. The ETST Hemp CBD suspension was then diluted in N-GRO media to contain the following concentrations of Hemp CBD: 5 M, 2.5 M, and 1 M CBD.
*Cells were incubated with ETST Hemp CBD for 48 hours prior to the addition of hydrogen peroxide.
*After 48 hours, ROS was induced by addition of H2O2 at concentration of 25 M to certain wells in quadruplicate. H2O2 was added to the wells of a 96 well plate according to the following design:


*Set up the Master Reaction Mix.
*Add 100 mL/well (96 well plate) Master Reaction Mix into the cell plate.
*Incubate the cells in a 5% CO2, 37 C incubator for one hour.
*Treat cells with 20 mL/well of H2O2 (96 well plate).
*For control wells (untreated cells), PBS was used.
*To induce ROS, incubate the cell plate in a 5% CO2, 37 C incubator for 16 hours.
*Measure the fluorescence intensity at lex = 490/lem = 525 nm for 1 second/well.

Experimental Procedure Lipid Peroxidation:

*Two 24-well plates were coated with Matrigel for 1 hour at 27 Celsius.
*The plates were seeded with human neural cells at a density of 2 X 104 cells/cm2. The cells were grown in neural media supplemented with 20 ng/ml of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).
*Media was changed every 2-3 days.
*ETST Hemp CBD/Neural-Growth media was prepared by diluting the ETST Hemp CBD in dimethyl sulfoxide (DMSO). The suspension was filtered with a 0.22 micron filter. The ETST Hemp CBD suspension was then diluted in N-GRO media to contain the following concentrations of Hemp CBD: 10 M, 5 M, 2.5 M, and 1 M CBD.
*After the cells reached 80-90% confluency, base media was replaced with media containing ETST Hemp CBD or control media; the controls were media only, media with DMSO 1:100, and media with hydrogen peroxide (H2O2) (H2O2 was added during induction phase).
*The cells were incubated under experimental conditions for 48 hours in media containing ETST Hemp CBD.
*After 48 hours, lipid peroxidation was induced by addition of H2O2 at concentration of 100 M to certain wells in duplicates. H2O2 was added according to the following protocol:

Well ID

A

B

C

D

E

F

1

Media only (control)

1 M CBD + H2O2

5 M CBD + H2O2

2

Media +DMSO (control)

Media+2.5M CBD only

Media+10M CBD only

3

Media + H2O2

2.5 M CBD + H2O2

10 M CBD + H2O2

4

Media+1M CBD only

Media+5M CBD only

*The cells were incubated with H2O2 for 120 minutes at 37 Celsius.
*Media was aspirated and the cells were lysed.
*Total protein was precipitated, and reaction to form MDA-TBA adduct was performed. The reactions were placed in a 96-well plate.
*MDA fluorometric detection was done by measuring fluorescence intensity (excitation 532 nm/ emission 553 nm) of samples and standard curve.
*Relative lipid peroxidation was measured by subtracting blank value from all readings.

Experimental Procedure Neutral Red Assay:

*Human prenatal neural cells were seeded in a 24 well flask coated with Matrigel.
*The cells were grown at 37 C, 90 % humidity, and 5.0 % CO2 until they reach 80% confluency.
*ETST Hemp CBD/Neural-Growth (N-GRO) media was prepared by diluting the ETST Hemp CBD in dimethyl sulfoxide (DMSO). The suspension was filtered with a 0.22 micron filter. The ETST Hemp CBD suspension was then diluted in N-GRO media to contain the following concentrations of Hemp CBD: 5 M, 2.5 M, and 1 M CBD.
*Cells were incubated with CBD for 48 hours prior to the addition of H2O2.
*After 48 hours, H2O2 was added at concentration of 25 M to certain wells in Duplicate. H2O2 was added according to the following protocol:

1

2

3

4

5

6

A

Control

1uM CBD + H2O2

5uM CBD + H2O2

B

DMSO control

2.5uM CBD

Blank

C

Media+ H2O2

2.5uM CBD + H2O2

D

1uM CBD

5uM CBD


*The cells were incubated with H2O2 for 16 hours at 37 C.
*After incubation, media was aspirated from all wells and 100uL of NR medium was added to each well.
*Incubate for 1 hour with Neutral red (NR) medium at 37 C.
*NR media was aspirated from all wells and washed each well with 150uL PBS
*100 l of lysis solution (ETOH/acetic acid) solution was added to all wells, including blanks.
*Plate was shaken rapidly on a microtiter plate shaker for 20 45 min to extract NR
*Lysis solution was transferred to a 96-well plate and absorbance was determined at 540nm in quadruplicate.

Results


Figure Legend:

Earth Science Tech (ETST) High Grade CBD (Cannabidiol) Oil rescues human brain cells from reactive oxygen species in vitro. Human brain cells were cultured and treated with ETST High Grade CBD (Cannabidiol) Oil diluted to concentrations indicated on the graph for 2 days (1.0 M CBD = 8 ppm CBD rich hemp Oil; 2.5 M CBD = 20 ppm CBD rich hemp oil; 5.0 M CBD rich hemp oil= 41 ppm Hemp Oil). The cells were treated with 25 M hydrogen peroxide (H2O2) for 16 hours and incubated with reactive oxygen species (ROS) master mix reagents for 1 hour to detect intracellular ROS. The amount of ROS was determined by fluorescence intensity at lex = 490/lem = 525 nm. The experiment is performed in quadruplicates, with error bars representing standard deviation error. Control: DMSO only. Positive control: Media + H2O2 + DMSO.

When brain cells are incubated with H2O2 + DMSO intracellular ROS accumulates and is much higher than normal physiological states as seen in the figure. ETST Hemp CBD on its own maintains intracellular ROS to similar levels as seen in physiological states. When ETST Hemp CBD is used prophylactically at all concentrations used (1, 2.5 and 5 M) there is a decrease in the formation of ROS.


Figure legend:

Earth Science Tech (ETST) High Grade CBD (Cannabidiol) Oil is protective against lipid peroxidation in human brain cells in vitro. Human brain cells were incubated with media (control), hydrogen peroxide (H2O2) to cause oxidative stress, DMSO which is the solvent to dilute ETST Hemp CBD in media and different concentrations of ETST Hemp CBD with and without H2O2. Error bar = Standard deviation. Control Media alone and DMSO alone. Positive control: H2O2.

When brain cells are incubated with H2O2 by itself lipid peroxidation occurs substantially as seen in the graph above. Lipid peroxidation as measured my MDA present is seen to reach greater than 2.5 nM as compared to controls of media and DMSO alone which register 0.5 nM or less. When human brain cells are treated with ETST High Grade CBD (Cannabidiol) in concentrations varying from 1 M to 10 M prior to the addition of H2O2 (prophylactically), human brain cells are protected against the damaging effects of lipid peroxidation.


Figure legend:

Earth Science Tech (ETST) High Grade CBD (Cannabidiol) Oil deters cell death in human brain cells in vitro. Human brain cells were cultured and treated with ETST Hemp CBDemp He diluted to concentrations indicated on the graph for 2 days (1.0 M CBD = 8 ppm CBD rich hemp oil; 2.5 M CBD = 20 ppm CBD rich hemp oil; 5.0 M CBD = 41 ppm CBD rich hemp oil). The cells were challenged with 25 M (0.85 ppm) H2O2 for 15 hours and stained with neutral red, which stains only live cells. The amount of Neutral Red is quantified by absorbance at 540 nm. The experiment is performed in quadruplicates, with error bars representing standard deviations. Control: Media. Positive control: Media + DMSO + H2O2.

When human brain cells are incubated with H2O2 cell death occurs as compared to normal physiological states of growing cells as measured using neutral red assay (see figure). When ETST Hemp CBD is used prophylactically at various concentrations, cell death is deterred even in the presence of H2O2.

DV Biologics Summary

Reactive oxygen species (ROS) and lipid peroxidation is damaging to human brain cells. ETST High Grade CBD (Cannabidiol) Oil in varying concentrations has shown to rescue the damaging effects of ROS and lipid peroxidation, two oxidative stress pathways that play a crucial role in various neurodegenerative disorders, cancer and aging. Moreover, ETST High Grade CBD (Cannabidiol) Oil deters cell death that occurs following the addition of hydrogen peroxide, a known ROS and lipid peroxidation inducer. This suggests a neuroprotective effect in vitro to human brain cells following the use of ETST Hemp CBD. This data merits the use of ETST High Grade CBD (Cannabidiol) Oil in human clinical studies for neurodegenerative disorders such as Alzheimers and Parkinsons.

*This is the end of the DV Biologics Study and Report*

Earth Science Tech (ETST) is focused on the science, research and studies of its High Grade Hemp CBD Oil as a nutraceutical and dietary supplement. In order to assure the public that ETST provides the best High Grade Hemp CBD (Cannabidiol) Oil, we continue to focus on providing the public with sound scientific research and keeping the public informed on the progression of studies being done on our High Grade Hemp CBD products. Science is what will progress the future of ETST High Grade Hemp CBD (Cannabidiol) Oil and its products. Cannabidiol (CBD) is a naturally occurring constituent of industrial hemp oil. It is the most abundant non-psychoactive cannabinoid in hemp/cannabis, and is commonly used as a dietary supplement to support general wellness. ETST all natural full spectrum High Grade Hemp CBD Oil is derived from Grade-A European Industrial Hemp Plants. ETST Hemp CBD oils are constantly analyzed for their purity and purity from potential contaminants. ETST encourage researching cannabidiol (CBD) from reliable informational sources to see what is being investigated and discussed about CBD oil. Numerous properties and benefits of CBD are being investigated at academic research centers in the United States and elsewhere. The FDA has not evaluated the validity or truthfulness of these claims; therefore, we encourage you to review published researches relating to the benefits and properties of Hemp CBD oils and other Hemp CBD products.

ETST invites all interested parties to contact the company at info@earthsciencetech.com in regards to securing its"High Grade Hemp CBD (Cannabidiol) Oil" in bulk for wholesale, resale, private labeling or distribution. CBD (Cannabidiol) is a natural constituent of hemp oil and legal to consume, sell and possess in the U.S.A. and over 40 countries as dietary supplement. From product formulating, mixing, labeling and launching ETST can do it all. ETST is always open to forming new strategic partnerships, joint ventures and/or supplying vendors, resellers and third party distribution companies with its High Grade Hemp CBD (Cannabidiol) Oil/Products at wholesale prices in order to better help supply consumers as well as the health and wellness markets on a large scale. You can view, research and purchase small quantities or bottles of ETST High Grade CBD (Cannabidiol) Rich Hemp Oil at www.earthsciencetech.com/cbd-rich-hemp-oil.

Humans are designed with a built-in Endocannabinoid System (ECS). Research shows that many scientists believe cannabinoids work synergistically to support the whole human endo-cannabinoid system (internal and natural human cannabinoid system). It is your body's way of communicating with itself and properly regulating the way every other body system functions from your brain to your toes. The Endocannabinoid System (ECS) has shown to control pain sensation, appetite, temperature regulation, stress reactivity, immune function, and sleep as well as other processes. Even more interesting is that muscle and fat tissue also utilize these receptors to control their processes. Consumers should think of the Endocannabinoid System as one of the body's main control and command centers for tweaking your metabolism's ability to react and adapt to the world around it. Like many other nutrients in our diet, Cannabinoids can become exhausted and in need of steady dietary replenishment. Scientific studies and data show that Hemp CBD (Cannabidiol) Oil as well as all micronutrients (phytonutrients) and phyto-cannabinoids found in the oil work in harmony with the human body's own endocannabinoid system making a positive effect for overall wellness. Hemp oil contains CBD -- a non-psychoactive compound of the plant. There are millions of hemp oil consumers around the world and this number is rapidly rising with an increasing number of reports and studies showing a variety of benefits of hemp oil.

ETST High Grade Hemp CBD (Cannabidiol) Oil: ETST High Grade Hemp CBD (Cannabidiol) Oil is all-natural, organic and formulated using a wide array of cutting-edge technologies. ETST's High Grade Hemp CBD Oil is all-natural and derived completely from the federally legal industrial hemp plant. Industrial Hemp (Hemp) is not marijuana and will not get you 'high' and it does not require a medical license of any kind to authorize purchase. It is lab tested multiple times during the manufacturing process, from seed to shelf. This also includes being tested for CBD content, other Cannabinoid content, yeast/mold/fungus, and bacteria like E. coli to ensure safety and top quality. ETST believes that Hemp CBD Oil awareness today is where Omega 3 and Omega 6 fatty acids were 15 to 20 years ago. ETST goal is to help consumers Worldwide with their overall wellness through advanced formulations with its High Grade Hemp CBD (Cannabidiol) Oil and other cutting edge nutraceuticals and dietary supplements. Hemp CBD Oil is posed to be the most renowned and effective natural compound and botanical alternative introduced to the nutritional and dietary supplement industry in decades. Consumers worldwide desperately need more effective safe botanical alternatives to help them with their overall health and internal wellness. Modern science has identified over 400 phytonutrients in various parts of the industrial hemp plant. These include hundreds of Terpenoids, Essential Oils and Antioxidants but perhaps the most dynamic are a group of 80 compounds called Cannabinoids that are exclusively found in Hemp and one other place; our bodies. There are roughly 80 different phyto-cannabinoids, most prominently CBD, CBC, CBG, CBN, and most likely others yet to be effectively identified. In addition to the cannabinoids present in High Grade Hemp CBD (Cannabidiol) Oil extracts, there are also many other types of natural molecules such as amino acids, fatty acids, flavonoids, ketones, nitrogenous compounds, alkanes, alcohols, glycosides, pigments and terpenes among other things. The most common terpenes in our Hemp CBD oils are Beta-caryophyllene, Caryophyllene Oxide, Myrcene, Terpinolene, Bisabolol, Pinene, Nerolidol, Ocimene and Phytol.

ABOUT ETST (www.earthsciencetech.com): ETST (ETST) is a unique Biotechnology company focused on delivering cutting edge Nutraceuticals, Bioceuticals and Dietary Supplements designed for excellence in the areas of Health, Wellness, Sports and Alternative Medicine and to improving the quality of life for Consumers Worldwide. ETST is also dedicated to providing Natural Alternatives to prescription medications through the use of its cutting edge Nutritional and Dietary Supplements. This may include products such as its High Grade Hemp CBD (Cannabidiol) Oil, Vitamins, Minerals, Herbs, Botanicals, Personal Care Products, Homeopathies, Functional Foods and other products. These products may be in various formulations and delivery systems including (but not limited to) capsules, tablets, soft gels, chewables, liquids, creams, sprays, powders, and whole herbs. ETST is focused on researching and developing innovative Hemp extracts and to make them accessible Worldwide. ETST plans to be the premier supplier of the highest quality and purity of High Grade Hemp CBD (Cannabidiol) Oil. ETST's primary goal is to advance different High Quality Hemp extracts with a broad profile of Cannabinoids and additional natural molecules found in Industrial Hemp and to identify their distinct properties. The company is dedicated in offering its consumers the finest and purest quality All Natural-Organic Hemp CBD Oil while never compromising on quality. ETST High Grade Hemp CBD (Cannabidiol) Oil is classified as "food based" and therefore perfectly permissible in all 50 states and more than 40 countries. Cannabinoids (Cannabidiol/CBD) are natural constituents of the Hemp plant and CBD is derived from Hemp stalk and seed. Hemp oil is a well-known dietary supplement and the naturally occurring CBD possesses no psychoactive qualities and presents a continuing stream of overwhelming evidence of significant Wellness benefits. With no psychoactive ingredient, Hemp CBD Oil is a ready-for-market Hemp-based Nutraceutical. The United States Food and Drug Administration (FDA) currently considers non-THC hemp based cannabinoids, including CBD, to be "food based" and therefore saleable. These new non-psychoactive CBD-rich hemp oil products that ETST has geared up to market and distribute are beyond reproach. CBD (cannabidiol), a naturally occurring constituent of the Industrial Hemp plant, promotes and supports the nutritional health of aging bodies in particular. Source: US Government Patent #6,630,507 "Cannabinoids as antioxidants and neuroprotectants." ETST does not grow, sell or distribute any substances that violate United States Law or the controlled substance act. ETST does sell and distribute hemp based products.

ABOUT DV BIOLOGICS (www.dvbiologics.com): DV Biologics is a global supplier of human biological tools to academic institutions and pharmaceutical companies engaging in cell and drug based discovery and development. Our mission is to provide biological tools needed to advance the innovation of technology that will ultimately be used to treat or prevent multiple human degenerative disorders and diseases. DV Biologics offers a diverse range of novel human biological tools and services that can be used to study various human pathological conditions in addition to an expanded product portfolio of unique cell types and tissue-derived products.

FOOD AND DRUG ADMINISTRATION (FDA) DISCLOSURE: These statements and products have not been evaluated by the FDA and are not intended to diagnose, treat or cure any disease. Always check with your physician before starting a new dietary supplement program.

FORWARD LOOKING DISCLAIMER: This release contains forward-looking statements that involve risks and uncertainties. Readers are referred to the Securities and Exchange Commission filings filed by the Company on EDGAR at http://www.sec.gov/edgar.shtml, specifically the most recent reports which identify important risk facts that could cause actual results to differ from those contained in the forward-looking statements. The Company undertakes no obligation to review or confirm analysts' expectations or estimates or to publicly release any revisions to any forward-looking statements. The information contained in this press release should not be construed as any indication of the Company's future stock price, its revenues or results of operations.

Contact:

Earth Science Tech, Inc. (ETST)
Boca Raton, FL
info@earthsciencetech.com
Phone: +1(305) 615-2118

DV Biologics
Biotechnology Company
Costa Mesa, CA

SOURCE: Earth Science Tech, Inc.