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Breakthrough Ashwaghanda formulation with increased activity from elevated actives.

 

Novel, patent pending AshWITH™ 

Breakthrough Ashwaghanda formulation with increased activity from elevated actives.
Novel, patent pending AshWITH™
 .

ASHWITH™ BENEFITS:

 

  • Helps reduce stress and anxiety 

  • Helps enhance memory and cognition 

  • Helps support healthy weight managment 

  • Helps promote muscle strength, size and recovery

  • Helps promote sexual function in men and women

  • Helps maintain healthy testosterone in men

  • Helps enhance cardiorespiratory endurance

  • Helps maintain normal thyroid

 

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AshWITH™Research Summary

Comparative efficacy analysis of Biologic’s AshWITH™ ashwagandha (Biologic’s engineered ashwagandha root extract with augmented active withanolide proportions) with non-branded and branded (Sensoril) ashwagandha extract.  

1. IN VITRO ANTIOXIDANT ACTIVITY ASSAY

 

Antioxidant activity of various ashwagandha samples was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. DPPH is a stable free radical which produces a violet solution in ethanol and upon reduction by antioxidant compound produces light yellow to a colorless solution.

Methods:

 

For comparative anti-oxidant activity analysis of Biologic’s AshWITH™, non-branded and branded (Sensoril) ashwagandha samples, two different dilutions (100 µg/ml and 1000 µg/ml) of each sample prepared in ethanol and 500 µl of each sample dilution mixed with 500 µl of 0.04mg/ml DPPH (Sigma #D9132-1G) ethanolic solution. Gallic acid and sucrose used as positive and negative controls, respectively. All samples and controls incubated for at least 30 min at room temperature in the dark followed by absorbance (Abs) reading at 517nm using a spectrophotometer. Percentage anti-oxidant activity was calculated using the formula: 100-(AbsSample-AbsBlank/AbsControlX100) where AbsSample=Sample dilution+DPPH solution; AbsBlank=Sample dilution+DPPH solvent; AbsControl=Sample solvent+DPPH solution.

 

Results:

 

Biologic’s AshWITH™ ashwagandha showed significantly higher antioxidant activity when compared to non-branded and branded (Sensoril) ashwagandha extract.

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2. CELL VIABILITY ASSAY

MTT based cell viability assay was performed to compare cytoprotection efficacy of various ashwagandha samples against oxidative stress-induced cellular damage.

 

Methods:

 

Hydrogen peroxide (H2O2) (IC50 0.4mM) was used to induce oxidative stress in cultured HEK293 (Human Embryonic Kidney) cells (Fig. 1). Approximately, 8000 HEK293 cells were seeded per well of a 96 well plate in 100ul DMEM and 10% FBS media. Cells were allowed to adhere overnight followed by treatment as follows:

 

1. Control (DMSO)

2. H2O2 (0.4mM) only

3. H2O along with Biologic’s AshWITH™ (15 µg/ml),

4. H2O2 along with Non-branded ashwagandha (15 µg/ml) and

5. H2O2 along with branded (Sensoril) ashwagandha (15 µg/ml) for 22hr.

 

MTT assay was performed as per standard protocol and absorbance of formazan crystals dissolved in DMSO measured at 540nm using SpectraMax i3X plate reader. The percentage cell viability in the above experimental groups was calculated as follows: [(AbsSample-AbsBlank/AbsControl-AbsBlank)X 100].

 

Results:

AshWITH™ ashwagandha co-treated cells showed better cytoprotection efficacy (1.33 fold) in comparison to non-branded (1.21 fold) and branded (Sensoril) (1.02 fold) ashwagandha co-treated cells (Fig. 2). 

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3.  REACTIVE OXYGEN SPECIES (ROS) LEVEL MEASUREMENT 

Reactive Oxygen Species (ROS) are a natural byproduct of aerobic respiration that are crucial for many cellular signaling pathways. However, elevation in ROS level due to imbalance between generation and detoxification - a condition termed as oxidative stress - leads to cell and tissue damage and has been implicated in the development of various pathological conditions such as premature aging, cancer, atherosclerosis, vascular diseases, etc.  

Methods:

 

ROS lowering potential of different ashwagandha samples was compared using DCFDA cellular ROS assay kit (Abcam ab113851). Two different compounds were used such as hydrogen peroxide (H2O2) and Sodium arsenite (SA) to induce oxidative stress by increasing cellular ROS in HEK293 cells. TBHP (tert-Butyl hydroperoxide) used as a positive control for ROS generation.

 

Results:

 

Biologic’s AshWITH™ ashwagandha co-treated cells showed higher potential to reduce intracellular ROS level caused by both H2O2 (Fig. 1) and SA (Fig. 2) as compared to non-branded and branded (Sensoril) ashwagandha co-treated cells. 

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4. EXPRESSION OF INTRACELLULAR ANTI-OXIDANT ENZYME GLUTATHIONE PEROXIDASE

Glutathione Peroxidase (GPx) is a major intracellular anti-oxidant enzyme whose main biological function is to protect the organism from oxidative damage. GPx was first identified in 1957 as an enzyme that protects red blood cells against hydrogen peroxide (H2O2).  GPx catalyzes the conversion of H2O2 in to H2 and O2 via oxidation of reduced GSH into its disulfide form (GSSG).

 

Methods:

 

Intracellular GPx1 level was compared in oxidative stress-induced HEK293 cells co-treated with different ashwagandha samples using the semi-quantitative dot blot method. Approximately, 10,000 HEK293 cells were seeded per well of 6-well plates and cultured until cells were 60-70% confluent.  Then, cells were treated as follows:

 

1. Control (DMSO)

2. H2O2 (0.4mM) only

3. H2O2 along with Biologic’s AshWITH™ (15 µg/ml),

4. H2O2 along with Non-branded ashwagandha (15 µg/ml) and

5. H2O2 along with branded ashwagandha (15 µg/ml) for 22hr.

 

Cells were harvested, total protein quantified and 2µg protein per sample spotted onto nitrocellulose membrane and probed with anti-GPX1 antibody (Abcam #ab22604). Dot blot membranes were imaged using BioRad’s Fluor-S Max multi imager and spot intensity quantified using Quantity One software.

 

Results:

 

Ashwagandha co-treated cells showed up-regulation in GPX1 expression as compared to H2O2 only group with highest GPX1 expression observed in Biologic’s AshWITH™ co-treated cells when compared to non-branded and branded (Sensoril) ashwagandha co-treated cells. An increase in GPX1 enzyme expression was possibly due to cellular recovery by increasing resistance to oxidative stress.

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Clearing the Path to Potent Longevity

 

Attain Healthy Essential Fatty Acid Status and Healthy Inflammatory Activity.

 

E-BOOK EXCERPT:   CURCUMIN— A POWERFUL ANTI-INFLAMMATORY

 

Curcumin displays strong anti-inflammatory potential in the entire body, and in the near future it will be recognized as one of the vital anti-aging nutrients. It plays a core role in any health program. I’ve been using extremely high doses of curcumin (turmeric 95% curcuminoids) for various reasons, which you’ll see disclosed throughout this book series.

 

Curcuminoids, extracted from turmeric, contribute a potent defense of DNA. Studies have compared its anti-inflammatory potential and

anti-cancer properties against common pharmaceutical therapies to reveal, in some cases, better results and in other cases conclusive synergistic value. Have pharmaceutical companies reported on this? No, they haven’t. The incredible facts rising out of this research reveal that curcumin, unlike a common nonsteroidal anti-inflammatory drug (NSAID), doesn’t induce gastrointestinal, liver, and/or kidney toxicity.

 

Having read reports of the potential of curcuminoids to down-regulate

cyclo-oxygenase-enzyme activity and eicosanoid-hormone biosynthesis, I was confident some time ago that they regulated gene expression of the enzymes involved in eicosanoid-hormone production, much like the protective NF-kappa-B gene influence of alpha lipoic acid. In this way curcuminoids prevent the overexpression of genes involved in the immune and inflammatory response.

 

After ongoing, relentless search, I found my answer. Curcumin had

been investigated for its ability to suppress the activation of HIV to the status of AIDS. In a 1993 Harvard University study it was demonstrated that curcumin inhibited the expression of a gene in the DNA of the virus. This gene was responsible for turning on HIV to initiate the assault on the body. Recently, through this historical work, curcumin has been shown to block the activation of NF-kappa-B. Not only is curcumin a powerful HIV inhibitor, it can be justifiably applied as part of the anti-aging and anti-inflammatory program with scientific proof that it works powerfully alongside or independent of alpha lipoic acid.  This insight, was the impetus for my push to study curcuminoids exclusively and define with more accuracy how they can be used and even engineered to increase their potential to bring general health benefits and disease treatment.

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