Description

Ashwagandha 5% Nootropic Powder

 

 

 

CAS Number	5119-48-2
Other Names	Withaferin A, Withaferine A, NSC-101088, Ashwagandha
IUPAC Name	(1S,2R,6S,7R,9R,11S,12S,15R,16S)-6-hydroxy-15-[(1S)-1-[(2R)-5-(hydroxymethyl)-4-methyl-6-oxo-2,3-dihydropyran-2-yl]ethyl]-2,16-dimethyl-8-oxapentacyclo[9.7.0.02,7.07,9.012,16]octadec-4-en-3-one
Molecular Formula	C₂₈H₃₈O₆
Molecular Weight	470.61
Purity	≥99% Pure (LC-MS)
Liquid Availability	30mL liquid (250mg/mL, 7500mg bottle)
Powder Availability	30 grams, 60 grams, 60 capsules (250mg/capsule, 15 grams bottle)
Gel Availability	N/A
Storage	Store in cool dry environment, away from direct sunlight.
Terms	All products are for laboratory developmental research USE ONLY. Products are not for human consumption.

 

What is Ashwagandha?

Withania somnifera, commonly referred to as Ashwagandha is a healing herb frequently used in Ayurveda, the traditional method of medicine practiced in India. Ashwagandha has shown the potential to elicit several anti-inflammatory, antitumor, antioxidant, anti-stress, anti-diabetic, and cardioprotective effects [1]. Current research is focused on isolating and amplifying the compound in order to direct its beneficial properties towards areas in the body damaged by inflammation and oxidative stress.

 

Main Research Findings

1) Treatment with Ashwagandha was found to prevent a rise in lipid peroxidation induced by lipopolysaccharide and peptidoglycan administered in doses meant to mimic the biological effects of everyday stressors.

2) Treatment with Ashwagandha was found to improve locomotor activity and cognitive functioning, ammonia levels, brain and hepatic histopathological alterations as well as brain and hepatic levels of MDA, GS, iNOS, GSH, Nrf2, and HO-1, while also downregulating the expression of p38, ERK1/2 mRNA expression NF-kB and TNF-alpha.

 

Selected Data

1) Lipopolysaccharide and peptidoglycan are known to modify pharmacokinetics of orally administered drugs and act in the body as internal stressors. The presence of these compounds is linked to increased lipid peroxidation and formation of free radicals resulting in cell damage. Researcher Jayan N. Dhuley et al examined the effects of Ashwagandha on lipid peroxidation induced by lipopolysaccharide and peptidoglycan in both rabbits and mice [2].

Male Belgian albino rabbits weighing 2.5-3.0 kg each, and Hindustan antibiotic strain mice weighing 20-30 grams each were used for the purpose of this study. All test subjects were housed in a relatively humid and air-conditioned room and maintained on a 10 hour light/14 hour dark cycle. The rabbits were fed a standard granular diet with water and leucern grass provided ad libitum and the mice were fed a standard granular diet with ad libitum access to water.

0.2 ug/kg of lipopolysaccharide or 100 ug/kg of peptidoglycan were dissolved in normal saline and injected into the marginal auricular vein of the rabbits and in the tail vein of the mice. 100 mg/kg of Ashwagandha was orally administered to one group of rabbits receiving lipopolysaccharide, one group of mice receiving lipopolysaccharide, one group of rabbit receiving peptidoglycan, and one group of mice receiving peptidoglycan. All treatments were administered to the experimental animals simultaneously, while the control group received 2% gum acacia orally, and saline intravenously. Blood samples were collected from the test subjects via cardiac punctuation before treatment was administered, as well as 1, 2, 4, 6,8,10, and 24 hours after treatment was administered [2].

Lipid peroxidation was evaluated by collecting blood samples from all test subjects and mixing them with sodium dodecyl sulfate, acetate buffer, and aqueous solution. The mixture was heated for 60 minutes and red pigment was extracted after cooling using n-butanol:pyridine. Tetramethoxypropane was used as an external standard and lipid peroxidation was expressed using an extinction coefficient in terms of malondialdehyde equivalents. Statistical differences between the acquired results were identified using Student’s t-test [2].

2) The research team of Khalil et al examine the ability of Ashwagandha to decrease the rapid profession of hepatocellular damage and encephalopathy associated with acute liver failure, as it is related to neuropsychiatric dysfunction characterized by cognitive deficits, impaired consciousness, and eventually death. For the purpose of this study 28 adult female Wistar rats each weighing 150-180 grams were utilized. The rats were maintained under standard laboratory conditions with ad libitum access to a commercial diet and water, and were allowed to acclimatize to their surroundings before the initiation of experimental testing [3].

Each rat was randomly assigned to one of 4 groups with 7 subjects each. Group 1 was a negative control group including normal rats given 2 ml/kg of water via oral gavage for 30 days. Group 2 was a positive control group including rats intraperitoneally injected with 350 mg/kg thioacetamide (TAA) on day 30 of the experiment. Group 3 included rats pretreated with 200 mg/kg of Ashwagandha via oral gavage for 30 days, followed by administration of 350 mg/kg TAA on day 30. Group 4 included rats pretreated with 400 mg/kg of Ashwagandha via oral gavage for 30 days, followed by administration of 350 mg/kg TAA on day 30. The behavioral tests performed by the subjects in this experiment were the open field test, the Y maze test, the modified elevated plus maze, and the novel object recognition test; behavioral testing took place on days 31 to 35 between 9 AM and 3PM.

The open field test took place first; the female rats were placed in the open field apparatus in order for the research to examine their locomotor activity and exploratory behavior. The open field apparatus was composed of a square wooden box with the floor divided into 16 equal squares. After the rats were gently placed in one corner of the box they were allowed to explore for 3 minutes while the total number of crossings and rearing activity was assessed as representation for locomotion and exploration, respectively. Between each trial the apparatus was cleaned with alcohol and allowed to air dry before the initiation of the next testing session [3].

The next behavioral assessment was the Y maze test that observed the spatial working memory in rats with induced hepatocellular damage and encephalopathy. The rats were placed into one corner of the Y maze apparatus and allowed to explore for 5 minutes while the researchers observed and recorded the number of arm entries and spontaneous alternation behavior. The measured parameters act as a representation of both motor activity and the natural ability of the rats to differentiate between different sections of the maze. The open field and Y maze tests were followed by the modified elevated plus maze test. This maze was used to assess long-term spatial memory utilizing a wooden elevated plus maze with two open arms and two enclosed arms connected by a central platform. The procedures of the modified elevated plus maze test includes two different phases that take place 24 hours apart [3].

The first phase is defined as the acquisition phase where the test subjects are placed at one end of the open arm facing the outside of the arm. The researcher then recorded and calculated the amount of time it takes for the rat to move to either one of the enclosed arms of the maze. This amount of time it took was referred to as transfer latency-1. The test subjects were then allowed to explore the closed arm for 30 seconds. If after 90 seconds an animal failed to exit the open arm to explore the closed arm, they were gently urged by the researchers into the closed arm and allowed to explore for 10 seconds. The second phase of the modified elevated plus maze was the testing phase where transfer latency-2 was measured by assessing how long it took for the animal to reach one of the enclosed arms before the 90 second time allotment was over.

The final behavioral exam was the novel object recognition test that evaluated the hippocampal-dependent memory and cognitive impairments associated with hepatocellular damage and encephalopathy. The test included a wooden square box that was involved in three different testing trials that took place 24 hours apart. The first trial was the habituation trial where the rats were placed inside of the apparatus and allowed to familiarize themselves for 3 minutes. The second trial was the acquisition trial where the rats were allowed to explore and familiarize themselves with two identical objects in the box for 5 minutes. For this purpose of this study, the identical objects were two 1.0 kg dumbbells. The third trial was the recall trial where the rats were allowed to explore and familiarize themselves with one familiar object, the 1.0 kg dumbbell, and a new object, a 1 L water bottle, in the box for 5 minutes. The research team observed exploratory activity of the animals that was defined as sniffing and touching the objects in the box. It is important to mention that the animals climbing on the objects was not considered exploratory activity. From the data collected, the discrimination ratio and recognition index was calculated [3].

24 hours after the behavioral testing took place, blot samples were collected from the retro-orbital plexus and left to clot followed by 10 minutes of centrifugation to separate the serum. The rats were then euthanized and samples from the liver and brain were collected and stored for the purpose of further gene expression and biochemical analyses. The various biochemical assessments that occurred included the determination of serum levels of ammonia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, and total protein levels. Additionally, homogenates from liver and brain tissues were used to quantify levels of reduced glutathione and lipid peroxidation while an ELISA assay quantified the amount of glutamine synthetase (GS), Nrf2, heme-oxygenase-1 (HO-1), and inducible nitric oxide synthase (iNOS) using homogenates from liver and brain tissues.

Histopathological and immunohistochemical investigations occurred by fixing liver and brain specimens in formalin, followed by preparation according to protocol and embedding in paraffin. 5 um thick slices were cut and stained using hematoxylin, eosin, and Masson trichrome stain in order for the stained slides to be examined via light microscope. Liver specimens were given a histological score ranging from 0 to 3 based on the severity of the inflammation, regional necrosis, extensive necrosis, and fibrosis present in the samples. Brain sections were also given a histological score on a scale of 0 to 3 based on the severity of neuronal edema, neuronal necrosis, and astrocyte infiltration in the samples [3].

 

Discussion

1) Endotoxins such as lipopolysaccharide and peptidoglycan are known to induce various neuro-endocrine physiological changes. This is due to a chain of cellular events involving the activation of cytokines such as interleukins and tumor necrosis factors that are heavily associated with inflammation and various pathological features in the body. The purpose of the study conducted by Dhuley was to assess how administration of Ashwagandha affects lipid peroxidation induced by lipopolysaccharide and peptidoglycan in an animal-based study model composed of rabbits and mice. It is important to mention that the lipopolysaccharide and peptidoglycan were given to the test subjects in doses that mimicked the conditions of stressful situations of everyday life. It was confirmed that lipid peroxidation occurred when both of the stressor compounds were intravenously administered to the animals [2].

There was a noted difference at the time of onset of induced lipid peroxidation by each compound; peak exertion of lipopolysaccharide was achieved 2-6 hours after administration while peak exertion of peptidoglycan was achieved 1 hour after administration. After administration of lipopolysaccharide there was a remarkable increase in lipid peroxidation in both rabbits and mice. When Ashwagandha was delivered to the animals at the same time the rise in lipid peroxidation levels was significantly prevented by the compound. Additionally, administration of peptidoglycan was found to result in an increase in lipid peroxidation in both rabbits and mice. Simultaneous administration of Ashwagandha was shown to significantly prevent this rise in lipid peroxidation levels [2].

Ashwagandha was also found to inhibit elevated lipid peroxidation by targeting the process of free radical scavenging rather than modify the glutathione system. This was an important note considering that the glutathione system is one of the main physiological antioxidant systems in the body. Overall, the study concluded that administration of Ashwagandha prevented increased lipid peroxidation caused by the presence of lipopolysaccharide and peptidoglycan [2].

2) The results of the study conducted by Khalil et al found that in rats intoxicated with TAA that underwent the open field test, there was a significant decrease in the number of crossings into other quadrants, as well as the frequency of rearing activities as a measurement of locomotion and exploratory behavior, respectively. When Ashwagandha was administered to the animals in doses of 200 mg/kg and 400 mg/kg, both exploratory behavior and locomotion activity experienced a remarkable increase in comparison to the TAA-intoxicated rats. It is important to mention that while both doses were effective, the observed behaviors and activities were enhanced to a greater degree in the animals treated with 400 mg/kg of Ashwagandha [3].

Next, the rat completed the Y maze test as a measurement of spatial working memory. The results reported that the animals intoxicated with TAA experienced a significantly reduced number of alternations compared to the control groups of rats. When the animals were pretreated with Ashwagandha, those who were administered a 400 mg/kg dose experienced a marked elevation in spontaneous alternations compared to the TAA-intoxicated group. However, it is important to mention that there were no significant changes in spontaneous alternations in the rats administered 200 mg/kg of Ashwagandha. In terms of the frequency with which the animals entered the different maze arms, both the 200 mg/kg and the 400 mg/kg pretreatment doses of Ashwaganha were shown to significantly increase the frequency of arm entries.

Figure 1: Changes in A) number of crossings and B) rearing frequency during the open field test, and changes in C) number of arm entries and D) spontaneous alternation percentage during the Y maze test, measured across the experimental treatment groups.

The modified elevated plus maze was completed by the test subjects next in order to assess the long term memory of the animals. The results report that there were no significant differences in the findings collected from different experimental groups in terms of the measurement of transfer latency-1. For the measurement of transfer latency-2, the rats intoxicated with TAA exhibited an increased amount of time taken to transfer to an enclosed maze arm from an open one, in comparison to the control group. When Ashwagandha was administered to the animals in doses of 200 mg/kg and 400 mg/kg, the amount of time it took for the animal to transfer to an enclosed maze arm was significantly shorter in comparison to the TAA-intoxicated rats. It is important to mention that while both doses were effective, transfer latency-2 was reduced to a greater degree in the animals treated with 400 mg/kg of Ashwagandha [3].

The novel object recognition test was the final behavioral examination the rats underwent in order to assess their non-spatial working memory. The recorded discrimination ratio was not found to experience any significant changes between the different experimental treatment groups. In the rats intoxicated with TAA, there was a significant decrease in the recognition index compared to the group of control rats. When Ashwagandha was administered to the animals in doses of 200 mg/kg and 400 mg/kg, the recognition index of the animals experienced a remarkable increase in comparison to the TAA-intoxicated rats. It is important to mention that while both doses were effective, the recognition index was enhanced to a greater degree in the animals treated with 400 mg/kg of Ashwagandha.

Figure 2: Changes in A) time latency-1 and B) time latency-2 during the modified elevated plus maze, and changes in C) discrimination ratio and D) recognition index during the novel object recognition test measure across the experimental treatment groups.

Hepatotoxicity was assessed by measuring changes in serum levels of ALT, AST, and ALP. After TAA was administered to intoxicate the rats, all serum levels were increased, in addition to a marked reduction in total protein and albumin levels. All levels of ALT, AST, ALP, total protein, and albumin were restored to baseline levels with pretreatment of both 200 mg/kg and 400 mg/kg doses of Ashwagandha. That being said, liver tissues were assessed for evidence of damage characterized by histopathological alterations. In animals administered TAA, there was noticeable hepatocellular vacuolar change and extensive necrosis indicative of intensive histopathological damage. Additionally, portal areas were infiltrated by portal fibroplasia and mononuclear inflammatory cells while sinusoidal dilatation and hemorrhagic areas were detectable in the hepatic parenchyma. When the animals were pretreated with Ashwagandha at a dose of 200 mg/kg there was a mild improvement in the liver histopathological impairments marked by lower degrees of fibroplasia, focal areas of necrosis, and limited hemorrhagic zones. When the animals were pretreated with Ashwagandha at a dose of 400 mg/kg, normal hepatic functioning was achieved with evidence of very little fibroplasia [3].

As for the samples collected from brain tissues, TAA-intoxicated rats were found to have extensive perivascular and neuronal edema with necrosis and neuronal degenerating encroaching on various brain regions. These findings are in comparison to the rats included in the control group that exhibited normal histological structuring of the different brain regions. When the animals were pretreated with 200 mg/kg of Ashwagandha prior to TAA-intoxication, there was only mild edema observed and very few degenerating neurons present in the cerebral cortex. When the rats were pretreated with 400 mg/kg of Ashwagandha prior TAA-intoxication, brain sections appeared normal, similar to the control group, with only mild edema observed in the striatum [3].

Oxidative stress was measured by levels of MDA and GSH stores within the liver and tissue. TAA-intoxicated rats experienced a noticeable increase in MDA levels associated with depleted GSH stores in the liver and brain in comparison to the control group of rats. When pretreated with 200 mg/kg of Ashwagandha, elevated levels of MDA were regulated and decreased and GHS content in the liver and brain was replenished, while pretreatment with 400 mg/kg of Ashwagandha was shown to enhance antioxidant activities. The enhanced antioxidant activities were further assessed by measuring levels of Nrf2 and HO-1; TAA-intoxicated rats experienced a significant decrease in these markers in the liver and brain, however pretreatment with 200 mg/kg of Ashwagandha significantly elevated levels of Nrf2 and HO-1 while 400 mg/kg restored them to their baseline [3].

Figure 3: Changes in A) MDA in the liver, B) GSH in the liver, C) MDA in the brain, and D) GSH in the brain measured across the experimental treatment groups.

Figure 4: Changes in A) Nrf2 in the liver, B) HO-1 in the liver, C) Nrf2 in the brain, and D) HO-1 in the brain measured across the experimental treatment groups

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Azab KS, Maarouf RE, Abdel-Rafei MK, El Bakary NM, Thabet NM. Withania somnifera (Ashwagandha) root extract counteract acute and chronic impact of γ-radiation on liver and spleen of rats. Hum Exp Toxicol. 2022 Jan-Dec;41:9603271221106344. doi: 10.1177/09603271221106344. PMID: 35656930.

[2] Dhuley JN. Effect of ashwagandha on lipid peroxidation in stress-induced animals. J Ethnopharmacol. 1998 Mar;60(2):173-8. doi: 10.1016/s0378-8741(97)00151-7. PMID: 9582008.

[3] Khalil HMA, Eliwa HA, El-Shiekh RA, Al-Mokaddem AK, Hassan M, Tawfek AM, El-Maadawy WH. Ashwagandha (Withania somnifera) root extract attenuates hepatic and cognitive deficits in thioacetamide-induced rat model of hepatic encephalopathy via induction of Nrf2/HO-1 and mitigation of NF-κB/MAPK signaling pathways. J Ethnopharmacol. 2021 Sep 15;277:114141. doi: 10.1016/j.jep.2021.114141. Epub 2021 Apr 24. PMID: 33905819.

 

 

Ashwagandha 5 Percent is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.

 

 

 

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