Main Research Findings

1) Investigation of spleen and liver tissues confirmed the ability of Ashwagandha to counter the effects of radiation hazards, indicating that the compound possesses radio-protective, antioxidant, and anti-inflammatory effects.

2) 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.

 

Selected Data

1) Exposure to ionizing radiation (IR) has become unavoidable due to the use of electronic equipment both in the medical field and in day to day life, as well as through the sterilization process of raw food material and industrial developmental equipment. This exposure to radiation can cause oxidative damage to tissues due to the production of reactive oxygen species, ultimately leading to lipid peroxidation, oxidation of DNA and proteins, and the activation of pro-inflammatory factors. The research team Azab et al examined the effect of Ashwagandha on damage induced by radiation to the spleen and liver due to the crucial role these organs play in metabolic activity, inflammatory responses, detoxification, and immunologic functioning [2].

For the purpose of this study, adult male Swiss Albino rats each weighing 120-150 grams were utilized. The animals were maintained under standard laboratory conditions with ad libitum access to pellet food and water, and were allowed to acclimatize to their new surroundings for 1 week. The rats were then randomly divided into 6 groups each including 6 rats: group 1 (C) was the control group receiving administration of physiological saline and no gamma irradiation treatment; group 2 (Ag) was orally administered a 300 mg/kg dose of Ashwagandha for 15 consecutive days; group 3 (Rs) received a single dose of gamma radiation on day 15; group 4 Ag + Rs) was orally administered 300 mg/kg of Ashwagandha for 15 days and received a single dose of gamma irradiation on day 15; group 5 (Rf) was exposed to gamma radiation every other day, 4 times per day, starting on day 9; and group 6 (Ag + Rf) was orally administered 300 mg/kg of Ashwagandha for 15 days and was exposed to gamma radiation every other day, 4 times per day, starting on day 9 [2].

After 15 days of experimental treatment the rats fasted overnight and were euthanized on day 16. Blood samples were collected via cardiac perforation and left to coagulate while the liver and spleen were dissected and washed in saline before being divided into two parts. One half of each organ was used for biochemistry measurements while the other half of each organ was used for histopathological examinations. Oxidative status of the spleen and liver tissues were evaluated by measuring levels of malondialdehyde as an indicator of lipid peroxidation and glutathione as an indicator of antioxidant levels. Additionally, activity levels of alanine amino-transferase and aspartate amino-transferase were observed as an indicator of liver functional status [2].

A homogenization lysis buffer was used to evaluate protein expression of MMP-2, MMP-9, TIMP-1 and alpha-7-nAchR in tissue samples collected from the livers and spleen of rats administered Ashwagandha and/or exposed to radiation. The protein levels of the samples were quantified by densitometric analysis followed by normalization to beta-actin protein expression and assaying. This procedure was followed by histopathological examination that began by fixing autopsy samples from the liver and spleen of rats of each experimental treatment group in a formalin saline for 24 hours. The samples were then washed and dehydrated followed by clearing with xylene and exposure to a hot air oven for 24 hours prior to being embedded in paraffin. The paraffin blocks were sectioned into 4 mm slices and collected on glass slides for staining with hematoxylin and eosin for further examination [2].

2) 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.

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 [3].

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 [3].

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 [3].

 

Discussion

1) When looking at changes in the levels of malondialdehyde as an indicator of lipid peroxidation, glutathione as an indicator of antioxidant levels and the production of reactive oxygen species in the liver there were no significant differences between the group administered Ashwagandha versus the control group. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of malondialdehyde experienced 1.55 fold and 1.77 fold increases, levels reactive oxygen species experienced 2.11 fold and 2.53 fold increases, and glutathione levels decreased by 40.25% and 42.05%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, malondialdehyde levels decreased by 42.47% and 38.02%, reactive oxygen species decreased by 31.37% and 35.18%, and glutathione experienced 1.43 fold and 1.71 fold increases, respectively [2].

Figure 1: The effects of Ashwaganha on liver reactive oxygen species, liver malondialdehyde levels, and liver glutathione levels after receiving a single dose or fractionated doses of gamma radiation.

When looking at changes in the levels of malondialdehyde as an indicator of lipid peroxidation, glutathione as an indicator of antioxidant levels and the production of reactive oxygen species in the spleen there were no significant differences between the group administered Ashwagandha versus the control group. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of malondialdehyde experienced 2.26 fold and 2.75 fold increases, levels reactive oxygen species experienced 1.58 fold and 2.28 fold increases, and glutathione levels decreased by 57.78% and 65.41%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, malondialdehyde levels decreased by 31.89% and 34.59%, reactive oxygen species decreased by 34.62% and 26.16%, and glutathione experienced 1.81 fold and 2.30 fold increases, respectively [2].

Figure 2: The effects of Ashwaganha on spleen reactive oxygen species, spleen malondialdehyde levels, and spleen glutathione levels after receiving a single dose or fractionated doses of gamma radiation.

When looking at changes in the levels of malondialdehyde as an indicator of lipid peroxidation, glutathione as an indicator of antioxidant levels and the production of reactive oxygen species in serum blood samples there were no significant differences between the group administered Ashwagandha versus the control group. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of malondialdehyde experienced 3.00 fold and 3.21 fold increases, levels reactive oxygen species experienced 2.09 fold and 2.69 fold increases, and glutathione levels decreased by 63.27% and 55.56%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, malondialdehyde levels decreased by 42.90% and 38.07%, reactive oxygen species decreased by 38.50% and 44.54%, and glutathione experienced 2.39 fold and 1.89 fold increases, respectively [2].

Figure 3: The effects of Ashwaganha on serum reactive oxygen species, spleen malondialdehyde levels, and spleen glutathione levels after receiving a single dose or fractionated doses of gamma radiation.

The presence of inflammation and the anti-inflammatory effects of Ashwagandha were assessed by observing changes in protein levels of IL-17, IL-10, and alpha-7-nAchR. When comparing the control group of rats to the group of rats administered 300 mg/kg of Ashwagandha, there were no significant changes seen in the livers between the two groups. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of IL-17 experienced 2.26 fold and 2.53 fold increases, IL-10 levels decreased by 56.11% and 59.78%, and alpha-7-nAchR levels decreased by 82.76% and 79.30%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, IL-17 levels decreased by 38.20% and 31.77%, IL-10 levels experienced 1.94 fold and 2.07 fold increases, and alpha-7-nAchR levels experienced 3.64 fold and 2.88 fold increases, respectively [2].

Figure 4: The effects of a 300 mg/kg dose of Ashwagandha on levels IL-17, IL-10, and alpha-7-nAchR in the liver after receiving a single dose or fractionated doses of gamma radiation

The presence of inflammation and the anti-inflammatory effects of Ashwagandha were assessed by observing changes in protein levels of IL-17, IL-10, and alpha-7-nAchR. When comparing the control group of rats to the group of rats administered 300 mg/kg of Ashwagandha, there were no significant changes seen in the spleens between the two groups. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of IL-17 experienced 1.83 fold and 2.17 fold increases, IL-10 levels decreased by 44.17% and 52.40%, and alpha-7-nAchR levels decreased by 76.49% and 72.19%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, IL-17 levels decreased by 33.72% and 31.64%, IL-10 levels experienced 1.43 fold and 1.93 fold increases, and alpha-7-nAchR levels experienced 2.82 fold and 2.75 fold increases, respectively [2].

Figure 5: The effects of a 300 mg/kg dose of Ashwagandha on levels IL-17, IL-10, and alpha-7-nAchR in the spleen after receiving a single dose or fractionated doses of gamma radiation

The presence of inflammation and the anti-inflammatory effects of Ashwagandha were assessed by observing changes in protein levels of IL-17 and IL-10. When comparing the control group of rats to the group of rats administered 300 mg/kg of Ashwagandha, there were no significant changes seen in the blood serum levels between the two groups. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of IL-17 experienced 2.68 fold and 3.09 fold increases, IL-10 levels decreased by 58.72% and 65.62%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, IL-17 levels decreased by 44.45% and 36.77%, IL-10 levels experienced 1.89 fold and 2.06 fold increases, respectively [2].

Figure 6: The effects of a 300 mg/kg dose of Ashwagandha on levels IL-17 and IL-10 in blood serum after receiving a single dose or fractionated doses of gamma radiation.

Levels of protein expression of MMP-2, MMP-9, and TIMP-1 in liver tissue are indicative of tissue damage and were assessed to study the effects of gamma radiation and Ashwagandha treatment in the liver. When comparing the control group of rats to the group of rats administered 300 mg/kg of Ashwagandha, there were no significant changes seen in the livers between the two groups. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of MMP-2 experienced 4.13 fold and 4.52 fold increases, levels of MMP-9 experienced 6.41 fold and 5.85 fold increases, and TIMP-1 levels decreased by 77.29% and 71.37%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, MMP-2 levels decreased by 53.95% and 46.81%, MMP-9 levels decreased by 54.51% and 50.48%, and TIMP-1 levels experienced 2.68 fold and 2.52 fold increases, respectively [2].

Figure 7: The effects of a 300 mg/kg dose of Ashwagandha on levels MMP-2, MMP-9 and TIMP-1 in the liver tissues after receiving a single dose or fractionated doses of gamma radiation.

Levels of protein expression of MMP-2, MMP-9, and TIMP-1 in splenic tissue are indicative of structural tissue damage and were assessed to study the effects of gamma radiation and Ashwagandha treatment in the spleen. When comparing the control group of rats to the group of rats administered 300 mg/kg of Ashwagandha, there were no significant changes seen in the spleens between the two groups. In the groups that received a single dose of gamma radiation and fractionated doses of gamma radiation, levels of MMP-2 experienced 6.47 fold and 6.98 fold increases, levels of MMP-9 experienced 7.61 fold and 7.70 fold increases, and TIMP-1 levels decreased by 43.79% and 70.00%, respectively. When 300 mg/kg of Ashwagandha was administered in combination with a single dose of gamma radiation and fractionated doses of gamma radiation, MMP-2 levels decreased by 54.89% and 53.63%, MMP-9 levels decreased by 50.00% and 49.59%, and TIMP-1 levels experienced 1.58 fold and 2.65 fold increases, respectively [2].

Figure 8: The effects of a 300 mg/kg dose of Ashwagandha on levels MMP-2, MMP-9 and TIMP-1 in the splenic tissues after receiving a single dose or fractionated doses of gamma radiation.

2) 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 [3].

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 [3].

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 [3].

 

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] 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.

[2] 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.

[3] 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.

 

 

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