Standardized Root Extract of Withania somnifera and Withanolide A Exert Moderate Vasorelaxant Effect in the Rat Aortic Rings by Enhancing Nitric Oxide Generation

Abstract

Ethno-pharmacological relevance:

With aniasomnifera (L.) Dunal, commonly known as Ashwagandha, is a key herb in Ayurveda, recognized as an adaptogen, rejuvenator, general health tonic, and remedy for various disorders such as cerebrovascular diseases, insomnia, asthma, and ulcers. Its steroidal lactones (withanolides: Withanolide A, Withaferin A, Withanolide D, Withanone, etc.) possess notable medicinal properties like anti-inflammatory and immune-stimulatory effects. A standardized root extract (NMITLI-118R, NM) was prepared and evaluated for biological activities, including neuroprotection in a rat model of middle cerebral artery occlusion (MCAO).

Aim of the study:

Given that endothelial dysfunction is a primary event in cerebrovascular and cardiovascular disorders, this study aimed to evaluate the vasoprotective potential of NM and its biomarker compound Withanolide A (WA) using rat aortic rings and EA.hy926 endothelial cells.

Materials and Methods:

Transverse aortic rings from 10-week-old Wistar rats were used to assess the effects of NM and WA on vasoreactivity. Mechanisms were investigated in EA.hy926 cells by measuring nitric oxide (NO) generation, nitrite content, Serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS), reduced/oxidized biopterin levels, and eNOS mRNA and protein expression.

Results:

HPLC fingerprinting confirmed the presence of WA in NM. Both NM and WA exhibited moderate vasorelaxant effects in endothelium-intact rat aortic rings, though less than acetylcholine (ACh). Both compounds enhanced ACh-induced relaxation. Their vasorelaxant effects were blocked by N-nitro-L-arginine methyl ester (L-NAME) or ODQ, indicating involvement of the NO/cGMP pathway. In EA.hy926 cells, NM and WA increased nitrite content, NO levels, eNOS expression, and eNOS phosphorylation at Serine 1177.

Conclusion:

NM and WA improve NO availability, likely through enhanced eNOS phosphorylation. WA appears to be an active constituent, though other vasoactive substances may also contribute. The vasorelaxant property of NM supports its neuroprotective potential.

Keywords: Withania somnifera (L.) Dunal; Withanolide A; Rat aortic rings; Vasorelaxation; Nitric oxide; eNOS.

1. Introduction

With aniasomnifera (Ashwagandha), also called “Indian ginseng” or “Indian winter cherry,” has been used in Indian traditional medicine for over 3,000 years. It is prescribed for insomnia, stress, nervous breakdown, memory loss, arthritis, diabetes, gastrointestinal problems, asthma, stroke, and more. The plant exhibits anti-inflammatory, antigenotoxic, anti-tumor, and antioxidant activities. Traditionally, its roots were used in rasayans to promote health and longevity by enhancing immunity.

Several classes of compounds have been isolated from Withania somnifera, including withanolides, cuseohygrine, anahygrine, tropine, anaferine, glycosides, sitoindosides, other alkaloids, starches, and amino acids. Withanolides are unique steroidal lactones with multiple biological activities.

To promote therapeutic uses, discrete chemotypes of Ashwagandha were developed. The aqueous ethanolic root extract (NMITLI-118R, NM) was characterized and evaluated for stability and neuroprotective potential in the MCAO rat model. NM exhibited neuroprotection against ischemia/reperfusion injury and showed immunostimulatory properties.

Stroke, a leading cause of mortality and morbidity, is associated with endothelial dysfunction and reduced NO availability. eNOS-mediated NO generation in vascular endothelial cells is vasoprotective and offers protection against ischemia-reperfusion injury. Improving endothelial function is a desirable approach for treating cerebrovascular and cardiovascular disorders. This study assessed the effect of NM on vasoreactivity and the involvement of NO in its vasorelaxant effect using aortic rings and EA.hy926 cells.

2. Materials and Methods
2.1 Materials

Acetylcholine chloride (ACh), N-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside (SNP), phenylephrine hydrochloride (PE), ODQ, and Withanolide A were obtained from Sigma. eNOS and peNOS antibodies were from Santa Cruz Biotechnology. Other chemicals were sourced from Merck, Glaxo, SRL, or Qualigens.

Standardized extract NM was provided by Dr. A.K. Dwivedi. The extract was prepared by extracting freshly harvested roots with ethanol-water, filtering, concentrating, and lyophilizing. HPLC fingerprinting confirmed the presence of WA.

2.2 Methods

Aortic Ring Vasoreactivity:

Aortic rings (4 mm) from male Wistar rats were used. After equilibration, rings were pre-contracted with PE before cumulative addition of NM/WA or ACh. NOS inhibitor (L-NAME) or sGC inhibitor (ODQ) was used to probe mechanisms. The influence of NM/WA on ACh- and PE-induced responses was also tested. Endothelium-denuded rings were used as controls.

In Vitro Studies (EA.hy926 Cells):

Cells were treated with NM or WA at various concentrations.Nitrite Estimation: Griess reagent was used to measure nitrite after treatment.Gene Expression: Real-time PCR assessed eNOS mRNA.Immunoblotting: eNOS and peNOS protein levels were determined by Western blot.Cell Proliferation and Apoptosis: Flow cytometry after PI staining.Fluorescence Microscopy: NO production was visualized using DAF-2-DA probe and DAPI nuclear staining.Free Radical Generation: DHE and DCF-DA probes assessed ROS and RONS by flow cytometry.Biopterin Levels: HPLC measured BH4/BH2 ratios.Statistical Analysis: Data were analyzed using ANOVA with Tukey-Kramer post-hoc test or unpaired t-test. Results are mean ± SEM, with significance at P < 0.05.

3. Results
3.1 Effect on Rat Aortic Ring Vasoreactivity

ACh induced maximal relaxation (90±3%). NM and WA induced moderate vasorelaxation (40±0.9% and 54.1±0.9%, respectively), significantly less than ACh but significant compared to control. WA had a greater effect than NM. L-NAME and ODQ completely blocked vasorelaxation by ACh, NM, and WA, indicating dependence on NO/cGMP pathway. NM and WA enhanced ACh-induced relaxation but did not affect PE-induced contraction. No effect was observed in endothelium-denuded rings, confirming the role of endothelium-derived NO.

3.2 Effect on NO Generation in EA.hy926 Cells

NM and WA significantly increased nitrite content in EA.hy926 cells, with maximal effect at 50 µg/ml (NM) and 5 µg/ml (WA). WA produced a greater increase than NM. Both compounds enhanced NO levels, as shown by increased DAF intensity in flow cytometry and fluorescence microscopy. This correlated with increased eNOS phosphorylation at Ser1177.

3.3 Effects on Cell Cycle, ROS Generation, and Biopterin Levels

NM and WA did not affect cell cycle progression or apoptosis, even at high concentrations. NM slightly increased the BH4/BH2 ratio, suggesting improved eNOS coupling. WA increased DHE and DCF-DA fluorescence (indicating ROS/RONS) at higher concentrations, but NM did not significantly affect these parameters.

4. Discussion

Reduced NO availability due to endothelial dysfunction is implicated in cardiovascular and cerebrovascular disorders. Therapeutic strategies often aim to enhance NO bioavailability. This study demonstrated that both NM and WA exert moderate vasorelaxant effects in rat aortic rings, mediated by endothelium-derived NO and the NO/cGMP pathway. They also potentiated ACh-induced relaxation.

In endothelial cells, NM and WA increased NO generation, eNOS expression, and phosphorylation. NM slightly improved BH4/BH2 ratio, which is important for eNOS function. Neither compound induced apoptosis or significant ROS production at effective concentrations, indicating safety.

Endogenous NO production by eNOS is crucial for vascular health, and impaired NO bioavailability is a hallmark of hypertension, diabetes, atherosclerosis, and stroke. By improving NO bioavailability, NM may be beneficial in these conditions.

5. Conclusion

With aniasomnifera root extract (NM) and Withanolide A enhance endothelial NO generation and exert moderate vasorelaxant effects in rat aortic rings. These findings support the use of NM in managing cerebrovascular and cardiovascular disorders.