Ashwagandha leaf derived withanone protects normal human cells against the toxicity of methoxyacetic acid, a major industrial metabolite.

The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative non-toxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.

Peroxiredoxin I is a negative regulator of Th2-dominant allergic asthma.

Peroxiredoxin (Prx) I, a ubiquitous antioxidant enzyme, is known to protect against inflammation; however, its role in the allergic inflammation remains unidentified. We determined whether intristic Prx I protects against allergic asthma traits using Prx-I knockout (-/-) mice. Prx I (-/-) and wild-type (WT) mice were immunized with ovalbumin (OVA) plus aluminum potassium sulfate (Alum: Th2 adjuvant) and subsequently challenged with OVA. Twenty-four hours after the last OVA challenge, leukocyte influx including eosinophils into bronchoalveolar lavage fluid was significantly greater in Prx I (-/-) mice compared to that in WT mice. On the other hand, when these mice were immunized with OVA+complete Freund's adjuvant (Th1 adjuvant), opposite phenomenon was observed. In the presence of OVA/Alum, peribronchial inflammatory leukocyte infiltration, cholinergic airway resistance, and the lung expression of interleukin (IL)-2 were significantly greater and that of interferon-gamma was significantly lesser in Prx I (-/-) than in WT mice. In vitro, OVA/Alum-sensitized Prx I (-/-) T cells proliferated more profoundly than WT T cells when they were cocultured with syngeneic bone marrow-generated dendritic cells. These results indicate that endogenous Prx I protects against allergen-related Th2-type airway inflammation and hyperresponsiveness, at least partly, via the suppression of the lung expression of IL-2 and regulation of the Th1/Th2 balance in addition to its antioxidative properties. Furthermore, Prx I can inhibit allergen-specific T-cell proliferation through immunological synapse. Our findings implicate an alternative therapeutic value of Prx I in the treatment of Th2-skewed allergic airway inflammatory diseases such as atopic asthma.

Deceleration of senescence in normal human fibroblasts by withanone extracted from ashwagandha leaves.

Ashwagandha is an Ayurvedic shrub that forms a common ingredient of health supplements, tonics, and Indian home remedies designed to promote health and quality of life. Though sustained through experience and history, there are only a limited laboratory studies and experimental evidence to its effects. In our efforts to characterize Ashwagandha activities and their molecular mechanisms, we initially prepared leaf extract of Ashwagandha (i-Extract) that showed tumor-inhibitory activity. In the present study, we demonstrate that a major component of i-Extract and withanone (i-Factor) protected the normal human fibroblasts against the toxicity caused by withaferin A. It increased the in vitro division potential of normal human cells that appeared to be mediated by decreased accumulation of molecular damage, downregulation of the senescence-specific beta-galactosidase activity and the senescence marker protein, p21(WAF-1), protection against oxidative damage, and induction of proteasomal activity. To the best of our knowledge, we provide the first example of phytochemical(s) (i-Extract and withanone) that have both anticancer and antiaging activities and point to the molecular link between aging and cancer.

Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: combinational approach for enhanced differentiation.

Ashwagandha (Withania somnifera) is widely used in the Indian traditional system of medicine, Ayurveda. Although it is claimed to have a large variety of health-promoting effects, including therapeutic effects on stress and disease, the mechanisms of action have not yet been determined. In the present study, we aimed to investigate the growth inhibition and differentiation potential of the alcoholic extract of Ashwagandha leaves (i-Extract), its different constituents (Withaferin A, Withanone, Withanolide A) and their combinations on glioma (C6 and YKG1) cell lines. Withaferin A, Withanone, Withanolide A and i-Extract markedly inhibited the proliferation of glioma cells in a dose-dependent manner and changed their morphology toward the astrocytic type. Molecular analysis revealed that the i-Extract and some of its components caused enhanced expression of glial fibrillary acidic protein, change in the immunostaining pattern of mortalin from perinuclear to pancytoplasmic, delay in cell migration, and increased expression of neuronal cell adhesion molecules. The data suggest that the i-Extract and its components have the potential to induce senescence-like growth arrest and differentiation in glioma cells. These assays led us to formulate a unique combination formula of i-Extract components that caused enhanced differentiation of glial cells.

Selective killing of cancer cells by leaf extract of Ashwagandha: components, activity and pathway analyses.

Ashwagandha, also called as "Queen of Ayurveda" and "Indian ginseng", is a commonly used plant in Indian traditional medicine, Ayurveda. Its roots have been used as herb remedy to treat a variety of ailments and to promote general wellness. However, scientific evidence to its effects is limited to only a small number of studies. We had previously identified anti-cancer activity in the leaf extract (i-Extract) of Ashwagandha and demonstrated withanone as a cancer inhibitory factor (i-Factor). In the present study, we fractionated the i-Extract to its components by silica gel column chromatography and subjected them to cell based activity analyses. We found that the cancer inhibitory leaf extract (i-Extract) has, at least, seven components that could cause cancer cell killing; i-Factor showed the highest selectivity for cancer cells and i-Factor rich Ashwagandha leaf powder was non-toxic and anti-tumorigenic in mice assays. We undertook a gene silencing and pathway analysis approach and found that i-Extract and its components kill cancer cells by at least five different pathways, viz. p53 signaling, GM-CFS signaling, death receptor signaling, apoptosis signaling and G2-M DNA damage regulation pathway. p53 signaling was most common. Visual analysis of p53 and mortalin staining pattern further revealed that i-Extract, fraction F1, fraction F4 and i-Factor caused an abrogation of mortalin-p53 interactions and reactivation of p53 function while the fractions F2, F3, F5 work through other mechanisms.

Selective killing of cancer cells by leaf extract of Ashwagandha: identification of a tumor-inhibitory factor and the first molecular insights to its effect.

PURPOSE: Ashwagandha is regarded as a wonder shrub of India and is commonly used in Ayurvedic medicine and health tonics that claim its variety of health-promoting effects. Surprisingly, these claims are not well supported by adequate studies, and the molecular mechanisms of its action remain largely unexplored to date. We undertook a study to identify and characterize the antitumor activity of the leaf extract of ashwagandha. EXPERIMENTAL DESIGN: Selective tumor-inhibitory activity of the leaf extract (i-Extract) was identified by in vivo tumor formation assays in nude mice and by in vitro growth assays of normal and human transformed cells. To investigate the cellular targets of i-Extract, we adopted a gene silencing approach using a selected small hairpin RNA library and found that p53 is required for the killing activity of i-Extract. RESULTS: By molecular analysis of p53 function in normal and a variety of tumor cells, we found that it is selectively activated in tumor cells, causing either their growth arrest or apoptosis. By fractionation, purification, and structural analysis of the i-Extract constituents, we have identified its p53-activating tumor-inhibiting factor as with a none. CONCLUSION: We provide the first molecular evidence that the leaf extract of ashwagandha selectively kills tumor cells and, thus, is a natural source for safe anticancer medicine.

Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers.

Inchinkoto (ICKT), a herbal medicine, has been recognized in Japan and China as a "magic bullet" for jaundice. To explore potent therapeutic agents for cholestasis, the effects of ICKT or its ingredients on multidrug resistance-associated protein 2 (Mrp2/ MRP2)-mediated choleretic activity, as well as on antioxidative action, were investigated using rats and chimeric mice with livers that were almost completely repopulated with human hepatocytes. Biliary excretion of Mrp2 substrates and the protein mass, subcellular localization, and mRNA level of Mrp2 were assessed in rats after 1-wk oral administration of ICKT or genipin, a major ingredient of ICKT. Administration of ICKT or genipin to rats for 7 days increased bile flow and biliary excretion of bilirubin conjugates. Mrp2 protein and mRNA levels and Mrp2 membrane densities in the bile canaliculi and renal proximal tubules were significantly increased in ICKT- or genipin-treated rat livers and kidneys. ICKT and genipin, thereby, accelerated the disposal of intravenously infused bilirubin. The treatment also increased hepatic levels of heme oxygenase-1 and GSH by a nuclear factor-E2-related factor (Nrf2)-dependent mechanism. Similar effects of ICKT on MRP2 expression levels were observed in humanized livers of chimeric mice. In conclusion, these findings provide the rationale for therapeutic options of ICKT and its ingredients that should potentiate bilirubin disposal in vivo by enhancing Mrp2/MRP2-mediated secretory capacities in both livers and kidneys as well as Nrf2-mediated antioxidative actions in the treatment of cholestatic liver diseases associated with jaundice.

Artificial cytokine storm combined with hyperthermia induces significant anti-tumor effect in mice inoculated with lewis lung carcinoma and B16 melanoma cells.

In cancer immunotherapies combined with hyperthermia, one or two cytokines have been tested to augment the anti-tumor effect. However, the therapies have not shown sufficient improvement. The aim of this study is to find a new potent tumor immunotherapy in order to augment antitumor effect of hyperthermia by the cytokine cocktails in vivo. We used a combination therapy of local hyperthermia (LH) and various cytokine cocktails composed of IFNs (IFN-alpha, -beta, and -gamma), Thl cytokines (IL-2, -12, -15, and -18), a Th2 cytokine (IL-4), inflammatory cytokines (IL-lalpha and TNF-alpha), and dendritic cell-inducible cytokines (IL-3 and GM-CSF). These cytokines in a proper combination augmented the anti-tumor effect of LH and prolonged survival time in Lewis lung carcinoma or B16 melanoma significantly. Moreover, the 12-cytokine cocktail suppressed B 16 metastasis to the lung and lymph nodes, and complete regression of the tumors without regrowth occurred in 3 of 5 mice. In the cured three B16 mice, there was hyperplasia of lymphatic organs with many CD3-positive T lymphocytes. The most effective cytokine combination should be able to augment the anti-tumor effect of other therapies besides hyperthermia that induce the necrosis of tumor cells.