Molecular mechanisms of Asparagus racemosus willd. and Withania somnifera (L.) Dunal as chemotherapeutic adjuvants for breast cancer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite various treatment modalities, the progression and metastasis of breast cancer (BC) are grave concerns due to the alarming disease-free survival rate (DFS) and overall survival rate (OS) of affected patients. Over the years, many antibiotics, synthetic compounds, medicinal plant isolates and polyherbal combinations have been used as adjuvants in therapy for the management of primary and secondary tumors. Paclitaxel (PTX)-based chemotherapy for breast cancer causes multiple adverse side effects in patients. Withania somnifera (L.) Dunal (WS) and Asparagus racemosus Willd. (AR) as Ayurveda-inspired plant-based adjuvants were investigated for their anticancer effects on MDA-MB-231 and 4T1 cells in mouse model systems. AIM OF THE STUDY: This study focused on evaluating the adjuvant properties of WS and AR plant extracts with PTX and their effectiveness over PTX alone in terms of tumor inhibition. MATERIALS AND METHODS: The effects of WS and AR on DNA double-strand breaks (DSBs), senescence induction and mitochondrial functions were evaluated in BC cells in vitro. The potential for cancer stem cell (CSC) inhibition was evaluated via mammosphere formation assays and CD44/CD24 immunostaining. In vivo tumor growth studies were conducted in athymic BALB/c mice for MDA-MB-231 cells and in BALB/c mice for 4T1 cells. RESULTS: Induction of senescence was evident due to DSBs induced by the WS and AR extracts. Mammosphere formation and CD44/CD24 CSC markers were reduced after treatment with WS, AR or the combination of both in MCF-7 cells. WS or AR inhibited epithelial-to-mesenchymal transition (EMT). In vivo studies demonstrated that tumor growth inhibition was more pronounced in the treated group than in the PTX alone group and the untreated control group. CONCLUSION: Our study showed that the use of WS or AR plant hydroalcoholic extracts in combination with paclitaxel (PTX) has better effects on sensitivity and efficacy than PTX alone, as demonstrated in in vitro BC cells and mouse models with BC cell grafts. Hence, scheduling adjuvant therapy with WS or AR alone or combined with PTX can be advantageous for the management of triple-negative BC (TNBC). Further studies are warranted in human clinical conditions to ascertain the efficacy of these treatments.

Ayurveda-based Botanicals as Therapeutic Adjuvants in Paclitaxel-induced Myelosuppression.

Chemotherapy-induced myelosuppression is one of the major challenges in cancer treatment. Ayurveda-based immunomodulatory botanicals Asparagus racemosus Willd (AR/Shatavari) and Withania somnifera (L.). Dunal (WS/Ashwagandha) have potential role to manage myelosuppression. We have developed a method to study the effects of AR and WS as therapeutic adjuvants to counter paclitaxel (PTX)-induced myelosuppression. Sixty female BALB/c mice were divided into six groups-vehicle control (VC), PTX alone, PTX with aqueous and hydroalcoholic extracts of AR (ARA, ARH) and WS (WSA, WSH). The myelosuppression was induced in mice by intraperitoneal administration of PTX at 25 mg/kg dose for three consecutive days. The extracts were orally administered with a dose of 100 mg/kg for 15 days prior to the induction with PTX administration. The mice were observed daily for morbidity parameters and were bled from retro-orbital plexus after 2 days of PTX dosing. The morbidity parameters simulate clinical adverse effects of PTX that include activity (extreme tiredness due to fatigue), behavior (numbness and weakness due to peripheral neuropathy), body posture (pain in muscles and joints), fur aspect and huddling (hair loss). The collected samples were used for blood cell count analysis and cytokine profiling using Bio-Plex assay. The PTX alone group showed a reduction in total leukocyte and neutrophil counts (4,800 ± 606; 893 ± 82) when compared with a VC group (9,183 ± 1,043; 1,612 ± 100) respectively. Pre-administration of ARA, ARH, WSA, and WSH extracts normalized leukocyte counts (10,000 ± 707; 9,166 ± 1,076; 10,333 ± 1,189; 9,066 ± 697) and neutrophil counts (1,482 ± 61; 1,251 ± 71; 1,467 ± 121; 1,219 ± 134) respectively. Additionally, higher morbidity score in PTX group (7.4 ± 0.7) was significantly restricted by ARA (4.8 ± 1.1), ARH (5.1 ± 0.6), WSA (4.5 ± 0.7), and WSH (5 ± 0.8). (Data represented in mean ± SD). The extracts also significantly modulated 20 cytokines to evade PTX-induced leukopenia, neutropenia, and morbidity. The AR and WS extracts significantly prevented PTX-induced myelosuppression (p < 0.0001) and morbidity signs (p < 0.05) by modulating associated cytokines. The results indicate AR and WS as therapeutic adjuvants in cancer management.

Withania somnifera (L.) Dunal: Opportunity for Clinical Repurposing in COVID-19 Management.

As the COVID-19 pandemic is progressing, the therapeutic gaps in conventional management have highlighted the need for the integration of traditional knowledge systems with modern medicine. Ayurvedic medicines, especially Ashwagandha (Withania somnifera (L.) Dunal, WS), may be beneficial in the management of COVID-19. WS is a widely prescribed Ayurvedic botanical known as an immunomodulatory, antiviral, anti-inflammatory, and adaptogenic agent. The chemical profile and pharmacological activities of WS have been extensively reported. Several clinical studies have reported its safety for use in humans. This review presents a research synthesis of in silico, in vitro, in vivo, and clinical studies on Withania somnifera (L.) Dunal (WS) and discusses its potential for prophylaxis and management of COVID-19. We have collated the data from studies on WS that focused on viral infections (HIV, HSV, H1N1 influenza, etc.) and noncommunicable diseases (hypertension, diabetes, cancer, etc.). The experimental literature indicates that WS has the potential for 1) maintaining immune homeostasis, 2) regulating inflammation, 3) suppressing pro-inflammatory cytokines, 4) organ protection (nervous system, heart, lung, liver, and kidney), and 5) anti-stress, antihypertensive, and antidiabetic activities. Using these trends, the review presents a triangulation of Ayurveda wisdom, pharmacological properties, and COVID-19 pathophysiology ranging from viral entry to end-stage acute respiratory distress syndrome (ARDS). The review proposes WS as a potential therapeutic adjuvant for various stages of COVID-19 management. WS may also have beneficial effects on comorbidities associated with the COVID-19. However, systematic studies are needed to realize the potential of WS for improving clinical outcome of patients with COVID-19.

Pre-conditioning of Mesenchymal Stem Cells with Piper longum L. augments osteogenic differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: The Indian Traditional Medicine, Ayurveda prescribes Piper longum L. popularly known as Long Pepper (Pippali) for the treatment of inflammatory and degenerative diseases. Therapeutic benefits of Piper longum L. are mainly attributed to the anti-inflammatory and arthritic potential. AIM OF THE STUDY: This study was aimed to explore the activity of Piper longum L. fruit extract on proliferation and osteogenic differentiation of human Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) to find out it's possible role as anti-osteoporotic agent. MATERIALS AND METHODS: Proliferation of WJMSCs treated with Piper longum L. fruit extract was assessed by MTT assay and Cell Cycle Analysis. Effect of Piper longum L. preconditioning on osteogenic differentiation was performed. Ca2+ accumulation and matrix mineralization (Von Kossa and Alizarin Red Staining), alkaline phosphatase (ALP) activity and gene expression of key mRNA (RT PCR) was analyzed. RESULTS: Significant increase in the proliferation of WJMSCs was observed upon treatment of Piper longum L. at 5 µg/mL (P < 0.001) which can be attributed to the significant decrease in apoptotic cells (P < 0.05) as evidenced by cell cycle analysis. Preconditioning of Piper longum L. (10-100 µg/mL) enhanced Ca2+ accumulation and matrix mineralization as observed by Von Kossa and Alizarin Red staining where ALP activity was elevated 3.6 folds as compared to untreated WJMSCs (P < 0.001). RT-PCR analysis exhibited up regulation of Runx2, Osterix, ALP and OPN mRNAs. CONCLUSIONS: We demonstrate for the first time that Piper longum L. fruit extract enhanced osteogenic differentiation of WJMSCs. This finding can be clinically translated into development of an anti-osteoporotic agent.

Withania somnifera (L.) Dunal: A potential therapeutic adjuvant in cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal (WS) is one of the moststudied Rasayana botanicals used in Ayurveda practice for its immunomodulatory, anti-aging, adaptogenic, and rejuvenating effects. The botanical is being used for various clinical indications, including cancer. Several studies exploring molecular mechanisms of WS suggest its possible role in improving clinical outcomes in cancer management. Therefore, research on WS may offer new insights in rational development of therapeutic adjuvants for cancer. AIM OF THIS REVIEW: The review aims at providing a detailed analysis of in silico, in vitro, in vivo, and clinical studies related to WS and cancer. It suggests possible role of WS in regulating molecular mechanisms associated with carcinogenesis. The review discusses potential of WS in cancer management in terms of cancer prevention, anti-cancer activity, and enhancing efficacy of cancer therapeutics. MATERIAL AND METHODS: The present narrative review offers a critical analysis of published literature on WS studies in cancer. The reported studies were analysed in the context of pathophysiology of cancer, commonly referred as 'cancer hallmarks'. The review attempts to bridge Ayurveda knowledge with biological insights into molecular mechanisms of cancer. RESULTS: Critical analysisof the published literature suggests an anti-cancer potential of WS with a key role in cancer prevention. The possible mechanisms for these effects are associated with the modulation of apoptotic, proliferative, and metastatic markers in cancer. WS can attenuate inflammatory responses and enzymes involved in invasion and metastatic progression of cancer.The properties of WS are likely to be mediated through withanolides, which may activate tumor suppressor proteins to restrict proliferation of cancer cells. Withanolides also regulate the genomic instability, and energy metabolism of cancer cells. The reported studies indicate the need for deeper understanding of molecular mechanisms of WS in inhibiting angiogenesis and promoting immunosurveillance. Additionally, WS can augment efficacy and safety of cancer therapeutics. CONCLUSION: The experimentally-supported evidence of immunomodulatory, anti-cancer, adaptogenic, and regenerative attributes of WS suggest its therapeutic adjuvant potential in cancer management. The adjuvant properties of withanolides can modulate multidrug resistance and reverse chemotherapy-induced myelosuppression. These mechanisms need to be further explored in systematically designed translational and clinical studies that will pave the way for integration of WS as a therapeutic adjuvant in cancer management.

Herbal pre-conditioning induces proliferation and delays senescence in Wharton's Jelly Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are multipotent stem cells which are being explored for various clinical applications. Isolation and in-vitro expansion of MSCs remain important in achieving desired cell number for the therapy. However, in-vitro proliferation of MSCs is often associated with senescence and early onset of apoptosis which limits its therapeutic ability and long term clinical use. Tinospora cordifolia and Withania somnifera are used widely in Ayurveda: the traditional Indian system of medicine and are reported to have rejuvenating and anti-aging potential. In the present study, we investigated the effect of Tinospora cordifolia and Withania somnifera on proliferation and senescence of wharton's jelly MSCs (WJMSCs) in-vitro. METHODS: WJMSCs were treated in culture medium with Tinospora cordifolia leaf and Withania somnifera root extracts to examine their effect on proliferation and senescence properties of WJMSCs. Proliferation of WJMSCs was assayed by cell count, MTT, BrdU incorporation assay, cell cycle analysis and Ki67 mRNA expression. Senescence was demonstrated using ß-galactosidase senescence assay and associated mRNA markers. RESULTS: Culture medium supplemented with Tinospora cordifolia leaf and Withania somnifera root extracts exhibited significant increase in proliferation of WJMSCs as evidenced by cell count and MTT assay. Cell cycle analysis using propidium iodide showed increase in G2/M phase and decrease in apoptotic cells. BrdU incorporation and upregulation of proliferation marker ki67 by RT PCR showed increased DNA synthesis/proliferation in Tinospora cordifolia and Withania somnifera extract treated MSCs. Delayed senescence was confirmed by ß-galactosidase senescence assay and down regulation of senescence marker p21. CONCLUSION: Our results demonstrate for the first time that Tinospora cordifolia and Withania somnifera extracts support proliferation and inhibit senescence in WJMSCs making them suitable candidates as supplements for in-vitro expansion without affecting the cell viability indicating its non-toxic nature.

Network Pharmacology of Ayurveda Formulation Triphala with Special Reference to Anti-Cancer Property.

Network pharmacology is an emerging technique, which integrates systems biology and computational biology to study multi-component and multi-targeted formulations. Ayurveda, the traditional system of Indian medicine, uses intelligent formulations; however, their scientific rationale and mechanisms remain largely unexplored. This paper presents the potential of network pharmacology to understand the rationale of a commonly used Ayurveda formulation known as Triphala. We have developed pharmacology networks of Triphala based on the information gathered from different databases and using the software Cytoscape. The networks depict the interaction of bioactives with molecular targets and their relation with diseases, especially cancer. The network pharmacology analysis of Triphala has offered new relationships among bioactives, targets and putative applications of cancer etiology. This pioneering effort might open new possibilities to know pharmacodynamics of Ayurvedic drugs like Triphala and also help in the discovery of new leads and targets for various diseases.

Cancer, inflammation, and insights from ayurveda.

A recent, exciting discovery relates to the concept of "shared pathology" between cancer and metabolic syndrome. One major pathway common to cancer and metabolic syndrome is chronic inflammation, which is a major driving force in carcinogenesis. Indeed, chronic inflammation precedes most cancers and is considered a "hallmark" of the neoplastic process. We discuss molecular and biochemical evidence which links diet, obesity, abnormal lipid metabolism, and type 2 diabetes mellitus with chronic inflammation. We also explain how each of these factors is linked with biochemical aberrations of carcinogenesis and the prevalence and risk of cancer. While there are reliable biomarkers for chronic inflammation, there are few markers for a mechanistic link between early inflammation and digestive disorders. Discovery of such a marker could lead to identification of a new subtype of patients with digestive disorders that predispose them to cancer and/or metabolic syndrome. In this context, we discuss the ayurvedic concept of "Ama" which is thought to be a toxic, proinflammatory waste-product of improper digestion. We then develop hypotheses and outline preclinical and clinical experiments designed to prove whether "Ama" can serve as a novel and reliable biomarker that links abnormal digestive status, with the onset of chronic inflammation.