Panchvalkala, a traditional Ayurvedic formulation, exhibits antineoplastic and immunomodulatory activity in cervical cancer cells and C57BL/6 mouse papilloma model.

ETHNOPHARMACOLOGICAL RELEVANCE: Panchvalkala, an Ayurvedic traditional formulation has references in Charak Samhita and Bhavaprakasha Nighantu for the treatment of women with endometriosis-related problems, leucorrhea and vaginal ailments. The formulation comprises of equal ratios of the barks from Ficus glomerata, Ficus virens, Ficus religiosa, Ficus benghalensis, and Thespesia populnea. AIM OF THE STUDY: The present study aimed to evaluate the anticancer and immunomodulatory activity of aqueous extract of Panchvalkala (PVaq) against cervical cancer in vitro and in vivo. MATERIALS AND METHODS: The effect of PVaq on disruption of mitochondrial membrane potential in cervical cancer cell lines, SiHa and HeLa, was studied by using JC1 dye. The expression of generic caspases in the cells after treatment with PVaq was evaluated by ELISA kit. The expression of pRb, p53, E6 and E7 proteins were evaluated by western blotting. Acute oral toxicity and DRF studies were performed in Swiss albino mice by following OECD guidelines 423 and 407, respectively. Tumor retardation study was done in C57BL/6 mouse papilloma model. The mice were divided into six groups: No tumor control (NTC), Tumor control (TC), Cisplatin (Cis) (4 mg/kg b.w.), PVaq 100, 200 mg/kg b.w and combination of PVaq (200 mg/kg b.w.) and Cisplatin (4 mg/kg b.w.). The mice were orally gavaged with PVaq daily for 14 days and cisplatin was given intravenously on every 1st, 5th and 9th day. Hematological and biochemical parameters were studied by using hematology analyzer and kits, respectively. E6 and E7 gene expression in the tumor samples was determined by qPCR. Th1 and Th2 cytokine levels were determined by ELISA. RESULTS: PVaq induced mitochondrial depolarization in SiHa and HeLa, and increased the expression of generic caspases, resulting into apoptosis. PVaq upregulated the expression of tumor suppressor proteins (p53 and pRb) and reduced the expression of viral oncoproteins (E6 and E7). Acute toxicity study displayed non-toxicity of PVaq while DRF study ensured its safe dose for further efficacy studies. PVaq reduced tumor volume and weight in mouse papilloma model and induced immunomodulation in the animals. It increased serum levels of IL-2 (Th1) with a concomitant decrease in IL-10 (Th2) cytokines. The drug did not affect body weight, food consumption and organ histopathology of the animals. CONCLUSIONS: PVaq exhibited anticancer and immunomodulatory activities against cervical cancer cells and female mouse papilloma model.

A polyherbal formulation, HC9 regulated cell growth and expression of cell cycle and chromatin modulatory proteins in breast cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: HC9, a polyherbal formulation, is based upon a traditional Ayurvedic formulation, Stanya Shodhana Kashaya (SSK, having 10 plant materials), formulated on Stanyashodhana gana, explained by Charaka in Charakasamhita Sutrasthana IV and mentioned in other texts as well. Stanyasodhana is the Sanskrit name for a group of medicinal plants, classified for "improving the quality of milk". SSK is used by Ayurvedic practitioners for the cleansing and detoxification of breast milk in lactating mothers as well as for the management of various clinical conditions. HC9 is composed of equal ratios of nine different medicinal plants that include Picrorhiza kurroa Royle ex Benth., Cyperus rotundus L., Zingiber officinale Roscoe, Cedrus deodara (Roxb. ex D.Don) G.Don, Tinospora cordifolia (Willd.) Miers, Holarrhena antidysenterica (Roth) Wall. ex A.DC., Swertia chirata Buch.-Ham. ex Wall., Cissampelos pareira L. and Hemidesmus indicus (L.) R. Br. ex Schult.. It differs from the SSK formulation by having one ingredient [Marsdenia tenacissima (Roxb.)Moon (Murva)] less, due to its unavailability since it is mostly found in tropical hilly tracts of peninsular India and Vindhya ranges as well as in lower Himalayan tracts. All the medicinal plants in the formulation have reported activity against different types of cancers. AIM OF THE STUDY: The present study is aimed at evaluating the anticancer activity of the polyherbal formulation (HC9) and its mechanism of action against breast cancer cell lines. MATERIALS AND METHODS: The effect of HC9 on the viability of breast cancer (MCF-7 and MDAMB231) and non-cancerous (MCF-10A) cell lines was evaluated by MTT assay. The effect on cell growth and colony formation potential of cancer cells was determined by trypan blue dye exclusion method and soft agar assay, respectively. Cell cycle arrest was determined by propidium iodide (PI) staining and analyzed by flowcytometer. Scratch wound assay was used for studying cell migration. Cell invasion was determined by using BD BioCoat Matrigel invasion chambers. The gene expression of HIF-1α was examined by RT-PCR. The expression of p53, SMAR1, p16, MMP-2, CDP/Cux, p21, Rb, phospo-Rb (ppRb), VEGF, NFқB and COX-2 proteins was determined by western blotting. RESULTS: HC9 significantly altered growth of breast cancer cell lines, MCF-7 and MDA MB-231. It blocked the cell cycle progression at S phase in MCF-7 by up regulating the expression of p53, p21 and p16 proteins. In MDA MB-231, HC9 induced G1 phase arrest by up regulating the expression of p53, p21 and pRb proteins with simultaneous decrease in ppRb. It significantly reduced migration and invasion in both the cell lines, accompanied by decrease in the expression of MMP-2/9, HIF-1α and VEGF. HC9 decreased the expression of inflammatory markers (NF-қB, COX-2), and modulated the expression of chromatin modulators (SMAR1 and CDP/Cux) in both MCF-7 and MDA MB-231. CONCLUSIONS: HC9 exhibited potent anticancer activity against breast cancer cells, thereby warranting further pre-clinical and clinical studies in future.

Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer.

Cervical cancer is the second leading cause of cancer death in women in India. Previously, we have reported that alpha linolenic acid (ALA), induced apoptosis in cervical cancer cell lines and reduced expression of E6 and E7 oncoproteins with simultaneous decrease in Cox2/VEGF/MAP kinase proteins. Here, we investigated the tumor retardation potential of flax oil (FO), rich in ALA, in mouse papilloma model. Flax oil significantly reduced tumor volume and weight in mice compared to the Tumor control (TC) group. Interestingly, compared to cisplatin (Cis) alone, there was slightly enhanced decrease in tumor weight when FO was given together with Cis (Cis + FO). A marked increase in plasma antioxidant levels in mice, and increase in lipid peroxidation in tumors with simultaneous decrease in liver tissues was observed in Cis + FO group compared to either TC or Cis groups. FO and Cis + FO significantly modulated immune response in mice by increasing CD8α and IFNγ and decreasing IL-4 expression. Interestingly, when given together with cisplatin, flax oil reduced HPV E6 and E7 oncoprotein expression with concomitant increase in the relative mRNA expression of tumor suppressor genes p53 and Rb. Thus, flax oil could be explored for its therapeutic potential in cervical cancer.

Triphala, Regulates Adipogenesis through Modulation of Expression of Adipogenic Genes in 3T3-L1 Cell Line.

BACKGROUND: Triphala, an Ayurvedic polyherbal formulation, is used for the treatment of various diseases including obesity. OBJECTIVE: The present study was planned to evaluate the anti-adipogenic potential of aqueous extract of Triphala (TPaq) using 3T3-L1 adipocyte cell line model. METHODS: The effect of aqueous extract of Triphala (TPaq) was tested on the viability of 3T3- L1 cells by MTT assay. The cells were treated with a cocktail of dexamethasone (DEX), isobutylmethylxanthine (IBMX) and insulin to induce adipogenesis. The cells were treated either with the induction cocktail or with the cocktail containing different concentrations (1, 10 and100 µg/ml) of TPaq. Intracellular lipid content was analyzed using Oil O Red stain and was quantified after extracting with isopropanol at 500 nm wavelength. The expression of early (PPAR-γ and C/EBP-α) and late (GLUT4 and FAS) phase adipogenic genes was studied by real time PCR. RESULTS: TPaq did not affect the viability of 3T3-L1 cell line. Interestingly, TPaq induced a concentration dependant decrease in the intracellular lipid content and expression of both early and late phase adipogenic genes. This decrease was statistically significant compared to cells treated with only induction cocktail. CONCLUSION: These results suggested that Triphala regulated lipid accumulation by down regulating expression of adipogenic genes, resulting into prevention of adipogenesis. SUMMARY: The purpose of this study was to evaluate the effect of an ayurvedic polyherbal drug Triphala on adipogenesis using 3T3-L1 cell line. The results suggested that Triphala regulated lipid accumulation by downregulating expression of adipogenic genes, resulting into the prevention of adipogenesis. Abbreviations used: TPaq: Aqueous extract of Triphala; DMEM: Dulbecco's Modified Eagle's medium; FBS: Fetal Bovine Serum; IBMX: Isobutyl methylxanthine; DMX: Dexamethasone; MTT: [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay; PPARγ: Peroxisome proliferator-activated receptor; C/EBP:Enhancer binding protein α, FAS:Fatty acid synthase; Glut-4: Glucose phosphate transporter 4.

Cinnamaldehyde, Cinnamic Acid, and Cinnamyl Alcohol, the Bioactives of Cinnamomum cassia Exhibit HDAC8 Inhibitory Activity: An In vitro and In silico Study.

BACKGROUND: The altered expression of histone deacetylase family member 8 (HDAC8) has been found to be linked with various cancers, thereby making its selective inhibition a potential strategy in cancer therapy. Recently, plant secondary metabolites, particularly phenolic compounds, have been shown to possess HDAC inhibitory activity. OBJECTIVE: In the present work, we have evaluated the potential of cinnamaldehyde (CAL), cinnamic acid (CA), and cinnamyl alcohol (CALC) (bioactives of Cinnamomum) as well as aqueous cinnamon extract (ACE), to inhibit HDAC8 activity in vitro and in silico. MATERIALS AND METHODS: HDAC8 inhibitory activity of ACE and cinnamon bioactives was determined in vitro using HDAC8 inhibitor screening kit. Trichostatin A (TSA), a well-known anti-cancer agent and HDAC inhibitor, was used as a positive control. In silico studies included molecular descriptor Analysis molecular docking absorption, distribution, metabolism, excretion, and toxicity prediction, density function theory calculation and synthetic accessibility program. RESULTS: Pharmacoinformatics studies implicated that ACE and its Bioactives (CAL, CA, and CALC) exhibited comparable activity with that of TSA. The highest occupied molecular orbitals and lowest unoccupied molecular orbitals along with binding energy of cinnamon bioactives were comparable with that of TSA. Molecular docking results suggested that all the ligands maintained two hydrogen bond interactions within the active site of HDAC8. Finally, the synthetic accessibility values showed that cinnamon bioactives were easy to synthesize compared to TSA. CONCLUSION: It was evident from both the experimental and computational data that cinnamon bioactives exhibited significant HDAC8 inhibitory activity, thereby suggesting their potential therapeutic implications against cancer. SUMMARY: Pharmacoinformatics studies revealed that cinnamon bioactives bound to the active site of HDAC8 enzyme in a way similar to that of TSAThe molecular descriptors of cinnamon compounds successfully correlated with TSA values. The binding interactions and energies were also found to be close to TSASynthetic accessibility values showed that cinnamon bioactives were easy to synthesize compared to TSA. Abbreviations used: ACE: Aqueous Cinnamon Extract; DFT: Density Function Theory; CAL: Cinnamaldehyde; CA: Cinnamic Acid; CALC: Cinnamyl Alcohol; MW: Molecular Weight; ROTBs: Rotatable Bonds; ROF: Lipinski's Rule of Five; TSA: Trichostatin A; PDB: Protein Data Bank; RMSD: Root Mean Square Deviation; HBA: Hydrogen Bond Acceptor; HBD: Hydrogen Bond Donor; ADMET: Absorption, Distribution, Metabolism, Excretion and Toxicity; FO: Frontier Orbital; HOMOs: Highest Occupied Molecular Orbitals; LUMOs: Lowest Unoccupied Molecular Orbitals; BE: Binding Energy.

Nardostachys jatamansi Root Extract Modulates the Growth of IMR-32 and SK-N-MC Neuroblastoma Cell Lines Through MYCN Mediated Regulation of MDM2 and p53.

AIM: The present study evaluated the effect of ethanolic extract of Nardostachys jatamansi roots (NJet) on MYCN mediated regulation of expression of MDM2 and p53 proteins in neuroblastoma cell lines, IMR-32 and SK-N-MC. MATERIALS AND METHODS: The effect of NJet on cell viability was determined by MTT; and on growth kinetics was evaluated by trypan blue dye exclusion method and soft agar assay. The expression of p53, MDM2 and MYCN proteins in response to NJet treatment was evaluated by immunoblotting. RESULTS: NJet decreased the viability of neuroblastoma cells without affecting the viability of non-cancerous, HEK-293 cells. It altered the growth kinetics of the cancer cells in a dose-dependent manner. NJet down regulated the expression of MYCN and MDM2 proteins with a simultaneous increase in the expression of tumor suppressor protein p53. CONCLUSIONS: The present data demonstrated that NJet regulated the growth of IMR-32 and SK-N-MC through reduction in MYCN expression that lead to down regulation of MDM2 protein and increase in p53 expression. These preliminary results warrant further in depth studies to explore the therapeutic potential of Nardostachys jatamansi in the management of neuroblastoma. SUMMARY: NJet reduced the viability of human neuroblastoma cell lines without affecting the viability of non-cancerous, HEK-293 cells.NJet regulated the growth kinetics of the cancer cells.NJet decreased the expression of MYCN and MDM2 proteins and simultaneously increased the expression of tumor suppressor protein p53. Abbreviation used: NJet: Ethanolic extract of Nardostachys jatamansi MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide HPTLC: High performance thin layer chromatography.

Evaluating the anti-inflammatory potential of Tectaria cicutaria L. rhizome extract in vitro as well as in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Tectaria cicutaria has been used in the folklore system of Indian traditional medicine (Ayurveda) for the treatment of various disorders such as rheumatic pain, chest complaints, burns, sprain, poisonous bites, tonsilitis, toothache, gum complaints, cuts and wounds. The present work has for the first time tried to elucidate the anti-inflammatory potential of aqueous extract of Tectaria cicutaria rhizome (TCRaq) in vitro as well as in vivo. MATERIALS AND METHODS: Anti-inflammatory potential of TCRaq was analyzed in vivo in carrageenan induced rat paw edema model. Serum antioxidant status in TCRaq-treated as well as untreated control rodents was measured by oxygen radical absorbance capacity (ORAC) assay. In vitro experiments for analyzing the anti-inflammatory potential of TCRaq were performed on murine macrophage cell line, RAW 264.7. Analysis of nitric oxide release in RAW 264.7 cells was done by Griess reaction. RT-PCR and western blotting experiment was performed to analyze the expression of iNOS. Expression of COX-2 and NFκB proteins was evaluated by western blotting. RESULTS: TCRaq significantly reduced the paw volume in Sprague-Dawley rats at a dose of 200mg/kg body weight, which was comparable with the standard diclofenac treatment. The rats treated with TCRaq showed a significant increase in the serum antioxidant levels compared to the untreated control animals. TCRaq was able to reduce the nitric oxide (NO) levels in RAW 264.7 cells that had been stimulated with lipopolysaccharide (LPS). This was accompanied by a corresponding decrease in iNOS expression at mRNA and protein level. Interestingly, TCRaq was found to decrease the expression of COX-2 as well as the nuclear translocation of NFκB in RAW 264.7 cells. CONCLUSION: Our study signifies the anti-inflammatory potential of Tectaria cicutaria and scientifically validates its traditional use in inflammatory conditions.