Genome-wide DNA methylation profiling after Ayurveda intervention to bronchial asthmatics identifies differential methylation in several transcription factors with immune process related function.

BACKGROUND: The Indian traditional medicinal system, Ayurveda, describes several lifestyle practices, processes and medicines as an intervention to treat asthma. Rasayana therapy is one of them and although these treatment modules show improvement in bronchial asthma, their mechanism of action, particularly the effect on DNA methylation, is largely understudied. OBJECTIVES: Our study aimed at identifying the contribution of DNA methylation changes in modulating bronchial asthma phenotype upon Ayurveda intervention. MATERIALS AND METHODS: In this study, genome-wide methylation profiling in peripheral blood DNA of healthy controls and bronchial asthmatics before (BT) and after (AT) Ayurveda treatment was performed using array-based profiling of reference-independent methylation status (aPRIMES) coupled to microarray technique. RESULTS: We identified 4820 treatment-associated DNA methylation signatures (TADS) and 11,643 asthma-associated DNA methylation signatures (AADS), differentially methylated [FDR (≤0.1) adjusted p-values] in AT and HC groups respectively, compared to BT group. Neurotrophin TRK receptor signaling pathway was significantly enriched for differentially methylated genes in bronchial asthmatics, compared to AT and HC subjects. Additionally, we identified over 100 differentially methylated immune-related genes located in the promoter/5'-UTR regions of TADS and AADS. Various immediate-early response and immune regulatory genes with functions such as transcription factor activity (FOXD1, FOXD2, GATA6, HOXA3, HOXA5, MZF1, NFATC1, NKX2-2, NKX2-3, RUNX1, KLF11), G-protein coupled receptor activity (CXCR4, PTGER4), G-protein coupled receptor binding (UCN), DNA binding (JARID2, EBF2, SOX9), SNARE binding (CAPN10), transmembrane signaling receptor activity (GP1BB), integrin binding (ITGA6), calcium ion binding (PCDHGA12), actin binding (TRPM7, PANX1, TPM1), receptor tyrosine kinase binding (PIK3R2), receptor activity (GDNF), histone methyltransferase activity (MLL5), and catalytic activity (TSTA3) were found to show consistent methylation status between AT and HC group in microarray data. CONCLUSIONS: Our study reports the DNA methylation-regulated genes in bronchial asthmatics showing improvement in symptoms after Ayurveda intervention. DNA methylation regulation in the identified genes and pathways represents the Ayurveda intervention responsive genes and may be further explored as diagnostic, prognostic, and therapeutic biomarkers for bronchial asthma in peripheral blood.

Ketorolac modulates Rac-1/HIF-1α/DDX3/ß-catenin signalling via a tumor suppressor prostate apoptosis response-4 (Par-4) in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20-30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/ß-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/ß-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.

Molecular pharmacology of multitarget cyclin-dependent kinase inhibitors in human colorectal carcinoma cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. OBJECTIVE: Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. METHOD: Protein expression was analyzed for key signaling proteins by western blotting. ß-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. RESULTS: CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. CONCLUSION: Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.

Isolation, Culture and Morphological Assessment of Primary Cell Lines from Human Primary Oral Squamous Cell Carcinoma Using Explant Technique.

BACKGROUND: Due to many uses of cell culture in cell biology, biotechnology, and medical research, this technique has evolved into a widely used and accepted methodology. The isolation of primary cells from primary cancer tissue is a crucial step in cell culture technology since it offers a trustworthy source for studying the biology, morphology, and molecular evaluation of cancer cells, just like in the oral cavity tissue of patients. Therefore, the technique used for the isolation, culture, and evaluation of these cells is crucial. AIM: The aim of the present study is to isolate and culture the cells from human primary Oral Squamous Cell Carcinoma [OSCC] tissue and evaluate them for morphological variations using an explant method. MATERIALS AND METHODS: The patients with OSCC who were undergoing surgery provided the tissue samples. An explant technique was used to achieve the isolation of cells from tissue samples. Following that, the cells were maintained, subcultured, and stored in accordance with the standard American Type Culture Collection [ATCC] protocol. Routine Hematoxylin & Eosin and crystal violet stains were used. These cells were morphologically studied, and the results were assessed for further studies. RESULTS: We were able to successfully isolate and culture cells from 4 different tissue samples using the explant method. Morphological analysis revealed that one tissue had a significantly distinct presentation of epithelial and stromal cells, whereas the other three tissues had only minor morphological differences predominantly stromal cells. Two tissues were discarded after showing contamination. CONCLUSION: Tissue culture should be done very meticulously specially when oral cavity tissue is used as it is house for millions of microorganisms. The technique must also be thoroughly followed and adjusted accordingly. Using common, inexpensive stains like Hematoxylin and Eosin and crystal violet, which are of great help for examining the morphology of cells routinely.

Efficacy and safety of polyherbal formulation as an add-on to standard-of-care in mild-to-moderate COVID-19: A randomized, double-blind, placebo-controlled trial.

Background: Novel corona virus disease-2019 (COVID-19) pandemic is a significant contributor to morbidity and mortality in affected individuals. Modulating the immune response in COVID-19 is now an established treatment approach. Polyherbal formulations have long been assessed for their potential immune modulating effects and are expected to be beneficial on COVID-19. Methods: This study aims at assessing the efficacy and safety of polyherbal formulation (referred as IP) in comparison to placebo, as add on to the standard of care (SOC), in patients with mild to moderate COVID-19 patients. Hospitalized RT-PCR positive patients were randomized to either SOC + IP or SOC + Placebo arm. The viral load (VL) was assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Immunological parameters were also assessed. The clinical improvement was assessed using a numeric rating scale (NRS) and WHO ordinal scale, and follow-up period was 30 days. Results: Seventy-two patients were randomized to SOC + IP (n = 39) and SOC + Placebo (n = 33) arms. There was significant reduction in VL in SOC + IP arm from day 0-4 (p = 0.002), compared to SOC + Placebo arm (p = 0.106). Change in the NRS score and WHO score was significant in both arms, however, the difference between the two arms was statistically significant in favour of IP arm. The increase in Th1 response was significant in SOC + IP arm (p = 0.023), but not in SOC + Placebo arm. COVID-19 specific antibodies were numerically higher in the SOC + IP arm. Conclusion: The study finds that polyherbal formulation significantly reduces VL and contributes to immunomodulation and improvement in clinical conditions without side effects.

Systemic explanation of Glycyrrhiza glabra's analyzed compounds and anti-cancer mechanism based on network pharmacology in oral cancer.

OBJECTIVES: Several studies suggest that Glycyrrhiza glabra (GG) extract could be a useful supplemental source for various cancer treatments. However, very few studies on oral cancer (OC) have been conducted. The present study was aimed at exploring the bioactive compounds (bioactives) along with the mode of action of GG against OC using network pharmacology. METHODS: Liquid chromatography-mass spectrometry/mass spectrometry was used to identify and analyze compounds from GG. Public databases were used to identify genes associated with the selected bioactives and OC. With the help of Cytoscape software, the association between bioactive and common genes was built, visualized, and investigated. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to investigate protein-protein interactions for intergenic interactions. Finally, the pathway enrichment analysis of common genes was done using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. RESULTS: Overall, 378 bioactives were identified in GG. Using public databases, an entire 254 bioactive-related genes and 734 OC-related genes were recognized, with 48 common genes. Cytoscape analysis showed wortmannin as the key bioactive and androgen receptor as the hub gene. The DAVID results revealed that the significant mechanism of action of GG against OC may be to induce apoptosis of cancer cells by deactivating the PI3K-AKT signaling pathway. CONCLUSION: The key active components and mechanisms of action of GG against OC were investigated. The present study provides scientific suggestions to support the clinical outcome of GG for OC along with a research foundation for additional elaboration on the important bioactives and mechanisms of GG against OC.

Prostate Apoptosis Response-4 (Par-4): A Novel Target in Pyronaridine-Induced Apoptosis in Glioblastoma (GBM) Cells.

Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma samples of GBM patients were significantly lower than normal healthy volunteers. Thus, our study demonstrates for the first time that PYR can be repurposed against GBM with a novel mechanism of action involving Par-4. Herewith, we discuss the role of upregulated Par-4 in a highly interconnected signaling network thereby advocating its importance as a therapeutic target.

Identification and characterization of a hydrophobin Vmh3 from Pleurotus ostreatus.

Hydrophobins (HPs) are relatively small surface-active proteins of fungal origin. Being an industrially important protein, isolation of new molecules from GRAS (Generally Regarded as Safe) strains like mushrooms is the need of the time. In the present work, hydrophobin Vmh3-1 is isolated, purified, and identified from a culture broth and vegetative mycelia of Pleurotus ostreatus grown in a Potato dextrose broth (PDB) in static culture conditions. Purified proteins from the broth and the cell wall showed bands of 11 kDa and 17 kDa when analyzed on SDS-PAGE. Hydrophobin Vmh3-1 was identified in purified protein samples by the Orbitrap-HR-LC-MS/MS analysis with a maximum of 66% sequence coverage. The amphipathic nature of the protein was revealed by an increase in the water contact angle (WCA) of the hydrophilic surface of glass by 87% as well as a decrease in the WCA of the hydrophobic surface of Teflon by 19%. The emulsification property was tested with food-grade oils and Hexane. A maximum activity (EI 24) of 87.64% was recorded for Sunflower oil. In CD (Circular dichroism) spectra, Vmh3-1 showed the typical spectra of hydrophobin with a dominance of ß-sheets (51%) in the secondary structure and a minimum percentage of the α-helix (2%). The protein did not show a self-aggregating property on vigorous shaking making it suitable for numerous industrial applications. The identification of Vmh3-1 with detailed amino acid sequencing and the characterization of the protein to evaluate its potential in surface modifications for various industrial applications is demonstrated herein for the first time.

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.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. OBJECTIVE: The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. METHODS: Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. RESULTS: Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. CONCLUSION: This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

Utility of serum homocysteine in oral squamous cell carcinoma patients as a potential biomarker.

Background: In India, Oral cancer is one of the most common cancers. Despite advances in treatments, prognosis for oral cancer has remained poor with a five-year survival rate of 40-50%. Therefore, it is necessary to develop effective diagnostic methods for early diagnosis and better prognosis. Homocysteine (Hcy) has been reported as a 'tumour marker' in various cancers such as breast cancer, colorectal cancer, lung cancer, cervical cancer. Aim: To study the levels of serum Hcy in oral squamous cell carcinoma (OSCC) patients. Objectives: To assess the clinical utility of serum Hcy as a potential tumour marker for OSCC cases. Methodology: Serum Hcy levels were studied and compared between patients with OSCC and healthy individuals. Results: Serum Hcy levels were higher in patients having OSCC. Conclusion: Serum Hcy levels could be utilized as a biological marker in the diagnosis and the prognosis of OSCC patients.

Exploring AyuGenomics approach for understanding COVID-19 predisposition and progression.

Recent reports on COVID-19 suggest that, the susceptibility to COVID-19 infection and its progression have a genetic predisposition. Majorly associated genetic variants are found in human leukocyte antigen (HLA), angiotensin convertase enzyme (ACE; rs1799752: ACE2; rs73635825), and transmembrane protease serine 2 (TMPRSS-2; rs12329760) genes. Identifying highly prone population having these variants is imperative for determining COVID-19 therapeutic strategies. Ayurveda (Indian traditional system of medicine) concept of Prakriti holds potential to predict genomic and phenotypic variations. Reported work on Prakriti correlates HLA-DR alleles with three broad phenotypes (Tridosha) described in Ayurveda (AyuGenomics). This is suggestive of differences in immune responses in individuals with specific constitutions. Therefore, the reported studies provide clues for clinically relevant hypotheses to be tested in systematic studies. The proposed approach of Ayurveda-based phenotype screening may offer a way ahead to design customized strategies for management of COVID-19 based on differences in Prakriti, immune response, and drug response. However, this needs clinical evaluation of the relation between Prakriti and genetic or phenotypic variants in COVID-19 prone and resistant populations.

Mesenchymal stem cells as a platform for research on traditional medicine.

The translation of Traditional Medicines (TMs) such as Ayurveda, and Traditional Chinese Medicine into clinical practice remains obstructed due to lack of scientific evidence by means of safety, quality, standardization, clinical efficacy, and mode of action. These limitations can be attributed to the lack of synonymous invitro models which reflect invivo features. Human mesenchymal stem cells (hMSCs) have emerged as an efficient cell source for regenerative medicine and tissue engineering. In this review, the authors discuss how hMSCs can be used as an invitro platform to screen herbs described in TMs using modern methods such as evaluation of its potential, safety, quality, mode of action, etc. Integration of traditional knowledge systems like Ayurveda and hMSCs as a platform to screen and study TMs using modern tools will effectively increase the validity of TMs as evidence-based medicine.

Mesenchymal stem cell conditioned medium ameliorates diabetic serum-induced insulin resistance in 3T3-L1 cells.

BACKGROUND: Pharmacological factors used to induce insulin resistance (IR) in in vitro models may not mimic the full in vivo features of type 2 diabetes mellitus (T2DM). This study aimed to examine the ability of diabetic serum (DS) to induce IR and investigate whether adipose-derived mesenchymal stem cell conditioned medium (ADMSC-CM) reverses DS-induced IR. METHODS: DS was obtained from newly diagnosed T2DM patients. IR was induced in differentiated 3T3-L1 cells by employing dexamethasone, tumor necrosis factor alpha (TNF-α), palmitate and DS. Glucose uptake (2-[N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino]-2-deoxyglucose(2-NBDG) uptake assay), intracellular levels of reactive oxygen species (ROS), and superoxide radicals (O2-) (fluorescence microscopy and fluorometry) were analyzed in control and experimental samples. mRNA expression of key genes involved in glucose transport and inflammation were analyzed by using reverse transcription polymerase chain reaction (RT-PCR). Pro-inflammatory cytokines and phospho-insulin receptor substrate (IRS) (Ser-307) protein expression were analyzed by fluorescence activated cell sorter analysis. Statistical significance was determined by using one-way ANOVA followed by Tukey's multiple comparison tests. RESULTS: ADMSC-CM significantly increased the DS-mediated decrease in 2-NBDG uptake (11.01 ± 0.50 vs. 7.20 ± 0.30, P < 0.01) and reduced DS-driven ROS (fluorescence count, 6.35 ± 0.46 vs. 9.80 ± 0.10, P < 0.01) and O2- (fluorescence count, 3.00 ± 0.10 vs. 4.60 ± 0.09, P < 0.01) production. Further, the ADMSC-CM restored DS-induced down regulation GLUT4 (1.52-fold, P < 0.05) as well as the up-regulation of PPARγ (0.35-fold, P < 0.01), and IKKß (0.37-fold, P < 0.01) mRNA, and phospho-IRS (Ser-307) protein expression compared to the baseline (median fluorescence intensity, 88,192 ± 2720 vs. 65,450 ± 3111, P < 0.01). DS induced IR, similar to the traditionally used pharmacological factors, namely dexamethasone, TNF-α, and palmitate, which can be attributed to the significantly higher pro-inflammatory cytokines levels (TNF-α (2.28 ± 0.03 pg/mL vs. 2.38 ± 0.03 pg/mL, P < 0.01), interleukin 6 (IL)-6 (1.94 ± 0.02 pg/mL vs. 2.17 ± 0.04 pg/mL, P < 0.01), IL-17 (2.16 ± 0.02 pg/mL vs. 2.22 ± 0.002 pg/mL, P < 0.05), and interferon gamma (IFN-γ) (2.07 ± 0.02 pg/mL vs. 2.15 ± 0.04 pg/mL, P < 0.05)) in DS. CONCLUSIONS: DS can be explored as a novel inducer of IR in in vitro studies with further standardization, substituting the conventionally used pharmacological factors. Our findings also affirm the validity of ADMSC-CM as a prospective insulin sensitizer for T2DM therapy.

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.

Two new polyoxygenated flavonoids from Blumea eriantha DC, methanol extract and their anti-proliferative activity.

Two new flavonoids, 3, 3', 4' -Trihydroxy- 6, 7, 8 -trimethoxy flavone (1) and 3-Hydroxy-6, 7, 8, 3', 4'-pentamethoxy flavone (2) have been isolated from the methanol extract of Blumea eriantha DC. The structures of these compounds were determined on the basis of spectroscopic techniques such as IR, MS, 1 D, and 2 D NMR spectroscopy. The compounds (1) and (2) were tested for their anti-proliferative activity on NCI-H23 cell line. A standard drug Paclitaxel showed potent anti-proliferative effect (56%) at 100 nM concentration after 24 h treatment whereas compound (2) did not show any anti-proliferative effect. Only compound (1) showed significant reduction in cell viability at concentration 25773 nM (89%) and 257731 nM (68%) after 24 h when compared with 0.1% DMSO. The compound (1) showed significant but much lower reduction in cell viability as compared to the standard drug Paclitaxel.