LC-MS/MS method for the simultaneous quantification of luteolin, wedelolactone and apigenin in mice plasma using hansen solubility parameters for liquid-liquid extraction: Application to pharmacokinetics of Eclipta alba chloroform fraction.

Eclipta alba (Bhringraj) in ayurveda has been widely used as a traditional medicine for its multi-therapeutic properties for ages. Luteolin (LTL), wedelolactone (WDL) and apigenin (APG) are the three main bioactive phytochemicals present in Eclipta alba extract. However there was a lack of sensitive bioanalytical method for the pharmacokinetics of these free compounds in plasma which majorly contributes for their activities after oral administration of Eclipta alba. The present study aims to develop a sensitive, rapid and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of mice plasma concentrations of LTL, WDL and APG using quercetin as an internal standard for the pharmacokinetic analysis. Analytes were separated on Phenomenex Luna C18 (150 × 4.6 mm, 3.0 µm) column with mobile phase containing methanol: acetonitrile (90: 10, v/v) and 0.1% formic acid in 10 mM ammonium formate buffer in the ratio of 70: 30 (v/v) in isocratic mode. Liquid-liquid extraction was optimized using Hansen solubility parameters and diethyl ether finalized as an extraction solvent for the recovery ranging from 61 to 76% for all analytes in mice plasma. The validated method has an accuracy and precision over the linearity range of 0.1-200 ng/mL with a correlation coefficient (r2) of ≥0.997. The intra and inter-day assay accuracy was between 98.17 and 107% and 95.83-107.89% respectively and the intra and inter day assay precision ranged from 0.37-6.05% and 1.85-10.76%, respectively for all the analytes. This validated method can be used for future clinical investigation studies of Eclipta alba extracts.

Anti-breast tumor activity of Eclipta extract in-vitro and in-vivo: novel evidence of endoplasmic reticulum specific localization of Hsp60 during apoptosis.

Major challenges for current therapeutic strategies against breast cancer are associated with drug-induced toxicities. Considering the immense potential of bioactive phytochemicals to deliver non-toxic, efficient anti-cancer therapeutics, we performed bio-guided fractionation of Eclipta alba extract and discovered that particularly the chloroform fraction of Eclipta alba (CFEA) is selectively inducing cytotoxicity to breast cancer cells over non-tumorigenic breast epithelial cells. Our unbiased mechanistic hunt revealed that CFEA specifically activates the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential, upregulating Hsp60 and downregulating the expression of anti-apoptotic protein XIAP. By utilizing Hsp60 specific siRNA, we identified a novel pro-apoptotic role of Hsp60 and uncovered that following CFEA treatment, upregulated Hsp60 is localized in the endoplasmic reticulum (ER). To our knowledge, this is the first evidence of ER specific localization of Hsp60 during cancer cell apoptosis. Further, our LC-MS approach identified that luteolin is mainly attributed for its anti-cancer activities. Moreover, oral administration of CFEA not only offers potential anti-breast cancer effects in-vivo but also mitigates tumor induced hepato-renal toxicity. Together, our studies offer novel mechanistic insight into the CFEA mediated inhibition of breast cancer and may potentially open up new avenues for further translational research.

Hematological malignancies: role of miRNAs and their in silico aspects.

Hematological malignancies is a broad term that includes blood cell cancers including chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), Myelodysplastic syndrome, acute lymphocytic leukemia (ALL), multiple myelomas (MM) and lymphomas. miRNAs are ~22-nt long non-coding RNAs that play a very important role in gene regulation by binding to mRNA at their complementary sequence. These miRNAs are conceptually connected with various signal and pathway networks that make them capable of regulating various diseases including hematological malignancies. These miRNAs are not only playing regulatory roles in hematological malignancies, but are also providing new potent markers for efficient diagnosis and prognosis for hematological malignancies patients. Since the discovery of very first miRNA, the importance and role of miRNAs have been established in various fields, and there is a need to search for new potent miRNAs and their targets. A large amount of sequence data have been generated in last few years, which has further generated the need to develop efficient and reliable computational tools to analyze and extract out relevant information promptly from raw data. Here, we review various possible roles played by miRNA in hematological malignancies, principles involved in miRNA gene identification, target prediction and their preceding role in hematological malignancies research.