Mahendra K. Verma

Simultaneous determination of etoposide and a piperine analogue (PA-1) by UPLC–qTOF-MS: Evidence that PA-1 enhances the oral bioavailability of etoposide in mice

In the present investigation, a UPLC-qTOF-MS/MS method has been developed for the simultaneous determination of etoposide and a piperine analogue, namely, 4-ethyl 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1). The analytes were separated on a reverse phase C18 column using methanol-water (72:28, v/v) mobile phase with a flow rate of 250 microL/min. The qTOF-MS was operated under multiple reaction monitoring mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions for etoposide and PA-1 were at m/z 185.1350 and 164.1581, respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique. The total run time was 6 min and the elution of etoposide and PA-1 occurred at 1.24 and 2.84 min, respectively. The calibration curves of etoposide as well as PA-1 were linear over the concentration range of 2-1000 ng/mL (r(2), 0.9829), and 1-1000 ng/mL (r(2), 0.9989), respectively. For etoposide intra-assay and inter-assay accuracy in terms of % bias was in between -7.65 to +6.26, and -7.83 to +5.99, respectively. For PA-1 intra-assay and inter-assay accuracy in terms of % bias was in between -7.01 to +9.10, and -7.36 to +6.71, respectively. The lower limit of quantitation for etoposide and PA-1 were 2.0 and 1.0 ng/mL, respectively. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). The method was used for a pharmacokinetic study which showed that PA-1 enhanced the oral bioavailability of etoposide in mice by 2.32-fold.

Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Monarda citriodora.

We have isolated an essential oil from Monarda citriodora (MC) and characterized its 22 chemical constituents with thymol (82%), carvacrol (4.82%), β-myrcene (3.45%), terpinen-4-ol (2.78%) and p-cymene (1.53%) representing the major constituents. We have reported for the first time the chemotherapeutic potential of MC in human promyelocytic leukemia HL-60 cells by means of apoptosis and disruption of the PI3K/AKT/mTOR signaling cascade. MC and its major constituent, thymol, inhibit the cell proliferation in different types of cancer cell lines like HL-60, MCF-7, PC-3, A-549 and MDAMB-231. MC was found to be more cytotoxic than thymol in HL-60 cells with an IC50 value of 22 μg/ml versus 45 μg/ml for thymol. Both MC and thymol induce apoptosis in HL-60 cells, which is evident by Hoechst staining, cell cycle analysis and immuno-expression of Bcl-xL, caspase-3,-8,-9 and PARP-1 cleavage. Both induce apoptosis by extrinsic and intrinsic apoptotic pathways that were confirmed by enhanced expression of death receptors (TNF-R1, Fas), caspase-9, loss of mitochondrial membrane potential and regression of Bcl-2/Bax ratio. Interestingly, both MC and thymol inhibit the downstream and upstream signaling of PI3K/AKT/mTOR pathway. The degree of apoptosis induction and disruption of the PI3K signaling cascade by MC was significantly higher when compared to thymol.

Essential oil composition of Artemisia dracunculus L. (Tarragon) growing in Kashmir - India.

Abstract Two distinct chemotypes of Artemisia dracunculus were found in Kashmir. The oils from these two distinct chemotypes were analysed by GC and GC-MS. The chemical constituents of local variety were α-pinene (0.7%), myrecene (1.2 %), limonene (3.5 %), Z-(β)-ocimene (12.7 %), E-(β)-ocimene (4.6 %), α-terpinolene (2.7 %), 5-phenyl-l,3-Pentadiyne (5.1 %), methyl eugenol (0.7 %), capillene (60.2 %), elemicin (2.3 %), bicyclogermacrene (0.5 %), iso-elemicin (2.1 %) and germacrene-B (0.6 %). The major chemical constituents found in exotic variety are α-pinene (1.4 %), β-pinene (0.3 %), myrecene (0.1 %), p-cymene (0.2 %), limonene (2.8 %), Z-(β)-ocimene (1.6 %), E-(β)-ocimene (1.0 %), γ-terpinene (0.3 %) linalool (1.2 %), β-thujone (0.4 %),camphor (0.4 %), methyl chavicol (71.3 %), iso-menthol (0.1 %), bornyl acetate (0.7 %), carvacraol (7.7 %), α-terpinyl acetate (0.2 %), hexyl hexanoate (0.5 %), α-copaene (2.2 %), β-caryophyllene (0.2 %). The chemical constituents of the volatile oil are similar to French Tarragon.

EXTRACTION STUDIES OF PODOPHYLLUM HEXANDRUM USING CONVENTIONAL AND NONCONVENTIONAL METHODS BY HPLC–UV–DAD

The composition of lignans extracted from Podophyllum hexandrum rhizomes was studied by sequential extraction with supercritical CO2, ethyl acetate modified CO2 and methanol modified CO2. The results were compared with the extracts obtained by Accelerated Solvent Extraction (ASE) and soxhlets methods. The lignan contents comprised of Podophyllotoxin, deoxypodophyllotoxin, 4′-demethylpodophyllotoxin, Picropodophyllotoxin, Isopicropodophyllotoxin, Podophllotoxin-β-D-glucopyranoside, and 4′-demethylpodophyllotoxin β-D-glucopyranoside in the extracts of Podophyllum hexandrum rhizomes obtained by different methods was studied. There was a variation in the concentration of lignan in the extracts obtained by different methods of extraction. Podophyllotoxin formed the major component (36.55%) of the extract obtained by SFE and picropodophyllotoxin, isopicropodophyllotoxin, deoxypodophyllotoxin, and 4′-demethylpodophyllotoxin were present in 6.82, 1.51, 1.46, and 0.85%, respectively, whereas 4′-demethylpodophyllotoxin β-D-glucopyranoside was 1.89% in the extract obtained by ASE, which is higher than SFE extracts. Soxhlets derived extract contains better concentration of isopicropodophyllotoxin (24.49%) than SFE. A simple, isocratic, and reliable analytical HPLC-UV (DAD) method was developed for simultaneous identification and quantification of different seven lignans in the extracts of different extraction techniques.

Development of a validated UPLC–qTOF-MS/MS method for determination of bioactive constituent from Glycyrrhiza glabra

Abstract An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC–qTOF-MS/MS) method was developed and validated for the simultaneous determination of glycyrrhizin and glycyrrhetic acid. These analytes were separated on a reverse phase C18 column using a mobile phase of acetonitrile:2% acetic acid in water (75:25, v/v) with a flow rate of 200μL/min. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using the electrospray ionization (ESI) technique with positive ion polarity. A comparison of three different extraction techniques i.e. accelerated solvent extraction (ASE), extraction under ultrasonic waves (USW) and the classical extraction by percolation (CE) method was done and quantification of these extracts was also carried out by the proposed method.

Concentration dependent Electrospray Ionisation Mass Spectrometry and Tandem Mass Spectrometry (MS/MS ) studies on (E,E)-1-[5-(1,3-benzodioxol-5yl)-1-oxo-2,4-pentadienyl]- piperidine (Piperine) and its analogues

Studies on piperine ((M1)) and its synthetic analogues (M2–18) by positive electrospray ionisation mass spectrometry were carried out in the flow injection mode of analysis in methanol. The MS experiments on these compounds at concentration 5 ng/μL or above yielded dimeric ionic species [2 M + Na]+ which revealed that piperine and its analogues exhibit clustering of ions when the solutions of these compounds at concentrations 5 ng/μL or above were allowed to move through the electrospray interface of the mass spectrometer. The same clustering of the ions was not observed when the solutions of the same compounds at concentrations below 5 ng/μL were used for similar studies. The formation of the clusters was further confirmed by tandem mass spectrometry (MS/MS) studies wherein the fragmentation of dimeric ionic species [2 M + Na]+ led to the formation of sodium adducted monomeric ionic species [M + Na]+. The MS measurements of these compounds by Atmospheric Pressure Chemical Ionisation (APCI) were on expected lines as there was no clustering of the ions in case of APCI-MS measurements.

Room Temperature, Metal-catalyzed Oxidative Acylation of Electron-deficient Heteroarenes with Alkynes, its Mechanism and Application Studies

Herein, we report an original one-step, simple, room-temperature, regioselective Minisci reaction for the acylation of electron-deficient heteroarenes with alkynes. The method has broad functional group compatibility and gives exclusively monoacylated products in good to excellent yields. The mechanistic pathway was analyzed based on a series of experiments confirming the involvement of a radical pathway. The 18O-labeling experiment suggested that water is a source of oxygen in the acylated product, and head space GC-MS experiment shows the C-C cleavage occurs via release as CO2.

In vitro acaricidal activity of Piper nigrum and Piper longum fruit extracts and their active components against Rhipicephalus (Boophilus) microplus ticks

In vitro acaricidal activity of Piper nigrum and P. longum fruit extracts and their active components (piperine for P. nigrum and piperine and piperlonguminine for P. longum) was evaluated against adults engorged females of Rhipicephalus (Boophilus) microplus using adult immersion test. Three concentrations of each extract with four replications were used in the bioassay. Extracts significantly affected mortality rates of ticks in dose-dependent manner ranged 12.5–95.8% for P. nigrum and 29.2–87.5% for P. longum, with an additional effect on the reproductive physiology of ticks by inhibiting oviposition (28.1–96.9% by P. nigrum and 36.1–89.3% by P. longum). However, the acaricidal and oviposition limiting properties were decreased significantly when the active component(s) of each extract was tested separately. However, the combination of piperine and piperlonguminine (obtained from P. longum extract) caused 79.2% mortality of ticks which is equivalent to the corresponding concentration (~ 5%) of the extract. It can be concluded that the fruit extracts of P. nigrum and P. longum had both acaricidal and oviposition limiting actions against the adults of R. (B.) microplus which could make it a valuable component of developing sustainable strategy for integrated tick management.

An endophytic Fusarium sp. isolated from Monarda citriodora produces the industrially important plant-like volatile organic compound hexanal

An endophytic fungus, MC_25L, has been isolated from the leaves of MonardacitriodoraCerv. ex Lag., a medicinal and aromatic herb from the northwestern Himalayas. It produces a fruity fragrance while growing on potato dextrose agar, suggesting that it is producing volatile organic compounds (VOCs). The endophyte inhibited the growth of plant pathogens such asSclerotiniasp. and Aspergillusflavus by virtue of VOCs. Identification of MC_25L based on morphological and microscopic features, as well as ITS-based rDNA sequence analysis, revealed that it is a Fusariumsp. GC-MS analysis revealed that this endophyte produces a unique array of VOCs, in particular hexanal, p-fluoroanisole, pentafluoropropionic acid 2-ethylhexyl, (5E)-5-ethyl-2-methyl-5-hepten-3-one, 2-butyl-2-hexanol, (7E)-2-methyl-7-hexadecene and acoradiene. Three major compounds were hexanal, (5E)-5-ethyl-2-methyl-5-hepten-3-one and acoradiene, and they account for around 84.57 % of the total VOCs. Moreover, of interest was the presence of hexanal, which has applications in the food and cosmetic industries, as well as in mycofumigation. This is the first report of a fungal endophyte producing the industrially important plant-like VOC hexanal. Hexanal is also active biologically. Thus this study indicates that Fusariumsp. (MC_25L) is a potential candidate for the up-scaling of hexanal.

Isolation, Chemical Fingerprinting and Simultaneous Quantification of Four Compounds from Tanacetum gracile Using a Validated HPLC-ESI-QTOF-Mass Spectrometry Method.

The present study was conducted to carry out the phytochemical investigation of Tanacetum gracile Hook. f. & Thomson and to develop a method for the simultaneous quantification of the isolated compounds in the extracts ofT. gracile growing in different locations. Cluster analysis rectangular similarity matrix was performed to understand the chemical fingerprinting variations in the extracts. High-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight-mass spectrometry (HPLC-ESI-QTOF-MS) was used to quantify four bioactive compounds, and separation of the compounds was achieved on a reverse-phase C8 column using a mobile phase of acetonitrile: 0.1% formic acid in water with a gradient elution by maintaining the flow rate of 300 μL/min. The QTOF-MS was operated using the electro-spray ionization technique with the positive ion polarity mode. The calibration curves of four marker compounds were linear over the concentration range of 3.12-100 ng/µL (R(2)> 0.996). A specific, accurate and precise HPLC-ESI-QTOF-MS method was optimized for the determination of kaempferol, ketoplenolide, tetramethoxyflavone and artemetin both individually and simultaneously. Quantification of these chemical markers in different extracts was carried out using this validated method. Kaempferol was isolated for the first time from T. gracile.