K.B. Rameshkumar

Structural characterization of flavonoid C- and O-glycosides in an extract of Adhatoda vasica leaves by liquid chromatography with quadrupole time-of-flight mass spectrometry.

RATIONALE Adhatoda vasica Nees is a well-known Ayurvedic medicinal plant, belonging to the family Acanthaceae. This study aims to seek identification and characterization of flavonoid C- and O-glycosides in the aqueous fraction of the plant leaves. METHODS A method was developed for simultaneous characterization of flavonoids and their glycosides using high-pressure liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC/ESI-QTOF-MS/MS). The chromatographic separation was carried on an Agilent Poroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 µm) operated with 0.1% formic acid aqueous solution and methanol as the mobile phase. RESULTS The fragmentations of the studied [M-H](-) ions of C-glycosides were shown to be cross-ring cleavages of the glycoside moiety [M-H-(60/90/120)](-) whereas O-glycosides were shown to eliminate the sugar moiety (Y0 (-) or [Y0 -H](-) ) from the aglycone unit; 6-C-glycosides exhibited [M-H-18](-) , a characteristic ion, and also a higher abundance of (0,3) X6 or 8 ions in comparison to 8-C glycosides; flavonoid 6,8-di-C-glycosides exhibited cross-ring cleavages of the sugar attached to the C-6 position preferentially. CONCLUSIONS This method was successfully applied for analysis of flavonoids and their glycosides in Adhatoda vasica leaves. A total of 29 compounds were tentatively identified including 17 C-, nine O-glycosides and three flavonoids.

Rapid fingerprinting of Rauwolfia species using direct analysis in real time mass spectrometry combined with principal component analysis for their discrimination

Medicinal plants of the genus Rauwolfia (Apocynaceae) are extensively used as folk medicines worldwide. Its antihypertensive activity is well known due to the presence of monoterpene indole alkaloids (MIAs). The therapeutic potential of the herbal medicines are affected due to variation of bioactive phytoconstituents. Therefore, a rapid and validated method was developed for fingerprinting of roots and leaves of six Rauwolfia species by direct analysis in real time mass spectrometry (DART-MS). Seventeen bioactive MIAs were tentatively identified on the basis of their exact mass measurement from the intact plant parts. Furthermore, principal component analysis (PCA) was used to analyze the DART-MS data of six Rauwolfia species to identify the chemical markers. Thirteen and twenty-three chemical markers were identified from the roots and leaves which were able to discriminate among six Rauwolfia species. This method was also cross-validated for the rapid identification, authentication and quality control of Rauwolfia species.

Rapid screening and quantitative determination of bioactive compounds from fruit extracts of Myristica species and their in vitro antiproliferative activity

Efficient and sensitive LC-MS/MS methods have been developed for the rapid screening and determination of bioactive compounds in different fruit parts of four Myristica species, viz., Myristica beddomeii, Myristica fragrans, Myristica fatua and Myristica malabarica. Twenty-one compounds were identified and characterized on the basis of their accurate mass and MS/MS fragmentation pattern using HPLC-QTOF-MS/MS and NMR analysis. Quantitative determination of five major bioactive compounds was performed using multiple-reaction monitoring mode with continuous polarity switching by UHPLC-QqQLIT-MS/MS. Moreover, in vitro antiproliferative activity of these Myristica species was evaluated against five human cancer cell lines A549, DLD-1, DU145, FaDu and MCF-7 using SRB assay. Seventeen phytoconstituents were identified and reported for the first time from M. beddomeii and sixteen from M. fatua. Quantification result showed highest total content of five major bioactive compounds in mace of M. fragrans. Evaluation of in vitro antiproliferative activity revealed potent activity in all investigated species except M. fragrans.

Ultra high performance liquid chromatography tandem mass spectrometry method for the simultaneous determination of multiple bioactive constituents in fruit extracts of Myristica fragrans and its marketed polyherbal formulations using a polarity switching technique

Fruits of Myristica fragrans Houtt. are the source of two valuable spices: nutmeg and mace, traditionally used for its flavoring and medicinal properties and found as an ingredient in many marketed polyherbal formulations and food products. In this study, a sensitive and efficient ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the rapid determination of 16 bioactive constituents in different parts of the fruit of M. fragrans and its marketed polyherbal formulations using a polarity switching technique. Chromatographic separation was achieved on an Aquity UPLC BEH C18 column in 9.4 min. Quantitative analysis was performed using multiple reaction monitoring mode with continuous polarity switching in a single analysis. The developed method was found to be accurate with overall recovery in the range from 95.95 to 102.07% (RSD ≤ 1.91%), precise (RSD ≤ 1.98%), and linear (r(2) ≥ 0.9992) over the concentration range of 0.1-200 ng/mL. Quantitative analysis indicated that the total content of the 16 bioactive constituents was highest in the mace of M. fragrans. Thus, this rapid and sensitive method could be utilized as a promising reference method for the quality control of M. fragrans and its marketed herbal formulations/food products.

Screening of tricyclic quinazoline alkaloids in the alkaloidal fraction of Adhatoda beddomei and Adhatoda vasica leaves by high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE Adhatoda beddomei and Adhatoda vasica are popular Ayurvedic medicinal plants in India, belonging to the family Acanthaceae. Tricyclic quinazoline alkaloids are found to be the most abundant in these plants which are responsible for broad-spectrum medicinal properties. This study aims to seek identification and characterization of those alkaloids based on their fragmentation patterns. METHODS A method was developed to elucidate the main fragmentation pathways of tricyclic quinazoline alkaloids in positive ion mode using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). Chromatographic separation was carried on a Supelco Discovery HS C18 column (15 cm × 4.6 mm, 3 µm) with 0.1% formic acid aqueous solution and acetonitrile as a mobile phase. RESULTS In full scan mass spectra, protonated molecules were observed for all the quinazoline alkaloids. Ring cleavages of the tricyclic quinazoline moiety were observed in MS(2) spectra and the characteristic ions provide valuable structural information of these alkaloids. Fragmentation pathways and fragment ion structures were proposed in two groups of quinazoline alkaloids. CONCLUSIONS The established fragmentation patterns have been successfully used to identify 23 tricyclic quinazoline alkaloids in the alkaloidal fraction of A. beddomei and A. vasica.

Comparative Phytochemical Evaluation and Antioxidant Assay of Piper longum L. and Piper chaba Hunter Used in Indian Traditional Systems of Medicine

Fruits of Piper longum (Indian tipali) and Piper chaba (Bangla tipali) are widely used in traditional Indian systems of medicine, and the latter is considered as a substitute for the former. Phytochemical evaluations of these two species were carried out with reference to the volatile chemical profiles of the leaves and fruits, and piperine content of the fruits and their antioxidant potential were compared to determine the propriety of using P. chaba as a substitute for P. longum. The leaf oil of P. longum was rich with the phenyl propanoids apiole (50.0%) and myristicin (26.9%), whereas sesquiterpene hydrocarbons β-caryophyllene (28.6%), α-humulene (22.8%), and germacrene D (14.6%) were the major constituents in P. chaba leaf oil. In the fruit oil of P. longum, aliphatic compounds predominated with n-pentadecane (15.8%), whereas P. chaba fruit oil mainly had sesquiterpene hydrocarbons such as germacrene D (21.5%), β-caryophyllene (18.5%), and α-humulene (11.4%). In P. longum fruits, piperine content was lower (0.03%) compared to P. chaba fruits (1.32%). In P. chaba, the piperamides were mainly piperine, whereas other piperamides were predominant in P. longum. Antioxidant activity was higher for P. longum fruits as compared to P. chaba fruits.

Fukugiside, a biflavonoid from Garcinia travancorica inhibits biofilm formation of Streptococcus pyogenes and its associated virulence factors

Purpose. Streptococcus pyogenes, a notorious human pathogen thatis responsible for various invasive and non‐invasive diseases, possesses multiple virulence armaments, including biofilm formation. The current study demonstrates the anti‐biofilm and anti‐virulence potential of fukugiside, a biflavonoid isolated from Garciniatravancorica, against S. pyogenes. Methodology. The anti‐biofilm activity of fukugiside was assessed and established using microdilution and microscopic analysis. Biochemical assays were performed to assess the effects of fukugiside on important virulence factors, which were further validated using quantitative real‐time PCR and in vivo analysis in Caenorhabditis elegans. Results. Fukugiside exhibited concentration‐dependent biofilm inhibition (79 to 96 %) against multiple M serotypes of S. pyogenes (M1, M56, M65, M74, M100 and st38) with a minimum biofilm inhibitory concentration of 80 &mgr;g ml−1. Electron microscopy and biochemical assay revealed a significant reduction in extracellular polymeric substance production. The results for the microbial adhesion to hydrocarbon assay, extracellular protease quantification and differential regulation of the dltA, speB, srv and ropB genes suggested that fukugiside probably inhibits biofilm formation by lowering cell surface hydrophobicity and destabilizing the biofilm matrix. The enhanced susceptibility to phagocytosis evidenced in the blood survival assay goes in unison with the downregulation of mga. The downregulation of important virulence factor‐encoding genes such as hasA, slo and col370 suggested impaired virulence. In vivo analysis in C. elegans evinced the non‐toxic nature of fukugiside and its anti‐virulence potential against S. pyogenes. Conclusion. Fukugiside exhibits potent anti‐biofilm and anti‐virulence activity against different M serotypes of S. pyogenes. It is also non‐toxic, which augurs well for its clinical application.

Quantification of coumarin and related phenolics in cinnamon samples from south India using UHPLC-ESI-QqQLIT-MS/MS method

ABSTRACT The presence of coumarin, a hepatotoxic phenyl propanoid, in samples of the spice, cinnamon, procured from the market has raised food safety concerns world over. A validated, ultra-performance liquid chromatography- linear ion trap triple quadrupole mass spectrometry method was developed in the present study for the estimation of coumarin and other phenolics in authentic Cinnamomum verum bark samples and market samples from south India. Coumarin contents in authentic bark samples were in the range of 12.3 to 143.0 mg/kg, whereas the coumarin contents in samples procured from the market were up to 3462.0 mg/kg. The high content of coumarin and cinnamaldehyde in cinnamon samples procured from the market suggested possible blending with substitutes like Cinnamomum cassia barks, which possess substantial amounts of coumarin and cinnamaldehyde.

Bioenergy potentials of three Euphorbia species from Western Ghats in India

ABSTRACT Bioenergy constituents of three Euphorbia species viz., Euphorbia vajravelui, E. tortilis, and E. trigona, were evaluated. These Euphorbia species showed moisture (79.5–91.9%), ash (4.06–6.80%), latex (0.08–0.26 g), oil (5.45–5.59%), polyphenol (3.12–8.39%), hydrocarbon (0.28–0.41%), carbon (42.40–44.5%), hydrogen (6.22–8.40%) contents and gross heat values (17.51–18.71 MJ/kg) comparable to other reported Euphorbia plants, energy crops and biomass sources. Thermal analyses (TG/DTG, DTA, DSC) produced comparable results. Trace elements such as Fe, Mg, K, Ca, P, Zn, Mn, Ba, Sr, Cu, Sc, and As were detected in these Euphorbia specimens. This study reveals the biomass/bioenergy potentials of these three Euphorbia species from Western Ghats, India.

Chemical composition and FtsZ GTPase inhibiting activity of the essential oil of Piper sarmentosum from Andaman Islands, India

Abstract Chemical composition of essential oils from the leaves, fruits and roots of Piper sarmentosum Roxb. (Piperaceae) growing wild in Andaman Islands, India was investigated by gas chromatography (GC), gas chromatography mass spectrometry (GC-MS) and 13C-nuclear magnetic resonance spectroscopy (13C-NMR). The phenyl propanoid myristicin was identified as the major compound comprising 83.4% in the leaf oil, 84.2% in the fruit oil and 81.2% in the root oil. The sesquiterpenoid β-caryophyllene was present in all the oils, 4.2% in the leaves, 5.7% in the fruits and 4.0% in the roots oil. The major constituent myristicin was isolated and structure was further confirmed by 13C-NMR analysis. The antimicrobial efficacy of myristicin was evaluated by both in vitro and in silico FtsZ inhibition studies. The in vitro assay showed 13.0% FtsZ inhibition by 200-μΜ myristicin and the molecular docking simulation supported the activity of myristicin.