Awantika Singh

Rapid profiling and structural characterization of bioactive compounds and their distribution in different parts of Berberis petiolaris Wall. ex G. Don applying hyphenated mass spectrometric techniques

RATIONALE Berberis petiolaris Wall. is a lesser known medicinal plant, belonging to the family Berberidaceae. The genus Berberis is known for many biological activities such as anti-microbial, anti-inflammatory and anti-diarrheal, etc. There are not many reports of the isolation of components from Berberis petiolaris. This study aims to seek identification, characterization and quantification of components. METHODS A method was developed for rapid screening of phytochemicals using high-pressure liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry (HPLC/ESI-QTOF-MS/MS). Suitable collision-induced dissociation mass spectrometry (CID-MS/MS) methods were developed for structural investigation of alkaloids, flavanoids and other classes of compounds using nine reference standards for authentication. Multiple reaction monitoring (MRM) methods were developed for quantitative study of five constituents using triple quadrupole-linear ion trap mass spectrometry (UPLC/QqLIT-MS/MS). RESULTS On the basis of HPLC retention behavior and fragmentation pathways obtained by high-resolution MS and MS/MS, 32 compounds were identified and characterized in different parts of Berberis petiolaris. Quantitative studies of chlorogenic acid, magnoflorine, jatrorrhizine, palmatine and berberine were also completed successfully. CONCLUSIONS Rapid and accurate HPLC/ESI-QTOF-MS/MS and UPLC/ESI-QqLIT-MS/MS methods were established for identification, characterization and quantification of phytochemicals in the ethanolic extract of Berberis petiolaris. These methods, therefore, can be used for studies on phytochemical variation in different parts of the plant. Principle components analysis (PCA) may be used for plant part discrimination.

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.

Access to Indole- And Pyrrole-Fused Diketopiperazines via Tandem Ugi-4CR/Intramolecular Cyclization and Its Regioselective Ring-Opening by Intermolecular Transamidation

An efficient approach for the synthesis of indole- and pyrrole-fused diketopiperazines has been developed. This protocol involves the Ugi four-component reaction (U-4CR) followed by an intramolecular cyclization of the Ugi products at room temperature to afford the desired products in good to excellent yields. In addition, it is interesting to report the subsequent regioselective ring-opening of diketopiperazine unit occurring via an intermolecular transamidation reaction under mild condition, resulting in the formation of highly functionalized indole-2-carboxamides and pyrrole-2-carboxamides.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT-MS/MS

INTRODUCTION The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. OBJECTIVE To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. METHODS Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. RESULTS Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. CONCLUSIONS Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers.

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.

Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC–ESI-QTOF-MS/MS

Phyllanthus species plants are a rich source of phenolics and widely used due to their medicinal properties. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed using high-pressure liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS) for the identification and characterization of quercetin, kaempferol, ellagic acid and their derivatives in ethanolic extracts of Phyllanthus species. The chromatographic separation was carried out on Thermo Betasil C8 column (250 mm×4.5 mm, 5 µm) using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. The identification of diagnostic fragment ions and optimization of collision energies were carried out using 21 reference standards. Totally 51 compounds were identified which include 21 compounds identified and characterized unambiguously by comparison with their authentic standards and the remaining 30 were tentatively identified and characterized in ethanolic extracts of P. emblica, P. fraternus, P. amarus and P. niruri.

Synthesis of biologically active pyridoimidazole/imidazobenzothiazole annulated polyheterocycles using cyanuric chloride in water

An efficient and mild protocol for rapid access to N-fused polyheterocycles via Pictet–Spengler type 6-endo cyclization using cyanuric chloride in an aqueous reaction medium has been developed. The protocol was successfully applied to a wide range of compounds including aryl/heteroaryl aldehydes (8a–o), ketones (10a–e), an electron-rich metallocene aldehyde (8e) and indoline-2,3-diones (12a–c) using cyanuric chloride (15–20 mol%) with tetra-n-butylammonium bromide (TBAB) (2.0 eq.) as an additive at 80–90 °C to give a good to excellent yield (66–92%) of polyheterocycles. Some of the synthesized compounds were found to exhibit antiplasmodial activity against chloroquine-sensitive (CQ-S) 3D7 and chloroquine-resistant (CQ-R) K1 strains of Plasmodium falciparum.

Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry☆

Rauwolfia species (Apocynaceae) are medicinal plants well known worldwide due to its potent bioactive monoterpene indole alkaloids (MIAs) such as reserpine, ajmalicine, ajmaline, serpentine and yohimbine. Reserpine, ajmalicine and ajmaline are powerful antihypertensive, tranquilizing agents used in hypertension. Yohimbine is an aphrodisiac used in dietary supplements. As there is no report on the comparative and comprehensive phytochemical investigation of the roots of Rauwolfia species, we have developed an efficient and reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ethanolic root extract of Rauwolfia species to elucidate the fragmentation pathways for dereplication of bioactive MIAs using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC–ESI–QTOF–MS/MS) in positive ion mode. We identified and established diagnostic fragment ions and fragmentation pathways using reserpine, ajmalicine, ajmaline, serpentine and yohimbine. The MS/MS spectra of reserpine, ajmalicine, and ajmaline showed C-ring-cleavage whereas E-ring cleavage was observed in serpentine via Retro Diels Alder (RDA). A total of 47 bioactive MIAs were identified and characterized on the basis of their molecular formula, exact mass measurements and MS/MS analysis. Reserpine, ajmalicine, ajmaline, serpentine and yohimbine were unambiguously identified by comparison with their authentic standards and other 42 MIAs were tentatively identified and characterized from the roots of Rauwolfia hookeri, Rauwolfia micrantha, Rauwolfia serpentina, Rauwolfia verticillata, Rauwolfia tetraphylla and Rauwolfia vomitoria. Application of LC–MS followed by principal component analysis (PCA) has been successfully used to discriminate among six Rauwolfia species.

Rapid screening for the adulterants of Berberis aristata using direct analysis in real-time mass spectrometry and principal component analysis for discrimination

Adulteration or substitution of commercial Berberis aristata and its herbal products with inferior-quality substituents is very common. Metabolic profiling of B. aristata, along with its common adulterants/contaminants/substituents such as B. asiatica, Mahonia borealis and Coscinium fenestratum, was rapidly carried out using direct analysis in real-time mass spectrometry (DART MS) to generate the chemical fingerprints for the differentiation of these species. Phytochemical analysis showed the presence of mainly alkaloids. The identified alkaloids were berberrubine, berberine, jatrorrhizine, ketoberberine, palmatine, dihydropalmatine or 7,8-dihydro-8-hydroxyberberine, berbamine and pakistanamine. Berberine, which was mainly reported from the root and stem bark of B. aristata, was also identified in the leaf along with chlorogenic acid. The DART MS data have been subjected to principal component analysis (PCA). The resulting score plots showed clustering and clear differentiation of the species and plant parts. It is thus apparent that the technique of DART MS followed by PCA is a quick and reliable method for the direct profiling of B. aristata and its adulterant plants and plant parts. The study reports the rapid analytical method to identify the possibility of illegal adulteration/contamination/substitution in potential plant materials and herbal extracts. Graphical Abstract

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.