Determination of bioactive compounds of Artemisia Spp. plant extracts by LC-MS/MS technique and their in-vitro anti-adipogenic activity screening.

Ultra Performance Liquid Chromatography coupled with hybrid triple quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLIT-MS/MS) method in multiple reaction monitoring (MRM) acquisition mode was developed and validated for identification and simultaneous determination of potential anti-diabetic and anti-malarial compounds in ethanolic extracts of different Artemisia species. The chromatographic separation was carried out on an Acquity BEH™ C18 column (1.7 µm, 2.1 × 50 mm) with 0.1 % (v/v) formic acid in water and acetonitrile as mobile phase under gradient condition in 6 min. The developed method was validated in terms of linearity, LOD, LOQ, precision, stability and recovery according to international conference on harmonization guidelines. The correlation coefficients of all the calibration curves were ≥0.9902 and recoveries ranged from 98.22 to 104.49% (RSD ≤2.18 %). Relative standard deviations of intra-day, inter-day precisions and stability were ≤ 1.04, 1.09 and 2.80 %, respectively. The quantitative results showed remarkable differences in the content of all the compounds in different Artemisia species. The quantitative values of each peak were summarized as mean ± SD. The statistical analysis for comparison of observed quantitative differences of each compound was done to show that they are statistically significant. In-vitro assessment of extracts of selected Artemisia species inhibited adipocyte differentiation in 3T3-L1 cells, hence it may have certain phytochemicals which are responsible for reducing obesity and related metabolic disorders.

Major bioactive phenolics in Bergenia species from the Indian Himalayan region: Method development, validation and quantitative estimation using UHPLC-QqQLIT-MS/MS.

Bergenia species are important medicinal plants used in indigenous systems of medicine for their antilithiatic and diuretic properties. An ultra-high performance liquid chromatography coupled to hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) method has been developed and validated for the estimation of quantitative variation of eight major bioactive phenolics in the rhizomes (150 samples) of four species of this herb, Bergenia (B. ciliata, B. ligulata, B. purpurascens and B. stracheyi). Chromatographic separation was obtained on a Waters ACQUITY UPLCTM BEH (ethylene bridged hybrid) C18 column with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile under a gradient elution manner. A hybrid linear ion trap triple quadrupole mass spectrometer was operated in negative electrospray ionization mode with multiple reactions monitoring for detection and quantification of the eight compounds. The validated method demonstrated good linearity (r2 ≥ 0.9991), precision (RSD ≤ 1.87%) and accuracy (95.16-102.11%, RSD ≤ 1.83%) for all reference analytes. The quantitative results revealed that B. ligulata contains the highest amount of the major active marker-bergenin. The results also suggest that sensitive UHPLC-QqQLIT-MS/MS method, a sensitive, accurate and convenient one, could be helpful in identification of potential accession(s), rapid quality control and establishing authenticity of Bergenia species as raw material for pharmaceutical industries.

Ameliorative Potential of Prepartal Trace Mineral and Vitamin Supplementation on Parturition-Induced Redox Balance and Myeloperoxidase Activity of Periparturient Sahiwal Cows.

Twelve apparently healthy multiparous parturient Sahiwal cows were allocated into two groups having six cows in each one. Six cows were supplemented with antioxidant mixture (mixture containing Cu, Mn, Cr, Zn, and vitamins A and D3) daily from 21 days before parturition till the day relative to calving. Whereas, remaining non-supplemented six cows were kept as the control group. Blood samples were obtained five times: at enrolment (21 days pre-partum), and again at days 0, +7, +14, and +21 relative to calving. In the non-supplemented control group, serum total antioxidant capacity (TAC) was significantly lower at days 0, +7, and +14 as compared to their own day -21 values. Likewise, significantly lower myeloperoxidase (MPO) activities were also exhibited by these cows at days 0 and +7. Conversely, serum malondialdehyde (MDA) and protein carbonyl (PC) levels were significantly higher in these cows at days 0, +7, +14, and +21. However, significant alterations in TAC content among the studied sampling days were not recorded in antioxidants supplemented group. Moreover, TAC content and MPO activities of supplemented group were significantly higher at days 0, +7, and +14 when compared with that of the non-supplemented control group. However, MDA and PC contents of supplemented group were significantly lower at days 0, +7, +14, and +21 as compared to that of the non-supplemented control group. In conclusion, periparturient Sahiwal cows experience substantial oxidative and immunological dents which can be potentially ameliorated by prepartal trace mineral and vitamin supplementation.

Quantitative determination of multi markers in five varieties of Withania somnifera using ultra-high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometer combined with multivariate analysis: Application to pharmaceutical dosage forms.

An ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for simultaneous quantification of six major bioactive compounds in five varieties of Withania somnifera in various plant parts (leaf, stem and root). The analysis was accomplished on Waters ACQUITY UPLC BEH C18 column with linear gradient elution of water/formic acid (0.1%) and acetonitrile at a flow rate of 0.3mLmin(-1). The proposed method was validated with acceptable linearity (r(2), 0.9989-0.9998), precision (RSD, 0.16-2.01%), stability (RSD, 1.04-1.62%) and recovery (RSD ≤2.45%), under optimum conditions. The method was also successfully applied for the simultaneous determination of six marker compounds in twenty-six marketed formulations. Hierarchical cluster analysis and principal component analysis were applied to discriminate these twenty-six batches based on characteristics of the bioactive compounds. The results indicated that this method is advance, rapid, sensitive and suitable to reveal the quality of Withania somnifera and also capable of performing quality evaluation of polyherbal formulations having similar markers/raw herbs.

Simultaneous Determination of Bioactive Monoterpene Indole Alkaloids in Ethanolic Extract of Seven Rauvolfia Species using UHPLC with Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometry.

INTRODUCTION: Rauvolfia serpentina is an endangered plant species due to its over-exploitation. It has highly commercial and economic importance due to the presence of bioactive monoterpene indole alkaloids (MIAs) such as ajmaline, yohimbine, ajmalicine, serpentine and reserpine. OBJECTIVE: To develop a validated, rapid, sensitive and selective ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (UHPLC-QqQLIT -MS/MS) method in the multiple reaction monitoring (MRM) mode for simultaneous determination of bioactive MIAs in ethanolic extract of seven Rauvolfia species and herbal formulations. METHODS: The separation of MIAs was achieved on an ACQUITY UPLC BEH™ C18 column (1.7 µm, 2.1 mm × 50 mm) using a gradient mobile phase (0.1% aqueous formic acid and acetonitrile) at flow rate 0.3 µL/min in 7 min. The validated method showed good linearity (r(2)  ≥ 0.9999), limit of detection (LOD) (0.06-0.15 ng/mL), limit of quantitation (LOQ) (0.18-0.44 ng/mL), precisions [intraday: relative standard deviation (RSD) ≤ 2.24%, interday: RSD ≤ 2.74%], stability (RSD ≤ 1.53%) and overall recovery (RSD ≤ 2.23%). RESULTS: The validated method was applied to quantitate MIAs. Root of Rauvolfia vomitoria showed a high content of ajmaline (48.43 mg/g), serpentine (87.77 mg/g) whereas high quantities of yohimbine (100.21 mg/g) and ajmalicine (120.51 mg/g) were detected in R. tetraphylla. High content of reserpine was detected in R. micrantha (35.18 mg/g) and R. serpentina (32.38 mg/g). CONCLUSION: The encouraging results of this study may lead to easy selection of suitable Rauvolfia species according to the abundance of MIAs. Copyright © 2016 John Wiley & Sons, Ltd.

Distribution and Discrimination Study of Bioactive Compounds from Berberis species using HPLC-ESI-QTOF-MS/MS with Principle Component Analysis.

Berberis specieshave been used extensively as raw material in various herbal medicines. In this work, a fast aid sensitive method based on reversed-phase high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandemmass spectrometry (HPLC-ESI-QTOF- MS/MS) was used to assist the distribution and discrimination study of bioactive compounds from Berberis species such as B. aristata, B. asiatica, B. chitria, B.jaeschkeana, B; koehneana, B. lyceum, B. petiolaris and B. pseudoumbellata. The HPLC separation was performed on a Thermo Betasil C8 column (250 mm x 4.5 mm; 5µ) using gradient elution with a mobile phase of 0.1% aqueous solution of formic acid (v/v) and acetonitrile. The high resolution (HR) and collision induced dissociation (CID) mass spectrometry experiments were conducted in order to provide molecular-mass information and MS/MS fragmentation patterns of the compounds for structural elucidation. A total of 59 compounds were tentatively identified and characterized including three acids, forty-one alkaloids and twelve flavonoids using seventeen reference standards for authentication. Principle component analysis (PCA) was also applied to discriminate eight Berberis species according to their plant parts i.e., leaf, stem and root. The highest total content of identified compounds was detected in the root of all the Berberis species wherein alkaloids were predominant.

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.

Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry / 药物分析学报

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.

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis.

INTRODUCTION: Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance. OBJECTIVE: To develop and validate a new, rapid, sensitive and selective UPLC-ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. METHODS: An UPLC-ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18 -column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes. RESULTS: The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09-104.84% (RSD ≤ 1.85%), precise (RSD ≤ 1.98%) and linear (r(2) ≥ 0.9971) over the concentration range of 0.5-1000 ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet. CONCLUSION: The method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations.

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.

Rapid screening and distribution of bioactive compounds in different parts of Berberis petiolaris using direct analysis in real time mass spectrometry.

Berberis petiolaris Wall. ex G. Don, an unexplored medicinal plant belonging to the family Berberidaceae, is a large deciduous shrub found in Western Himalaya between 1800-3000 m. Chemical profiling of fruit, leaf, root and stem was done by direct analysis in real time mass spectrometry followed by multivariate analysis for discrimination among the plant parts. The bioactive compounds, including magnoflorine, berberine, jatrorrhizine, thalifendine/berberrubine, demethyleneberberine, reticuline, 8-oxoberberine, N-methyltetrahydroberberine, tetrahydropalmatine, tetrahydroberberine and palmatine, were identified by their exact mass measurement and the corresponding molecular formula of each compound. A comparative study of distribution pattern for all these bioactive alkaloids showed qualitative and quantitative variations in different parts of B. petiolaris. Principal component analysis clearly discriminated each part of B. petiolaris plant.

Rapid screening and distribution of bioactive compounds in different parts of Berberis petiolaris using direct analysis in real time mass spectrometry / 药物分析学报

Berberis petiolaris Wall. ex G. Don, an unexplored medicinal plant belonging to the family Berberidaceae, is a large deciduous shrub found in Western Himalaya between 1800-3000 m. Chemical profiling of fruit, leaf, root and stem was done by direct analysis in real time mass spectrometry followed by multivariate analysis for discrimination among the plant parts. The bioactive compounds, including magnoflorine, berberine, jatrorrhizine, thalifendine/berberrubine, demethyleneberberine, reticuline, 8-oxoberberine, N-methyltetrahydroberberine, tetrahydropalmatine, tetrahydroberberine and palmatine, were identified by their exact mass measurement and the corresponding molecular formula of each compound. A comparative study of distribution pattern for all these bioactive alkaloids showed qualitative and quantitative variations in different parts of B. petiolaris. Principal component analysis clearly dis-criminated each part of B. petiolaris plant.

In vitro, in silico and in vivo studies of ursolic acid as an anti-filarial agent.

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (-8.6) with respect to the standard antifilarial drugs, ivermectin (IVM -8.4) and diethylcarbamazine (DEC-C -4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's 'Metadrug' tool screening predictions.