Role of cytokines in the clinical manifestation of exophthalmia in newborn calves with tropical theileriosis.

The present study aimed to evaluate the pathology of the exophthalmia and the host-immune response in naturally Theileria annulata-infected calves. The newborn calves detected positive for theileriosis were grouped into calves with theileriosis and absence of exophthalmia (n = 30), and calves with theileriosis and the presence of exophthalmia (n = 13). Sixteen healthy calves, free from any haemoprotozoal infection, were kept as healthy controls. A significantly (P ≤ .001) higher circulating levels of tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were estimated in diseased calves with and without exophthalmia as compared to healthy controls. Contrarily, significantly (P ≤ .01) lower interferon-γ (IFN-γ) level was estimated in diseased calves. The diseased calves with exophthalmia revealed significantly higher levels of TNF-α (P ≤ .001) and IL-10 (P ≤ .006) as compared to the diseased calves without exophthalmia. The diseased calves were not found to have an elevated intraocular pressure; rather they had significantly (P ≤ .001) lower intraocular pressure compared to the healthy controls. An elevated systemic TNF-α level might be attributed to the exophthalmia in calves with tropical theileriosis. The elevated circulatory IL-10 and reduced IFN-γ levels could be one of the strategies of Theileria annulata to escape the host immunity.

A walk on the wild side: gut bacteria fed to mass-reared larvae of Queensland fruit fly [Bactrocera tryoni (Froggatt)] influence development.

BACKGROUND: The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera, Tephritidae) is the most significant insect pest of Australian horticulture. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). Mass-rearing and irradiation of pupae in SIT can reduce the fitness and quality of the released sterile insects. Studies have also showed reduced microbial gut diversity in domesticated versus wild tephritids. RESULTS: Transmission electron microscopy confirmed the presence of the bacterial isolates in the mid-gut of mass-reared larvae, and plate counts from individual larval guts showed increased numbers of bacteria in supplemented larvae. Several developmental and fitness parameters were tested including larval development time (egg-hatch to pupation), pupal weight, emergence, flight ability, sex-ratio, and time to adult eclosion (egg-hatch to adult eclosion). Enterobacter sp. and Asaia sp. shortened larval development time, while this was delayed by Lactobacillus sp., Leuconostoc sp. and a blend of all four bacteria. The mean time from egg hatch to adult eclosion was significantly reduced by Leuconostoc sp. and the blend for males and females, indicating that the individual bacterium and consortium affect flies differently depending on the life stage (larval or pupal). There was no impact of bacterial supplemented larvae on pupal weight, emergence, flight ability, or sex ratio. CONCLUSIONS: Our findings show that bacteria fed to the larval stage of B. tryoni can impart fitness advantages, but the selection of probiotic strains (individual or a consortium) is key, as each have varying effects on the host. Bacteria added to the larval diet particularly Leuconostoc sp. and the blend have the capacity to reduce costs and increase the number of flies produced in mass-rearing facilities by reducing time to adult eclosion by 1.3 and 0.8 mean days for males, and 1.2 and 0.8 mean days for females.

Comparison of different PCR protocols and respective primer sets from pool of TAMS 1 gene for diagnosis of calf theileriosis from semi arid India.

The polymorphic nature of Theileria annulata merozoite surface antigen (TAMS 1) attributes to limitation in PCR based detection of various T. annulata genotypes present in different geographical domains across the globe. Multiple reports of failure of detection of tropical theileriosis using classical N516/517 primer set in the studied area were noticed. Hence, three single PCR protocols using N516/517, TAMS F/R and NTA F/R primer sets encoding different portions of TAMS 1 gene and two nested protocols, using combinations of these three primers, were compared to find out the most suitable primer set for diagnosis of calf theileriosis in studied area. The studied area constitutes the semi-arid theileriosis endemic area of Northern India. The various PCR protocols were tested on 75 clinically confirmed cases of calf theileriosis. Alongside, 25 confirmed theileriosis negative blood samples and DNA of other haemoprotozoa were also tested for specificity of these primer sets. Results revealed that the primer set NTA F/R to be more suitable in detecting the circulating T. annulata genotypes in the studied area in comparison to the classical N516/517 primer set. None of the primers gave false positive amplification with negative samples and/or DNA of other haemoprotozoa.

Decline in seminal quality in Indian men over the last 37 years.

BACKGROUND: Since the first report of a decline in semen quality in 1974, there have been several reports of similar declines across populations. Despite some scattered reports of declining semen quality in the Indian sub-continent, comprehensive studies analyzing semen quality over the last few decades have not been undertaken. We undertook the present study to investigate the temporal trend in semen parameters in Indian populations over a period of 37 years (1979-2016). METHODS: Publications providing semen analysis details for fertile and infertile men from the Indian sub-continent were collected by a thorough literature search. Semen quality data for 6466 normal fertile or presumptive normal men (from 119 studies/data sets) and 7020 infertile men (from 63 studies/data sets) published between 1979 and 2016 were retrieved. We undertook systematic review and quantitative analysis of mean sperm count, motility, normal morphology and other available parameters. Data were analyzed to estimate semen parameters reference values for Indian men and to assess temporal trends in infertile, fertile and all subjects. RESULTS: Seminal quality shows a decreasing temporal trend and the decrease is higher in infertile than fertile males. In pooled analysis for all individuals, significant (p < 0.05 or < 0.001) declines in sperm concentration and normal morphology are observed; however, isolated analysis for each group shows declines without statistical significance. The mean (± SD) semen volume, sperm concentration, total motility, rapid linear progressive motility, normal sperm morphology and sperm viability for Indian fertile men are 2.88 ± 0.77 ml, 81.08 ± 29.21 million/ml, 66.37 ± 10.95%, 52.64 ± 15.78%, 56.68 ± 20.23% and 72.63 ± 8.31%, respectively, whereas in infertile these are 3.07 ± 1.27 ml, 37.94 ± 26.41 million/ml, 40.22 ± 13.76%, 26.79 ± 15.47%, 36.41 ± 21.66% and 55.25 ± 11.99%, respectively. The mean seminal parameter values were significantly lower (p < 0.001) in infertile as compared to fertile men, except semen volume. CONCLUSIONS: Semen parameters in Indian men have declined with time and the deterioration is quantitatively higher in the infertile group. The study also provides reference values for semen parameters in Indian men.

Variation in cardiac markers and electrocardiographic alterations in young calves naturally infected with bovine tropical theileriosis.

The present study was designed to assess the deleterious effects of bovine tropical theileriosis on the cardiovascular system and the consequent myocardial involvement in young calves. Myocardial effects in parasitic diseases are often neglected. Hemolytic anemia, associated secondary hypoxia, and vasculitis are cardinal features of bovine theileriosis. In the present study, electrocardiogram (ECG) alongside serum cardiac troponin I (cTnI) and creatinine phosphokinase-myocardial band (CPK-MB) concentrations were analyzed in infected, treated, and control groups of young calves. Non-significant alterations were noticed in ECG. However, certain signs like sinus tachycardia, first-degree AV block, atrial premature complex, left atrial hypertrophy, and right atrial hypertrophy were found on consistent basis in infected calves. A significant increase in the serum concentration levels of cTnI and CPK-MB was noticed in infected calves followed by significant fall in both these biomarkers post treatment. cTnI and CPK-MB can definitely be used as myocardial markers in theileriosis-affected animals.

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.

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.

Reinvestigating nanoprecipitation via Box-Behnken design: a systematic approach.

This work demonstrates Box-Behnken design (BBD)'s capability in exploring scientific principles governing a process, different from its use in process optimisation. We have investigated nanoprecipitation (NP) of temozolomide with polycaprolactone. Five factors, surfactant, stirring speed (SS), dropping rate (DR), phase-volume ratio (PVR) and drug-polymer ratio (DPR) were varied over three levels. Corresponding particle size (238.9 ± 42.24 nm), zeta potential (ZP, -5.92 ± 3.15 mV), poly dispersity index (PDI, 0.176 ± 0.06) and entrapment efficiency (EE, 65.74 ± 9.83%) were put into different polynomial equations. Analysis of variance, lack of fit tests and regression analysis was applied on these equations to determine the one with best fit. This selected equation was subsequently adapted as the model to describe influence of factors on NP. 3D response surface plots corresponding to models and diagnostic plots relatable to normality of residuals were also constructed. In conclusion, application of BBD efficiently strategised experimental foray conducted to elucidate NP.

Phase III, Randomized, Double-blind, Placebo controlled trial of Efficacy, Safety and Tolerability of Antiviral drug Umifenovir vs Standard care of therapy in non-severe COVID-19 patients.

OBJECTIVE: To test efficacy, safety and tolerability of Umifenovir in non-severe COVID-19 adult patients. METHODS: We carried out randomized, double-blind, placebo-controlled, multicenter, phase III trials involving adult (18-75 years), non-severe COVID19 patients, randomized 1:1 on placebo or Umifenovir (800 mg BID, maximum 14 days) respectively along with standard-of-care. The primary endpoint for Asymptotic-mild patients was time to nasopharyngeal swab RT-PCR test negativity. For Moderate patients, the average change in the ordinal scale from the baseline scores on the eight-point WHO ordinal scale was assessed. RESULTS: 132 patients were recruited between 3rd October to 28th April 2021, of which 9 discontinued due to various reasons. In Mild-asymptomatic patients (n=82), we found that 73% patients in the Umifenovir arm were RT-PCR negative, while 40% patients in the placebo arm were negative (P=0.004) on day 5. However, in the moderate group (n=41), the WHO scores for the Umifenovir arm was not statistically significant (P=0.125 on day 3), while it was statistically significant in the Mild-asymptomatic group (P=0.019 on day 5). CONCLUSION: Umifenovir meets the primary and secondary endpoint criteria and exhibits statistically significant efficacy for Mild-asymptomatic patients. It is efficacious, safe and well-tolerated at the tested dosage of 800mg BID, maximum 14 days.

In-vitro culture of Plasmodium falciparum: utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay.

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the ((3)H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC(50) values of standard anti-malarials obtained by both the ((3)H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.

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.

Polyglucosan inclusions (Lafora bodies) in a gray-headed flying fox (Pteropus poliocephalus).

Polyglucosan bodies (Lafora bodies) were identified in a juvenile gray-headed flying fox (Pteropus poliocephalus) with neurological signs. The structures were only noted in the brain stem, and no associated degenerative changes were present. These structures have not been previously identified in any species in the order Chiroptera.

Chronic myositis in an Australian alpaca (Llama pacos) associated with Sarcocystis spp.

An alpaca (Llama pacos), born and raised in Australia, was presented with multiple subcutaneous abscesses. Histological findings indicated a severe necrotizing and histiocytic myositis and cellulitis associated with central caseation and multiple sarcocysts. Ultrastructural examination supported the diagnosis; however, cyst wall characteristics were not consistent with the 2 known species found in alpacas. While seroconversion in camelids is reported to be near ubiquitous, myositis is rare, and this is the first case reported outside of the Americas.

Rapid diagnosis of Theileria annulata by colorimetric loop-mediated isothermal amplification (LAMP) assay.

Theileria annulata the causative agent of bovine tropical theileriosis (BTT) is globally distributed. Rapid and accurate detection of the parasite is essential for the implementation and evaluation of mass drug administration and planned vaccination programs for controlling BTT. Loop mediated isothermal amplification (LAMP) amplifies targeted nucleic acid with a high efficacy, sensitivity and rapidity under isothermal conditions. In the present study, the internal transcriber space (ITS) gene of T. annulata was targeted for the development of a LAMP assay using pH-sensitive dye (phenol red) for enhanced visual detection of amplification. This method employed a set of specially designed four primers that recognized six distinct regions on the targeted gene. No amplification was detected with the DNA of other tested haemoprotozoans. Positive LAMP products were identified by a colour change from pink to yellow, and then rechecked by specific ladder pattern upon agarose gel electrophoresis. LAMP was able to detect infection in 63 out of 85 animals compared with blood microscopy, simple PCR and nested PCR which detected infection in 40, 49 and 64 animals, respectively. No difference in detection was seen in the colorimetric assay and the classical UV based LAMP assay.

Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi-arid India.

BACKGROUND: Knowledge of local isolates and strains is a prerequisite for the development of either effective mass vaccination strategy or a suitable molecular marker-based diagnostic tool. PURPOSE: The pathogenesis of Bovine tropical theileriosis (BTT), caused by Theileria annulata in susceptible ruminants, is known to vary depending upon the nature of isolate and strain involved. Therefore, RFLP and sequencing-based characterization of Indian isolates of T. annulata were attempted using TAMS gene. METHOD: In the present study, TAMS 1 gene of T. annulata was amplified from 25 naturally infected calves from the BTT endemic semi-arid zone of Northern India. The amplified products were then digested with three restrictions enzymes viz., Taq I, Rsa I, and Alu I to find out the variations in pattern of restriction digests, so as to have an idea about the various isolates of T. annulata present in the studied area. Around 14 samples covering all the variants (from the PCR-RFLP patterns) were sequenced and submitted in NCBI (MH277607-MH277620). RESULT: Coexistence of 4 variant genotypes was detected upon in-silico analysis of RFLP and sequence variations. CONCLUSION: The nucleotide variations alongside the chromatogram analysis revealed point mutations leading to presence of noticeable genetic diversity among the isolates.

A case control study of clinical and biochemical parameters of metabolic syndrome with special attention among young and middle aged population.

BACKGROUND: Metabolic syndrome (MS) increases the risk of heart disease, stroke, and other complications. AIM: The aim of this study was to assess the clinical and biochemical parameters of MS and its complications (cerebrovascular accidents, cardiovascular accidents, DN or chronic kidney disease (CKD) compared with healthy controls especially among the younger population in Northern India. MATERIAL AND METHODS: A total of 245 (healthy, MS and it's complicated) aged 18-70 years participated in the Open-Label, Single Centered; hospital-based random selection case-control comparative study. All anthropometric and biochemical assessment was done after proper consent. The metabolic syndrome was determined by IDF criteria. RESULTS: The key risk parameters in three groups i.e. Control, Metabolic syndrome, and Complicated was TG (96.5 ±â€¯46.9, 194.1 ±â€¯87.8, 148.0 ±â€¯102.2). LDL (91.2 ±â€¯27.2, 114.0 ±â€¯31.8, 69.1 ±â€¯42.5, BP (120.1 ±â€¯9.9, 139.3 ±â€¯13.3, 132.1 ±â€¯15.0) and high fasting glucose (81.1 ±â€¯13.7, 164.5 ±â€¯84.3, 138.0 ±â€¯74.5). The hs-CRP is also significantly increased in the complicated group. The subanalysis of data also indicates that younger middle age (36-55 years) group both male and female is obese, hypertensive, diabetic with lipid abnormality according to IDF criteria. CONCLUSION: The risk factors like high TG, low HDL, high BP, and high fasting glucose were found higher particularly in younger population which may lead to diagnosis & complications of diabetes, hypertension and lipid abnormality. Due to changing physiology in young and middle age population these individuals are moving towards metabolic syndrome easily and needs frequent monitoring, preventive checkups, and lifestyle changes to prevent complications.

Identification of hexose diastereomers by means of tandem mass spectrometry of oxocarbenium ions followed by neural networks analysis.

The ion abundances in the tandem mass spectra of oxocarbenium ions generated from aldohexoses and ketohexoses under electrospray ionization conditions depend on their stereochemistry. The ratios of the abundances of two of the major ions m/z 85 and 91 are different in the aldohexoses and the ketohexoses. Principal component analysis (PCA) allowed visual differentiation of the hexose isomers. However, identification of an individual hexose was difficult. The data were subjected to neural network analysis (NNA) using a multilayer perceptron (MLP) model. The model was first trained using the data from the 11 hexoses and then used to identify the hexoses using fresh data acquired later. There was 100% identification of the various hexoses. The hexose units in methyl glucoside, mannoside and galactoside could also be identified accurately. The method can therefore be used for the characterization of hexose units from monosaccharides and glycosides.

Identification of a novel nidovirus as a potential cause of large scale mortalities in the endangered Bellinger River snapping turtle (Myuchelys georgesi).

In mid-February 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, Myuchelys georgesi, in a coastal river in New South Wales, Australia. Mortalities continued for approximately 7 weeks and affected mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. At necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. Histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. A virus was isolated in cell culture from a range of tissues. Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. In contrast, in the affected turtles, the most significant pathological changes were in the kidneys. Real time PCR assays developed to detect this virus demonstrated very high virus loads in affected tissues. In situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. Collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. Bellinger River Virus is the name proposed for this new virus.

Emergent multisystemic Enterococcus infection threatens endangered Christmas Island reptile populations.

Multisystemic infections with a morphologically unusual bacterium were first observed in captive critically endangered Lister's geckos (Lepidodactylus listeri) on Christmas Island in October 2014. Since then the infection was identified in another captive critically endangered lizard species, the blue-tailed skink (Cryptoblepharus egeriae) and two species of invasive geckos; the four clawed gecko (Gehyra mutilata) and Asian house gecko (Hemidactylus frenatus), in a wide geographic range across the east side of the island. The Gram and periodic acid-Schiff positive cocci to diplococci have a propensity to form chains surrounded by a matrix, which ultrastructurally appears to be formed by fibrillar capsular projections. The bacterium was associated with severe and extensive replacement of tissues, but minimal host inflammatory response. Attempts to grow the organism in culture and in embryonated eggs were unsuccessful. Molecular characterisation of the organism placed it as a novel member of the genus Enterococcus. Disease Risk Analyses including this organism should now be factored into conservation management actions and island biosecurity.

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.