GC-MS Assisted Determination of 26 Compounds in Phlomis stewartii Extract Exhibiting Antioxidant, Antifungal, and Antibacterial Properties.

Medicinal plants have long been studied for their therapeutic benifits. The present research aims to unveil complex phytochemical profile and therapeutic properties of ethyl acetate fraction of Phlomis stewartii, an important medicinal plant. In this context, the Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the fraction identified 26 compounds. Additionally, the fraction exhibited concentration dependent antioxidant activity with an IC50 value lower than the standard antioxidant butylated hydroxytoluene. The antifungal activity of the fraction examined against F. oxysporum, A. alternate, and R. solani resulted in almost complete inhibition (>90%) of fungal growth. Furthermore, the fraction exhibited significant antibacterial potential against B. subtilus, S. aureus, E. coli, and S. dysenteriae, with inhibition zones of 18±0.22, 17±0.22, 12±0.11, and 10±0.12, respectively. Briefly, the plant extract was found to be highly potent, particularly in its antifungal action. Further studies, including natural products isolation coupled with bioassays, are recommended for promising drug candidates discovery.

A bibliometric analysis of minimally invasive dentistry: A review of the literature from 1994 to 2021.

STATEMENT OF PROBLEM: Minimally invasive dentistry is a technique for the maximal preservation of healthy tooth structures and has been a focus of attention in dental research. However, a detailed bibliometric analysis focusing on research related to minimally invasive dentistry is lacking. PURPOSE: The purpose of this bibliometric analysis was to analyze leading countries and organizations and to identify the preferred journals, the most productive authors, and the most commonly used keywords in the field of minimally invasive dentistry. MATERIAL AND METHODS: The search was performed by using the Scopus database, and publications pertinent to the field of minimally invasive dentistry from 1994 to 2021 were identified. A bibliometric analysis with reference to citations and documents, authors, journals, and keywords was performed. A total of 391 articles published in Scopus indexed journals between 1994 and January 2021 were analyzed by using a software program. RESULTS: A continuing yet steady increase in research publications was identified, with a substantial increase in publications since 2013. The United States and Germany were the countries that published the most articles (98 and 46, respectively), articles that received 1877 and 806 citations, respectively. Cardiff University, UK, was the most productive organization that received 212 citations. The most highly cited articles were from the Q1 category. Blum from King's College London was the highest-cited author in this field. CONCLUSIONS: Considerable advancement has been made in minimally invasive dentistry, as demonstrated by the increase in the number of publications linked with collaboration among various authors, nations, and institutes. This citation analysis gives a perspective on the progress of research in the field of minimally invasive dentistry and allows identification of the most significant and pertinent research areas.

A state-of-the-art technique to perform cloud-based semantic segmentation using deep learning 3D U-Net architecture.

Glioma is the most aggressive and dangerous primary brain tumor with a survival time of less than 14 months. Segmentation of tumors is a necessary task in the image processing of the gliomas and is important for its timely diagnosis and starting a treatment. Using 3D U-net architecture to perform semantic segmentation on brain tumor dataset is at the core of deep learning. In this paper, we present a unique cloud-based 3D U-Net method to perform brain tumor segmentation using BRATS dataset. The system was effectively trained by using Adam optimization solver by utilizing multiple hyper parameters. We got an average dice score of 95% which makes our method the first cloud-based method to achieve maximum accuracy. The dice score is calculated by using Sørensen-Dice similarity coefficient. We also performed an extensive literature review of the brain tumor segmentation methods implemented in the last five years to get a state-of-the-art picture of well-known methodologies with a higher dice score. In comparison to the already implemented architectures, our method ranks on top in terms of accuracy in using a cloud-based 3D U-Net framework for glioma segmentation.

Synthesis, characterization, antioxidant, antileishmanial, anticancer, DNA and theoretical SARS-CoV-2 interaction studies of copper(II) carboxylate complexes.

Copper(II) carboxylate complexes [Cu2(OOCR)4L2] (1) and [Cu2(OOCR`)4OCO(R`)CuL2]n (2), where L = 2-methyl pyridine, R = 2-chlorophenyl acetate and R` = 2-fluorophenyl acetate were synthesized and characterized by FT-IR spectroscopy and single crystal X-ray analysis. Complex 1 exhibits the typical paddlewheel array of a dinuclear copper(II) complex with carboxylate ligands. In complex 2, this scaffold is further extended into a polymeric arrangement based on alternate paddlewheel and square planar moieties with distinct coordination spheres. The complexes showed better 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activities and have been found to be more potent antileishmanial agents than their corresponding free ligand acid species. UV-Vis absorption titrations revealed good DNA binding abilities {Kb = 9.8 × 104 M-1 (1) and 9.9 × 104 M-1 (2)} implying partial intercalation of the complexes into DNA base pairs along with groove binding. The complexes displayed in vitro cytotoxic activity against malignant glioma U-87 (MG U87) cell lines. Computational docking studies further support complex-DNA binding by intercalation. Molecular docking investigations revealed probable interactions of the complexes with spike protein, the nucleocapsid protein of SARS-CoV-2 and with the angiotensin converting enzyme of human cells.

Binder-Free Fabrication of Prussian Blue Analogues Based Electrocatalyst for Enhanced Electrocatalytic Water Oxidation.

Developing a cost-effective, efficient, and stable oxygen evolution reaction (OER) catalyst is of great importance for sustainable energy conversion and storage. In this study, we report a facile one-step fabrication of cationic surfactant-assisted Prussian blue analogues (PBAs) Mx[Fe(CN)5CH3C6H4NH2]∙yC19H34NBr abbreviated as SF[Fe-Tol-M] (where SF = N-tridecyl-3-methylpyridinium bromide and M = Mn, Co and Ni) as efficient heterogeneous OER electrocatalysts. The electrocatalysts have been characterized by Fourier transform infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) analysis, and X-ray photoelectron spectroscopy (XPS). In the presence of cationic surfactant (SF), PBAs-based electrodes showed enhanced redox current, high surface area and robust stability compared to the recently reported PBAs. SF[Fe-Tol-Co] hybrid catalyst shows superior electrochemical OER activity with a much lower over-potential (610 mV) to attain the current density of 10 mA cm-2 with the Tafel slope value of 103 mV·dec-1 than that for SF[Fe-Tol-Ni] and SF[Fe-Tol-Mn]. Moreover, the electrochemical impedance spectroscopy (EIS) unveiled that SF[Fe-Tol-Co] exhibits smaller charge transfer resistance, which results in a faster kinetics towards OER. Furthermore, SF[Fe-Tol-Co] offered excellent stability for continues oxygen production over extended reaction time. This work provides a surface assisted facile electrode fabrication approach for developing binder-free OER electrocatalysts for efficient water oxidation.

A New MBH Adduct as an Efficient Ligand in the Synthesis of Metallodrugs: Characterization, Geometrical Optimization, XRD, Biological Activities, and Molecular Docking Studies.

This article reports the synthesis, characterization, geometrical optimization, and biological studies of new MBH-based organometallic compounds of medicinal significance. The ligand (MNHA) was prepared via the Morita-Baylis-Hillman (MBH) synthetic route, from aromatic aldehyde containing multiple functional groups. Metal complexes were prepared in an alkaline medium and under other suitable reaction conditions. Spectral and elemental analyses were used to identify the structural and molecular formulas of each compound. Optimized geometry was determined through density functional theory (DFT) B3LYP and 6-311++ G (d,p) basis set for the MBH adduct, whereas structures of novel complexes were optimized with the semi-empirical PM6 method. Powder XRD analysis furnished the crystal class of complexes, with Co3+, Cr3+, and Mn2+ being cubic, while Ni2+ was hexagonal, and Cu2+ was orthorhombic. Moreover, the ligand, along with Ni2+ and Co3+ complexes, showed profound antibacterial action against S. aureus, E. coli, B. pumilis, and S. typhi. Additionally, all of the complexes were shown to persist in the positive antioxidant potential of the ligand. Contrarily, not a single metal complex conserved the antifungal potentials of the ligand.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

Computer Vision-Based Microcalcification Detection in Digital Mammograms Using Fully Connected Depthwise Separable Convolutional Neural Network.

Microcalcification clusters in mammograms are one of the major signs of breast cancer. However, the detection of microcalcifications from mammograms is a challenging task for radiologists due to their tiny size and scattered location inside a denser breast composition. Automatic CAD systems need to predict breast cancer at the early stages to support clinical work. The intercluster gap, noise between individual MCs, and individual object's location can affect the classification performance, which may reduce the true-positive rate. In this study, we propose a computer-vision-based FC-DSCNN CAD system for the detection of microcalcification clusters from mammograms and classification into malignant and benign classes. The computer vision method automatically controls the noise and background color contrast and directly detects the MC object from mammograms, which increases the classification performance of the neural network. The breast cancer classification framework has four steps: image preprocessing and augmentation, RGB to grayscale channel transformation, microcalcification region segmentation, and MC ROI classification using FC-DSCNN to predict malignant and benign cases. The proposed method was evaluated on 3568 DDSM and 2885 PINUM mammogram images with automatic feature extraction, obtaining a score of 0.97 with a 2.35 and 0.99 true-positive ratio with 2.45 false positives per image, respectively. Experimental results demonstrated that the performance of the proposed method remains higher than the traditional and previous approaches.

Evaluation of the Antimicrobial Effect of Thymoquinone against Different Dental Pathogens: An In Vitro Study.

This study aimed to evaluate the antimicrobial effect of Thymoquinone (TQ) on four different oral microorganisms. Minimum Bactericidal Concentration (MBC), Minimum Inhibition Concentration (MIC), Broth microdilution, and Well diffusion tests were used to determine the optimum antimicrobial concentrations of TQ against Streptococcus salivarius, Streptococcus oralis, Streptococcus mutans, and Staphylococcus aureus over 1, 3, 6, 12 and 24 h. Chlorhexidine 0.12% was selected as a positive control. The inhibitory effect of TQ on bacterial growth was most noticeable with S. salivarius, while the least affected was S. aureus. TQ's MBC and MIC for S. oralis and S. aureus were comparable 2 mg/mL and 3 mg/mL, respectively. S. salivarius was most resistant to TQ and displayed a value of 5 mg/mL and 4 mg/mL for MIC and MBC, respectively. The viable count of different strains after exposure to TQ's MBC values was most noticeable with S. aureus followed by S. oralis and S. mutans, while S. salivarius was least affected. This study emphasized the promising antimicrobial effect of TQ against the four main oral microorganisms. It has a potential preventive effect against dental caries as well as other oral diseases.

Effect of Temperature on 1H NMR Spectra, Antitrypanosomal Activity, Conformational Analysis, and Molecular Docking of Curine Derivatives from Berberis brevissima.

The ethanolic root extract of Berberis brevissima afforded a new bisbenzylisoquinoline alkaloid, 13-nitrochondrofoline (2), and two known bisbenzylisoquinoline alkaloids, chondrofoline (1) and curine (4). The acetylation of chondrofoline (1) gave O-acetylchondrofoline (3). The dimeric structures of 1 and 2 were studied through variable-temperature 1H NMR spectroscopy at 25, 40, 60, and 80 °C and conformational analysis, using density functional theory employing the M06-2X functional and the 6-31G* basis set. The in vitro antitrypanosomal activity of compounds 1, 2, 3, and 4 against Trypanosoma brucei showed significant potential with MIC values of 2.6, 2.2, 2.3, and 3.8 µM, respectively. Molecular docking evaluation of alkaloids 1, 2, 3, and 4 against known T. brucei protein targets revealed T. brucei phosphodiesterase B1 to be the preferred target. The docking energies of the alkaloids with Tb6PGL (PDB 3EB9) ranged from -88.8 to -106.0 kJ/mol and was comparable to the cocrystallized ligand, citrate (Edock = -78.3 kJ/mol). It seems reasonable that the curine alkaloids may compete with the natural substrates for these protein targets and serve as leads in designing and developing more potent and selective drugs against T. brucei.

Estimating global injuries morbidity and mortality: methods and data used in the Global Burden of Disease 2017 study.

BACKGROUND: While there is a long history of measuring death and disability from injuries, modern research methods must account for the wide spectrum of disability that can occur in an injury, and must provide estimates with sufficient demographic, geographical and temporal detail to be useful for policy makers. The Global Burden of Disease (GBD) 2017 study used methods to provide highly detailed estimates of global injury burden that meet these criteria. METHODS: In this study, we report and discuss the methods used in GBD 2017 for injury morbidity and mortality burden estimation. In summary, these methods included estimating cause-specific mortality for every cause of injury, and then estimating incidence for every cause of injury. Non-fatal disability for each cause is then calculated based on the probabilities of suffering from different types of bodily injury experienced. RESULTS: GBD 2017 produced morbidity and mortality estimates for 38 causes of injury. Estimates were produced in terms of incidence, prevalence, years lived with disability, cause-specific mortality, years of life lost and disability-adjusted life-years for a 28-year period for 22 age groups, 195 countries and both sexes. CONCLUSIONS: GBD 2017 demonstrated a complex and sophisticated series of analytical steps using the largest known database of morbidity and mortality data on injuries. GBD 2017 results should be used to help inform injury prevention policy making and resource allocation. We also identify important avenues for improving injury burden estimation in the future.

Global injury morbidity and mortality from 1990 to 2017: results from the Global Burden of Disease Study 2017.

BACKGROUND: Past research in population health trends has shown that injuries form a substantial burden of population health loss. Regular updates to injury burden assessments are critical. We report Global Burden of Disease (GBD) 2017 Study estimates on morbidity and mortality for all injuries. METHODS: We reviewed results for injuries from the GBD 2017 study. GBD 2017 measured injury-specific mortality and years of life lost (YLLs) using the Cause of Death Ensemble model. To measure non-fatal injuries, GBD 2017 modelled injury-specific incidence and converted this to prevalence and years lived with disability (YLDs). YLLs and YLDs were summed to calculate disability-adjusted life years (DALYs). FINDINGS: In 1990, there were 4 260 493 (4 085 700 to 4 396 138) injury deaths, which increased to 4 484 722 (4 332 010 to 4 585 554) deaths in 2017, while age-standardised mortality decreased from 1079 (1073 to 1086) to 738 (730 to 745) per 100 000. In 1990, there were 354 064 302 (95% uncertainty interval: 338 174 876 to 371 610 802) new cases of injury globally, which increased to 520 710 288 (493 430 247 to 547 988 635) new cases in 2017. During this time, age-standardised incidence decreased non-significantly from 6824 (6534 to 7147) to 6763 (6412 to 7118) per 100 000. Between 1990 and 2017, age-standardised DALYs decreased from 4947 (4655 to 5233) per 100 000 to 3267 (3058 to 3505). INTERPRETATION: Injuries are an important cause of health loss globally, though mortality has declined between 1990 and 2017. Future research in injury burden should focus on prevention in high-burden populations, improving data collection and ensuring access to medical care.

COVID-19 outbreak and its monetary implications for dental practices, hospitals and healthcare workers.

The novel COVID-19 came under limelight few months back (December 2019) and has recently been declared a pandemic by WHO. It has resulted in serious financial implications being faced by dental practices, hospitals and healthcare workers. Dental practice currently is restricted to provision of emergency dental care whereas, many hospitals have also cancelled elective procedures to save finances for COVID-19 treatment which is expensive and unpredictable. In addition, healthcare workers are also facing financial challenges in this difficult time. Competent authorities should step in to help dental practices, hospitals and healthcare workers in order to ensure the provision of all types of healthcare efficiently in these testing times and beyond.

Comparative evaluation of two chelating agents in collagen fiber network modification over dentinal tubules: An in vitro analysis.

OBJECTIVE: To compare effectiveness of ethylenediaminetetraacetic acid (EDTA) and citric acid in removing collagen fiber network covering dentinal tubules of human teeth. MATERIALS AND METHODS: Eighteen dentin discs were divided in three groups; Gp 1: discs received no treatment (control), Gp 2: discs etched with 17% EDTA (pH = 7.1), and Gp 3: discs etched with 6 wt% citric acid (pH = 4.0). Scanning electron microscopy (SEM) was performed to assess collagen fiber removal and X-ray diffraction (XRD) was implemented to analyse crystal peaks of discs. RESULTS: The SEM analysis demonstrated more collagen removal with EDTA treatment compared to citric acid treated specimens. Grade 6 (81% to 100% fiber removal) was mostly achieved for Gp 2 samples whereas grade 2 (1% to 20% fiber removal) was mostly achieved for Gp 3 samples and inter-group comparisons between these groups were statistically significant (p < 0.05). X-ray diffractogram of control and experimental samples demonstrated absence of calcite phase in experimental groups. The change in peak shapes and intensities were observed and citric acid treated samples revealed more intense peaks than EDTA group. CONCLUSION: Our study found 17% EDTA to be more effective in removing collagen fibers when matched with 6% citric acid.

COVID-19 outbreak, disruption of dental education, and the role of teledentistry.

The novel coronavirus disease 2019 (COVID-19) has affected the whole world and has now been declared a Pandemic by the World Health Organization (WHO). Although the mortality rate of this virus is low, it is especially potent against people with underlying systemic conditions. Dentistry is a profession where the doctor, as well as the dental staff, works in close vicinity to the patient's mouth. Dental education has two core components; didactic and clinical training (including patient care). Dental education has been interrupted in the past due to certain events (Arab Spring and SARS outbreak). Currently, the pandemic of COVID-19 has disrupted dental education globally as most of the dental schools and universities in the world have closed amidst the COVID-19 outbreak. Teledentistry is a subspecialty of telemedicine that helps in the provision of educational activities, advice, and diagnosis about treatment over a distance with the help of technology like video conferencing. The current overview summarizes the potential role of teledentistry in continuing the dental educational process in terms of delivery of didactic components, clinical training, and patient care. It can be concluded that with modern updated devices and tools, teledentistry can be an effective way to prevent disruption of dental education and it can be utilized in continuing the dental educational process in this critical time of the COVID-19 outbreak.

Risk Assessment of Healthcare Workers at the Frontline against COVID-19.

The novel coronavirus disease 2019 (COVID-19) is a global pandemic. Healthcare workers (HCWs) are on the frontline of treating patients infected with COVID-19. However, data related to its infection rate among HCWs are limited. The aim was to present evidence associated with the number of HCWs being infected with COVID-19 from most viral affected countries (Italy, China, United States, Spain, and France). Furthermore, we looked into the reasons for HCWs COVID 19 infections and strategies to overcome this problem. Early available evidence suggested that HCWs are being increasingly infected with the novel infection ranging from 15% to 18% and in some cases up to 20% of the infected population. Major factors for infection among HCWs include lack of understanding of the disease, inadequate use and availability of Personal Protective Equipment (PPE), uncertain diagnostic criteria, unavailability of diagnostic tests and psychological stress. Therefore the protection of HCWs by authorities should be prioritized through education and training, the readiness of staff, incentives, availability of PPEs, and psychological support.

The chemistry and pharmacology of alkaloids and allied nitrogen compounds from Artemisia species: A review.

Several reviews have been published on Artemisia's derived natural products, but it is the first attempt to review the chemistry and pharmacology of more than 80 alkaloids and allied nitrogen compounds obtained from various Artemisia species (covering the literature up to June 2018). The pharmacological potential and unique skeleton types of certain Artemisia's alkaloids provoke the importance of analyzing Artemisia species for bioactive alkaloids and allied nitrogen compounds. Among the various types of bioactive Artemisia's alkaloids, the main classes were the derivatives of rupestine (pyridine-sesquiterpene), lycoctonine (diterpene), pyrrolizidine, purines, polyamine, peptides, indole, piperidine, pyrrolidine, alkamides, and flavoalkaloids. The rupestine derivatives are Artemisia's characteristic alkaloids, whereas the rest are common alkaloids found in the family Asteraceae and chemotaxonomically links the genus Artemisia with the tribes Anthemideae. The most important biological activities of Artemisia's alkaloids are including hepatoprotective, local anesthetic, ß-galactosidase, and antiparasitic activities; treatment of angina pectoris, opening blocked arteries, as a sleep-inducing agents and inhibition of HIV viral protease, CYP450, melanin biosynthesis, human carbonic anhydrase, [3H]-AEA metabolism, kinases, and DNA polymerase ß1 . Some of the important nitrogen metabolites of Artemisia include pellitorine, zeatin, tryptophan, rupestine, and aconitine analogs, which need to be optimized and commercialized further.

In silico and in vivo investigation of ferrocene-incorporated acyl ureas and homoleptic cadmium carboxylate derivatives for anticonvulsant, anxiolytic, and sedative potential.

In this study different derivatives of ferrocene-incorporated acyl ureas and homoleptic cadmium carboxylates were investigated for potential anticonvulsant, anxiolytic and sedative properties, using in-silico and in-vivo techniques. The molecular docking studies reveled that ferrocene compounds derivative 1-(4-bromobenzoyl)-3-(4-ferrocenylphenyl) urea (PB1) and cadmium compounds derivative bis (diphenylacetato) cadmium (II) (DPAA) exhibit binding affinities against various neurotherapeutic molecular targets involved in epilepsy, anxiety, and sedation. Both PB1 and DPAA showed high binding affinities against protein targets like mammalian shaker voltage dependent potassium channel beta subunit complex, calcium release-activated calcium channel, sodium channel 2A inactivation gate, human sodium/hydrogen exchanger regulatory factor, and gamma amino butyric acid A receptor associated protein. PB1 (2-10 mg/kg) and DPAA (1-5 mg/kg) delayed onset time of pentylenetetrazole-induced myoclonic jerks and tonic-clonic seizures in mice while decreased duration of tonic-clonic seizures, determining the anticonvulsant effect of these compounds. PB1 and DPAA (0.5-1 mg/kg) exhibited anxiolytic effect by increasing time spent and number of animals entries into open arms, while decreasing time spent in dark compartment. Furthermore, PB1 (0.5-1 mg/kg) and DPAA (0.1-1 mg/kg) reduced onset time of sleep and increased duration time of sleep in mice, showing sedative effect. Taken together, our results indicate that aforementioned derivatives of ferrocene and cadmium are potent neurotherapeutic agents possessing anticonvulsant, anxiolytic and sedative properties.

Antimicrobial and antiurease potential of Diorganotin(IV) Schiff bases.

The total phenolic content, flavonoid content, in vitro xanthine oxidase (XOD) inhibitory activity and antioxidant activity (AA) of Eucommia ulmoides Oliver leaf extracts were investigated. The AA investigations included 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, ß-carotene/linoleic acid bleaching assay and oxygen radical absorbance capacity (ORAC) test. The ethyl acetate fraction (EE) showed the highest AA and xanthine oxidase inhibitory activity. Whilst the lowest 50% inhibition (IC50) value of this fraction for DPPH free radical scavenging was 0.045mg/mL, its highest ORAC value was 10.57 µmol TE/mg. The highest inhibition rate against linoleic acid oxidation observed was 69.41%, and the lowest IC50 value for xanthine oxidase activity inhibition was 2.47mg/mL. These results show that E. ulmoides leaf extract is a promising source of natural antioxidants because it contains high contents of bioactive compounds, including chlorogenic acid, rutin, hyperin and astragalin, as detected by high-performance liquid chromatography coupled to HPLC-DAD-ESI-MS.