Liquid Chromatography-mass Spectrum Research Articles

Characterization of ribostamycin and its impurities using a nano-quantity analyte detector: Systematic comparison of performance among three different aerosol detectors

Characterization of aminoglycoside antibiotics like ribostamycin is important due to the complex composition and common toxic impurities. Aerosol detectors are often employed for determination of these non-absorbent analytes. In this work, a robust and cost-effective method was developed for simultaneous detection of ribostamycin and its related substances using high-performance liquid chromatography (HPLC) with a relative new aerosol detector named nano-quantity analyte detector (NQAD). With the introduction of less toxic but more compatible ion-pairs pentafluoropropionic acid (PFPA) and trifluoroacetic acid (TFA) in the eluent, an optimized separation effect was achieved. Compared with the other two aerosol detectors namely ELSD (evaporative light scattering detector) and CAD (charged aerosol detector), method verification and quantitative detection results revealed that NQAD had higher sensitivity than ELSD with a 0.8 μg/mL limit of detection, as well as wider linear range (from 2 μg/mL to 1000 μg/mL) than both CAD (from 2 μg/mL to 200 μg/mL) and ELSD (from 8 μg/mL to 200 μg/mL) detector. The performance of NQAD helped to realize detection of ribostamycin and its impurities with significant concentration differences in a single run. With a cation suppressor to eliminate the ion-suppression caused by the ion-pairs in the eluent, the structure of nine impurities in ribostamycin sample was characterized by liquid chromatography-mass spectrum (LC-MS). Both external standard and area normalization calculation were investigated, and NQAD obtained more accurate results due to its full-range linear response-to-concentration relationship, providing an alternative for routine quality control of multi analyte systems.

Phytochemical Profile of Rhizophora apiculata, Bruguiera gymnorhiza, and Bruguiera cylindrica for Wood Identification

Identification of wood species that are difficult to distinguish from their anatomical structure can be done through phytochemical (extractive substance) profiling. This research aimed to analyze the phytochemical profile as a sorter for three species of mangrove wood from Indramayu and Cilacap Regencies, Indonesia, using the liquid chromatography-mass spectrum. The phytochemical markers of taxonomic were the dominant compounds only found in one wood species. The results showed that the three types of wood are dominated by phytochemicals dissolved in ethanol. The results of LCMS analysis of the ethanol extract showed that the phytochemical markers were triterpenoid, flavonoid, and fatty acyls glycoside groups for Rhizophora apiculata, steroid and naphthalene groups for Bruguiera gymnorhiza, and alkaloid as well as fatty amide group for Bruguiera cylindrica. The dominant compounds that characterize these can be used in sorting between mangrove wood species. Keywords: characterizer, compound, mangrove, phytochemicals

Synthesis, characterization and evaluation of a pH-responsive molecular imprinted polymer for Matrine as an intelligent drug delivery system

Abstract To address the undesirable reactions associated with matrine (MAT) injection in clinical settings, a high-loading drug delivery system (DDS) based on pH-sensitive molecularly imprinted polymer (MAT@MIPs) was prepared for the first time. The imprinted materials containing recognition sites for the matrine were formed by using carboxyl-functionalized multiwalled carbon nanotubes as a supportive matrix and dopamine as a cross-linker due to its exceptional biocompatibility. Subsequently, the optimal reaction conditions and adsorption performance of MAT@MIPs were systematically investigated. The obtained polymers were characterized and evaluated by Fourier transform infrared spectrometry, scanning electron microscopy, elemental analysis, and thermogravimetric analysis. Results indicated that the MIPs demonstrated a favorable imprinting factor (2.36) and a high binding capacity (21.48 mg·g−1) for matrine. In vitro studies, we performed cell counting kit-8 assays in HepG2 cells, then the drug delivery capabilities of MAT-loaded MIPs were validated through light microscopy analyses, and the matrine content in culture medium was quantified using ultra high-performance liquid chromatography-mass spectrum synchronously. The facile fabrication of MAT@MIPs presents a viable solution for designing high-loading and pH-responsive DDS, which can offer a novel administration approach for drugs requiring injection in clinical applications.

Endocrine disrupting chemical Bisphenol A and its association with cancer mortality: a prospective cohort study of NHANES.

There is evidence suggesting that Bisphenol A (BPA) is associated with increased all-cause mortality in adults. However, the specific nature of the relationship between BPA exposure and cancer mortality remains relatively unexplored. The National Health and Nutrition Examination Survey (NHANES) dataset was used to recruit participants. Urinary BPA was assessed using liquid chromatography-mass spectrum (LC-MS). Through the use of multivariable Cox proportional hazard regressions and constrained cubic splines, the relationships between urine BPA and death from all causes and cancer were investigated. This study has a total of 8,035 participants, and 137 died from cancers after a 7.5-year follow-up. The median level of BPA was 2.0 g/mL. Urinary BPA levels were not independently associated with all-cause mortality. For cancer mortality, the second quartile's multivariable-adjusted hazard ratio was 0.51 (95% confidence interval: 0.30 to 0.86; p = 0.011) compared to the lowest quartile. The restricted cubic splines showed that the association was nonlinear (p for nonlinearity = 0.028) and the inflection point was 1.99 ng/mL. Urinary BPA exposure was U-shaped associated with the risk of cancer mortality, and a lower level of BPA less than 1.99 ng/mL was associated with a higher risk of cancer mortality.

EVALUATION OF THE ANTI-TYROSINASE-ANTI-AGING POTENTIAL AND METABOLITE PROFILING FROM THE BIOACTIVE FRACTION OF CORN COB (ZEA MAYS L.)

Objective: Maize (Zea mays L.) is a crop that has been widely cultivated in Indonesia. Using corn kernels on a large scale will produce much corn cob waste, usually unused. According to the literature search, corn cobs’ phytochemical studies and pharmacological activities still need to be improved. This study aims to determine the content of secondary metabolites (metabolite profiling) and their antityrosinase and anti-aging potential.
 Methods: Corn cobs were macerated with methanol and fractionated with n-hexane, ethyl acetate, and butanol. The phytochemical profiling approach of the methanol extract was performed by liquid chromatography-mass spectra (LC-MS/MS). Anti-tyrosinase and anti-aging bioactivity were evaluated by thin layer chromatography (TLC)-bioautography and IC50 spectrophotometrically.
 Results: The evaluation results show that the butanol fraction leads to a potential value (IC50 99.92 µg/ml). Several compounds, especially flavonoid compounds (including catechin; kaempferol 3-arabinofuranoside 7-rhamnoside; 6,8-Di-C-beta-D-arabino pyranosyl apigenin; 5,7-Dihydroxy-8,4’-dimethoxyisoflavone) were identified by LC-MS/MS by comparing the molecular mass of MS/MS data with literature data.
 Conclusion: Based on this study, it can be concluded that butanol is the fraction that most actively inhibits tyrosinase, elastase, and collagenase enzymes, which means it potentially becomes a new anti-aging candidate.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. N/A. This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Solid-state fermentation of food waste by Serratia marcescens NCHU05 for prodigiosin production

BackgroundProdigiosin is a red colored pigment with applications in antibacterial, antifungal, anticancer, and immunosuppressive activities. It is produced as a secondary metabolite by bacteria genera such as Serratia, Pseudomonas, and Streptomyces. The high-value prodigiosin offers a promising opportunity for the valorization of local food wastes. MethodsThe newly found red pigment-producing colony was isolated, sequenced, and identified. The red pigment was characterized using UV–VIS and liquid chromatography mass spectrum. Two cultivation methods, submerged culture and solid-state fermentation (SSF), were employed to produce prodigiosin. Three food waste sources, including rice bran, fish meal, and soybean dreg, were employed as both nutrition and solid matrices for SSF. Significant findingsA new red pigment-producing Serratia marcescens NCHU05 was successfully isolated and identified. The red pigment was characterized as prodigiosin. S. marcescens NCHU05 was employed for prodigiosin production. SSF outperformed submerged cultures in prodigiosin production. Specifically, SSF using LB1 agar plate (1 g/L NaCl) yielded 22 mg/kg-dry-solid, while submerged cultures in LB1 medium produced only 3.2 ± 0.2 mg/L. Remarkably, when rice bran, fish meal, and soybean dregs were used as nutrients and solid matrices for SSF, prodigiosin productions of 50, 54, and 79 mg/kg-dry-solid were achieved, respectively. This study illustrates the feasibility of utilizing locally isolated microorganisms to contribute to the valorization of local wastes.

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue.

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

Discovery of anti-allergic components in Guomingkang Formula using sensitive HEMT biochips coupled with in vitro and in vivo validation

BackgroundAllergic rhinitis (AR) is a prevalent allergic disease, which seriously affects the sufferers’ life quality and increases the socioeconomic burden. Guominkang (GMK), a well-known prescription for AR treatment, showed satisfactory effects; while its anti-allergic components remain to be disclosed. AlGaN/GaN HEMT biochip is more sensitive and cost-effective than other binding equipments, indicating its great potential for screening of active ingredients from herbal medicines. MethodsAR mouse models were first established to test the anti-allergic effect of GMK and discover the ingredients absorbed into blood by ultra-high performance liquid chromatography-mass spectra (UHPLC-MS). Then, novel Syk/Lyn/Fyn-functionalized high electron mobility transistor (HEMT) biochips with high sensitivity and specificity were constructed and applied to screen the active components. Finally, the results from HEMT biochips screening were validated via in silico (molecular docking and molecular dynamics simulation), in vitro (RBL-2H3 cells), and in vivo (PCA mice model) assays. ResultsGMK showed a potent therapeutic effect on AR mice, and fifteen components were identified from the medicated plasma. Furthermore, hamaudol was firstly found to selectively inhibit the Syk and Lyn, and emodin was to selectively inhibit Lyn, which were further confirmed by isothermal titration calorimetry, molecular docking, and molecular dynamics simulation analyses. Suppression of the activation of FcεRI-MAPK signals might be the possible mechanism of the anti-allergic effect of hamaudol. ConclusionsThe targets of emodin and hamaudol were discovered by HEMT biochips for the first time. This study provided a novel and effective strategy to discover active components in a complex herbal formula by using AlGaN/GaN HEMT biochips.

Survey of Acetylation for Thermoanaerobacter tengcongensis.

Non-histone protein acetylation is involved in key cellular processes both in eukaryotes and prokaryotes. Acetylation in bacteria is used to modify proteins involved in metabolism and allow the bacteria to adapt to their environment. TTE (Thermoanaerobacter tengcongensis) is an anaerobic, thermophilic saccharolytic bacterium that grows at extreme temperature range between 50 and 80℃. The annotated TTE proteome contains less than 3000 proteins. We analyzed the proteome and acetylome of TTE using 2DLC-MS/MS (2-dimensional liquid chromatography mass spectrum). We evaluated the ability of mass spectrometry technology to cover a relatively small proteome as much as possible. And we also observed wide spread of acetylation in TTE, which changed under different temperatures. A total of 2082 proteins were identified, which accounts for about 82% of the database. A total of 2050 (~ 98%) proteins were quantified in at least one culture condition and 1818 proteins were quantified in all 4 conditions. The result also consisted 3457 acetylation sites corresponding to 827 distinct proteins, which covered 40% of the proteins identified. Bioinformatics analysis reported that proteins related to replication, recombination, repair, and extracellular structure cell wall biogenesis had more than half members acetylated, while energy production, carbohydrate transport, and metabolism related proteins were least acetylated. Our result suggested that acetylation affects the ATP-related energy metabolism and energy-dependent biosynthesis process. Comparing the enzymes related with lysine acetylation and acetyl-CoA (acetyl-coenzyme A) metabolism, we suggested that the acetylation of TTE took a non-enzymatic mechanism and affected by abundance of acetyl-CoA.

Thermal decomposition synthesis of cylindrical rod-like MoO3 and irregular sphere-like Ag2MoO4 nanocrystals for accelerating photocatalytic degradation of industrial reactive dyes and biosensing application

This paper reports the thermal decomposition method to prepare a facile, one-pot synthesis of cylindrical rod-like MoO3 and irregular sphere-like Ag2MoO4 nanocrystals. The characteristic properties of MoO3 and Ag2MoO4 nanocrystals were investigated by XRD, FESEM, HRTEM, EDS, IR, UV-Vis, PL, EIS, XPS, and CV techniques. Ag2MoO4 nanocrystals result in an optical band gap to redshift, assessed by Tauc plots of the ultraviolet-visible (UV–vis) spectra. When compared to other MWCNTs modified and unmodified CPEs for the detection of the 5-Flououracil drug, the MoO3/MWCNTs/CPE showed excellent electrocatalytic activity in terms of a larger anodic peak current and an inferior peak potential. Ag2MoO4 nanocrystals shown in an excellent photocatalytic degradation efficiency of 91.39% and 57.89% for the Cibacron Deep Red C-D (CR) and Cibacron Orange C-RN (CO) dyes as compared to MoO3 as evaluated by UV–visible spectrophotometric analysis in 150 mins under 100 W visible-light irradiation. Electrochemical impedance spectroscopy has shown that the improved separation and transport of photogenerated electron-hole pairs account for Ag2MoO4’s greater efficiency than MoO3. The effects of experimental parameters for the photocatalytic degradation of dyes, including initial dye concentration of 10 mg/L, catalyst dose 75 mg, pH 7, and catalyst recycling, have been investigated to optimize reaction conditions. The primary active species were obtained to be photogenerated holes (h+) and superoxide radical anions (O2•‾). The cyclic photocatalytic degradation of CR and CO testified to Ag2MoO4’s good extent of reusability. The details of the electrochemical and photocatalytic mechanisms were then provided. Also, liquid chromatography-mass spectra (LC-MS) were used to find out the photocatalytic degradation intermediate products.

Beneficial effect of ζ-carotene-like compounds on acute UVB irradiation by alleviating inflammation and regulating intestinal flora.

ζ-Carotene is a key intermediate in the carotenoid pathway, but owing to its low content and difficulties in isolation, its application is restricted. In this study, three genes (pnCrtE, pnCrtB, and pnCrtP) in the carotenoid pathway of Antarctic moss were identified, recombined, and expressed in Escherichia coli (E. coli) BL21(DE3). The expression product was identified as one of the ζ-carotenes by UV absorbance spectrum, thin layer chromatography (TLC), and super-high-performance liquid chromatography-mass spectrum (UPLC-MS), and was called a ζ-carotene-like compound (CLC). Excessive exposure to ultraviolet B (UVB) irradiation is one of the main risk factors for skin photodamage. The purpose of this study was to investigate the preventive and therapeutic effects of CLC on UVB-induced skin photodamage in mice. In this paper, through histological examinations (hematoxylin-eosin, HE; Masson and TdT-mediated dUTP Nick-End Labeling, Tunel), biochemical index detection (reactive oxygen species, ROS; inflammatory factors; cyclobutyl pyrimidine dimers, CPDs and hyaluronic acid, HA), quantitative real time polymerase chain reaction (qRT-PCR), immunohistochemistry and intestinal content flora, etc., it is concluded that CLC has the potential to enhance skin antioxidant capacity by activating the nuclear transcription factor/antioxidant reaction element (Nrf2/ARE) pathway and also reduce skin inflammation and aging by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Moreover, the regulation of intestinal flora may potentially mitigate skin damage induced by UVB radiation. This research not only developed a green and sustainable platform for the efficient synthesis of CLC but also laid a foundation for its application in functional food and medicine for skin resistance against UVB damage.

Analgesic and Anti-inflammatory Activities of Urena lobata L. Leaf Extracts

Pulutan (Urena lobata) is a medicinal plant used to treat fever, infection and swelling traditionally, although the pre-clinical study about the herbs as analgesic and anti-inflammatory are still limited. The objective of this study is to measure analgesic and anti-inflammatory activity of U. lobata leaf extract through in vivo study and in silico. This study was performed using male wistar rats divided into eight groups (two control groups and six test groups) (n=5). U. lobata leaf was extracted by water and ethanol, each prepared in dose of 125, 250, 500 mg/kg body weight. Anti-inflammatory activity was evaluated by plethysmometer for 6 hours, meanwhile analgesic activity was examined by analgesymeter for 4 hours. The data was analyzed using one way ANOVA test continued with LSD test (p < 0.05). Following active compound identification using Liquid Chromatography-Mass Spectra (LC-MS/MS), several compounds were used in an in silico study using docking server. Both aqueous and ethanolic extract of U.lobata at 125 and 250 mg/kg bw inhibited paw edema with an Area Under Curve (AUC) volume about 10 % and 5%, respectively, compared to control group (p<0.05). For analgesic activity, aqueous extract of U. lobata were able to increase the AUC for pain threshold of about 30-100 % compared to control group (p<0.05), whereas the activity of ethanolic extract lower than aqueous extract. Stigmasterol and beta-sitosterol in U. lobata potentially inhibited Cycloxygenase-2 (COX-2) as a pro-inflammatory mediator and pain. Aqueous and ethanolic extract of U. lobata have analgesic activity and anti-inflammation, the mechanism was predicted through inhibitory activity of COX-2.

Effect of dissolved silicate on the degradation of sulfamethoxazole by nZVI@D201 nanocomposite

Dissolved silicate (Si), which is omnipresent in natural water and wastewater, is a key factor mediating the separation and transformation of contaminants by forming complexes on the surface of zero-valent iron (ZVI) and iron oxides. In this study, we prepared an nZVI@D201 nanocomposite and studied the redistribution of sulfamethoxazole (SMX) on nZVI@D201 in the pre-equilibration and Si-SMX simultaneous addition systems with 0.1 mM and 1.0 mM Si at an initial pH of 3.0–11.0. The results revealed that both Si pre-equilibration and Si-SMX simultaneous addition exhibited excellent SMX removal efficiency. Si pre-equilibration altered the adsorbed SMX to degradative SMX at an initial pH of 5.0–9.0. Moreover, the simultaneous Si addition at increasing concentrations decreased the degradative SMX. X-ray diffraction, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy were used to characterize nZVI@D201 after pre-equilibration and simultaneous addition of Si and SMX at an initial pH of 5.0. The results showed that Si pre-equilibration inhibited the transformation of ZVI to magnetite and decreased the crystallinity of the minerals. In addition, the simultaneous addition of Si and SMX impeded the transformation of nZVI to magnetite, whereas the formation of Fe-O-Si complexes hindered the crystallinity. Moreover, scavenger experiments and electron spin resonance tests proved that addition of Si didn’t change the reactive species formation and ·OH was the mainly species for SMX degradation. Based on the detected intermediates via ultra-high performance liquid chromatography-mass spectra, the possible degradation pathways of SMX were proposed.

Screening and characterizing the quality markers of Selaginella tamariscina (P. Beauv.) Spring using metabonomics and molecular networking

Quality control of traditional Chinese medicine (TCM) should be linked with the authentication and efficacy of TCM. Selaginella tamariscina is a frequently used traditional Chinese herbal medicine. However, its quality control is still difficult due to its multiple adulterants. We established quality markers (Q-markers) of S. tamariscina by using metabolomics, molecular networking and network pharmacology to improve the authenticity study and quality control of S. tamariscina. In this study, ultra high performance liquid chromatography-mass spectrum (UHPLC-MS) coupled with multivariate statistical analyses was applied to distinguish between S. tamariscina samples and their confusing adulterants. Principal component analysis, hierarchical clustering analysis (PCA), hierarchical clustering analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were employed to screen the distinguishing markers from S. tamariscina samples and their adulterants. The top-2 distinguishing markers were isolated from S. tamariscina and identified by molecular networking together with nuclear magnetic resonance spectroscopy (NMR). Network pharmacology predicted the bioactivity and cytotoxicity of the top-2 distinguishing markers. The top-2 distinguishing markers were adopted as Q-markers of S. tamariscina for content determination. Based on the results of ultra performance liquid chromatography-quardrupole-time of flight mass spectrometry (UHPLC-QTOF-MS) metabolomics, we revealed that selaginellins could only be detected in S. tamariscina samples and contributed greatly to discriminating S. tamariscina samples from their confused species. The top-2 distinguishing markers were isolated and purified from S. tamariscina extract. Then, they were further identified as selaginellin and selaginellin A by molecular networking and NMR. Network pharmacology predicted the antitumor activity of selaginellin and selaginellin A, while the cytotoxicity assay verified their bioactivity. In conclusion, selaginellin and selaginellin A were selected as Q-markers for the determination and quality evaluation of S. tamariscina based on ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS). The ranges of the concentrations of selaginellin and selaginellin A were 41.57–44.89 μg/g and 15.09–16.75 μg/g, respectively. This study provides a novel strategy combining Ultra performance liquid chromatography mass spectrometry based (UHPLC-MS-based) metabolomics with molecular networking for rapid species identification of S. tamariscina and discovery of the Q-markers of TCM.

Lactate Dehydrogenase-Inhibitors Isolated from Ethyl Acetate Extract of Selaginella doederleinii by Using a Rapid Screening Method with Enzyme-Immobilized Magnetic Nanoparticles.

Lactate dehydrogenase (LDH) is one of the important enzyme systems for glycolysis and gluconeogenesis. It can catalyze the reduction and oxidation reaction between propionic acid and L-lactic acid, which is usually overexpressed in cancer cells. Therefore, inhibiting the activity of LDH is a promising way for the treatment of cancer. In this study, an effective method based on ligand fishing and ultra performance liquid chromatography-mass spectrum (UPLC-MS) was established to screen and identify active ingredients from Selaginella doederleinii with potential inhibitory activity for LDH. Firstly, LDH was immobilized on the magnetic nanoparticles (MNPs), three immobilization parameters including LDH concentration, immobilization time and pH were optimized by single factor and response surface methodology for maximum (max) immobilization yield. Then, a mixed model of galloflavin and chlorogenic acid (inhibitors and non-inhibitors of LDH) was used to verify the specificity of immobilized LDH ligand fishing, and the conditions of ligand fishing were further optimized. Finally, combined with UPLC-MS, immobilized LDH was used to simultaneously screen and identify potential LDH inhibitors from the ethyl acetate extract of Selaginella doederleinii. The prepared fishing material was comprehensively characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared spectrometer (FT-IR). The optimal immobilization conditions were obtained as LDH concentration of 0.7 mg/mL, pH value of 4.5, and immobilization time of 3.5 h. Under these conditions, the max immobilization yield was (3.79 ± 0.08) × 103 U/g. The specificity analysis showed that immobilized LDH could recognize and capture ligands, and the optimal ligand fishing conditions included that the incubation time was 30 min, the elution time was 20 min, and the concentration of methanol as eluent was 80%. Finally, two LDH inhibitors, amentoflavone and robustaflavone, were screened by immobilized LDH from the ethyl acetate extract of Selaginella doederleinii. The study provided a meaningful evidence for discovering the bioactive constituents in ethyl acetate extract of Selaginella doederleinii related to cancer treatment, and this ligand fishing method was feasible for screening enzyme inhibitors from similar complex mixtures.

In Silico Study of Pulutan (Urena lobata) Leaf Extract as Anti Inflammation and their ADME Prediction

Inflammation is the basic for the pathogenesis of several diseases both of degenerative and non-degenerative disease. Urena lobata is a medicinal plant that can be found in Indonesia and has been used traditionally to cure influenza, inflammation and fever. However, there have been few reports about their anti-inflammatory activity and their mechanism action are still unclear. The aim of study to evaluate the anti-inflammatory activities of active substances from U.lobata leaf and their pharmacokinetic property through in silico study. lobata leaf was extracted by digeration methods using ethanol solvent. Therefore, the active substances in the extract was analyzed by Liquid Chromatography-Mass Spectra (LC-MS). Pharmacokinetic property and physicochemical of active compounds were evaluated using pkCSM online tool. Anti-inflammatory activity of U. lobata active compound on phospholipase-A2 (PLA-2), cyclooxygenase-2 (COX-2) and lipoxygenase-5 (LOX-5) were evaluated by in silico study. Ethanolic extract of U. lobata contained five active compound, there are stigmasterol, ?-sitosterol mangiferin, gossypetin and chrysoeriol. Molecular docking study indicated stigmasterol and ?-sitosterol of U. lobata have a strong activity as anti-inflammatory based on the estimation of inhibition constant (Ki) value against PLA2 and COX-2. Meanwhile, mangiferin and gossypetin have a stronger anti-inflammatory effect on LOX-5 among others compound. U. lobata has anti-inflammatory activity through inhibition on COX-2 greater than on PLA2 and LOX-5.

The metabolomics variations among rice, brown rice, wet germinated brown rice, and processed wet germinated brown rice

Germination and processing are always accompanied by significant changes in the metabolic compositions of rice. In this study, polished rice (rice), brown rice, wet germinated brown rice (WGBR), high temperature and pressure-treated WGBR (WGBR-HTP), and low temperature-treated WGBR (WGBR-T18) were enrolled. An untargeted metabolomics assay isolated 6 122 positive ions and 4 224 negative ions (multiple difference ≥1.2 or ≤0.8333, P<0.05, and VIP≥1) by liquid chromatography-mass spectrum. These identified ions were mainly classified into three categories, including the compounds with biological roles, lipids, and phytochemical compounds. In addition to WGBR-T18 vs. WGBR, massive differential positive and negative ions were revealed between rice of different forms. Flavonoids, fatty acids, carboxylic acids, and organoxygen compounds were the dominant differential metabolites. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, there 7 metabolic pathways (phenylalanine/tyrosine/tryptophan biosynthesis, histidine metabolism, betalain biosynthesis, C5-branched dibasic acid metabolism, purine metabolism, zeatin biosynthesis, and carbon metabolism) were determined between brown rice and rice. Germination changed the metabolic pathways of porphyrin and chlorophyll, pyrimidine, and purine metabolisms in brown rice. In addition, phosphonate and phosphinate metabolism, and arachidonic acid metabolism were differential metabolic pathways between WGBR-HTP and WGBR-T18. To sum up, there were obvious variations in metabolic compositions of rice, brown rice, WGBR, and WGBR-HTP. The changes of specific metabolites, such as flavonoids contributed to the anti-oxidant, anti-inflammatory, anti-cancer, and immunomodulatory effects of GBR. HTP may further improve the nutrition and storage of GBR through influencing specific metabolites, such as flavonoids and fatty acids.

Isolation and Identification of New Pigment From Monascus purpureus

This research was aimed to isolate and identify the new pigment from the main pigments of Monascus purpureus, which derive from five different isolates and through with solid fermentation used IR-64 rice as substrate. The measurement of pigment was done by extracting t samples using methanol in the 14th day of fermentation process. The absorbance of main pigment was measured by a UV-Visible spectrophotometer at the wavelengths of 364, 365, 375, 400, 480 and 500 nm. The results showed that the absorbance of each pigments isolate varied within ranges from 0.219 to 0.559. Result followed of Thin Layer Chromatography (TLC) method with chloroform: acetone (9: 1). TLC results were followed by isolating IPB-B samples using Thin Layer Chromatography- Preparative (TLC-P), whereas the band obtained was identified by using Liquid Chromatography-Mass Spectrum (LC-MS) instrument. The isolated and identified IPB-B samples showed that there was found a new compound of Molecular Weight (MW), which was Monaphilol A with Molecular Weight (MW) of 384.47.

ASSESSMENT OF PHENOLIC COMPOUNDS, ANTIMICROBIAL ACTIVITY AND FREE RADICAL SCAVENGING POTENCY OF THREE SELECTED VEGETABLES

Phytochemicals are compounds derived from plants that are assumed to have defensive role against certain disease. They have antioxidants, anticancer, antimicrobial, antifungal, antiviral, antithrombic and anti-inflammatory properties. They have a high specificity to boost the immune system and play important role in the metabolism of hormones. The current study is based on qualitative and quantitative evaluation of total phenolics contents, phenolic compounds, antioxidant potential, free and bound phenolic acids in selected vegetables available at the local market of Hyderabad, Sindh, Pakistan. Two different extraction procedures ultrasonic-assisted base hydrolysis extraction and sonication extraction were used. Total 13 phenolic compounds were found and quantified by high-performance liquid chromatography (HPLC), in which Ferulic acid was quantified in a higher amount of 16.71 mg/g in bitter gourd. Total phenolic contents were determined by using Perklin-Elmer lambda UV/Visible spectrophotometer and higher concentration was found in Bitter gourd 92.56 mg 100/g as compared to Luffa and Brinjal with 79.03 and 66.56 mg 100/g, respectively. The antioxidant activity (DPPH assay) was measured at ?max of 517 nm, results revealed that Bitter gourd possessed the higher antioxidant activity with 182.61 µMol/g followed by Luffa and Brinjal with 112.94 and 82.96 µMol/g. The total Flavonoid contents were higher in Brinjal with 44.32 mg g-1 whereas Luffa and Bitter gourd possess the Flavonoid concentration in the range 38.02 and 34.64mg g-1 respectively, the total tannin contents also higher in Brinjal 31.40 mg/g follwed by in Luffa and Bitter gourd with 25.17 and 21.19 mg/g respectively. Antimicrobial activity showed that, all the extracts are the highly effective against S. aureus as compared to E. coli. Finally, it is concluded that all the selected vegetables are very good sources of Phenolic compounds as well as phytochemicals and should be included in the daily human diet for good health. On the basis of obtained results, it is also suggested that these samples will be further investigated for the determination of fatty acids by GC-MS and liquid chromatography-mass spectrum (LC-MS).