Ultra-performance Liquid Chromatography Analysis Research Articles

Fungal Diversity and Gibberellin Hormones Associated with Long Whips of Smut-Infected Sugarcanes.

Sugarcane smut, caused by the fungus Sporisorium scitamineum (Sydow), significantly affects sugarcane crops worldwide. Infected plants develop whip-like structures known as sori. Significant variations in these whip lengths are commonly observed, but the physiological and molecular differences causing these morphological differences remain poorly documented. To address this, we employed conventional microbe isolation, metagenomic, and metabolomic techniques to investigate smut-infected sugarcane stems and whips of varying lengths. Metagenomics analysis revealed a diverse fungal community in the sugarcane whips, with Sporisorium and Fusarium genera notably present (>1%) in long whips. Isolation techniques confirmed these findings. Ultra-performance liquid chromatography analysis (UHPLC-MS/MS) showed high levels of gibberellin hormones (GA3, GA1, GA4, GA8, and GA7) in long whips, with GA4 and GA7 found exclusively in long whips and stems. Among the prominent genera present within long whips, Fusarium was solely positively correlated with these gibberellin (GA) hormones, with the exception of GA8, which was positively correlated with Sporisorium. KEGG enrichment analysis linked these hormones to pathways like diterpenoid biosynthesis and plant hormone signal transduction. These findings suggest that Fusarium may influence GA production leading to whip elongation. Our study reveals fungal dynamics and gibberellin responses in sugarcane smut whips. Future research will explore the related molecular gibberellin synthesis mechanisms.

Spatiotemporal expression analysis of jasmonic acid and saponin-related genes uncovers a potential biosynthetic regulation in Panax notoginseng.

Sanqi, the root of Panax notoginseng, has long been recognized for its therapeutic effects on cardiovascular diseases. Saponins, including ginsenosides and notoginsenosides, are the main bioactive components of P. notoginseng. The biosynthesis of saponins is closely related to the defense responses orchestrated by endogenous hormones. To provide new insights into the underlying role of phytohormone jasmonic acid (JA) in the synthesis and regulation of saponins, we performed an ultra-performance liquid chromatography analysis of different tissues of P. notoginseng aged 2-4 years. Moreover, by combined evaluation of saponin content and transcriptome profiling of each tissue, the spatial and temporal distribution of saponins was analyzed. N notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rd accumulated in the underground tissues, including the root, tuqi, fibril and rhizome. In agreement with this data, the corresponding genes of the endogenous hormone JAs, especially coronatine insensitive 1 (COI1) and myelocytomatosis proteins 2 (MYC2), were predominantly expressed in the underground tissues. The tissue- and age-specific distribution of saponins was consistent with the expression of genes involved in JA biosynthetic, metabolic and signaling pathways. The present study has revealed the temporal and spatial effects of endogenous phtohormones in the synthesis and regulation of notoginsenosides, which will provide a significant impact on improving the ecological planting technology, cultivating new high-quality varieties and protecting the rare resources of medicinal P. notoginseng. © 2024 Society of Chemical Industry.

Glycan clock of ageing-analytical precision and time-dependent inter- and i-individual variability.

Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10years, respectively. Samples were collected every 3months and 3weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.

Assessing the quality ecology of endemic tree species in China based on machine learning models and UPLC methods: The example of Eucommiaulmoides Oliv.

Climate change significantly affects the suitability distributions of natural populations, especially relict species such as Eucommia ulmoides Oliv. (E. ulmoides). However, the driving eco-factors of the distribution of E. ulmoides and the relationship between geo-distribution patterns and quality are still unclear. Here, a new approach was established by combining machine learning models (MaxEnt, Random Forest, and Biomod2 model), ultra-performance liquid chromatography (UPLC) and spatial analysis to address this question. The results reveal that temperature is the most critical variable influencing the distribution of E. ulmoides including Bio1, Bio10, and Bio11, followed by precipitation. Multi-model predictions of suitable habitats for E. ulmoides cover an area ranging from 113.33 × 104 km2 to 285.98 × 104 km2, mainly distributed in the mountainous areas of southwestern China, including the eastern regions of the Dalou, Wuling, and Qinling-Daba Mountains. Under different future scenarios, suitable habitat shifted northward by 184.90–424.88 km, and the contraction area is greater than the expansion area, with the difference ranging from 49.28 × 104 km2 to 98.39 × 104 km2. Eight common components (e.g., chlorogenic acid and pinoresinol doglucoside) and two endemic components (caffeic acid and geniposidic acid) were found in the leaves of the northern and southern medicinal districts (SDM and NMD). The contents of eight key secondary metabolites of E. ulmoides leaves were better distributed in SDM than in NDM. Among these, Xinyang in Henan, Lu'an and Fuyang in Anhui, and the southeastern region of Zhejiang are suitable growing areas for the high-quality cultivation of E. ulmoides, which deserve special attention. The seasonal precipitation (Bio15) was negatively correlated with chlorogenic acid and pinoresinol diglucoside while longitude significantly positively correlated with pinoresinol diglucoside. Overall, this study provides a novel assessment pattern for quality assessment and effective protection of E. ulmoides.

Newly Isolated Trichoderma spp. Show Multifaceted Biocontrol Strategies to Inhibit Potato Late Blight Causal Agent Phytophthora infestans both In Vitro and In Planta

Potato growers worldwide have been at war for more than 150 years with an enemy whose lifecycle, genome size and architecture, infection rate, and economic impacts are the epitome of a plant pathogen. Phytophthora infestans is an oomycete that causes the notorious late blight infection in potato and tomato fields. This study explored the benefits of the multitalented plant symbiotic fungi Trichoderma spp. and their metabolites as potential biopesticides against P. infestans. Eleven strains of Trichoderma spp. were obtained from soil and tree barks and were identified using DNA sequence analysis of three molecular markers. The antagonistic potential of the strains against P. infestans was first evaluated in vitro. In dual-culture assays, P. infestans growth was significantly inhibited (53 to 95%) by different Trichoderma spp. through direct mycoparasitism, competition for space and nutrients, or antibiosis. The cell-free filtrates (CFFs) of different Trichoderma strains were obtained and characterized for anti- Phytophthora activities as well as biochemical stability. The obtained results indicated that Trichoderma CFFs were chemically stable and strongly decreased P. infestans’ mycelial growth and zoospore motility and viability. Similarly, in leaf-disk assays, Trichoderma CFFs showed significant protection against P. infestans infection. Ultraperformance liquid chromatography analysis revealed the presence of harzianic acid, iso-harzianic acid, and 6-pentyl-2H-pyran-2-one as major compounds in different Trichoderma CFFs. Furthermore, selected Trichoderma strains significantly protected potato plants against soil-mediated late blight infection. Finally, Trichoderma spp. showed high compatibility with a copper-based fungicide, especially at lower concentrations, suggesting that both protective agents could be combined in integrated pest management programs. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Abnormal functional connectivity within the prefrontal cortex is associated with multiple plasma lipid species in major depressive disorder

BackgroundAbnormalities in functional connectivity (FC) in major depressive disorder (MDD) have been widely reported. Analysis of the relationship between FC and plasma lipid profiles would be meaningful in the exploration of pathophysiological mechanisms and helpful for the identification of biomarkers for MDD. MethodsPatients with MDD (n = 49) and healthy controls (HC, n = 87) were recruited. Resting-state functional magnetic resonance imaging (rs-fMRI) data were collected for FC construction. The plasma lipid profiles were acquired using ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) analysis and clustered as co-expression modules. The differential FC and lipid modules between HCs and patients with MDD were identified, and then the association between FC and lipid co-expression modules was analyzed using correlation analysis. The modules associated molecular function was explored using metabolite set enrichment analysis (MSEA). ResultsMDD-associated FC and lipid co-expression modules were identified. One module was associated with FC values between the right orbital part of the middle frontal gyrus and the opercular part of the left inferior frontal gyrus, which was enriched in lipid sets of diacylglycerols and fatty alcohols; another module was associated with FC values between the right middle frontal gyrus and the right anterior cingulate and paracingulate gyri, which was enriched in lipid sets of glycerophosphocholines and glycerophosphoethanolamines. ConclusionOur results indicated that abnormal FC in the prefrontal cortex is associated with multiple plasma lipid species, which may provide novel clues for exploring the pathophysiology of MDD.

Biochemical assessment of spironolactone oral suspension in human plasma using ultra‐performance liquid chromatography‐tandem mass spectrometry: Application toward pharmacokinetic study

AbstractSpironolactone is one of the non‐selective mineralocorticoid receptor antagonists acting as anti‐diuretic drugs. Spironolactone is reported to possess mild to severe hyperkalemia which could often result in fatal conditions. Although several analytical methods for evaluating spironolactone as tablet dosage forms have been established in human biological systems, it is crucial to highlight that these approaches are still limited to the suspension dosage form. In this study, we have quantified spironolactone suspension in human plasma with an efficient, sensitive, and optimized ultra‐performance liquid chromatography (UPLC)‐tandem mass spectrometry‐based bioanalytical method using a deuterated internal standard method. A reverse phase UPLC analysis and mass spectrometric detection was performed using electrospray ionization in positive ion mode as an interface, and multiple reaction monitoring as a mode of acquisition. The retention of Spironolactone (SL) and Spironolactone d7 (SL‐d7) was found to be around 6.15 and 6.07 min, respectively. The linearity range was 1.007–100.224 ng/mL for SL with 0.1 mL sample volume. The method was successfully applied to the pharmacokinetic study of spironolactone in healthy male volunteers by administrating 25 mg/5 mL oral suspension and is in accordance with bioanalytical guidelines.

In vitro production of gibberellic acid and fusaric acid by Fusarium spp. and their role in bakanae disease development

Gibberellic acid (GA3) and fusaric acid (FA) are directly correlated with the symptom development of bakanae disease in rice plants. The role of GA3 and FA, produced in vitro by several Fusarium species associated with bakanae disease, was studied to understand the disease’s etiology and its impact on symptom development. In total, 121 Fusarium strains were obtained from bakanae-infected rice plants collected from various rice cultivation regions in Bangladesh. Finally, 18 highly virulent Fusarium strains were selected based on virulence assay and further evaluated for GA3 and FA production through ultra-performance liquid chromatography analysis. Among the Fusarium strains, Fusarium fujikuroi strains produced a high amount of GA3 and a low amount of FA. In contrast, Fusarium proliferatum and Fusarium verticillioides strains produced a high amount of FA and a low amount of GA3. In exception, the Fusarium strain BD117R of F. fujikuroi produced no GA3 but a high amount of FA. Interestingly, Fusarium commune produced only FA in high concentration. In bakanae disease development, GA3 production was positively related to elongation symptoms whereas FA contributes to stunting symptoms. This is the first record of the production of GA3 and FA by Fusarium species causing bakanae disease of rice in Bangladesh.

Ultra-Performance Liquid Chromatography (UPLC) Analysis of Heme Biosynthesis Intermediates.

The utilization of ultra-performance liquid chromatography (UPLC) to analyze the various intermediates in the heme biosynthetic pathway is presented. The first product, ALA, was derivatized to a highly fluorescent pyrrolizine; PBG, the second intermediate, was enzymatically converted to uroporphyrinogen, and all the porphyrinogen intermediates were oxidized in acid to form fluorescent porphyrins. Heme was measured as hemin. The stable porphyrinforms of the intermediates, are then resolved and quantified by UPLC. Further details about the various methods are discussed to promote successful UPLC analyses. Method variations that may be preferable in certain situations are also presented.

Red bioactive pigment from Himalayan Janthinobacterium sp. ERMR3:09: optimization, characterization, and potential applications.

Prodigiosin is a red pigment commonly produced as a secondary metabolite by Serratia marcescens. It exhibits inherent bioactivities, including antimicrobial and anticancer, with low to no toxic effects on normal cells. The present study investigates a bioactive prodigiosin production from an atypical, red-pigmented, potentially novel Janthinobacterium sp. ERMR3:09 isolated from a glacial moraine. Statistically optimized culture parameters, i.e., w/v 1.0% glucose and 0.08% peptone as carbon and nitrogen sources, temperature 20°C, and media pH 7, resulted in a four-fold increase in the pigment yield. The upscaled production in an 8 L volume resulted in higher pigment production within a shorter period of 48h. The ultra-performance liquid chromatography (UPLC) analysis validated the identity of the purified pigment as prodigiosin that showed thermostability at 75°C for 3h. Evaluation of antimicrobial activity showed potent inhibitory effects (> 50%) against the opportunistic pathogenic fungal and Gram-positive bacterial strains. The pigment showed significant cytotoxicity (p < 0.05) towards A549 and HeLa cell lines with IC50 values of 42.2μM and 36.11μM, respectively. The study demonstrated that microbial communities from extreme niches can be ideal sources of bioactive pigments with immense pharmaceutical potential vital for the development of non-synthetic therapeutic agents.

A serum metabolomics study of vascular cognitive impairment patients based on Traditional Chinese medicine syndrome differentiation.

Objective: Vascular cognitive impairment (VCI) accounts for approximately 50%-70% of all dementia cases and poses a significant burden on existing medical systems. Identifying an optimal strategy for preventing VCI and developing efficient symptomatic treatments remains a significant challenge. Syndrome differentiation represents a fundamental approach for personalized diagnosis and treatment in Traditional Chinese Medicine (TCM) and aligns with the principles of precision medicine. The objective of this study was to elucidate the metabolic characteristics of VCI based on TCM syndrome differentiation, thus providing novel insights into the diagnosis and treatment of VCI. Methods: A 2-year cross-sectional cognitive survey was conducted in four communities in Beijing between September 2020 and November 2022. The syndrome differentiation of participants was based on the Kidney-Yang Deficiency Syndrome Scale (KYDSS), which was originally developed by Delphi expert consultation. The identification of serum metabolites was performed by Ultra performance liquid chromatography (UPLC) analysis coupled with an electrospray ionization quadruple time-of-flight mass spectrometer (ESI-QTOF MS). Multivariate, univariate, and pathway analyses were used to investigate metabolic changes. Logistic regression models were also used to construct metabolite panels that were capable of discerning distinct groups. Phospholipase A2 (PLA2) levels were measured by a commercial ELISA kit. Results: A total of 2,337 residents completed the survey, and the prevalence of VCI was 9.84%. Of the patients with VCI, those with Kidney-Yang deficiency syndrome (VCIS) accounted for 70.87% of cases and exhibited more severe cognitive impairments. A total of 80 participants were included in metabolomics study, including 30 with VCIS, 20 without Kidney-Yang deficiency syndrome (VCINS), and 30 healthy control participants (C). Ultimately, 45 differential metabolites were identified when comparing the VCIS group with group C, 65 differential metabolites between the VCINS group and group C, and 27 differential metabolites between the VCIS group and the VCINS group. The downregulation of phosphatidylethanolamine (PE), and phosphatidylcholine (PC) along with the upregulation of lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), phosphatidic acid (PA) and phospholipase A2 (PLA2) can be considered as the general metabolic characteristics associated with VCI. Dysfunction of glycerophospholipids, particularly LPEs and PCs, was identified as a key metabolic characteristic of VCIS. In particular Glycerophospho-N-Arachidonoyl Ethanolamine (GP-NArE) was discovered for the first time in VCI patients and is considered to represent a potential biomarker for VCIS. The upregulation of PLA2 expression was implicated in the induction of alterations in glycerophospholipid metabolism in both VCIS and VCINS. Moreover, robust diagnostic models were established based on these metabolites, achieving high AUC values of 0.9322, 0.9550, and 0.9450, respectively. Conclusion: These findings contribute valuable information relating to the intricate relationship between metabolic disorders in VCI, neurodegeneration and vascular/neuroinflammation. Our findings also provide a TCM perspective for the precise diagnosis and treatment of VCI in the context of precision medicine.

Anonna crassiflora suppresses colonic carcinogenesis through its antioxidant effects, bioactive amines, and phenol content in rats

Marolo (Annona crassiflora) is an underutilized Brazilian Cerrado fruit with few reports in the literature about its bioactive compounds and functional properties. In this context, the chemoprevention against the carcinogen 1,2-dimethylhydrazine (DMH)-induced pre-neoplastic lesions in Wistar rat colon was investigated and correlated with marolo’s antioxidant activity and the contents of phenolic compounds and bioactive amines. Total phenolic compounds (TPC) and total flavonoids compounds (TFC) were determined in the marolo pulp extract by spectrophotometric and Ultra-Performance Liquid Chromatography and diode array detection (UPLC-DAD) analysis. Free bioactive amines were determined by High Performance Liquid Chromatography and fluorescence detection (HPLC-FLD) after post column derivatization with o-phthalaldehyde. In addition, the in vitro antioxidant activity was determined by DPPH, and ABTS. Wistar rats were treated orally with marolo pulp at 0.7, 1.4 and 2.8 g/kg body weight (bw)/day added to a standard ration. Four subcutaneous injections of DMH (40 mg/kg bw) were used to induce a pre-neoplastic lesion that was assessed by the aberrant crypt foci (ACF) assay. The marolo pulp (fresh weigh) showed high content of total phenolic compounds (9.16 mg GAE/g), with predominance of chlorogenic acid (1.86 µg/g) and epicatechin (0.99 µg/g), and total flavonoids (7.26 mg CE/g), ∼85 % of the TPC. The marolo pulp had significant contents of tyramine (31.97 mg/kg), putrescine (20.65 mg/kg), and spermidine (6.32 mg/kg). The marolo pulp inhibited (p < 0.05) pre-neoplastic lesions induced by DMH administration at the all concentrations tested. These findings indicate that marolo pulp has a colon carcinogenesis chemopreventive effect, which could be due to, at least in parts, its antioxidant action associated with its phenolics and flavonoids content as well of spermidine.

Fresh Chokeberry (Aronia melanocarpa) Fruits as Valuable Additive in Extruded Snack Pellets: Selected Nutritional and Physiochemical Properties.

In this paper, the nutritional value and (selected) physiochemical properties of extruded snack pellets enriched with fresh chokeberry (Aronia melanocarpa) fruits were analyzed from the perspective of being a new product for the functional food sector. The purpose of this study was to determine the effect of the addition of fresh chokeberry and variation in content and screw speed on extruded snack pellet basic compositions, fatty acid profiles, antioxidant activity, as well as water absorption and solubility indexes, fat absorption and color profiles. The obtained results revealed a significant increase in antioxidant activity for all samples (above 90% of free radical scavenging) in comparison to potato-based control samples (just over 20% of free radical scavenging). The total phenolic content assay revealed the most valuable results for samples enriched with 30% chokeberry, while Ultra Performance Liquid Chromatography (UPLC) analysis allowed the determination of the most important phenolic acids. Of interest, chokeberry addition decreased the fat absorption index (FAI) after expansion by frying. Moreover, the highest values of crude protein and crude ash were observed in snack pellets supplemented by the application of 30% chokeberry. In such samples, the crude protein content was at the level of 4.75-4.87 g 100 g-1 and crude ash content at 4.88-5.07 g 100 g-1. Moreover, saturated fatty acids (SFA) content was lower in snack pellets with chokeberry addition, and increasing the amount of chokeberry additive from 10% to 30% in extruded snack pellet recipes resulted in more than double an increase in polyunsaturated fatty acids (PUFA) proportion in the total fatty acids.

Bacterial chemotaxis of herbicide atrazine provides an insight into the degradation mechanism through intermediates hydroxyatrazine, N–N-isopropylammelide, and cyanuric acid compounds

In recent times, the herbicide atrazine (ATZ) has been commonly used before and after the cultivation of crop plants to manage grassy weeds. Despite its effect, the toxic residues of ATZ affect soil fertility and crop yield. Hence, the current study is focused on providing insight into the degradation mechanism of the herbicide atrazine through bacterial chemotaxis involving intermediates responsive to degradation. A bacterium was isolated from ATZ-contaminated soil and identified as Pseudomonas stutzeri based on its morphology, biochemical and molecular characterization. Upon ultra-performance liquid chromatography analysis, the free cells of isolated bacterium strain was found to utilize 174 μg/L of ATZ after 3-days of incubation on a mineral salt medium containing 200 μg/L of ATZ as a sole carbon source. It was observed that immobilized based degradation of ATZ yielded 198 μg/L and 190 μg/L by the cells entrapped with silica beads and sponge, respectively. Furthermore, the liquid chromatography-mass spectroscopy revealed that the secretion of three significant metabolites, namely, cyanuric acid, hydroxyatrazine and N– N-Isopropylammelide is responsive to the biodegradation of ATZ by the bacterium. Collectively, this research demonstrated that bacterium strains are the most potent agent for removing toxic pollutants from the environment, thereby enhancing crop yield and soil fertility with long-term environmental benefits.

Vitro UPLC analysis and mass method identification, and in vivo or cellular immune anti-inflammatory function of Sanhuang Xiexin Decoction (SHXD)

Ethnopharmacological relevanceSanhuang Xiexin Decoction (SHXD), consisting of Coptis chinensis Franch., Scutellaria baicalensis Georgi and Rheum palmatum L., is traditionally used for relieving fever, purging fire for removing toxins, eliminating phlegm and haemostasis, eliminating the wetness-evil from the upper warmer, clearing away the heat-evil and expelling superficial evils. Each of the three herbs contained in SHXD has been indicated to have anti-inflammatory effects in vivo, but its effects on rat NK-cell phenotypes remain unexplored, and the comprehensive mechanism of this compound SHXD in curing the inflammation induced by lipopolysaccharides (LPS) remains to be revealed. Aim of the studyThe study aim was to assess the effect of SHXD on LPS-induced fever and inflammation in a rat model, reduce NLRP3 activation in NK cells expressing specific cell phenotype antibodies and determine the therapeutic value of this approach in vivo. Materials and methodsSHXD extract was prepared and analysed by the developed ultra-performance liquid chromatography (UPLC) method for the simultaneous detection of 14 compounds. The main peaks were firstly identified on an Orbitrap via high resolution tandem mass spectrometry (MS). Then, the extract was used in the rat model of LPS-induced inflammation and fever for pharmacologically study the effects of drug treatment. Peripheral blood lymphocyte cells were isolated from the animals, including those subjected to the SHXD extract treatment, and the cell phenotype was determined prior to cell culture and after treating the cell cultures with the extract. The phenotypes of cells harvested using CD3, CD4, CD8a, CD81, CD161 and CD86 antibodies were used to verify the enhanced memory of the peripheral blood lymphocytes cells (PBMC) that were induced into nature killer (NK) cells. ResultsThe SHXD extract was prepared, analysed and identified via quality control equipment and was observed to have pharmacological effects that reduced NLRP3 activation and fever in rats. The production of NK cells and peripheral blood lymphocytes was induced by the SHXD extract, which manifested as increased levels of CD4+, CD8a+, CD81+, CD161+ and CD86+ cells. The levels of CD3+ cells were significantly different between the model group and the drug-treated or control groups (p < 0.01) with dose independence, while the levels of CD4+ cells were not significantly different between the drug-treated and control groups, with a trend towards lower levels in the model group with dose independence. The levels of CD4+ cells was significantly different between the drug-treated group and the model groups with dose independence (p < 0.05). The levels of CD86+ cells were not significantly different between the drug-treated group and the model and control groups. The levels of CD8a + cells was significantly different between the model group and the drug and control groups (p < 0.05, dose 2.0 μg/ml), with higher levels in the drug-treated group. The levels of CD3+, CD4+, CD8a + cells in the drug treated group have dose dependence with SHXD. ConclusionsThis experiment revealed that SHXD reduced NLRP3 activation in the blood of LPS-treated rats, which occurred through the activation of NK cells that expressed CD3, CD8a and CD161. SHXD may possess anti-inflammatory effect via activacting the one of major pharmacology effcet of NK cells that expressed CD3, CD8a and CD161 phenotypes expression. This result demonstrates that SHXD may possess ability to enhance the memory of peripheral blood lymphocytes and natural killer cells.

A Review on Ultra High Performance liquid Chromatography Method

Abstract: UPLC is a modern technique which gives a new direction for liquid Chromatography. UPLC refers to ultra performance liquid chromatography, Which enhance mainly in three areas: “speed, resolution and sensitivity. Ultra Performance liquid chromatography (UPLC) applicable for particle less than 2µm in diameter to acquire better resolution, speed, and sensitivity compared With high-performance liquid chromatography (HPLC). In twenty first centenary Pharmaceutical industries are focusing for new ways to in economy and shorten Time for development of drugs. UPLC analysis at the mean time gives the better Quality of their products and analytical laboratories are not exception in this trend. The separation and quantification in UPLC is done under very high pressure (up To 100M Pa). As compare to HPLC, under high pressure it is observed that not Any negative influence on analytical column and also other components like Time and solvent consumption is less in UPLC.

Network pharmacology-based prediction and validation of the active ingredients and potential mechanisms of the Huangxiong formula for treating ischemic stroke

Ethnopharmacological relevanceHuangxiong Formula (HXF) is composed of four herbs: Rheum palmatum L., Ligusticum striatum DC., Curcuma aromatica Salisb., and Acorus gramineus Aiton. HXF is clinically used for the treatment of ischemic stroke (IS). However, its molecular mechanism remains unclear. Aim of the studyA network pharmacology-based strategy combined with experimental study in vivo and in vitro to were used to investigate the bioactive components, potential targets, and molecular mechanisms of HXF in the treatment of IS. Materials and methodsThe components of HXF were detected by ultra-performance liquid chromatography (UPLC). The potential active ingredients of HXF were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature, and corresponding targets were discerned through the Swiss TargetPrediction database. IS-related targets were obtained from Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and DisGeNET. The intersection of ingredient and disease targets was screened, and a herbal-compound-target network was constructed. A protein-protein interaction (PPI) network was created, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Based on these analyses, we established a compound-target-pathway (C-T-P) network. A cerebral ischemia-reperfusion (I/R) animal model was established, and the cerebral protective effect of HXF was assessed. The accuracy of the predicted targets was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Hippocampal neuronal injury cell model induced by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of α-Asarone. Furthermore, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to verify whether α-Asarone can bind to PI3K. ResultsA total of 44 active ingredients and 795 gene targets were identified through network pharmacology. Network analysis showed that naringenin, eupatin, kaempferol, and α-Asarone were possible drug candidates. SRC, AKT1, TP53, MAPK3, STAT3, HRAS, CTNNB1, EGFR, VEGFA, PIK3R1 could serve as potential drug targets. KEGG analysis implied that the PI3K/AKT signaling pathway might play an important role in treating IS by HXF. Moreover, HXF significantly reduced neurological impairment, cerebral infarct volume, brain index, and brain histopathological damage in I/R rats. The mRNA expression of the top 10 potential targets was verified in the brain tissue. The C-T-P network and UPLC analysis suggested that α-Asarone might be an important component of HXF and can inhibit oxidative stress and apoptosis in HT22 cells by activating the PI3K/AKT signaling pathway. Molecular docking, DARTS, and CETSA assay analysis confirmed that there were direct interactions between α-Asarone and PI3K. ConclusionHXF had a therapeutic effect in IS with multi-component, multi-target, and multi-approach features. α-Asarone, identified as one of the major active components of HXF, could alleviate oxidative stress and apoptosis by targeting PI3K/AKT pathway.

Phenolic Compounds and Antioxidant and Antimicrobial Activities of Pulicaria Odora Extract

Background: Asteraceae family, of which Pulicaria genus, plays a remarkable role in the development of drugs used in modern medicine. Leaf extracts of Pulicaria odora harvested in Bejaia were evaluated for their antioxidant and antimicrobial activities. Methods: HCl method at three different wavelengths 280, 320 and 360 nm was carried out for total phenolics content and characterized by ultra-performance liquid chromatography (UPLC) analysis. Antimicrobial activity was evaluated using four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria innocua) and three molds (Aspergillus flavus, Aspergillus niger and Trichoderma reesei). Four antioxidant tests (ORAC, ABTS, DPPH and FRAP) were performed. Results: The extracts had high total polyphenols (305 mg catechin equivalents [CE]), flavonols (56 mg quercetin equivalents [QE]), tannins (77 mg CE) and tartaric acids (38 mg caffeic acid equivalents [CAE]) per g dry matter contents. UPLC analysis of the 70 % ethanol extracts showed abundant phenolic acids (protocatechuic, chlorogenic and caffeic acids). Pulicaria odora extracts exhibited good antimicrobial activity against bacterial (~18 mm inhibition zones) and molds (11.3-17.3 mm) strains. Conclusion: All extracts displayed good antioxidant activity and effective antimicrobial activity, which allow its use as a conservative agent or therapeutic remedy.

Plant Growth-Promoting Attributes of Zinc Solubilizing Dietzia maris Isolated from Polyhouse Rhizospheric Soil of Punjab.

Zinc solubilizing rhizobacteria (ZSR) enhance the phyto-availability of Zn by converting its insoluble forms into usable forms that are essential for the growth and nutritional quality of crops. In the present study, a potential ZSR, hereafter referred to as strain N14, was isolated from the polyhouse rhizospheric soil of Punjab, India. The isolated rhizobacteria was found to be Gram-positive, aerobic, rod-shaped, and demonstrated a solubilization index of 63.75 on the Bunt Rovira (BR) medium. The 16S rRNA gene sequence analysis revealed that isolated strain N14 matches substantially with type strain Dietzia maris DSM 43672T. In its ZnO broth assay, a significant amount of soluble Zn was detected along with a simultaneous decrease in pH of the broth. Ultra-performance liquid chromatography analysis revealed the release of organic acids, specifically, lactic acid and acetic acid by D. maris strain N14 which could be the reason for the decrease in broth pH. The production of indole acetic acid (29.91µg/ml), gibberellic acid (4.72µg/ml), ammonia (38.87µg/ml), siderophore (0.89%), along with the release of HCN and appearance of phosphate solubilization zone (14.4mm) with this strain suggested its possible plant growth-promoting (PGP) characteristics. Therefore, this strain was employed in the formulation of pellets which were applied for in vivo PGPstudies using tomato plants. The developed bioformulated pellets showed a significant enhancement in plant growth as compared to control and vermicompost treated plants. To the best of our knowledge, this is the first report describing the Zn solubilizing and PGP characteristics of D. maris.

Investigation of the hemostatic mechanism of Gardeniae fructus Praeparatus based on pharmacological evaluation and network pharmacology.

As documented in the Chinese Pharmacopoeia, Gardeniae fructus Praeparatus (GFP) can cool the blood during hemostasis and treat various internal hemorrhagic diseases. However, the underlying mechanisms are not yet well understood. This work was designed to decipher the possible mechanism by which GFP prevents hemorrhage. The integration of pharmacodynamics-based and bioinformatics-based methods provided evidence to support the clinical effects of GFP in treating bleeding. Using ultra-performance liquid chromatography (UPLC) analysis, we quantified the main active ingredients for a preliminary quality assessment of GFP. The pharmacology study was conducted to confirm the essential antihemorrhagic effects of GFP. A rat model of ethanol-induced gastric hemorrhage was established and was followed by intervention with GFP in low, middle, and high doses (4.5, 9, 18 g/kg). Gastric tissues were harvested for macroscopic and histological evaluation of lesions. The contents of thromboxane B2 (TXB2) and 6-keto-prostaglandin-F1α (6-keto-PGF1α) in the serum were determined. Additionally, network pharmacology was proposed to illuminate the potential mechanisms. Following the collection of GFP compositions, the compound- and hemorrhage-related targets were retrieved from public databases. The protein-protein interaction (PPI), gene ontology, pathways analysis, and molecular docking were performed for targets of GFP in gastrointestinal bleeding. The study found ten main active ingredients that could be used for quality control of GFP. Importantly, the middle and high doses of GFP were found to promote the healing of gastric bleeding. The content of 6-keto-PGF1α was significantly degraded in the middle and high treated groups (P<0.05). The level of TXB2 was augmented by a middle (P<0.05) and high dose of GFP. Further, we constructed the network of candidate ingredients and hemorrhage-related targets. Pathway analysis predicted the mechanisms associated with interleukin 4 and interleukin 13 signaling and platelet activation. PPI analysis identified subnetworks with biological functions and also sifted hub targets that affected the antihemorrhagic progress. The candidate proteins had a good binding force with major components. GFP exhibits a promising effect in ameliorating bleeding, with the relevant molecular mechanisms possibly being related to the regulation of the immune system and platelet activation. Therefore, GFP can potentially exert a protective effect on gastrointestinal bleeding in clinic.