High-performance Liquid Chromatography Analysis Research Articles

Alpinia galanga induces caspase-dependent apoptotic cell death in human lung and cervical cancer cells

Introduction: Alpinia galanga (L.) Willd is a rhizomatous, recurrent herb used to treat numerous diseases, including cancer. This study examines the efficiency of the different parts of A. galanga for their phytochemical, antioxidant, and anti-cancer properties. Methods: The powdered leaf, stem, and rhizome of A. galanga were extracted using hexane, ethyl acetate and methanol; the phytochemical compositions of the extracts were characterized using high performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS). Their antioxidant potentials were evaluated using different assays, including 2,2-Diphenyl-1picrylhydrazil (DPPH), 2-Azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), Superoxide anion (O2-), Hydroxyl radical (OH), and Phosphomolybdenum assays. The in vitro anticancer activity was studied using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Comet assay was used to determine the level of DNA damage. Inductions of morphological alterations were studied using the AO/Et-Br dual staining method. The ELISA kits were used to measure caspase activities. Results: The cytotoxicity results showed that the various extracts had inhibitory and cytotoxic effects on cancer cell lines. The leaf hexane (LH) extract of A. galanga induced DNA damage with prominent increased tail length and tail moment followed by the induction of apoptotic cells and up-regulation of the caspase activities. HPLC and GC-MS analysis allowed the identification of bioactive compounds, recognized as apoptotic inducers in human cancer cells by activating caspase-dependent pathways. Conclusion: This work reports for the first time the effect of LH extract of A. galanga in triggering apoptosis in A-549 and HeLa cells through the upregulation of caspase-8 and caspase-3 activities.

Physicochemical, structural, and biological properties of novel water-soluble polysaccharide derived from the Tunisian Hammada scoparia plant and its application on beef meat preservation

This work aims to characterize a novel water-soluble polysaccharide from Hammada scoparia leaves named PSP. The Infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra confirmed the presence of different polysaccharide functional bands. The High-Performance Liquid Chromatography (HPLC) analysis identified a heteropolysaccharide composed of two monosaccharides. A semi-crystalline structure of PSP was proved using the X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis. The evaluation of the antioxidant activity revealed an interesting potential to prevent oxidative stress.Additionally, PSP showed interesting functional propreties such as good oil and water retention abilities, higher foaming stability, and higher emulsifying capacity and stability. However, the effect of PSP on the oxidation of lipids in the ground beef meat was established during nine days at 4 °C. Obtained data revealed a significant decrease in malondialdehyde levels, inhibition of metmyoglobin (MetMb) accumulation, and significant inhibition of microbial growth compared with the control sample during storage. Moreover, incorporating PSP in minced meat proved color pH and moisture stability. Overall, the findings in the present study confirmed that PSP could be considered a natural bioactive polymer for food applications.

Fluorescence switching sensor for sensitive detection of curcumin/Mn2+ using biomass carbon quantum dots: A combination of experimental and theoretical insights

Compared to high-performance liquid chromatography, capillary electrophoresis, and electrochemical analysis, fluorescent carbon dots derived from biomass materials have garnered significant attention due to their non-toxic, eco-friendly, and renewable advantages. In this research, CQDs-NH2 was synthesized in one step by hydrothermal method using banana peel as biomass carbon source and ethylenediamine (EDA) as dopant. The blue fluorescence of CQDs-NH2 was found to be quenched by the addition of curcumin through the synergistic effect of static quenching and internal filtering effect (IFE). The response to curcumin showed good linearity in the concentration range of 1-15μM with a limit of detection (LOD) of 18.56nM. The fluorescence of CQDs-NH2 was partially restored in the presence of curcumin and Mn2+, leading to the establishment of a curcumin@CQDs-NH2 switch sensor for the detection of Mn2+ in the linear range of 5-60μM with a LOD of 20.35nM. Furthermore, density functional theory (DFT) calculations suggest that the interaction between Mn2+ and curcumin is stronger than the interaction between Mn2+ and functionalized CQDs (f-CQDs), elucidating the introduction of Mn2+ disrupts the interaction between curcumin and CQDs-NH2, leading to the restoration of CQDs-NH2 fluorescence. Finally, the CQDs-NH2 fluorescence switch sensor demonstrated high accuracy and precision in liquid food and water quality testing with recoveries ranging from 95.19% to 105.54%.

Enantioselective Binding of Proton Pump Inhibitors to Alpha1-Acid Glycoprotein and Human Serum Albumin-A Chromatographic, Spectroscopic, and In Silico Study.

The enantioselective binding of three proton pump inhibitors (PPIs)-omeprazole, rabeprazole, and lansoprazole-to two key plasma proteins, α1-acid glycoprotein (AGP) and human serum albumin (HSA), was characterized. The interactions between PPI enantiomers and proteins were investigated using a multifaceted analytical approach, including high-performance liquid chromatography (HPLC), fluorescence and UV spectroscopy, as well as in silico molecular docking. HPLC analysis demonstrated that all three PPIs exhibited enantioseparation on an AGP-based chiral stationary phase, suggesting stereoselective binding to AGP, while only lansoprazole showed enantioselective binding on the HSA-based column. Quantitatively, the S-enantiomers of omeprazole and rabeprazole showed higher binding affinity to AGP, while the R-enantiomer of lansoprazole displayed greater affinity for AGP, with a reversal in the elution order observed between the two protein-based columns. Protein binding percentages, calculated via HPLC, were greater than 88% for each enantiomer across both transport proteins, with all enantiomers displaying higher affinity for AGP compared to HSA. Thermodynamic analysis indicated that on the HSA, the more common, enthalpy-controlled enantioseparation was found, while in contrast, on the AGP, entropy-controlled enantioseparation was observed. The study also identified limitations in using fluorescence titration due to the high native fluorescence of the compounds, whereas UV titration was effective for both proteins. The determined logK values were in the range of 4.47-4.83 for AGP and 4.02-4.66 for HSA. Molecular docking supported the experimental findings by revealing the atomic interactions driving the binding process, with the predicted enantiomer elution orders aligning with experimental data. The comprehensive use of these analytical methods provides detailed insights into the enantioselective binding properties of PPIs, contributing to the understanding of their pharmacokinetic differences and aiding in the development of more effective therapeutic strategies.

Exogenous MgH2-derived hydrogen alleviates cadmium toxicity through m6A RNA methylation in rice

Cadmium (Cd) contamination poses a substantial threat to crop yields and human health. While magnesium hydride (MgH2) has been reported as a hydrogen (H2) donor that promotes plant growth under heavy metal contamination, its role in rice remains elusive. Herein, seedlings of Oryza sativa L. Japonica variety Zhonghua 11 (ZH11) were selected and exposed to 20 µL of 1-mol/L cadmium chloride (CdCl2) solution via hydroponics to simulate Cd stress. Meanwhile, 0.1 mg of MgH2 was used to slow-release H2 to the experimental group to explore its potential effects on rice over a 2-week period. The results indicated that Cd exposure severely inhibited the growth and development of ZH11 rice seedlings. However, the exogenous slow-release of H2 from MgH2 effectively mitigated this inhibitory effect by restoring the balance of reactive oxygen species (ROS), maintaining endogenous H2 homeostasis, and supporting the photosynthetic system. High-performance liquid chromatography analysis revealed that exogenous H2 reduces m6A RNA methylation levels in mRNA under Cd stress. Consequently, MeRIP-seq was conducted to investigate the effect of Cd exposure in rice in the presence and absence of H2. The m6A modifications were enriched at the start codon, stop codon, and 3′ UTR. By integrating RNA-seq data, 118 transcripts were identified as differentially methylated and expressed genes under Cd stress. These gene annotations were associated with ROS, biological stress, and hormonal responses. Notably, 297 differentially methylated and expressed genes were identified under Cd stress in the presence of H2, linked to heavy metals, protein kinases, and calcium signaling regulation. Cd strongly activates the MAPK pathway in response to stress. Exogenous H2 reduces Cd accumulation as well as enhances plant tolerance and homeostasis by lowering m6A levels, thereby decreasing the mRNA stability of these genes. Our findings indicate that MgH2, by supplying H2, regulates gene expression through m6A RNA methylation and confers Cd tolerance in rice. This study provides potential candidate genes for studying the remediation of heavy metal pollution in plants.

Chromatographic Profiles of Polyphenols in the Herbs of Artemisia campestris L. and Artemisia ludoviciana Nutt.

This study aimed to analyze the compositions of flavonoids and phenolic acids in the aerial parts of two Artemisia L. species (Artemisia ludoviciana Nutt. and Artemisia campestris L.) when grown in Ukraine. High-performance liquid chromatography analysis (HPLC) detected the presence of 11 flavonoids and 10 phenolic acids in the plant materials under study. Among the phenolic acids, chlorogenic acid was the most abundant in the raw material of both species (14.503 mg/g in Artemisia ludoviciana and 4.504 mg/g in Artemisia campestris). The main flavonoids in the Artemisia ludoviciana herb decreased in the following order: flavanone-7-O-glycoside (naringin) (21.924 mg/g) > fisetin (13.068 mg/g) > kaempferol-3-b-glucoside (5.119 mg/g) > rutin (1.295 mg/g). In comparison, in Artemisia campestris raw material the order was: flavanone-7-O-glycoside (7.525 mg/g) > fisetin (2.933 mg/g) > rutin (1.355 mg/g). Since the predominant polyphenols have demonstrated valuable therapeutic potential, the data obtained could be considered for further exploration of their biological activities.

Effects of Pleurotus Eryngii (DC.) Quél. Mushroom Extracts on Cell Proliferation in Breast and Colon Cancer Cell Lines

This study was aimed to collect Pleurotus eryngii (DC.) Quél. edible/medicinal mushroom from Çanakkale/Kumkale and to evaluate the antiproliferative effects of alcoholic extracts on breast and colon cancer cell lines. For this purpose, the Soxhlet method prepared methanol and ethanol:H2O (70:30) extracts by gradually increasing solvent polarities. Then, breast (MDA-MB-231, BT-549, BT-20, MCF-7) and colon (HT-29) cancer cell lines were treated with the extracts in increasing concentrations (0.05-0.5 mg/mL) for 48 h. In conclusion, methanol and aqueous ethanol extracts exhibited significant antiproliferative effects in cancer cell lines, according to the MTT assay. The cell viability in the triple-negative breast cancer (TNBC) MDA-MB-231 cell line was highly decreased by methanol extract at a very low concentration (0.1 mg/mL). Furthermore, methanol extract at 0.3 mg/mL reduced the percentage of cell viability in the HT-29 cell line. Aqueous ethanol extract showed antiproliferative activity in breast and colon cancer cell lines at 0.25 mg/mL concentration for 48 h applications. In addition, some bioactive components such as 4-hydroxy flavone, chrysin, and tannic acid of aqueous ethanol extract have been determined by High-Performance Liquid Chromatography (HPLC) analysis. As a result, this study may increase interest in the studies on the anti-cancer activities and the mechanisms of P. eryngii mushroom.

Crop Improvement of Moringa oleifera L. through Genotype Screening for the Development of Clonal Propagation Techniques of High-Yielding Clones in Malaysia.

Moringa oleifera L. is a valuable multipurpose tree species widely planted for centuries due to its high medicinal value and antifungal, antiviral, antidepressant, and anti-inflammatory properties in the food industry. However, its cultivation is hindered by production constraints such as the unavailability of planting material and the inadequate number of high-yielding clones. Thus, a study was initiated to select high-yielding clones in terms of growth and chemical content for the mass propagation of superior moringa trees. Screening on high-yielding clones with high astragalin content was conducted through the high-performance liquid chromatography (HPLC) analysis of moringa leaf extract. Selected genotypes were evaluated for their anti-inflammatory potential through in vitro bioactivity assays of leaf methanol extract. The effects of the rooting hormone, rooting substrates, and size of the cutting on the rooting response of branch cuttings of moringa were investigated. Results found that samples collected from different ecological zones of Peninsular Malaysia show significant variation in terms of astragalin content. The extracts were observed to show considerable variation in biological activity against the pro-inflammatory enzymes. The size of the cuttings had significant effects on the rooting of the cuttings as longer cuttings with bigger diameters rooted better than shorter cuttings with smaller diameters. Several genotypes of M. oleifera with superior phenotypic characteristics and bioactive compounds have been identified. Factors affecting the rooting efficiency and optimal conditions of rooting are suggested, which provides valuable information for the propagation of the superior planting material of moringa. This effort will ensure the sustainable production and supply of good quality raw materials for the production of quality end-products for the food and pharmaceutical industry.

Antithrombotic activity of Punica granatum, L (Pomegranate): Experimental investigation and exploration of its mechanism of action on primary and secondary hemostasis in vitro and ex vivo

Hemostasis is the physiological process that stops bleeding by forming a thrombus (or blood clot). However, the inappropriate formation of a thrombus can constitute a pathophysiological event involving the same steps as those of hemostasis. Despite some adverse effects, antithrombotic therapy remains an effective means in the prevention and treatment of thrombotic diseases. In this regard, scientific research aimed at discovering new remedies of natural origin is particularly active.This study aims to evaluate the antithrombotic activity of the aqueous extract of Punica granatum peels in vivo. To understand the mechanism of action of this activity, we will investigate primary and secondary hemostasis in vitro and ex vivo. Furthermore, we will examine its antioxidant activity, toxicity, and phytochemical composition to identify the molecules responsible for the observed effects.The antithrombotic activity is assessed in vivo using the acute pulmonary thromboembolism model in mouse. Thrombosis is induced by injecting the thrombogenic solution (epinephrine + collagen) into the lateral caudal vein. Platelet aggregation, both in vitro and ex vivo, is performed on washed platelets isolated by centrifugation from rat blood. The platelet suspension is pre-incubated with the extract for 1 min. Aggregation is triggered by adding the following agonists: ADP, thrombin, collagen, and arachidonic acid. The bleeding time is measured in the absence and presence of the extract following an apical section of the mouse tail. This time corresponds to the duration of flow between the first and last drops of blood. To evaluate plasma coagulation, activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured under conditions both with and without the extract, using appropriate reagents. Acute toxicity is conducted by administering a single oral dose of the extract to mice (5 and 10 g kg-1). Possible changes in animal behavior are observed for fifteen days. The identification of polyphenols and flavonoids is performed by High Performance Liquid Chromatography (HPLC).The results obtained show that P. granatum exerted a significant preventive antithrombotic action (80 % protection) in mice. Furthermore, the extract inhibited platelet aggregation induced in vitro by all agonists in a dose-dependent manner, prolonged bleeding time, and coagulation times in rats. The results of the ex vivo study in rats confirmed these effects. We also noted that the extract did not exhibit toxicity following administration, and HPLC analysis revealed a richness of polyphenols and flavonoids in the extract.P. granatum exhibited an interesting antithrombotic activity, which could be attributed to its antiplatelet and anticoagulant actions. The phytochemical compounds identified in this extract could, at least in part, be responsible for these effects. Therefore, the demonstrated activities of this plant on hemostasis could lead to the development of more effective remedies and represent a means of prevention against thrombotic events.

High performance liquid chromatographic analysis of capsaicinoids in fruits of Capsicum annuum L grown in soil treated with Rhodopseudomonas sps

High performance liquid chromatographic analysis of capsaicinoids in fruits of Capsicum annuum L grown in soil treated with Rhodopseudomonas sps

Development and Characterization of Gingerol-Assisted Silver Nanoparticle and Chitosan Alginate Membrane for Antibacterial and Wound Healing Activity.

This study synthesized and characterized gingerol-loaded silver nanoparticles (Ag-NPs), which were incorporated into chitosan-alginate (Cs/Alg) films for advanced wound care applications. Gingerol was isolated with a 9.81% yield, forming pale-yellow needle-shaped crystals with a melting point of 32ºC. The purity and structure of gingerol were confirmed through thin-layer chromatography (TLC), UV-visible, fourier-transform infrared spectroscopy (FTIR), and high-performance liquid chromatography (HPLC) analyses. The synthesized Ag-NPs, particularly from the GN3 batch, exhibited a small particle size (36.52 ± 3.52 nm), high stability (zeta potential of -23.20 mV), and notable encapsulation efficiency (76.35%) and yield (74.11%). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed their spherical and smooth morphology. Stability tests over three months showed no substantial changes in particle size, ZP, or %EE. The Cs/Alg films demonstrated appropriate thickness, weight, water uptake, and pH changes, with GN-CM4 showing the best performance. In-vitro drug release studies indicated increased release rates, especially for GN-CM4, which also exhibited the highest antibacterial activity. In-vivo studies on excision and burn wound models showed that GN-CM4 promoted complete wound closure by day 21, enhanced hydroxyproline levels, reduced myeloperoxidase content, and facilitated superior tissue regeneration. These findings underscore the potential of gingerol-loaded Ag-NPs in enhancing wound healing through their anti-inflammatory and antibacterial properties.

Lipase-catalyzed synthesis of erythorbyl oleate and its characterization as a multifunctional emulsifier.

Erythorbyl oleate (EO), a novel emulsifier with multifunctional properties, was synthesized via lipase-catalyzed solvent-free esterification between erythorbic acid and oleic acid. The reaction produced EO of 30.75±1.65mM at 60h, monitored by high-performance liquid chromatography (HPLC) analysis. Purified EO was chemically identified as 6-O-oleoyl-erythorbic acid by HPLC-electrospray ionization/mass spectrometry (ESI-MS), 1H nuclear magnetic resonance (NMR), and 13C NMR analyses, and further investigations on its multifunctionalities were conducted. EO displayed antioxidant activity equivalent to erythorbic acid in 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities. Investigation of the antibacterial activity of EO against foodborne pathogens showed MICs of 0.27±0.09 and 0.40mM for Bacillus cereus ATCC 10876 and Staphylococcus aureus ATCC 12692, respectively. EO also displayed interfacial properties, lowering interfacial tension between medium-chain triglyceride oil and water to 3.40±0.04 mN/m with a critical micelle concentration of 2.54±0.03mM. Dynamic light scattering analyses of the emulsion stabilized by EO demonstrated a similar polydispersity index (0.15±0.00) to Tween 80-stabilized emulsion (0.28±0.01), with an average droplet size of 173.2±1.8nm. In addition, the EO-stabilized emulsion exhibited a negative surface charge (-56.83±4.30mV) in zeta potential measurements, indicating sufficient electrostatic repulsion between droplets. These results suggest that EO is a multifunctional emulsifier to simultaneously control oxidative rancidification and bacterial spoilage in emulsion-based foods. Practical Application: EO was synthesized via an enzymatic esterification reaction between erythorbic acid and oleic acid. The synthetic process excluded any use of organic solvent, diminishing potential health hazards or environmental pollution associated with chemical reactions. The interfacial properties of the resultant EO, along with excellent antioxidant and antibacterial activities, propose its potential application in emulsion-based foods. Simultaneous control of bacterial spoilage and oxidative rancidification by EO would fulfill the industrial needs for extending shelf life and enhancing consumer safety.

Feasible biosynthesis of biologically active metabolites in in vitro culture of Macrotyloma uniflorum

Macrotyloma uniflorum, commonly called “Horse gram” is an underutilized pulse crop recognized for its great nutritional significance and a broad range of biological properties. There have been no in vitro studies for the biosynthesis of enhanced bioactive metabolites in the callus culture of M. uniflorum. In the study reported here, we have designed a feasible protocol for high-frequency callus induction and maintenance utilizing a leaf as an explant grown on MS media enriched with various concentrations of different plant growth regulators (PGRs) including α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and thidiazuron (TDZ) either alone or in combination. Among all the tested PGRs, NAA alone resulted in high-frequency callus induction (97%), and maximum biomass accumulation (Fresh weight (FW): 200 g/L: Dry weight (DW): 20.4 g/L). Moreover, hormonally optimized callus cultures exhibit maximum production of phenolic compounds (166.6 mg/L) and flavonoid compounds (351.6 mg/L). The antioxidant potential of calli extracts was also determined by utilizing various antioxidant activities. Maximum antioxidant activities ((2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) ABTS = 497.9; TEAC μM; (Ferric reducing antioxidant power) FRAP = 823 TEAC μM; (Cellular Antioxidant Activity) CAA: 58.2%) were recorded in leaf-derived calli supplemented with different PGRs treatments. High-performance liquid chromatography (HPLC) analysis further revealed maximum biosynthesis of caffeic acid (1.63 mg/g DW), gallic acid (8.92 mg/g DW), kaempferol (0.71 mg/g DW), myricetin (0.39 mg/g DW), apigenin (0.64 mg/g DW) at 10 mg/L BAP and isorhamnetin (0.68 mg/g DW) at 1 mg/L TDZ + 10 mg/L NAA. The objective of this study was to explore in vitro biosynthesis of enhanced bioactive metabolites in the callus culture of M. uniflorum, leveraging a feasible protocol for high-frequency callus induction and maintenance. The results showcase the remarkable efficacy of NAA in callus induction and biomass accumulation, highlighting the hormonally optimized callus cultures as a potent source for enhanced biosynthesis of bioactive metabolites, paving the way for further applications in pharmaceutical and commercial industries.

Phytochemical Extraction, Analysis, and Characterization of Madhuca longifolia Leaf Compounds.

Objective: This study aimed to perform the extraction, phytochemical analysis, and compound characterization of Madhuca longifolia leaf extracts. Materials and Methods: Sequential extraction of M. longifolia leaves was performed using petroleum ether, chloroform, methanol, ethanol, and water. The yield of the extracts was measured and reported as 1.3 ± 0.10% for petroleum ether, 3.85 ± 0.15% for chloroform, 20.25 ± 1.22% for methanol, 30.1 ± 1.38% for ethanol, and 26.7 ± 0.21% for water. Qualitative phytochemical analysis was conducted to determine the presence of various compounds, including alkaloids, glucose-alkaloids, steroids, tannins, phenols, proteins, and flavonoids. The absence of ethyl and methyl alkalis in petroleum ether and chloroform extracts was noted. Additionally, the presence of saponins and alkaloids was observed only in methanol extracts, with cresol absent from these extracts. The isolated compounds were further characterized using thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), ultraviolet (UV) spectroscopy, and Fourier-transform infrared (FTIR) spectroscopy. Results: The Rf value of the isolated compound was 0.49 using TLC. HPLC analysis identified major constituents with retention times of 4.576 and 10.461 minutes. These findings highlight the diverse phytochemical composition of M. longifolia leaf extracts and provide a detailed analysis of the active compounds present. Conclusion: This comprehensive study of M. longifolia leaf extracts offers valuable insights into the plant’s medicinal potential. The identified active compounds could serve as a reference for future medicinal use and research.

Comprehensive Determination of the Phenolic Compound Contents and Antioxidant Potentials of Leaves and Roots of Peucedanum japonicum Harvested from Different Accessions and Growth Periods.

Peucedanum japonicum Thunberg, a medicinal plant, remains understudied despite its potential therapeutic benefits. This study aimed to determine the phytochemical profiles and antioxidant capacities in the extracts of different accessions of P. japonicum by measuring the total polyphenol and flavonoid content of the P. japonicum extracts coupled with DPPH and ABTS+ assays. In addition, phytochemical screening via LC-MS/MS and high-performance liquid chromatography analysis quantified nine compounds wherein chlorogenic acid (CA) was found to be the most abundant in all compounds while hyperoside and peucedanol were the least. Results showed variation in these compounds' content among accessions (2.01-21.31 mg/g CA) and plant parts (0.34-19.57 mg/g CA), with leaves generally showing higher antioxidant activity. The abundance of these compounds These integrated analyses provide insights into the phytochemical composition and antioxidant activity of this understudied plant, contributing to advances in natural product chemistry and potential therapeutic applications.

Biological transformation of herbal extracts through Lactic Acid Bacteria fermentation: A study on Milk thistle and liquorice root extract

Herbal extracts have recently gained attention in pharmaceuticals, supplements, and cosmetics. This study explored the impact of Lactic Acid Bacteria (LAB) fermentation on the functional properties of milk thistle and licorice root extracts to develop health-enhancing beverages. Biological transformation using microorganisms or enzymes was employed to improve the stability and efficacy of these extracts. After 36 h of fermentation, bacterial growth in both extracts surged approximately two-fold. The licorice root extract showed a particularly significant increase in total phenolic content and antioxidant activity, attributed to bacterial enzymes breaking down complex, high-molecular-weight phenolic compounds into simpler, more potent free phenols. Despite a reduction in total flavonoid content across all samples, this did not indicate a decline in biological activity. High-Performance Liquid Chromatography (HPLC) analysis revealed a notable increase in silymarin concentration in the milk thistle extract, rising from 0.59 mg mL−1 to 2.5 mg mL−1 after fermentation with Lactiplantibacillus plantarum. Glycyrrhizic acid in the licorice root extract slightly decreased following fermentation with L. plantarum, while no significant changes were observed in extracts fermented with L. acidophilus. Cell viability studies during cold storage showed that the bacterial count in the licorice root extract remained within acceptable limits (106 CFU mL−1) for four weeks, ensuring ongoing health benefits. However, in the milk thistle-fermented extract, no viable cells were detected by the third week. These findings demonstrate the potential of LAB-fermented extracts for developing functional beverages with enhanced health benefits and therapeutic properties.

An efficient protocol for the extraction of pigment-free active polyphenol oxidase and soluble proteins from plant cells.

The elimination of brownish pigments from plant protein extracts has been a challenge in plant biochemistry studies. Although numerous approaches have been developed to reduce pigments for enzyme assays, none has been able to completely remove pigments from plant protein extracts for biochemical studies. A simple and effective protocol was developed to completely remove pigments from plant protein extracts. Proteins were extracted from red anthocyanin-rich transgenic and greenish wild-type tobacco cells cultured on agar-solidified Murashige and Skoog medium. Protein extracts from these cells were brownish or dark due to the pigments. Four approaches were comparatively tested to show that the diethylaminoethyl (DEAE)-Sephadex anion exchange gel column was effective in completely removing pigments to obtain transparent pigment-free protein extracts. A Millipore Amicon® Ultra 10K cut-off filter unit was used to effectively desalt proteins. Moreover, the removal of pigments significantly improved the measurement accuracy of total soluble proteins. Furthermore, enzymatic assays using catechol as a substrate coupled with high-performance liquid chromatography analysis demonstrated that the pigment-free proteins not only showed polyphenol oxidase (PPO) activity but also enhanced the catalytic activity of PPO. Taken together, this protocol is effective for extracting pigment-free plant proteins for plant biochemistry studies. A simple and effective protocol was successfully developed to not only completely and effectively remove anthocyanin and polyphenolics-derived quinone pigments from plant protein extracts but also to decrease the effects of pigments on the measurement accuracy of total soluble proteins. This robust protocol will enhance plant biochemical studies using pigment-free native proteins, which in turn increase their reliability and sensitivity.

Hydrogen sulfide alleviates cadmium stress in germinating carrot seeds by promoting the accumulation of proline

Carrot (Daucus carota L.), a widely cultivated economically vegetable from the Apiaceae family, is grown globally. However, carrots can be adversely impacted by cadmium (Cd) pollution in the soil due to its propensity to accumulate in the fleshy root, thus impeding carrot growth and posing health hazards to consumers. Given the potential of hydrogen sulfide (H2S) to improve plant resistance against Cd stress, we treated germinating carrot seeds with varying concentrations of sodium hydrosulfide (NaHS), aiming to alleviate the toxic impacts of Cd stress on carrot seed germination. The results revealed that carrot seeds treated with a concentration of 0.25 mM NaHS displayed better seed germination-associated characteristics compared to seeds treated with NaHS concentrations of 0.1 mM and 0.5 mM. Further investigation revealed a rise in the expression levels of L-cysteine desulfhydrase and D-cysteine desulfhydrase, along with enhanced activity of L-cysteine desulfhydrase and D-cysteine desulfhydrase among the NaHS treatment group, thereby leading to H2S accumulation. Moreover, NaHS treatment triggered the expression of pyrroline-5-carboxylate synthase and pyrroline-5-carboxylate reductase and promoted the accumulation of endogenous proline, while the contents of soluble sugar and soluble protein increased correspondingly. Interestingly, since the application of exogenous proline did not influence the accumulation of endogenous H2S, suggesting that H2S served as the upstream regulator of proline. Histochemical staining and biochemical indices revealed that NaHS treatment led to elevated antioxidant enzyme activity, alongside a suppression of superoxide anion and hydrogen peroxide generation. Furthermore, high performance liquid chromatography analysis revealed that NaHS treatment reduced Cd2+ uptake, thereby promoting germination rate, seed vitality, and hypocotyl length of carrot seeds under Cd stress. Overall, our findings shed light on the application of NaHS to enhance carrot resistance against Cd stress and lay a foundation for exploring the regulatory role of H2S in plants responding to Cd stress.

A simple and efficient method for betalain quantification in RUBY-expressing plant samples.

The RUBY reporter system has demonstrated great potential as a visible marker to monitor gene expression in both transiently and stably transformed plant tissues. Ectopic expression of the RUBY reporter leads to bright red pigmentation in plant tissues that do not naturally accumulate betalain. Unlike traditional visual markers such as β-glucuronidase (GUS), luciferase (LUC), and various fluorescent proteins, the RUBY reporter system does not require sample sacrifice or special equipment for visualizing the gene expression. However, a robust quantitative analysis method for betalain content has been lacking, limiting accurate comparative analyses. In this work, we present a simple and rapid protocol for quantitative evaluation of RUBY expression in transgenic plant tissues. Using this method, we demonstrate that differential RUBY expression can be quantified in transiently transformed leaf tissues, such as agroinfiltrated Nicotiana benthamiana leaves, and in stable transgenic maize tissues, including seeds, leaves, and roots. We found that grinding fresh tissues with a hand grinder and plastic pestle, without the use of liquid nitrogen, is an effective method for rapid betalain extraction. Betalain contents estimated by spectrophotometric and High-Performance Liquid Chromatography (HPLC) analyses were highly consistent, validating that our rapid betalain extraction and quantification method is suitable for comparative analysis. In addition, betalain content was strongly correlated with RUBY expression level in agroinfiltrated N. benthamiana leaves, suggesting that our method can be useful for monitoring transient transformation efficiency in plants. Using our rapid protocol, we quantified varying levels of betalain pigment in N. benthamiana leaves, ranging from 110 to 1066 mg/kg of tissue, and in maize samples, ranging from 15.3 to 1028.7 mg/kg of tissue. This method is expected to streamline comparative studies in plants, providing valuable insights into the effectiveness of various promoters, enhancers, or other regulatory elements used in transgenic constructs.

Design and fabrication of acorn-like Janus molecularly imprinted materials for highly specific separation and enrichment of oxytetracycline from restaurant oily wastewater

Molecularly imprinted polymer (MIP) is dedicated to the adsorption of target substances in the aqueous phase, but ignores the adsorption in a more complex environment (oily wastewater). In order to explore the application field of existing MIPs, acorn-like Janus particles were fabricated by photo-initiated seed swelling polymerization. A novel amphiphilic Janus-MIP was prepared with the acorn-like Janus particles as matrix, methacrylic acid, ethylene dimethacrylate and oxytetracycline (OTC) as functional monomers, crosslinking agents and template molecules via surface initiated-atom transfer radical polymerization (SI-ATRP). For comparison, the poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly (GMA-co-EDMA)) microspheres were also utilized as the matrix to prepare common spherical-MIP. The adsorption capacity of Janus-MIP for OTC was 23.8 mg g−1 in oil-water system, while the adsorption capacity of spherical-MIP for OTC was only 12.6 mg g−1 in the same system. At the same time, through high performance liquid chromatography (HPLC) analysis, Janus-MIP can specifically recognize and adsorb trace OTC in restaurant oily wastewater samples, and the proposed method exhibited a lower limit of detection (LOD, 3 ng mL−1) and a higher OTC recovery rate (94.2 %–98.4 %). This work demonstrated great potential for the detection and control of OTC contamination from real samples in an oil-water mixed environment.