Quantification of chlorinated paraffins by chromatography coupled to high-resolution mass spectrometry - Part B: Influence of liquid chromatography separation.

The analysis of chlorinated paraffins (CPs) is today an analytical challenge. Indeed, it is still impractical to describe their real composition in terms of polychlorinated alkanes (PCAs) homologue groups, which dominate technical mixtures. The co-elution of PCA congeners generates interferences due to the competition phenomena which occur during the ionisation process as well as to the dependence of the ionisation sources on the PCA chemistry. Therefore, the aim of this study was to investigate the influence of chromatographic separation, by LC-ESI-HRMS coupling, on the PCA homologue group pattern and, eventually, on their determination in food samples from interlaboratory studies. For this, three different mobile phases and six LC chromatographic columns were studied in order to optimise the analysis of CP mixtures. The first results showed that the use of a MeOH/H2O mobile phase reveals more appropriately the higher chlorinated PCAs. However, using ACN/H2O led to less ion species, with almost exclusively [M + Cl]- adducts, formed using post-column dichloromethane addition. Regarding the choice of the stationary phases, Hypercarb column provided a completely different homologue group pattern from the other chromatographic columns, in relation with the stronger retention of PCAs. Among the other columns, the C30 column better highlighted the short-chain PCAs compared to the C18 column conventionally used. Because the regulations now concern short-chain CPs, the quantification of food samples was then carried out on the C30 column. The optimised LC-ESI-HRMS conditions using C30 column and MeOH/H2O solvent mixture led to a quantification of PCAs in samples from interlaboratory studies with satisfactory accuracy (|Z-score| ≤ 2) and precision (<15%).

Cytotoxic and antiviral activities of Jatropha variegata and Jatropha spinosa in relation to their metabolite profile.

Jatropha variegata and Jatropha spinosa (family: Euphorbiaceae) are utilized in Yemeni traditional medicine to treat respiratory tract infection and in different skin conditions such as wound healing, as antibacterial and hemostatic. In this study, we evaluated the cytotoxicity and the antiviral activities of the methanolic J. variegata (leaves: Ext-1, stems: Ext-2, and roots: Ext-3), and J. spinosa extracts (aerial parts: Ext-4 and roots: Ext-5), in addition to their methylene chloride fractions of roots extracts (F-6 and F-7, respectively). All samples were tested against three human cancer cell lines in vitro (MCF-7, HepG2, and A549) and two viruses (HSV-2 and H1N1). Both plants showed significant cytotoxicity, among them, the methylene chloride fractions of roots of J. variegata (F-6) and J. spinosa roots (F-7) showed the highest activity on MCF-7 (IC50 = 1.4 and 1 µg/mL), HepG2 (IC50 = 0.64 and 0.24 µg/mL), and A549 (IC50 = 0.7 and 0.5 µg/mL), respectively, whereas the IC50 values of the standard doxorubicin were (3.83, 4.73, and 4.57 µg/mL) against MCF-7, HepG2, and A549, respectively. These results revealed that the roots of both plants are potential targets for cytotoxic activities. The in vitro results revealed potential antiviral activity for each of Ext-3, Ext-5, F-6, and F-7 against HVS-2 with IC50 of 101.23, 68.83, 4.88, 3.24 µg/mL and against H1N1 with IC50 of 51.29, 27.92, 4.24, and 3.06 µg/mL respectively, whereas the IC50 value of the standard acyclovir against HVS-2 was 83.19 µg/mL and IC50 value of the standard ribavirin against H1N1 was 52.40 µg/mL .The methanol extracts of the roots (Ext-3 and Ext-5) of both plants were characterized using UPLC/MS. A total of 73 metabolites were annotated, including fourteen diterpenoids, eleven flavonoids, ten phenolic acid conjugates, twelve fatty acids and their conjugates, five triterpenes and steroids, two sesquiterpenes, and six coumarins. The cytotoxicity and antiviral activities determined in the present work are explained by the existence of flavonoids, coumarins and diterpenes with commonly known cytotoxicity and antiviral activities.

Plant Extracts and Phytochemicals from the Asteraceae Family with Antiviral Properties.

Asteraceae (Compositae), commonly known as the sunflower family, is one of the largest plant families in the world and includes several species with pharmacological properties. In the search for new antiviral candidates, an in vitro screening against dengue virus (DENV) was performed on a series of dichloromethane and methanolic extracts prepared from six Asteraceae species, including Acmella bellidioides, Campuloclinium macrocephalum, Grindelia pulchella, Grindelia chiloensis, Helenium radiatum, and Viguiera tuberosa, along with pure phytochemicals isolated from Asteraceae: mikanolide (1), eupatoriopicrin (2), eupahakonenin B (3), minimolide (4), estafietin (5), 2-oxo-8-deoxyligustrin (6), santhemoidin C (7), euparin (8), jaceidin (9), nepetin (10), jaceosidin (11), eryodictiol (12), eupatorin (13), and 5-demethylsinensetin (14). Results showed that the dichloromethane extracts of C. macrocephalum and H. radiatum and the methanolic extracts prepared from C. macrocephalum and G. pulchella were highly active and selective against DENV-2, affording EC50 values of 0.11, 0.15, 1.80, and 3.85 µg/mL, respectively, and SIs of 171.0, 18.8, >17.36, and 64.9, respectively. From the pool of phytochemicals tested, compounds 6, 7, and 8 stand out as the most active (EC50 = 3.7, 3.1, and 6.8 µM, respectively; SI = 5.9, 6.7, and >73.4, respectively). These results demonstrate that Asteraceae species and their chemical constituents represent valuable sources of new antiviral molecules.

The ethnopharmacological study of plant drugs used traditionally in Djibouti for malaria treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Djibouti was a country where malaria has been endemic for centuries. The local population use the plants as repellents or first aid for uncomplicated malaria. AIM OF THE STUDY: The aim was, for the first time, to collect and identify plants used by the local population to treat malaria and select the most interesting plants (those that are more commontly used, more available, and have fewer studies). These plants were evaluated for their antiplasmodial activity as well as their cytotoxicity on human cell lines for the most active ones. MATERIALS AND METHODS: A semi-structured questionnaire was developed for this study to collect information about the use and identity of botanical drugs used to treat malaria. The use-reports (percentage) of each plant were recorded to determine their use importance. Also, the availability status of the plants was assessed; and those in critical condition were discarded excluded from further study. Fifteen plants, out of the 41 listed, were extracted with hydro alcohol, ethyl acetate, and dichloromethane for biological testing. Chloroquine-resistant strain FcB-1 of P. falciparum and a human diploid embryonic lung cell line were used for the antiplasmodial test, and to assess the cytotoxicity for human cells respectively. Preliminary analysis of extract constituents was carried out using thin layer chromatography (TLC). RESULTS: This study identifies 41 plant taxa belonging to 32 families and records their use against malaria. Balanites rodunfolia, belonging to the Zygophyllaceae family, was the most commonly used plant, representing 44 % of use-reports. It was followed by Cadaba rodunfolia (15 %) from the Capparaceae family, and then the three species of Aloe: Aloe djiboutiensis (8.2 %), Aloe ericahenriettae (3.4 %), and Aloe rigens (3.4 %) from the Asphodelaceae family. The leaves are the most commonly used part of the plants to treat malaria, accounting for 76 % of usage. The preparation methods were decoction (52 %), maceration (29 %), and boiling (19 %). The administration routes were by oral (80 %), inhalation 19 %), and bathing (1 %). The best antiplasmodial activities were observed in the dichloromethane extracts of Cymbopogon commutatus and the ethyl acetate extracts of Aloe rigens and Terminalia brownii, with IC50 values of 9.8, 5, and 7.5 µg/mL, respectively. Their toxicity/activity levels were very favorable with selectivity indices of 5.6, 8.1, and 11.8 for C. commutatus, A. rigens, and T. Brownii, respectively. CONCLUSION: Forty-one species of botanical drugs were listed as being used to treat malaria in Djibouti. All fifteen selected species showed antiplasmodial activity (IC50 < 50 µg/mL). This work will help guide the valorization of botanical drugs used to treat malaria in Djibouti.

A small-scale silica gel column chromatography method for separating carbazole compounds from highly mature crude oil.

The concentration of carbazoles in highly mature crude oil is quite low, making it challenging to separate carbazole compounds for the gas chromatography-mass spectrometry (GC-MS) detection. This study presents a small-scale column chromatography method for separating carbazoles from highly mature crude oil using silica gel as a solid phase adsorbent and a Pasteur pipette as a separation device. The carbazole-rich crude oil from the Pearl River Mouth Basin was selected to explore the impact of reagent polarity and injection mode on the separation of carbazoles. The oil sample was eluted with solvents mixed with different volume proportions of n-hexane and dichloromethane and each eluted fraction was collected for GC-MS testing. The results indicated that increasing the reagent polarity caused the aromatic hydrocarbons and carbazole compounds in crude oil to be eluted sequentially. Most aromatic compounds in the crude oil could be selectively eluted using a reagent polarity ratio of 9:1 (Vn-hexane: Vdichloromethane), with no carbazole compounds. A significant amount of carbazole compounds were eluted in the polar segments of 8:2-6:4, with the eluted carbazoles concentration accounting for more than 98 % of the total concentration. Moreover, the concentration and recovery of carbazoles eluted by direct injection mode were about 10 % higher than those after adsorption by silica gel. The standard deviation of the parameter ratio for the separated carbazole compounds in the three groups of repeatable parallel experiments was less than 0.2 %. Our method is superior to traditional two-step method and C18 column method in separation efficiency and damage to human body. This method can be applied to both highly mature crude oil and other kinds of oils including biodegradable oil. It could be a versatile method for the carbazoles separation and provide technical support in unveiling the geochemical implications of these compounds in complex areas.

Efficient degradation of aqueous dichloromethane by an enhanced microbial electrolysis cell: Degradation kinetics, microbial community and metabolic mechanisms.

Dichloromethane (DCM) has been listed as a toxic and harmful water pollutant, and its removal needs attention. Microbial electrolysis cells (MECs) are viewed as a promising alternative for pollutant removal, which can be strengthened from two aspects: microbial inoculation and acclimation. In this study, the MEC for DCM degradation was inoculated with the active sludge enhanced by Methylobacterium rhodesianum H13 (strain H13) and then acclimated in the form of a microbial fuel cell (MFC). Both the introduction of strain H13 and the initiation in MFC form significantly promoted DCM degradation. The degradation kinetics were fitted by the Haldane model, with Vmax, Kh, Ki and vmax values of 103.2 mg/L/hr, 97.8 mg/L, 268.3 mg/L and 44.7 mg/L/hr/cm2, respectively. The cyclic voltammogram implies that DCM redox reactions became easier with the setup of MEC, and the electrochemical impedance spectrogram shows that the acclimated and enriched microbes reduced the charge transfer resistance from the electrode to the electrolyte. In the biofilm, the dominant genera shifted from Geobacter to Hyphomicrobium in acclimation stages. Moreover, Methylobacterium played an increasingly important role. DCM metabolism mainly occurred through the hydrolytic glutathione S-transferase pathway, given that the gene dcmA was identified rather than the dhlA and P450/MO. The exogenous electrons facilitated the reduction of GSSG, directly or indirectly accelerating the GSH-catalyzed dehalogenation. This study provides support for the construction of an efficient and stable MEC for DCM removal in water environment.

Evaluation of alternate hosts for recombinant expression of a reductive dehalogenase.

Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl3, Na2S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H2-oxidizing C. necator H16, a novel host for the expression of RDases.

Induction of apoptosis and autophagy by dichloromethane extract from Patrinia scabiosaefolia Fisch on acute myeloid leukemia cells.

Patrinia scabiosaefolia Fisch (PS), a perennial herb belonging to the genus Pinus in the family Pinnacle Sauce, has been previously known for its analgesic, anti-inflammatory, antibacterial, and antitumor properties. However, the specific mechanism behind its antileukemic effect remains unknown. This study focused on the cytotoxicity and potential modes of action of the dichloromethane extract from PS (DEPS) in acute myeloid leukemia (AML) cells. Our results demonstrated that DEPS reduced cell viability, arrested the cell cycle in the G2/M phase, disrupted the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and upregulated the expression of Bax/Bcl-2 and Cleaved caspase-3. However, the impact of DEPS on cell viability and the expression of apoptosis-associated proteins was reversed upon pretreatment with the caspase-3 inhibitor (Z-DEVD-FMK) in HL-60 cells, which demonstrated that DEPS could induce apoptosis through the mitochondria-associated apoptotic pathway. Interestingly, DEPS also influenced autophagy by upregulating the expression of LC3II/I, P62, and Beclin-1 proteins, and the autophagy inhibition chloroquine(CQ) could attenuate the apoptotic effects of DEPS in HL-60 cells. Furthermore, SMART 2.0 analysis predicted that the main components present in DEPS were likely terpenoids. In conclusion, DEPS possibly exerts antileukemic effects by downregulating the PI3K/AKT and ERK pathways, thereby promoting intracellular ROS production, activating the mitochondrial apoptotic pathway, and affecting autophagy, providing valuable insights for the potential future application of PS in the treatment of AML.

Multiple liquid-liquid extraction of dissolved compounds in immortelle hydrosol with four different solvents.

Immortelle, a revered Mediterranean medicinal plant, is celebrated for its potent essential oil renowned in the cosmetic industry for its skin-enhancing properties. Yet, immortelle hydrosol, an often-overlooked byproduct, holds promise in cosmetics due to its compatibility with polar active ingredients. This study investigates the chemical composition of immortelle hydrosol by employing liquid-liquid extraction (LLE) to transfer volatile organic components into nonpolar solvents. Four solvents - chloroform, dichloromethane, hexane, and benzene - were assessed through ten consecutive extractions from industrially produced immortelle hydrosol. Quantification was achieved using GC analysis with tetradecane as an internal standard. Chloroform emerged as the most efficient solvent, yielding 2447.0 mg/L of volatile compounds, surpassing dichloromethane, hexane, and benzene. Key compounds in immortelle hydrosol included 3-pentanone, 2-methyl-1-butanol, and γ-terpineol. Importantly, the study revealed that a portion of essential oil compounds persists in the hydrosol even after ten LLE cycles, with optimal results achievable in five extractions (~92 % in most cases).

Antinociceptive and anti-inflammatory potential, and chemical characterization of the dichloromethane fraction of Solanum lycocarpum (Solanaceae) ripe fruits by LC-DAD-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum lycocarpum A. St. Hil. (Solanaceae) is a species from the Brazilian Cerrado, exhibiting several medicinal properties, being used by the population in the treatment of ulcers, bronchitis, asthma and hepatitis, which involve inflammatory processes. AIM OF THIS STUDY: This study aimed to chemically characterize the dichloromethane fraction (DCM), as well as verify its antinociceptive, anti-inflammatory and antioxidant potential. MATERIALS AND METHODS: The DCM fraction was obtained by partitioning the ethanol extract. The chemical constituents of the DCM fraction were characterized by LC-DAD-MS. The DPPH and FRAP assays were used to evaluate the antioxidant potential. The carrageenan-induced paw edema model was used to assess the anti-inflammatory effects, and the inflammatory infiltrate was evaluated by qualitative and quantitative histological analyses. The antinociceptive action of the DCM fraction was evaluated by acetic acid-induced abdominal writhing test, formalin-induced nociception and hot-plate test. RESULTS: Steroidal alkaloids solasonine, solasodine and solamargine, as well as the alkaloid peiminine/imperialine and caffeoylquinic acids, were annotated in DCM fraction by LC-DAD-MS. The DCM fraction showed antioxidative action in the in vitro DPPH and FRAP tests, as well as an anti-inflammatory effect for the three evaluated doses of 30, 100 and 300 mg/kg in the fourth and sixth hours after the administration of carrageenan. The histological analyses evidenced considerably reduction in leukocyte migration and the number of polymorphonuclear leukocytes. The study also demonstrated antinociceptive activity for the DCM fraction, which reduced abdominal writhing at three concentrations evaluated, as well as a decrease in paw licking in the formalin-induced nociception test both in the neurogenic phase and the inflammatory phase, with greater effectiveness compared to the anti-inflammatory indomethacin. The DCM fraction also increased the latency time of the animals in the hot plate test 60 min after treatment, although it did not seem to involve the opioidergic system. CONCLUSION: This work evidenced that the dichloromethane fraction of S. lycocarpum fruit possesses antinociceptive and anti-inflammatory potential, which supports its use in folk medicine for management inflammatory conditions.

Determination of Volatile Organic Compounds and Endogenous Extracts and Study of Expression Patterns of TPS and BSMT in the Flowers of Seven Lilium Cultivars.

Lily is one of the most important cut flowers in the world, with a rich floral fragrance. To further explore the fragrance emission mechanisms of lily cultivars, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and organic solvent extraction-gas chromatography-mass spectrometry (OSE-GC-MS) were used to unveil the volatile organic compounds (VOCs) and endogenous extracts of seven lily cultivars. Furthermore, real-time quantitative PCR (qRT-PCR) was used to determine the expression levels of two key genes (TPS and BSMT) related to the biosynthesis of monoterpenoids and methyl benzoate. The results show that forty-five VOCs were detected in the petals of seven lily cultivars, and the main compounds were monoterpenoids and phenylpropanoids/benzenoids. Dichloromethane was the best solvent for extracting the endogenous extracts of Lilium 'Viviana' petals and eighteen endogenous extracts were detected using dichloromethane to extract the petals of seven lily cultivars. Each compound's emission ratio (natural logarithm of the ratio of VOC content to endogenous extract content) was calculated, and linear regression analyses between emission ratios and boiling points were conducted. Significant linear negative correlations existed between the emission ratios and boiling points of compounds, and the regression equations' coefficients of determination (R2) were all greater than 0.7. TPS was expressed highly in 'Viviana', 'Pink News', and 'Palazzo', and BSMT was expressed highly in 'Pink News' and 'Palazzo'. Correlation analyses between the gene expression levels and the monoterpenoids and methyl benzoate contents found that the TPS expression levels have strong positive correlations with monoterpenoids content, while no correlations were found between the expression levels of BSMT and the contents of methyl benzoate. This study lays the foundation for research on the release patterns of VOCs in the flowers of Lilium, and the breeding of lilies for their floral fragrance.

The Reactions of Alkenes with Phenyl-N-triflylimino-λ3-iodane: Solvent and Oxidant Impact.

The reactions of alkenes with phenyl-N-triflylimino-λ3-iodane PhI=NTf (1) have been studied in different conditions. In methylene chloride, in the presence of N-halosuccinimides, the products of mono and bis-triflamidation were obtained. In MeCN, the product of bromotriflamidation (with NBS) with solvent interception or of bis-triflamidation (with NIS) is formed. The reaction with trans-stilbene in acetonitrile with NBS gave rise to cyclization to 2-methyl-4,5-diphenyl-1-triflyl-4,5-dihydro-1H-imidazole. In contrast, with NIS as an oxidant, both in CH2Cl2 and MeCN, the major product was 2,3-diphenyl-1-triflylaziridine formed in good yield. With NBS, aziridine is also formed but as a minor product, the major one being a mixture of diastereomers of the product of bromotriflamidation. The reaction of compound 1 with vinylcyclohexane in methylene chloride affords the mixtures of regioisomers of the products of halotriflamidation, whereas in acetonitrile, the products of solvent interception and cyclization to the imidazoline are formed. A mechanism explaining the formation of all isolated products is proposed.

1H NMR Mixture Design-Fingerprints and ASCA Analysis in Ilex paraguariensis: Model Stability in Search of a Global Metabolome.

The effects of experimental repetitions and solvent extractors on the 1H NMR fingerprinting of yerba mate extracts, obtained from two genders and two light environments, were analyzed in-depth by ANOVA-simultaneous component analysis (ASCA). Different solvents were used according to a mixture design based on ethanol, dichloromethane, and hexane and their combinations. The number of experimental repetitions significantly affected the ASCA results. Increasing repetitions led to decreases in the percentage effect variance values and an increase in the percentage residual variance. However, secondary sexual dimorphism, light availability, and their interaction effects became more significant with decreasing p-values at or above the 95% confidence level. The choice of a solvent extractor significantly affects the chemical profile and can lead to distinct conclusions regarding the significance of effect values. Pure solvents yielded different conclusions about the significance of factorial design effects, with each solvent extracting unique metabolites and maximizing information for specific effects. However, the use of binary solvent mixtures, such as ethanol-dichloromethane, proved more efficient in extracting sets of compounds that simultaneously differentiate between different experimental conditions. The mixture design-fingerprint strategy provided satisfactory results expanding the range of extracted metabolites with high percentage of residual variances and low explained percentage effect variances in the ASCA models. Ternary and even higher-ordered mixtures could be good alternative extracting media for work-intensive procedures. Our study underscores the significance of experimental design and solvent selection in metabolomic analysis, improving the accuracy, robustness, and interpretability of metabolomic models, leading to a better understanding of the chemical composition and biological implications of plant extracts.

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.

Phytochemical Analysis and Evaluation of Genotoxic, Antigenotoxic Effects in Bacterial Models, and Hypoglycemic Activity of Epilobium angustifolium L. with Bioguided Isolation of Active Compounds.

This study examined the effects of methanol extract and its sub-extracts from Epilobium angustifolium on α-glucosidase and α-amylase activity. Secondary metabolites and amino acids were quantified using LC-MS/MS. Dichloromethane sub-extract displayed the highest activity and was chosen for further investigation. Despite the widespread use of E. angustifolium, genotoxicity studies were conducted to assess its safety. Dichloromethane significantly inhibited α-glucosidase (IC50 =17.340 µg/mL), making it approximately 293 times more effective than acarbose. Six known compounds, including gallic acid (1), a mixture of quercetin-3-O-α-galactoside (2a) and quercetin-3-O-α-glucoside (2b), quercetin-3-O-α-glucuronic acid (3), quercetin-3-O-α-rhamnoside (4), and kaempferol-3-O-α-rhamnoside (5) were identified. Quercetin-3-O-α-rhamnoside exhibited the highest inhibition of α-glucosidase (IC50 =1735±85 µM), making it 3.70 times more effective than acarbose. Dichloromethane also showed significant antigenotoxic activity against mutagenesis induced by NaN3, 9-AA, 4-NPD, and MNNG. Gallic acid was found in the highest abundance (13253.6931 ng/mL) in the methanolic extract. Furthermore, L-Aspartic acid was the most concentrated amino acid (363.5620 nmol/mL) in the methanolic extract.

Combinative effects of akarkara root-derived metabolites on anti-inflammatory and anti-alzheimer key enzymes: integrating bioassay-guided fractionation, GC-MS analysis, and in silico studies.

BACKGROUND: Anacyclus pyrethrum L. (Akarkara root), a valuable Ayurvedic remedy, is reported to exhibit various pharmacological activities. Akarkara root was subjected to bioassay-guided fractionation, to isolate its active constituents and discover their potential bioactivities, followed by computational analysis. METHODS: The methanol extract and its fractions, methylene chloride, and butanol, were assessed for their antioxidant, anti-inflammatory, and anticholinergic potentials. The antioxidant activity was determined using DPPH, ABTS, FRAP, and ORAC assays. The in vitro anticholinergic effect was evaluated via acetyl- and butyryl-cholinesterase inhibition, while anti-inflammatory effect weas determined using COX-2 and 5-LOX inhibitory assays. The methylene chloride fraction was subjected to GC/MS analysis and chromatographic fractionation to isolate its major compounds. The inhibitory effect on iNOS and various inflammatory mediators in LPS-activated RAW 264.7 macrophages was investigated. In silico computational analyses (molecular docking, ADME, BBB permeability prediction, and molecular dynamics) were performed. RESULTS: Forty-one compounds were identified and quantified and the major compounds, namely, oleamide (A1), stigmasterol (A2), 2E,4E-deca-2,4-dienoic acid 2-phenylethyl amide (A3), and pellitorine (A4) were isolated from the methylene chloride fraction, the most active in all assays. All compounds showed significant in vitro antioxidant, anticholinergic and anti-inflammatory effects. They inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in activated RAW macrophages. The isolated compounds showed good fitting in the active sites of acetylcholinesterase and COX-2 with high docking scores. The ADME study revealed proper pharmacokinetics and drug likeness properties for the isolated compounds. The isolated compounds demonstrated high ability to cross the BBB and penetrate the CNS with values ranging from 1.596 to -1.651 in comparison with Donepezil (-1.464). Molecular dynamics simulation revealed stable conformations and binding patterns of the isolated compounds with the active sites of COX-2 and acetyl cholinesterase. CONCLUSIONS: Ultimately, our results specify Akarkara compounds as promising candidates for the treatment of inflammatory and neurodegenerative diseases.

Advancing carotenoid Quantification: A new method for semi-quantitative assessment of ß -Carotene and lycopene content in food extracts.

Carotenoids, such as lycopene and ß-carotene, have been widely recognized for their antioxidant properties and potential health benefits. Accurate quantification of carotenoids in plant extracts is essential for nutritional assessment, quality control, and research investigations. This study introduces an innovative method for quantifying lycopene and ß-carotene, in plant extracts and aims to bridge the gap between complex and expensive carotenoid quantification techniques and the need for accessible methods that can be widely adopted. The primary difference between HPLC and HPTLC lies in the medium used for separation. HPLC employs a liquid phase within columns, while HPTLC utilizes a thin layer of adsorbent on a plate. This distinction impacts factors like equipment, cost, and analysis time. The VisionCats software, combined with the CAMAG Visualizer-2, allows the semi-quantification of metabolites using an image-based evaluation method enabling the simultaneous assessment of qualitative and semi-quantitative information from the HPTLC images. Sample preparation involves washing and drying the vegetal material, followed by dichloromethane extraction. HPTLC analysis is performed using the CAMAG Advanced Herbal System, and the validation studies include establishing calibration curves and determining the detection threshold and minimum quantification threshold for lycopene and ß-carotene. Specificity and precision were evaluated to ensure accurate identification and repeatability of the method. Data analysis involves selecting the regression method based on the nature of the data and assessing the goodness of fit using the R2 value. The results showed distinct peaks corresponding to lycopene and ß-carotene in the chromatograms of the plant extract samples. The visualizer-based method demonstrates good specificity and precision, with no interfering peaks observed and low relative standard deviation. The method shows promising results regarding specificity, precision, and reliability. It has the potential for broader implementation in carotenoid research and rapid monitoring of carotenoid content in various agricultural and food products, particularly in resource-limited settings. Further optimization and validation on a wider range of samples would enhance the applicability of this method in carotenoid research. Sample preparation involves washing and drying the vegetal material, followed by dichloromethane extraction. HPTLC analysis is performed using the CAMAG Advanced Herbal System, and the validation studies include establishing calibration curves and determining the detection threshold and minimum quantification threshold for lycopene and ß-carotene. Specificity and precision were evaluated to ensure accurate identification and repeatability of the method. Data analysis involves selecting the regression method based on the nature of the data and assessing the goodness of fit using the R2 value. The results showed distinct peaks corresponding to lycopene and ß-carotene in the chromatograms of the plant extract samples. The visualizer-based method demonstrates good specificity and precision, with no interfering peaks observed and low relative standard deviation. The method shows promising results regarding specificity, precision, and reliability. It has the potential for broader implementation in carotenoid research and for rapid screening and monitoring of carotenoid content in various agricultural and food products, particularly in resource-limited settings. Further optimization and validation on a wider range of samples would enhance the applicability of this method in carotenoid research.

Antioxidant and anti-Alzheimer activities of Clivia miniata (Lindl) roots, bulbs, and aerial parts: In-vitro and in-silico studies.

Clivia miniata (Lindl) is a member of the family Amaryllidaceae known for its chemically diverse alkaloids with a wide range of biological activities. Many reports revealed a direct role of oxidative stress in the early stage of Alzheimer's disease (AD). Meanwhile, ß-site amyloid precursor protein cleavage enzyme 1 (BACE-1) is a molecular target for the treatment of AD. We aimed to investigate C. miniata root, bulb, and aerial part chemical profiling, antioxidant, BACE-1, and AChE enzyme inhibitory activities. Results showed that the total root had the most potent radical scavenging activity as compared to the total bulb and aerial part, respectively. Ethanol root extract had the most potent BACE-1 inhibitory activity (IC50 = 0.02 ± 0.001 µg/mL) as compared to the bulb and aerial part (IC50 = 0.93 ± 0.13, 1.80 ± 0.24 µg/mL), respectively. Moreover, the total root extract mitigated AChE enzyme activity more than total bulb and aerial fractions with IC50 values of (0.06 ± 0.02, 0.58 ± 0.3, and 1.89 ± 0.42 µg/mL, respectively. Bioassay-guided acid-base fractionation confirmed superior BACE-1 inhibitory activity of the root fractions particularly, methylene chloride and ethyl acetate fractions with (IC50 values of 0.21 ± 0.60 and 0.01 ± 0.001 µg/mL), respectively. UPLC-MS analysis of ethyl acetate and methylene chloride fractions of C. miniata root led to the identification of eight phenolics and thirteen alkaloids, respectively. Molecular docking studies against BACE-1 protein revealed that lycorine di-hexoside, miniatine, and cliviaaline were the most promising hits. Further investigation of anti-AD potential of the aforementioned small molecules is required.

Sensory-directed isolation and identification of an intense salicin-like bitter compound in infected teas with bird's eye spot disease.

Teas infected with bird's eye spot disease generally exhibited a lingering and long-lasting, salicin-like bitter taste, which was unpalatable to consumers. Sensory-directed isolation processes have been performed in this study to investigate the salicin-like bitter compounds in infected teas. Results showed that infected teas were extracted using a 70% methanol aqueous solution to produce methanol extract, which was then further separated by sequential solvent extraction (SSE) to obtain dichloromethane extract, which contained the salicin-like bitter compounds. The dichloromethane extract was then isolated by flash chromatography to produce two salicin-like bitter fractions, eluted using 60% and 65% methanol aqueous solution. Finally, these two salicin-like bitter fractions were analyzed by RP-HPLC using 60-68% and 70-75% methanol aqueous solution, respectively, affording the location of the salicin-like bitter compounds in RP-HPLC chromatograms. Moreover, a new ursane-type triterpenoid, camellisin A methyl ester, was identified from infected teas. This study has provided preliminary isolation methods of salicin-like bitter compounds from the infected teas, which were essential to designing targeted debittering strategies for infected teas and improving the quality of the finished tea and the effective utilization of fresh tea leaves.

Anti-osteoporosis bioactivity evaluation in zebrafish model of raw and salt-processed Achyranthes bidentata followed by liquid chromatography-mass spectrometry analysis and correlation analysis.

Achyranthes bidentata has been found to possess beneficial effects against osteoporosis, but there is still a lack of comprehensive studies on its anti-osteoporotic compounds. Therefore, in this study, we established a zebrafish osteoporosis model to evaluate the anti-osteoporotic effect of different fractions of raw and salt-processed A. bidentata. Among these fractions, the dichloromethane fraction showed the most promising anti-osteoporotic effect. To further investigate the active compounds responsible for the anti-osteoporosis effects, we prepared and analyzed the dichloromethane fraction of 10 batches of raw and salt-processed A. bidentata using liquid chromatography-mass spectrometry. As a result, we tentatively identified 19 compounds, including 11 saponins, three phenolic amides, three unsaturated fatty acids and two other compounds. To further narrow down the potential active compounds, we employed both orthogonal partial least squares discriminant analysis and gray relationship analysis. Through these analyses, we were able to identify eight compounds that showed a high correlation with the anti-osteoporosis effects of the dichloromethane fraction. Furthermore, we validated the anti-osteoporotic effects of ß-ecdysterone, wogonin, ginsenoside Ro, oleanolic acid, linoleic acid and palmitic acid using the zebrafish model. These compounds demonstrated significant anti-osteoporotic effects, further supporting their potential as active compounds in A. bidentata.