Biological and metabolomics-guided isolation of tetrahydrofurofuran lignan from Croton spp. with antiproliferative activity against human melanoma cell line.

The Croton genus (Euphorbiaceae) is recognized as a promising source for identifying bioactive compounds with antiproliferative activity. However, knowledge on the chemical composition and activity of Croton floribundus, Croton echinocarpus, and Croton zehntneri is limited. Thus, this study aimed to investigate the antiproliferative activity of these species on cells derived from tumoral breast, lung, and melanoma cells, and primary fibroblasts derived from human skin. Metabolomic strategies were applied via ultra-performance liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to target the main active compound. The C. floribundus leaf extract exhibited the highest activity, with an IC50 value lower than that of the reference drug - temozolomide - in the most responsive cell line - SK-MEL-147 - and in all the evaluated melanoma cell lines (SK-MEL-147, CHL-1 and WM-1366). Four tetrahydrofurofuran lignans were isolated for the first time from the most promising fraction of the C. floribundus extract. According to the metabolomic and multivariate statistical analyses, the isolated lignan epi-yangambin constituted the main antiproliferative compound against SK-MEL-147; furthermore, it exhibited selective antiproliferative activity for this cell line (IC50 = 13.09 µg/mL and selectivity index = 3.82; temozolomide, IC50 = 121.50 µg/mL) due to, at least in part, its ability to inhibit cell cycle progression at G2/M. This is especially relevant considering the high resistance of melanoma cells to available drugs. Thus, epi-yangambin can serve as a prototype for further antiproliferative investigations.

Study on the correlation and difference of qualitative information among three types of UPLC-HRMS and potential generalization in metabolites annotation.

The variation of qualitative information among different types of mainstream hyphenated instruments of ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) makes data sharing and standardization, and further comparison of results consistency in metabolite annotation not easy to attain. In this work, a quantitative study of correlation and difference was first achieved to systematically investigate the variation of retention time (tR), precursor ion (MS1), and product fragment ions (MS2) generated by three typical UPLC-HRMS instruments commonly used in metabolomics area. In terms of the findings of systematic and correlated variation of tR, MS1, and MS2 between different instruments, a computational strategy for integrated metabolite annotation was proposed to reduce the influence of differential ions, which made full use of the characteristic (common) and non-common fragments for scoring assessment. The regular variations of MS2 among three instruments under four collision energy voltages of high, medium, low, and hybrid levels were respectively inspected with three technical replicates at each level. These discoveries could improve general metabolite annotation with a known database and similarity comparison. It should provide the potential for metabolite annotation to generalize qualitative information obtained under different experimental conditions or using instruments from various manufacturers, which is still a big headache in untargeted metabolomics. The mixture of standard compounds and serum samples with the addition of standards were applied to demonstrate the principle and performance of the proposed method. The results showed that it could be an optional strategy for general use in HRMS-based metabolomics to offset the difference in metabolite annotation. It has some potential in untargeted metabolomics.

Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Sphingomyelin is involved in regulating UCP1-mediated nonshivering thermogenesis.

Adipogenesis is one of the major mechanisms for adipose tissue expansion, during which spindle-shaped mesenchymal stem cells commit to the fate of adipocyte precursors and differentiate into round-shaped fat-laden adipocytes. Here, we investigated the lipidomic profile dynamics of ex vivo-differentiated brown and white adipocytes derived from the stromal vascular fractions of interscapular brown (iBAT) and inguinal white adipose tissues. We showed that sphingomyelin was specifically enriched in terminally differentiated brown adipocytes, but not white adipocytes. In line with this, freshly isolated adipocytes of iBAT showed higher sphingomyelin content than those of inguinal white adipose tissue. Upon cold exposure, sphingomyelin abundance in iBAT gradually decreased in parallel with reduced sphingomyelin synthase 1 protein levels. Cold-exposed animals treated with an inhibitor of sphingomyelin hydrolases failed to maintain core body temperature and showed reduced oxygen consumption and iBAT UCP1 levels. Conversely, blockade of sphingomyelin synthetic enzymes resulted in enhanced nonshivering thermogenesis, reflected by elevated body temperature and UCP1 levels. Taken together, our results uncovered a relation between sphingomyelin abundance and fine-tuning of UCP1-mediated nonshivering thermogenesis.

Non-Volatile Component and Antioxidant Activity: A Comparative Analysis between Litsea cubeba Branches and Leaves.

Litsea cubeba, which is found widely distributed across the Asian region, functions as both an economic tree and a medicinal plant with a rich historical background. Previous investigations into its chemical composition and biological activity have predominantly centered on volatile components, leaving the study of non-volatile components relatively unexplored. In this study, we employed UPLC-HRMS technology to analyze the non-volatile components of L. cubeba branches and leaves, which successfully resulted in identifying 72 constituents. Comparative analysis between branches and leaves unveiled alkaloids, organic acids, and flavonoids as the major components. However, noteworthy differences in the distribution of these components between branches and leaves were observed, with only eight shared constituents, indicating substantial chemical variations in different parts of L. cubeba. Particularly, 24 compounds were identified for the first time from this plant. The assessment of antioxidant activity using four methods (ABTS, DPPH, FRAP, and CUPRAC) demonstrated remarkable antioxidant capabilities in both branches and leaves, with slightly higher efficacy observed in branches. This suggests that L. cubeba may act as a potential natural antioxidant with applications in health and therapeutic interventions. In conclusion, the chemical composition and antioxidant activity of L. cubeba provides a scientific foundation for its development and utilization in medicine and health products, offering promising avenues for the rational exploitation of L. cubeba resources in the future.

Fine-Scale Characterization of Plant Diterpene Glycosides Using Energy-Resolved Untargeted LC-MS/MS Metabolomics Analysis.

Plant diterpene glycosides are essential for diverse physiological processes. Comprehensive structural characterization proved to be a challenge due to variations in glycosylation patterns, diverse aglycone structures, and the absence of comprehensive reference databases. In this study, a method for fine-scale characterization was proposed based on energy-resolved (ER) untargeted LC-MS/MS metabolomics analysis using steviol glycosides as a demonstration. Energy-dependent fragmentation patterns were unveiled by a series of model compounds. Distinct glycosylation sites were discerned by leveraging varying fragmentation energies for the precursor ions. The sugar moiety linkage at C19OOH (R1) exhibited facile and intact cleavage at low collision energies, while the sugar moiety at C13-OH (R2) demonstrated consecutive cleavage with increasing energy. Aglycone ions exhibited a higher relative intensity at NCE 50, with relative intensities ranging from 95% to 100%. Subsequently, aglycone candidates, R1 sugar composition, and R2 sugar sequence were deduced through ER-MS/MS analysis. The developed method was applied to Stevia rebaudiana leaves. A total of 91 diterpene glycosides were unambiguously identified, including 16 steviol glycosides with novel acetylglycosylation patterns. This method offers a rapid alternative for glycan analysis and the structural differentiation of isomers. The developed method enhances the understanding of diterpene glycosides in plants, providing a reliable tool for the in-depth characterization of complex metabolite profiles.

The Study on Timolol and Its Potential Phototoxicity Using Chemical, In Silico and In Vitro Methods.

Timolol (TIM) is a non-selective ß-adrenergic receptor antagonist used orally for the treatment of hypertension and heart attacks, and topically for treating glaucoma; lately, it has also been used in some specific dermatological problems. In the present study, its photodegradation and potential risk of phototoxicity were examined using chemical, in silico and in vitro methods. The UV/VIS irradiated solutions of TIM at pH 1-13 were subjected to LC-UV and UPLC-HRMS/MS analyses showing pseudo first-order kinetics of degradation and several degradation products. The structures of these photodegradants were elucidated by fragmentation path analysis based on high resolution (HR) fragmentation mass spectra, and then used for toxicity evaluation using OSIRIS Property Explorer and Toxtree. Potential risk of phototoxicity was also studied using chemical tests for detecting ROS under UV/VIS irradiation and in vitro tests on BALB/c 3T3 mouse fibroblasts (MTT, NRU and Live/Dead tests). TIM was shown to be potentially phototoxic because of its UV/VIS absorptive properties and generation ROS during irradiation. As was observed in the MTT and NRU tests, the co-treatment of fibroblasts with TIM and UV/VIS light inhibited cell viability, especially when concentrations of the drug were higher than 50 µg/mL.

In vivo pharmacokinetic study of vanillic acid in monocrotaline-induced pulmonary arterial hypertension rats and its tissue distribution.

Vanillic acid (VA) is a bioactive chemical present in many food plants and fruits. It has been shown to have a protective effect on pulmonary tissues in monocrotaline-induced pulmonary arterial hypertension, as well as an intervention effect on right ventricular remodeling. The purpose of this study was to develop and test a reliable method for assessing VA utilizing ultra-performance liquid chromatography-high resolution mass spectrometry using caffeic acid as the internal standard. Across diverse substrates, the correlation coefficient for VA ranged from 0.9992 to 0.9995. The method's intraday precision was <13.53% (RSD), and its accuracy (RE) ranged from -9.88 to 4.35%. The precision across days was <13.69% (RSD), while the accuracy ranged from 2.16 to 10.94% (RE). The extraction recoveries ranged from 80.30 to 118.81%, with a lower limit of quantification of 20 ng/mL. The approach was successfully applied to pharmacokinetic and tissue distribution studies of VA in rat plasma after gavage administration, and the pharmacokinetic parameters of VA in the plasma of the monocrotaline-induced pulmonary arterial hypertension were significantly different from those of the control group.

Determination of alkaloid-inspired molecule vindeburnol in rabbit plasma by UPLC-HRMS and its application to pharmacokinetic studies and preliminary metabolite identification.

The eburnamine-vincamine alkaloids exhibit a range of pharmacological activities. There is a limited understanding of the pharmacokinetics and pharmacodynamics of vindeburnol, a synthetic derivative of this chemical class of alkaloids. A fast and reliable UPLC-HRMS method was developed and validated to quantify vindeburnol in Soviet Chinchilla rabbit plasma from pharmacokinetics studies. An ultra-performance liquid chromatography system equipped with a Waters Acquity UPLC HSS T3 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and acetonitrile. An Impact II QqTOF high-resolution mass spectrometer equipped with an Apollo II electrospray ionization source was used for analysis in positive mode; the ions [M+H]+m/z 269.1648 ± 0.003 and m/z 351.2067 ± 0.003 were monitored for vindeburnol and internal standard (vinpocetine), respectively. Preliminary metabolite profiling was also performed, and the pharmacokinetics of the identified metabolites were evaluated. The mean retention times for vindeburnol and vinpocetine were 2.0 and 3.5 min. The UPLC-HRMS method was validated with accuracy and precision within the 15% acceptance limit (8.2% and 11.0%, respectively). The mean extraction recovery value of vindeburnol from rabbit plasma was 77%. Pharmacokinetic evaluation of vindeburnol revealed that the compound is distributed rapidly with a short elimination half-life. Vindeburnol undergoes extensive first-pass metabolism and is metabolized into hydroxyvindeburnol and vindeburnol glucuronide.

Surface multi-walled carbon nanotube modified quaternary amine-functionalized polymers for purification and determination of glyphosate and its four metabolites in plasma samples.

The present study focused on the pretreatment and detection of GLY and its four metabolites AMPA, N-acetyl AMPA, N-methyl GLY and N-acetyl GLY in plasma samples. Multi-walled carbon nanotube-modified quaternary amine-functionalized polymers (QA-PDNV@MWCNTs) were synthesized in a controlled manner by self-assembly, and its morphology and composition were extensively characterized. The QA-PDNV@MWCNTs microspheres were then used as an SPE adsorbent for the preparation and rapid determination of GLY and its four metabolites in plasma samples combined with ultra-performance liquid chromatography-high resolution mass spectrometry (UPLCHRMS). The SPE conditions based on QA-PDNV@MWCNTs were optimized for GLY and its metabolites to obtain the best purification efficiency. The experimental results show that when the adsorbent contains 8% MWCNTs, it can balance the adsorption of target analytes and the purification performance of the adsorbent for impurities. In addition, this study compared the QA-PDNV@MWCNTs based SPE method with the commercial Waters Oasis MAX SPE cartridge and the results showed that the developed method in this study has better resistance to matrix interference. Under optimal conditions, the recoveries of GLY and its metabolites spiked in plasma were 82.6-99.4 % with relative standard deviations (RSDs) of 1.0-7.8 %. The limits of detection (LODs, S/N ≥ 3) and limits of quantification (LOQs, S/N ≥ 9) of the method were 0.05-0.33 µg/L and 0.15-1.00 µg/L, respectively. Finally, the developed QA-PDNV@MWCNTs based SPE-UPLCHRMS method was used to confirm GLY poisoning not only on the basis of the detection of the GLY prototype, but also on the basis of its four metabolites.

Untargeted metabolomics-based molecular networking for chemical characterization of selected Apiaceae fruit extracts in relation to their antioxidant and anti-cellulite potentials.

Stress, obesity, hormonal changes, and aging have been connected to cellulite aggravation resulting in skin dimpled appearance, a very common painless skin disorder with a female preponderance. Several Apiaceae plants have been traditionally used for cosmetic applications. However, their screening for anti-cellulite potential has not been deeply investigated. In this work, UPLC-HRMS/MS coupled with molecular networking was employed to glean a holistic overview of the chemodiversity of the metabolome of nine Apiaceae fruits. Additionally, the extracts were screened for in vitro antioxidant and anti-cellulite activities. Apium graveolens and Petroselinum crispum revealed excellent free radical scavenging activity, remarkably increased lipolysis, and decreased adipogenesis. Furthermore, apigenin and its glycosides were identified to be the major components in both extracts, which might be responsible for the antioxidant activity and anti-cellulite potential. Conclusively, these results signify the potent antioxidant and anti-cellulite properties of A. graveolens and P. crispum fruit extracts, holding potential for the development of plant derived products for cellulite management.

Hippocampal metabolic profile during epileptogenesis in the pilocarpine model of epilepsy.

Temporal lobe epilepsy (TLE) is a common form of refractory epilepsy in adulthood. The metabolic profile of epileptogenesis is still poorly investigated. Elucidation of such a metabolic profile using animal models of epilepsy could help identify new metabolites and pathways involved in the mechanisms of epileptogenesis process. In this study, we evaluated the metabolic profile during the epileptogenesis periods. Using a pilocarpine model of epilepsy, we analyzed the global metabolic profile of hippocampal extracts by untargeted metabolomics based on ultra-performance liquid chromatography-high-resolution mass spectrometry, at three time points (3 h, 1 week, and 2 weeks) after status epilepticus (SE) induction. We demonstrated that epileptogenesis periods presented different hippocampal metabolic profiles, including alterations of metabolic pathways of amino acids and lipid metabolism. Six putative metabolites (tryptophan, N-acetylornithine, N-acetyl-L-aspartate, glutamine, adenosine, and cholesterol) showed significant different levels during epileptogenesis compared to their respective controls. These putative metabolites could be associated with the imbalance of neurotransmitters, mitochondrial dysfunction, and cell loss observed during both epileptogenesis and epilepsy. With these findings, we provided an overview of hippocampal metabolic profiles during different stages of epileptogenesis that could help investigate pathways and respective metabolites as predictive tools in epilepsy.

Correlation between secondary metabolites of Iris confusa Sealy and Iris pseudacorus L. and their newly explored antiprotozoal potentials.

BACKGROUND: In the last few decades, the use of plant extracts and their phytochemicals as candidates for the management of parasitic diseases has increased tremendously. Irises are aromatic and medicinal plants that have long been employed in the treatment of different infectious diseases by traditional healers in many cultures. This study aims to explore the potential of three common Iris species (I. confusa Sealy, I. pseudacorus L. and I. germanica L.) against infectious diseases. Their in vitro antiprotozoal potency against Plasmodium falciparum, Trypanosoma brucei brucei, T. b. rhodesiense, T. cruzi and Leishmania infantum beside their cytotoxicity on MRC-5 fibroblasts and primary peritoneal murine macrophages were examined. METHODS: The secondary metabolites of the tested extracts were characterized by UPLC-HRMS/MS and Pearsons correlation was used to correlate them with the antiprotozoal activity. RESULTS: Overall, the non-polar fractions (NPF) showed a significant antiprotozoal activity (score: sc 2 to 5) in contrast to the polar fractions (PF). I. confusa NPF was the most active extract against P. falciparum [IC50 of 1.08 µg/mL, selectivity index (S.I. 26.11) and sc 5] and L. infantum (IC50 of 12.7 µg/mL, S.I. 2.22 and sc 2). I. pseudacorus NPF was the most potent fraction against T. b. rhodesiense (IC50 of 8.17 µg/mL, S.I. 3.67 and sc 3). Monogalactosyldiacylglycerol glycolipid (18:3/18:3), triaceylglycerol (18:2/18:2/18:3), oleic acid, and triterpenoid irridals (spirioiridoconfal C and iso-iridobelamal A) were the top positively correlated metabolites with antiplasmodium and antileishmanial activities of I. confusa NPF. Tumulosic acid, ceramide sphingolipids, corosolic, maslinic, moreollic acids, pheophytin a, triaceylglycerols, mono- and digalactosyldiacylglycerols, phosphatidylglycerol (22:6/18:3), phosphatidylcholines (18:1/18:2), and triterpenoid irridal iso-iridobelamal A, were highly correlated to I. pseudacorus NPF anti- T. b. rhodesiense activity. The ADME study revealed proper drug likeness properties for certain highly corelated secondary metabolites. CONCLUSION: This study is the sole map correlating I. confusa and I. pseudacorus secondary metabolites to their newly explored antiprotozoal activity.

[Detection of three metabolites of xylene in urine samples by solid-phase extraction coupled with liquid chromatography-mass spectrometry].

Objective: To establish a method for the rapid determination of the three metabolites of xylene, 2-methylmarmaluronic acid, 3-methylmarmaluronic acid and 4-methylmarmaluronic acid, in urine of occupationally exposed workers by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) . Methods: In July 2022, urine samples were diluted and extracted with pH=6.86 phosphate cache solution, cleaned up by a MAX solid-phase extraction (SPE) column and separated by an Accucore Ph/Hexyl column (100 mm×2.1 mm, 2.6 µm) with a gradient of 5 mmol/L ammonium formate-0.1% formic acid aqueous solution and methanol as mobile phases. The analysis was carried out in electrospray ionization mode and full mass-data dependent secondary mass spectrometry mode, and quantified by external standard method. The characteristics of each index of this method were analyzed. Results: A good linearity was obtained in the concentration range of 1.0-200.0 µg/L for 2-methylmuramic acid, 3-methylmuramic acid and 4-methylmuramic acid with the correlation coefficients of 0.9979-0.9993. The limits of detection of the method were 0.18-0.24 µg/L. While the spiked recoveries at the three concentrations (1.0 µg/L, 100.0 µg/L, and 180.0 µg/L) were in the range of 83.0%-93.7%, with the relative standard deviations of 2.2%-7.9%. Conclusion: The UPLC-HRMS method is simple, rapid, sensitive and accurate, and is suitable for the simultaneous determination of the three metabolites of xylene in the urine of occupationally exposed workers.

A classical herbal formula alleviates high-fat diet induced nonalcoholic steatohepatitis (NASH) via targeting mitophagy to rehabilitate dysfunctional mitochondria, validated by UPLC-HRMS identification combined with in vivo experiment.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) has caused a significant burden on public health care systems, the economy and society. However, there has still been no officially approved pharmacotherapy for NASH. It has been suggested that oxidative stress and mitochondrial dysfunction play vital roles in NASH pathological progression. Shugan Xiaozhi (SG) formula, as a kind of classical herbal formula, was shown to attenuate NASH. PURPOSE: This study aimed to explore the potential mechanisms of SG formula treating NASH. STUDY DESIGN AND METHODS: Ultra-high-performance liquid chromatography-high resolution mass spectrometry combined with bioinformatics analysis was applied to explore the therapeutic targets and main components of SG formula. Moreover, in vivo NASH model was utilized to confirmed the therapeutic effects of SG formula. Molecular docking analysis and further validation experiments were conducted to verify the results of bioinformatics analysis. RESULTS: The in vivo experiments confirmed SG formula significantly attenuated hepatic pathological progression and relieved oxidative stress in high-fat diet (HFD) induced - NASH model. Ultra-high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) combined with bioinformatics analysis expounded the components of SG formula and revealed the mitochondrial regulation mechanism of SG formula treating NASH. Further in vivo experiments validated that SG formula could alleviate oxidative stress by rehabilitating the structure and function of mitochondria, which was strongly related to regulating mitophagy. CONCLUSION: In summary, this study demonstrated that SG formula, which could attenuate NASH by regulating mitochondria and might be a potential pharmacotherapy for NASH.

Study on the Common Molecular Mechanism of Metabolic Acidosis and Myocardial Damage Complicated by Neonatal Pneumonia.

Pneumonia is a common clinical disease in the neonatal period and poses a serious risk to infant health. Therefore, the understanding of molecular mechanisms is of great importance for the development of methods for the rapid and accurate identification, classification and staging, and even disease diagnosis and therapy of pneumonia. In this study, a nontargeted metabonomic method was developed and applied for the analysis of serum samples collected from 20 cases in the pneumonia control group (PN) and 20 and 10 cases of pneumonia patients with metabolic acidosis (MA) and myocardial damage (MD), respectively, with the help of ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The results showed that compared with the pneumonia group, 23 and 21 differential metabolites were identified in pneumonia with two complications. They showed high sensitivity and specificity, with the area under the curve (ROC) of the receiver operating characteristic curve (ROC) larger than 0.7 for each differential molecule. There were 14 metabolites and three metabolic pathways of sphingolipid metabolism, porphyrin and chlorophyll metabolism, and glycerophospholipid metabolism existing in both groups of PN and MA, and PN and MD, all involving significant changes in pathways closely related to amino acid metabolism disorders, abnormal cell apoptosis, and inflammatory responses. These findings of molecular mechanisms should help a lot to fully understand and even treat the complications of pneumonia in infants.

Ameliorating effect of Citrus trifoliata L. fruits extract on motor incoordination, neurodegeneration and oxidative stress in Parkinson's disease model.

BACKGROUND: Citrus trifoliate fruit (also known as Trifoliate orange) is one of the commercially-cultivated Citrus genus of plants belonging to the Rutaceae family. It has been traditionally-utilized in treatment of neurodegenerative disorders. However, the scientific evidence verifying this utilization needs further elucidation. AIM OF THE STUDY: Characterization of the bioactive constituents of C. trifoliata L. fruits extract and evaluating its effect on Parkinson's disease (PD) model. MATERIAL AND METHODS: Rats were classified into 5 groups; control, PD, PD-treated by L-dopa/Carpidopa and PD-treated by oral Citrus trifoliata L. fruits extract (50 and 100 mg/kg). Deterioration in brain functions was evaluated through an in vivo open field, grid and catalepsy tests. The study also assessed the striatal neurotransmitters, oxidative stress markers and histopathological changes. RESULTS: Citrus trifoliata L. fruit extract has revealed motor improvement comparable to L-dopa and carbidopa. It has also effectively-improved oxidative stress via reduction of striatal malondialdehyde & nitric oxide along with replenishment of the striatal glutathione and superoxide dismutase. The extract caused significant reduction of the striatal myeloperoxidase activity and restoration of dopamine, γ-amino butyric acid (GABA), and acetylcholinesterase. This effect was further confirmed by amelioration of neuronal apoptosis, microgliosis and peri-neuronal vacuolation. Metabolite profiling revealed 40 constituents, with flavonoids representing the main identified class. CONCLUSION: The neuro-protective effect of Citrus trifoliata extract was achieved through the antioxidant and anti-inflammatory activities of its flavonoids, particularly hesperidin and naringin. This neuro-protective effect was evident at the behavioral, histological and neurotransmitter levels.

The Impact of Acute Low-Dose Gamma Irradiation on Biomass Accumulation and Secondary Metabolites Production in Cotinus coggygria Scop. and Fragaria × ananassa Duch. Red Callus Cultures.

Cotinus coggygria Scop. (smoketree) and Fragaria × ananassa Duch. (strawberry) are two industrially important species due to their composition in bioactive compounds. In this study, we investigated the effects of acute low-dose gamma irradiation (15, 20, 25, 30, 35 and 40 Gy) on two red callus cultures established in smoketree and strawberry. The biomass production, dry weight, content of phenols, flavonoids, monomeric anthocyanins', index of anthocyanins polymerization and antioxidant activity were evaluated. For the smoketree callus, a negative correlation between irradiation doses and callus biomass accumulation was observed. For the strawberry callus, irradiation did not significantly affect the accumulation of the biomass. An increased dry weight was observed in irradiated smoketree callus, while for treated strawberry callus, a decrease was recorded. Irradiation with 30 Gy was stimulative for polyphenols' accumulation in both cultures; however, the increase was significant only in the strawberry callus. The flavonoids increased in the 30 Gy strawberry variants, while it significantly decreased in smoketree callus irradiated with 35 and 40 Gy. In irradiated strawberry callus, except for the 25 Gy variant (1.65 ± 0.4 mg C-3-GE/g DW), all treatments caused an increase in anthocyanins' accumulation. In smoketree, except for the 15 Gy variant (2.14 ± 0.66 mg C-3-GE/g DW), the irradiation determined an increase in anthocyanins synthesis, with the highest value being seen in the 20 Gy variant (2.8 ± 0.94 mg C-3-GE/g DW). According to UPLC-HRMS investigations, an unidentified compound increased by 99% at the 30 Gy dose in strawberry callus, while in smoketree, maslinic acid increased by 51% after irradiation with 40 Gy. The results of this study showed, for the first time, the differential response of two performant callus cultures to low-dose gamma irradiation, a biotechnological method that can be used to stimulate the synthesis of important flavonoids and triterpenes.

Discriminative Metabolomics Analysis and Cytotoxic Evaluation of Flowers, Leaves, and Roots Extracts of Matthiola longipetala subsp. livida.

Matthiola longipetala subsp. livida is an annual herb in Brassicaceae that has received little attention despite the family's high reputation for health benefits, particularly cancer prevention. In this study, UPLC-HRMS-MS analysis was used for mapping the chemical constituents of different plant parts (i.e., flowers, leaves, and roots). Also, spectral similarity networks via the Global Natural Products Social Molecular Networking (GNPS) were employed to visualize their chemical differences and similarities. Additionally, the cytotoxic activity on HCT-116, HeLa, and HepG2 cell lines was evaluated. Throughout the current analysis, 154 compounds were annotated, with the prevalence of phenolic acids, glucosinolates, flavonol glucosides, lipids, peptides, and others. Predictably, secondary metabolites (phenolic acids, flavonoids, and glucosinolates) were predominant in flowers and leaves, while the roots were characterized by primary metabolites (peptides and fatty acids). Four diacetyl derivatives tentatively assigned as O-acetyl O-malonyl glucoside of quercetin (103), kaempferol (108 and 112), and isorhamnetin (114) were detected for the first time in nature. The flowers and leaves extracts showed significant inhibition of HeLa cell line propagation with LC50 values of 18.1 ± 0.42 and 29.6 ± 0.35 µg/mL, respectively, whereas the flowers extract inhibited HCT-116 with LC50 24.8 ± 0.45 µg/mL, compared to those of Doxorubicin (26.1 ± 0.27 and 37.6 ± 0.21 µg/mL), respectively. In conclusion, the flowers of M. longipetala are responsible for the abundance of bioactive compounds with cytotoxic properties.

Phytochemical investigation of Pistacia lentiscus L. var. Chia leaves: A byproduct with antimicrobial potential.

Pistacia lentiscus L. var. Chia belongs to the Anacardiaceae family, and it is cultivated only in the south part of Chios island, in Greece. Even though it is renowned for its unique resin, Chios mastic gum (CMG), the tree leaves have also been used in traditional medicine, while the annual pruning generates a large biomass of unused by-products. Thus, the aim of the present study was the detailed phytochemical investigation of P. lentiscus var. Chia leaves towards the search of antimicrobial agents. UPLC-HRMS & HRMS/MS based dereplication methods led to the detailed characterization of the aqueous leaf extract. In addition, twelve compounds were isolated and purified from the methanol extract and were identified using spectroscopic and spectrometric methods (NMR, HRMS) belonging to phenolic acids, tannins, flavonoids and terpenes, with the most interesting being 2-hydroxy-1,8-cineole ß-D-glucopyranoside which was isolated for the first time in the Anacardiaceae family. Remarkably, based on NMR data, methanol and aqueous extracts were found to be particularly rich in shikimic acid, a valuable building block for the pharmaceutical industry, for instance in the synthesis of the active ingredient of Tamiflu®, oseltamivir. Finally, extracts (EtOAc, MeOH, H2O) and major compounds i.e., shikimic acid, 2-hydroxy-1,8-cineole ß-D-glucopyranoside and myricitrin were evaluated for their antimicrobial properties. MeOH and H2O mastic leaf extracts as well as myricitrin and, particularly, 2-hydroxy-1,8-cineole ß-D-glucopyranoside showed significant selective activity against pathogenic Mucorales, but not against Aspergilli (Aspergillus nidulans, Aspergillus fumigatus), Candida albicans or bacteria (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis).