Integrated non-targeted metabolomics and network pharmacology to reveal the mechanisms of berberine in the long-term treatment of PTZ-induced epilepsy.

AIMS: The increasing resistance to anti-seizure medications (ASMs) and the ambiguous mechanisms of epilepsy highlight the pressing demand for the discovery of pioneering lead compounds. Berberine (BBR) has received significant attention in recent years within the field of chronic metabolic disorders. However, the reports on the treatment of epilepsy with BBR are not systematic and the mechanism remains unclear. MAIN METHODS: In this study, the seizure behaviors of mice were recorded following subcutaneous injection of pentetrazol (PTZ). Non-targeted metabolomics was used to analyze the serum metabolites based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Meanwhile, multivariate statistical methods were used for metabolite identification and pathway analysis. Furthermore, network pharmacology, molecular docking, and quantitative real-time PCR assay were used for the target identification. KEY FINDINGS: BBR had anti-seizure effects on PTZ-induced seizure mice after long-term treatment. Tryptophan metabolism and phenylalanine metabolism were involved in regulating the therapeutic effects of BBR. SIGNIFICANCE: This study reveals the potential mechanism of BBR for epilepsy treatment based on non-targeted metabolomics and network pharmacology, which provides evidence for uncovering the pathogenesis of epilepsy, suggesting that BBR is a potential lead compound for anti-epileptic treatment.

Proteomic Analysis of Rat Duodenum Reveals the Modulatory Effect of Boron Supplementation on Immune Activity.

The proper supplementation of boron, an essential trace element, can enhance animal immune function. We utilized the method of TMT peptide labeling in conjunction with LC-MS/MS quantitative proteomics for the purpose of examining the effects of boric acid on a rat model and analyzing proteins from the duodenum. In total, 5594 proteins were obtained from the 0, 10, and 320 mg/L boron treatment groups. Two hundred eighty-four proteins that exhibit differential expression were detected. Among the comparison, groups of 0 vs. 10 mg/L, 0 vs. 320 mg/L, and 10 vs. 320 mg/L of boron, 110, 32, and 179 proteins, respectively, demonstrated differential expression. The results revealed that these differential expression proteins (DEPs) mainly clustered into two profiles. GO annotations suggested that most of the DEPs played a role in the immune system process, in which 2'-5'-oligoadenylate synthetase-like, myxovirus resistance 1, myxovirus resistance 2, dynein cytoplasmic 1 intermediate chain 1, and coiled-coil domain containing 88B showed differential expression. The DEPs had demonstrated an augmentation in the signaling pathways, which primarily include phagosome, antigen processing, and presentation, as well as cell adhesion molecules (CAMs). Our study found that immune responses in the duodenum were enhanced by lower doses of boron and that this effect is likely mediated by changes in protein expression patterns in related signaling pathways. It offers an in-depth understanding of the underlying molecular mechanisms that lead to immune modulation in rats subjected to dietary boron treatment.

Effects of konjac glucomannan/pomegranate peel extract composite coating on the quality and nutritional properties of fresh-cut kiwifruit and green bell pepper.

The effects of an edible coating, based on konjac glucomannan (KG) incorporated with pomegranate peel extracts (PE), on the physicochemical and nutritional properties of fresh-cut kiwifruit and green bell pepper during storage were investigated. The optimal extract time (40.6 min), temperature (54.5 °C), and ultrasound power (255.5 W) with response surface method, provided a high total antioxidant activity (TAA) of (92.31 ± 1.43)%. Fresh-cut kiwifruit and green bell pepper were coated by dipping using five treatments (distilled water, ascorbic acid, KG, PE, KG + PE), packed into polymeric film and stored for 8 days at 10 °C. Distilled water treatment was used as control. KG + PE treatment resulted in the highest total soluble solid and titratable acidity in fresh-cut kiwifruit, while the maximum firmness in fresh-cut green bell pepper. The weight loss was both effectively decreased in samples treated with KG or KG + PE. All samples treated with KG + PE had significantly higher contents of chlorophyll, ascorbic acid, total phenolic and TAA than others. Moreover, the KG + PE group had the lowest counts of microorganisms in all samples. KG coating incorporated with PE was proved to be efficient in maintaining the physico-chemical and nutritional properties of fresh-cut kiwifruit and green bell pepper during low temperature storage compared with control. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05006-7.

Transcriptome Profiling of Duodenum Reveals the Importance of Boron Supplementation in Modulating Immune Activities in Rats.

As an essential trace element, appropriate boron supplementation can promote immune function of animals. To illustrate the effects of boron in a rat model, RNA-Seq was conducted for the RNA from duodenum after treatment with different concentration of boron in which boron was given in the form of boric acid. More than 47 million reads were obtained in 0, 10, and 320 mg/L boron (0, 57.21, and 1830.66 mg/L boric acid) treatment groups that produced 58 965 402, 48 607 328, and 46 760 660 clean reads, respectively. More than 95% of the clean reads were successfully matched to the rat reference genome and assembled to generate 32 662 transcripts. A total of 624 and 391 differentially expressed candidate genes (DEGs) were found between 0 vs.10 and 0 vs. 320 mg/L boron comparison groups. We also identified transcription start site, transcription terminal site, and skipped exons as the main alternative splicing events. GO annotations revealed most of DEGs were involved in the regulation of immune activity. The DEGs were enriched in influenza A, herpes simplex infection, cytosolic DNA-sensing pathway, and antigen processing and presentation signaling pathways. The expression levels of genes enriched in these signaling pathways indicate that lower doses of boron could achieve better effects on promoting immune response in the duodenum. These effects on the immune system appear to be mediated via altering the expression patterns of genes involved in the related signaling pathways in a dose-dependent pattern. These data provide more insights into the molecular mechanisms of immune regulation in rats in response to dietary boron treatment.

Neuroprotective effects of macamide from maca (Lepidium meyenii Walp.) on corticosterone-induced hippocampal impairments through its anti-inflammatory, neurotrophic, and synaptic protection properties.

The present study aims to investigate the protective effects of N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (M 18:3) on corticosterone-induced neurotoxicity. A neurotoxic model was established by subcutaneous injection of corticosterone (40 mg per kg bw) for 21 days. Depressive behaviors (the percentage of sucrose consumption, the immobility time in the forced swimming test, and the total distance in the open field test) were observed. The levels of the brain-derived neurotrophic factor, the contents of tumor necrosis factor-α and interleukin-6, and the numbers of positive cells of doublecortin and bromodeoxyuridine in the hippocampus were measured. The density of hippocampal neurons was calculated. The morphological changes of hippocampal neurons (the density of dendritic spines, the dendritic length, and the area and volume of dendritic cell bodies) were observed. The expression levels of synaptophysin, synapsin I, and postsynaptic density protein 95 were measured. Behavioral experiments showed that M 18:3 (5 and 25 mg per kg bw) could remarkably improve the depressive behaviors. The enzyme-linked immunosorbent assay showed that M 18:3 could considerably reduce hippocampal neuroinflammation and increase hippocampal neurotrophy. Nissl staining showed that M 18:3 could remarkably improve the corticosterone-induced decrease in the hippocampal neuron density. Immunofluorescence analysis showed that M 18:3 could considerably promote hippocampal neurogenesis. Golgi staining showed that M 18:3 could remarkably improve the corticosterone-induced changes in the hippocampal dendritic structure. Western blotting showed that M 18:3 could considerably increase the expression levels of synaptic-structure-related proteins in the hippocampus. In conclusion, the protective effects of M 18:3 may be attributed to the anti-inflammatory, neurotrophic, and synaptic protection properties.

Different absorption and metabolism of ginsenosides after the administration of total ginsenosides and decoction of Panax ginseng.

RATIONALE: Panax ginseng C.A. Meyer (PG), which contains polysaccharides and ginsenosides as the major bioactive components, has been used to promote health and treat diseases for thousands of years in China. Total ginsenosides were extracted from a decoction of Panax ginseng (GD), which included both ginsenosides and polysaccharides, and dissolved in water to obtain a total ginsenosides aqueous solution (TGAS). To study their absorption and metabolism, the pharmacokinetics (PK) and metabolites of ginsenosides in vivo were investigated after the administration of GD and TGAS. METHODS: Rat and mice plasma samples were collected after the administration of GD and TGAS. Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry was used with the UNIFI platform to identify metabolites in the plasma sample. The pharmacokinetic parameters were calculated using a noncompartmental method in the Drug and Statistics software package. RESULTS: Thirty ginsenoside metabolites were identified in mice plasma, of which only seven were found in the rat plasma after the administration of GD. The PK of ginsenosides Rb1 , Rc, and Rd were also determined after the oral administration of GD and TGAS and showed significant differences in the pharmacokinetic parameters. CONCLUSIONS: There was no difference in the biotransformation pathways after the oral administration of GD and TGAS, indicating that there was no influence of polysaccharides on the biotransformation of ginsenosides in vivo. However, the pharmacokinetic parameters were different after the administration of GD and TGAS, possibly because of the polysaccharides in GD. This study should be of significance in exploring the basis of PG bioactivities and lays the foundation for the further development of new drugs using PG.

Effects of Boron on Cytotoxicity, Apoptosis, and Cell Cycle of Cultured Rat Sertoli Cells In vitro.

The present study aimed to investigate the effects of the administration of boron on viability, apoptosis, and cell cycle of primary rat Sertoli cells (SCs) in vitro. SCs were aseptically isolated from 18-22-day-old male Sprague-Dawley (SD) rats. SCs were identified with immunofluorescence using anti-vimentin antibody. Further, to investigate the effects of boron on Sertoli cells, SCs of the boron treatment group were exposed to different concentrations (0.25, 0.5, 1, 5, 10, 40, and 80 mmol/L) of boric acid. Using MTT and Cell Counting Kit-8 assays, the impact of boron on SCs viability was analyzed. Cell apoptosis and cycle of SCs were analyzed using flow cytometry. A concentration of 0.5 mmol/L boric acid resulted in the highest viability and lowest necrosis and apoptosis. Above this concentration (even 1.0 mmol/L) showed lower viability and higher levels of necrosis and apoptosis. Administration of < 0.5 mmol/L boron significantly promoted the viability of Sertoli cells (P < 0.01); however, the exposure to high dose (> 10 mmol/L) of boron exhibited significant adverse effects on Sertoli cells (P < 0.01) and even toxic effects, inhibiting cell viability compared to the control group. Flow cytometry analysis showed that treatment with 0.5 mmol/L of boron significantly inhibited the apoptosis of Sertoli cells and the proportion of cells in S and G2/M phases was markedly increased; however, a higher concentration of 40 and 80 mmol/L of boron promoted Sertoli cell apoptosis and cells were completely arrested at G0/G1 phase. Boron at doses below 0.5 mmol/L could significantly improve the viable capacity of testicular Sertoli cells in vitro and inhibit their apoptosis. However, high dose of boron (at a concentration higher than 5.0 mmol/L) exhibited noticeable toxic effects, inhibiting cell viability, accelerating apoptosis of Sertoli cells, and arresting cell cycle at G0/G1 phase.

The ameliorative effect of the Pyracantha fortuneana (Maxim.) H. L. Li extract on intestinal barrier dysfunction through modulating glycolipid digestion and gut microbiota in high fat diet-fed rats.

Pyracantha fortuneana fruits are consumed as a dietary supplement in China and attenuate obesity and metabolic disorders. Obesity is known to be associated with intestinal barrier dysfunction driven by hyperglycemia and gut dysbiosis. However, whether the health benefits of P. fortuneana fruits are linked with the intestinal barrier function (IBF) remains unknown. This study aimed to evaluate the restorative effects of P. fortuneana fruit extract (PFE) on the IBF. Sprague Dawley rats were fed with a chow, a high-fat diet (HFD), or a PFE-supplemented diet for 8 weeks. Results showed that PFE intervention ameliorated HFD-induced intestinal barrier dysfunction by attenuating impaired structural integrity, reducing the elevated lactulose/mannitol ratio, and improving the mRNA and protein expression levels of tight junction proteins in HFD-fed rats. The ameliorations were associated with a beneficial effect on glycolipid homeostasis, as evidenced from the PFE decreasing intestinal absorptive capacity based on the d-xylose excretory rate, lowering the expression of GLUT2 and inhibiting digestive enzyme activities. The proanthocyanidins in the PFE showed greater in vitro inhibition on α-amylase, α-glucosidase, and lipase compared with triterpenoid saponins. Furthermore, the ameliorations on the IBF were also associated with effects on the microbial composition based on 16S rRNA gene sequence analysis. Several bacterial groups, which were linked with gut barrier integrity, were modulated after PFE administration, that is, Actinobacteria, Bacteroidaceae, Corynebacteriaceae, Lactobacillaceae, and S24-7 were elevated and the HFD-induced increase in Clostridia, Ruminococcaceae, Oscillospira, and Flexispira was restored. These data provide evidence for the ameliorative effect of the PFE on diet-induced intestinal barrier functional alternations in association with its capacity to modulate glycolipid digestion and gut microbiota in HFD-fed obese rats.

Fecal Metabolomics of Type 2 Diabetic Rats and Treatment with Gardenia jasminoides Ellis Based on Mass Spectrometry Technique.

Modern studies have indicated Gardenia jasminoides Ellis (G. jasminoides) showed positive effect in treating type 2 diabetes mellitus (T2DM). In this study, 60 streptozotocin-induced T2DM rats were divided into four groups: type 2 diabetes control group, geniposide-treated group, total iridoid glycosides-treated group, and crude extraction of gardenlae fructus-treated group. The other ten healthy rats were the healthy control group. During 12 weeks of treatment, rat's feces samples were collected for the metabolomics study based on mass spectrometry technique. On the basis of the fecal metabolomics method, 19 potential biomarkers were screened and their relative intensities in each group were compared. The results revealed G. jasminoides mainly regulated dysfunctions in phenylalanine metabolism, tryptophan metabolism, and secondary bile acid biosynthesis pathways induced by diabetes. The current study provides new insight for metabonomics methodology toward T2DM, and the results show that feces can preferably reflect the liver and intestines disorders.

Characterisation of water-soluble proanthocyanidins of Pyracantha fortuneana fruit and their improvement in cell bioavailable antioxidant activity of quercetin.

Proanthocyanidins (PCs) with poor bioavailability were argued for their health benefits. In this study, water-soluble polymeric polyphenolic PCs fractions from Pyracanthafortuneana fruit were used to investigate whether the presence of PCs is correlated with the increased cell antioxidant activities (CAA) of quercetin (Q). The results indicated that the most decrement in the values of EC50, which Q inhibited peroxyl radical-induced DCFH oxidation effective in the HepG2 cells, was observed to be 2.91 (vs. control 5.97) in the present of the fraction with 15.8 of the average degree of polymerisation of PCs (ADP). Also, the order of efficacy was the same with the ADP of PCs. Further, this effect is associated with the improvement of the solubility and stability of Q after the addition of the PCs. Our current study suggests that the additive effects of PCs on small molecular polyphenols may be responsible for their antioxidant benefits in vivo.

Two novel dammarane-type compounds from the leaves and stems of Panax quinquefolium L.

Two new dammarane-type compounds were isolated from the leaves and stems of Panax quinquefolium L. The new compounds were named as pseudo-ginsenoside RT6 (1) and pseudoginsengenin R1 (2). The structures of the new compounds were elucidated by the combined analysis of NMR and HR-ESI-MS as (20S,24R)-6-O-ß-d-glucopyranosyl-dammar-3-one-20,24-epoxy-6α,12ß,25-triol (1) and (20S,24R)-dammar-3-one-20,24-epoxy-6α,12ß,25-triol (2).

Screening and determination for potential α-glucosidase inhibitory constituents from Dalbergia odorifera T. Chen using ultrafiltration-LC/ESI-MS(n).

In the present study, it was demonstrated that ethyl acetate soluble fraction partitioned from heartwood of Dalbergia odorifera T. Chen (HEF) had a remarkable inhibitory effect on α-glucosidase. Therefore HEF was selected as a starting material for screening the potential α-glucosidase inhibitors using ultrafiltration liquid chromatography/mass spectrometry (UF-LC/MS). Twenty-six compounds were identified with analysis of LC/MS. UF assay indicated that 18 compositions might be α-glucosidase inhibitors in HEF; eight of them were estimated for their α-glucosidase inhibitory activity, and the results showed that (2S)-liquiritigenin, (2S)-4',6-dihydroxy- 7-methoxyflavanone and isoliquiritigenin displayed obvious inhibition of yeast α-glucosidase. In addition, in order to control the quality of HEF, the content of five compounds in HEF was simultaneously determined for the first time. These results provide an important theoretical base for the further application of HEF to treat type 2 diabetes in the clinic and development of natural α-glucosidase inhibitors with low toxicity.

[Study on excretion of pseudo-ginsenoside GQ].

OBJECTIVE: To determine the pseudo-ginsenoside GQ (PGQ) in rat bile, feces and urine, and to study on the excretion of pseudo-ginsenoside GQ. METHOD: Reverse phase high-performance liquid chromatography (RP-HPLC) method with an evaporative light-scattering detector (ELSD) was performed on Diamonsil C18 column (4.6 mm x 250 mm, 5 microm), and the mobile phase was consisted of methanol-water (24: 7) with flow rate of 1.0 mL x min(-1). ELSD parameters were set as follows: nitrogen gas pressure 3.0 bar, drift tube temperature 50 degrees C. RESULT: The method fulfilled all the standard requirements of precision, accuracy and linearity. The main way of excretion of PGQ in rat administrated through sublingual vein was at the bile. The bile excretion ratio of PGQ was 41.60%, and feces excretion ratio was 9.97%. Only trace amount of PGQ was excreted in urine. CONCLUSION: Almost all unchanged PGQ was excreted in bile, feces and urine.

[LC-ESI-MS metabolic profiling analysis of taxanes from the extracts of Taxus chinensis cell cultures].

AIM: To develop a rapid analytical method for small amount biological samples of taxanes by using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS) in small amount biological samples. METHODS: A solution containing five given taxane constituents and the extract from cell cultures of Taxus chinensis were analysed separately. According to the performance of the given taxanes in ESI-MS/MS, run parameters of the mass spectrometer were optimized. Positive and negative electrospray modes were employed to simultaneously scan the cell cultures sample, and the full ion chromatogram and the molecular weight of individual peak were obtained. The qualitative analysis of taxanes was achieved by comparison of the retention time and molecular weight with those of the reference substances or was based on the interpretation of the MS/MS spectra of the analytes and the knowledge of the concerning genetic backgrounds of taxanes published in literatures. RESULTS: The taxanes with several acetyl substituents tended to produce ammonium adduct ions peak, while multi-hydroxy taxanes were subject to give protonized molecular ion peaks in positive ion ESI-MS. Thirteen taxanes in cell samples were assigned. Eight compounds of them were identified to be 1 -acetyl-5, 7, 10-deacetyl-baccatin I (DAB-I, 1) , baccatin III (B-III, 3), baccatin VI (B-VI, 8), taxol (9), yunnanxane (10 ), taxuyunnanine C (Tc, 11), sinenxane B (12), sinenxane C (13), separately. For the other five constituents, character of taxane and the number of substituents were deduced. CONCLUSION: The results confirm the feasibility of characterizing taxanes in biological samples by LC-ESI-MS analysis. The analytical methodology provided a rapid, conventional and reliable tool to study metabolic profiling of taxanes for structural elucidation in taxol biosynthesis.