Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors.

Increasing attention is being focused on the use of polypeptide-based N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of nervous system disorders. In our study on Achyranthes bidentata Blume, we identified an NMDA receptor subtype 2B (NR2B) antagonist that exerts distinct neuroprotective actions. This antagonist is a 33 amino acid peptide, named bidentatide, which contains three disulfide bridges that form a cysteine knot motif. We determined the neuroactive potential of bidentatide by evaluating its in vitro effects against NMDA-mediated excitotoxicity. The results showed that pretreating primary cultured hippocampal neurons with bidentatide prevented NMDA-induced cell death and apoptosis via multiple mechanisms that involved intracellular Ca2+ inhibition, NMDA current inhibition, and apoptosis-related protein expression regulation. These mechanisms were all dependent on bidentatide-induced inhibitory regulation of NR2B-containing NMDA receptors; thus, bidentatide may contribute to the development of neuroprotective agents that would likely possess the high selectivity and safety profiles inherent in peptide drugs.

Lactobacillus plantarum TWK10-fermented soymilk improves cognitive function in type 2 diabetic rats.

BACKGROUND: The brain is especially sensitive to diabetes-induced damage. Chronic hyperglycemia can potentially lead to brain dysfunctions, affecting spatial learning and memory. RESULTS: The type 2 diabetes (T2D) rats were administered TWK10-fermented soy milk water extract (WE) and ethanol extract (EE) for 6 weeks. WE and EE treatment attenuated T2D-induced alteration in cognitive function assessed using the Morris water maze. Moreover, administration of WE and EE significantly elevated superoxide dismutase activity (166.96% and 181.21%, P < 0.05, respectively) and reduced malondialdehyde concentration (35.03% and 43.97%, P < 0.05, respectively) in the hippocampus of the rats. Additionally, the calmodulin level and nitric oxide concentration were regulated by WE and EE. CONCLUSION: This study provides scientific evidence that WE and EE enhance anti-oxidative enzyme activity, which subsequently regulates factors associated with cognitive function in T2D rats. © 2020 Society of Chemical Industry.

Prevention of hypertension-induced vascular dementia by Lactobacillus paracasei subsp. paracasei NTU 101-fermented products.

CONTEXT: Numerous etiological studies have established positive clinical association between hypertension and vascular dementia (VaD). Lactobacillus paracasei subsp. paracasei NTU 101-fermented products have been shown to decrease vascular risk factors such as hypertension, atherosclerosis, hyperlipidemia and obesity. OBJECTIVE: This study investigated the effect of ethanol extract of Lactobacillus paracasei subsp. paracasei NTU 101-fermented products (NTU101F) in hypertension-induced VaD in rats. MATERIALS AND METHODS: Hypertension was promoted by subcutaneous injection of deoxycorticosterone acetate (DOCA, 25 mg/kg body weight/day, twice a week) and substitution of drinking water with 1.0% NaCl and 0.2% KCl. The NTU101F groups (0.5, 1.0, and 5.0) administered NTU101F at the concentrations 11, 22, and 110 mg/kg body weight/day, respectively, starting from day 51 day of DOCA-salt treatment. Morris water maze (MWM) was used for testing learning and memory. Different biochemical estimations were used to assess oxidative stress and inflammatory response in hippocampus. RESULTS: Oral administration of NTU101F in DOCA-salt hypertension-induced VaD rats resulted in a significant decrease in blood pressure by 18.3-23.2% (p < 0.001), which was regulated by increasing eNOS density (about 3-fold) in the aorta, promoting NO production, and decreasing of matrix metallopeptidase 9 activity (about 2-fold) in the hippocampus, in addition to improve the kidney function and structure, decrease escape latency and increase the times spent in the target quadrant by 23.5-27.8% (p < 0.05). CONCLUSION: Overall, our findings suggest that NTU101F could exert neuroprotection in the brain and attenuate hypertension-induced VaD.

Acute nephrotoxicity of aristolochic acid in vitro: metabolomics study for intracellular metabolic time-course changes.

Time-course metabolic changes of aristolochic acid nephrotoxicity (AAN) was investigated using acute AAN HK-2 model. And the AAN-related biomarkers were selected. In the results, 11 potential identified biomarkers were selected and validated using multivariate method combined with time-course analysis. Several metabolic pathways, including vitamin metabolism, lipids acalytion, trytophan metabolism and protein degradation were found to be associated with AAN pathology. This research will provide a valuable reference for the discovery of more potential biomarkers of AAN progression in clinic.

Pharmacokinetic study of Gastrodia elata in rats.

The pharmacokinetics of parishin, gastrodin, Gastrodia elata extract and Rhizoma Gastrodiae capsule was investigated by intragastric and/or intravenous administration to rats. Parishin was metabolized into nine metabolites after intravenous administration, and the area under the curve (AUC0-∞) of parishin and its metabolites (except parishin G and parishin E) increased nonlinearly from 72.5 to 220 mg/kg. When combining regression equation with the AUC0-∞ and dose of gastrodin injection, the percent conversion of parishin to gastrodin was obtained as 50 %. Based on multi-active metabolites of parishin in vivo, integrated pharmacokinetic mode was established. It is notable that each metabolite from parishin shares the similar metabolic process at three dosages of parishin and the bioavailability of parishin was approximately 14 %. The integrated pharmacokinetic mode was successfully applied to evaluate the holistic pharmacokinetics of gastrodin injection, G. elata extract and Rhizoma Gastrodiae capsule. The results showed that the holistic pharmacokinetics of gastrodin injection and G. elata extract was closed to that of gastrodin, but for parishin and Rhizoma Gastrodiae capsule, integrated pharmacokinetic parameters were more suitable to evaluate its holistic pharmacokinetics. Graphical abstract Pharmacokinetic study of Gastrodia elata in rats.

Metabolomic Responses of Human Hepatocytes to Emodin, Aristolochic Acid, and Triptolide: Chemicals Purified from Traditional Chinese Medicines.

The present study was undertaken to investigate the metabolic responses of human liver cells HL-7702 on chemicals purified from traditional Chinese medicine: emodin, triptolide, and aristolochic acid. Cytotoxicity tests demonstrated a dose-dependent toxic effect of emodin, triptolide, and aristolochic acid on HL7702 cells for 48 h. Emodin (14 µM), aristolochic acid (12 µg/mL), or triptolide (18 nM) was individually administrated to HL7702 and cell samples were collected after 48 h for metabolites extraction and analysis. Pattern recognition analysis reflected the significant difference in metabolic profiles between chemical-treated groups and the control group. Finally, eight metabolites including N1 -acetylspermidine, Glu Gly, N-undecanoylglycine, C16 sphinganine, sphinganine, glutathione, l-palmitoylcarnitine, and elaidic carnitine were detected as potential common biomarkers. Three pathways including sphinganine metabolism, fatty acid oxidation, and oxidative stress were identified. Our findings indicated that metabolomics would be an efficient approach to understand the molecular mechanism of hepatotoxicity induced by chemicals.

A cell-based model of bone remodeling for identifying activity of icarrin in the treatment of osteoporosis.

The activity of icarrin (a flavonoid from Herba epimedii) was investigated in the regulation of bone remodeling, a process coupled by osteoblast-mediated bone forming and osteoclast-mediated bone resorption. By directly co-culturing mouse bone marrow stromal cells and mouse preosteoclastic RAW264.7, and transwell co-culturing rat ovarian follicular granulosa cells (FGC), a 30 % increase in alkaline phosphatase (ALP) activity and 25 % increase in estradiol level occurred. Compared with the antiresorptive drug, alendronate, and an anabolic drug, PTH1-34, icarrin possessed all of the positive effects on the co-culture by increasing ALP activity, estradiol production and decreasing tartrate-resistant acid phosphatase activity. A similar action of icarrin occurred on co-culture of mesenchymal stem cells, mouse peripheral blood mononuclear cells, and FGC. Overall, by using a co-cultured cell-based in vitro screening assay, icarrin is suggested as a new class of dual-action therapeutic agent for osteoporosis.

Analysis of the metabolic profile of parishin by ultra-performance liquid chromatography/quadrupole-time of flight mass spectrometry.

Parishin is a dominant active ingredient originating from Gastrodia elata Blume, and has good neuroprotective effects against brain disorders. In the present study, the metabolic profile of parishin by in vitro and in vivo experiments was investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC/Q-TOF MS) combined with an automated MS(E) technique. By comparison with reference compounds, accurate mass measurement, the characteristic fragmentation patterns of the parent drug parishin and gastrodin and relevant bio-transformation knowledge, 14 metabolites (seven hydrolyzates and seven derivatives of gastrodin) were detected and identified in rat plasma and urine after intragastric administration of parishin, including processes of hydrolyzation, oxidation, sulfation and glucuronidation. According to the proposed metabolic pathways of parishin, in vitro hydrolytic experiments and metabolic study of gastrodin in rat plasma, it can be inferred that parishin mainly functions as a prodrug and undergoes hydrolysis before being absorbed into the blood. The hydrolyzate, mainly gastrodin, was involved in further metabolism, which was responsible for pharmacological activities of parishin. In conclusion, this work provides valuable information on parishin metabolism using a rapid and reliable UHPLC/Q-TOF MS method, which could be widely used for the metabolic investigation of natural product.

[Antitumor components screening of Stellera chamaejasme L. under the case of discrete distribution of active data].

This is to report the screening, extracting and validating antitumor components and compounds from Stellera chamaejasme L. under the case of discrete distribution of active data. In this work, different components from Stellera chamaejasme L. were collected by HPD macroporous resin and polyamide resin column, and their antitumor activity on A549 were tested by MTT assay. Activity results indicate that activity of components at 30-39 min is more potent than that of Stellera chamaejasme L. extract, and the activity of components at 33.97 min is equivalent to positive drug, cis-platinum at 100 microg x mL(-1), but with totally different mode of action. Under the case of discrete activity, the weight analysis is capable of screening active components and compounds from natural products.

Structure elucidation of degradation products of Z-ligustilide by UPLC-QTOF-MS and NMR spectroscopy.

Z-Ligustilide, a major phthalide isolated from a widely used traditional Chinese medicine Ligusticum chuanxiong, possesses various pharmacological activities including neuroprotective, anti-inflammatory, antiproliferative and vasorelaxing effects. However, it is unstable and inclined to degrade in natural conditions, which limits its study and application greatly. In this study, degradation behavior of Z-ligustilide and its degradation products stored at room temperature under direct sunlight were investigated and structure elucidated by HPLC-UV, UPLC-QTOF-MS and NMR. Z-ligustilide degradation and total five degradation products were generated and detected. Two degradation products were unequivocally identified as senkyunolide I and senkyunolide H by comparison with reference compounds. Another two degradation products were further isolated by semi-preparative HPLC and structure elucidated as (E)-6, 7-trans-dihydroxyligustilide and (Z)-6, 7-epoxyligustilide by 1H and 13C NMR, respectively. The degradation pathways of Z-ligustilide were finally proposed. Oxidation, hydrolysis and isomerization are the major degradation reactions.

[Analysis of chemical constituents of chuanxiong rhizoma absorbed into rat brain tissues by UPLC-Q-TOF-MS].

OBJECTIVE: To analyze and identify the chemical constituents in rat brain tissues after oral administration of Chuanxiong Rhizoma extracts. METHOD: The dosed and blank rat brain tissues were analyzed by UPLC-Q-TOF-MS. Different peaks were observed in total ion chromatograms and then identified according to their retention time, accurate mass weight, MS and MS/MS data. RESULT: After oral administration of Chuanxiong Rhizoma extracts, 3 compounds were absorbed into rat brain tissues through BBB. They were identified as senkyunolide I, senkyunolide A and ligustilide. CONCLUSION: The study is helpful for interpreting effective substance of Ligusticum chuanxiong.

Discovery of a cysteine-rich peptide with glycation modification from Achyranthes bidentata Blume.

Cysteine-rich peptides (CRPs) are stable molecules that contain multiple disulphide bonds. Various CRPs are found in plants and animals, representing potential compounds for drug development with diverse activities. Modification of CRPs, such as glycation, has attracted increased attention due to its special structural and functional properties. Hence, this study explored a CRP isolated from the Chinese herb Achyranthes bidentata Blume, which contains a glycation modification. Herein, a reverse phase high-performance liquid chromatography system with mobile phases was used to extract and purify the peptide. The eluted peptide was detected using high resolution mass spectrometry and structurally identified using high resolution mass spectrometry and nuclear magnetic resonance. The effect of the peptide on the viability of N-methyl-D-aspartic acid (NMDA)-induced HT22 cells was determined using a cell assay. Here, a new cysteine-rich glycation peptide, termed glycation-bidentatide (Gly-BTP), with three pairs of disulphide bonds and a glycation modification at the N-terminus linked to cysteine, was discovered. Cell bioactivity assay results suggested that Gly-BTP might be a potential therapeutic and provide a neuroprotective effect in NMDA-induced HT22 murine hippocampal neuronal cells. The discovery of Gly-BTP will promote the understanding of the role of CRPs in neuroprotection.

Computational exploration of natural peptides targeting ACE2.

Interaction between the SARS-COV-2 (2019 novel coronavirus) spike protein and ACE2 receptors expressed on cellular surfaces initialises viral attachment and consequent infection. Blocking this interaction shows promise for blocking or ameliorating the virus' pathological effects on the body. By contrast to work focusing on the coronavirus, which has significant potential diversity through possible accumulation of mutations during transmission, targeting the conserved ACE2 protein expressed on human cells offers an attractive alternative route to developing pharmacological prophylactics against viral invasion. In this study, we screened a virtual database of natural peptides in silico, with ACE2 as the target, and performed structural analyses of the interface region in the SARS-COV-2 RBD/ACE2 complex. These analyses have identified 15 potentially effective compounds. Analyses of ACE2/polypeptide interactions suggest that these peptides can block viral invasion of cells by stably binding in the ACE2 active site pocket. Molecular simulation results for Complestatin and Valinomycin indicate that they may share this mechanism. The discovery of this probable binding mechanism provides a frame of reference for further optimization, and design of high affinity ACE2 inhibitors that could serve as leads for production of drugs with preventive and therapeutic effects against SARS-COV-2. Communicated by Ramaswamy H. Sarma.

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS.

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.

A new diterpenoid from Rhizophora apiculata.

A new diterpenoid, 15(S)-isopimar-7-en-1-oxo-15,16-diol (1), was isolated from the stems of mangrove plant Rhizophora apiculata. The structure of the new compound was elucidated by MS, IR, 1D, and 2D NMR techniques, including HMQC, HMBC, and NOESY correlations. In addition, seven known constituents were isolated from this plant for the first time.

Plasma nontargeted peptidomics discovers potential biomarkers for major depressive disorder.

PURPOSE: There are great demands for identifying biomarkers of major depressive disorder (MDD), a common mental illness with a prevalence of approximately 6%. Finding potential biomarkers to aid MDD diagnosis is in high demand. EXPERIMENTAL DESIGN: In this study, a combination of pretreatment methods named salt-out assisted liquid-liquid extraction (SALLE) and nontargeted peptidomics based on nano-LC-Orbitrap/MS was primarily employed to discover the candidate peptide markers from the plasma of 238 subjects. RESULTS: Many peptides were enriched and identified from the plasma, 42 of which showed significant differences between MDD patients and controls by univariate statistical analysis. A binary logistic regression (BLR) model combined four peptide markers (P1, P9, P17, P29) was established, yielding an overall prediction accuracy of 91.7% and 82.2% in the discovery and validation sets, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, the excellent performance of the BLR model in both discovery and validation sets demonstrates the robustness of the four peptide markers panel. It is very valuable for quantification of the absolute content of four peptides and further verification.

Peptides as Potential Biomarkers for Authentication of Mountain-Cultivated Ginseng and Cultivated Ginseng of Different Ages Using UPLC-HRMS.

The growth conditions and age of Panax ginseng are vital for determining the quality of the ginseng plant. However, the considerable difference in price according to the cultivation method and period of P. ginseng leads to its adulteration in the trade market. We herein focused on ginseng peptides and the possibility of these peptides to be used as biomarker(s) for discrimination of P. ginseng. We applied an ultraperformance liquid chromatography-high resolution mass spectrometry-based peptidomics approach to characterize ginseng peptides and discover novel peptide biomarkers for authentication of mountain-cultivated ginseng (MCG). We identified 52 high-confidence peptides and screened 20 characteristic peptides differentially expressed between MCG and cultivated ginseng (CG). Intriguingly, 6 differential peptides were expressed significantly in MCG and originated from dehydrins that accumulated during cold or drought conditions. In addition, 14 other differential peptides that were significantly expressed in CG derived from ginseng major protein, an essential protein for nitrogen storage. These biological associations confirmed the reliability and credibility of the differential peptides. Additionally, we determined several robust peptide biomarkers for discrimination of MCG through a precise selection process. These findings demonstrate the potential of peptide biomarkers for identification and quality control of P. ginseng in addition to ginsenoside analysis.

[Differential analysis of peptides in Panax ginseng C. A. Meyer root by ultra-performance liquid chromatography-high resolution mass spectrometry].

An ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based peptidomics approach was developed for evaluating the similarities and differences between peptides in different morphological regions of Panax ginseng C. A. Meyer root. A total of 62 high-confidence peptides were characterized through database search, and four parts of ginseng root were all rich in peptides. The results of the differential analysis showed that the peptides were differentially expressed between the main root (MR), branch root (BR), rhizome (RH), and fibrous root (FR). Besides, a total of 25 potential peptide markers enabling the differentiation were discovered. In particular, the difference between the MR and the other parts was the most significant, and it led to a new viewpoint for the study of the difference in pharmacodynamics between the MR and the non-MR. These findings illustrate the structural diversity of ginseng peptides and reveal the similarities and differences between the peptides in ginseng root, thus providing chemical evidence for the quality control and rational application of ginseng.

Various Multicharged Anions of Ginsenosides in Negative Electrospray Ionization with QTOF High-Resolution Mass Spectrometry.

When characterizing components from ginseng, we found a vast number of multicharged anions presented in the liquid chromatography-mass spectrometry (LC-MS) chromatograms. The source of these anions is unclear yet, while ginsenosides, the major components of ginseng, are the main suspected type of molecules because of their sugar moiety. Our investigation using 14 pure ginsenosides affirmed that the multicharged anions were formed by ginsenosides rather than other types of ingredients in ginseng. Various anions could be observed for each ginsenoside. These anions contain ions ([M-2H]2-, [M+Adduct]2-), as well as those formed by polymerization of at least two ginsenosides, such as [nM-2H]2-, [nM-H+Adduct]2-, and [nM-3H]3-. The presence of so different types of ions from a ginsenoside explains the reason for the large number of anions in the LC-MS analysis of ginseng. We further found that formation of [nM-2H]2- ions was influenced by the number of sugar chains: ginsenosides containing two sugar chains produced all [nM-2H]2- ion types, whereas ginsenosides containing one sugar chain did not produce [2M-2H]2-. Thus, [2M-2H]2- and [3M-2H]2- can be utilized to rapidly identify monodesmosidic and/or bidesmosidic ginsenosides as joint diagnostic anions. The position of the glycosyl radical might be the key factor affecting the formation of multicharged multimer ions from monodesmosidic ginsenosides. Consequently, three groups of ginsenoside isomers were differentiated by characteristic [nM-2H]2- anions. Using concentration-dependent characteristics and collision-induced dissociation (CID), we confirmed that [nM-2H]2- ions are non-covalently bound multimers whose aggregation has marked distinction between monodesmosidic and bidesmosidic ginsenosides, accounting for the differentiated formation of [nM-2H]2- between them. Graphical Abstract.

Effect of ethanol extract from Lactobacillus plantarum TWK10-fermented soymilk on wound healing in streptozotocin-induced diabetic rat.

Wound healing is a highly dynamic phenomenon comprising numerous coordinated steps including homeostasis/coagulation, inflammation, migration, proliferation, and remodeling. Diabetes mellitus (DM) is a multisystem chronic epidemic that prolongs inflammation in wounds and is associated with impaired healing. This study aimed to investigate the effect of an ethanol extract from Lactobacillus plantarum TWK10 (TWK10)-fermented soymilk on wound healing. The anti-inflammatory effects of the ethanol extract of TWK10-fermented soymilk on lipopolysaccharide-stimulated RAW264.7 macrophage cells were examined. The ethanol extract of TWK10-fermented soymilk (100 µg/mL) significantly decreased nitric oxide production from 11.34 ± 0.74 µM to 8.24 ± 2.02 µM (p < 0.05) and enhanced proliferation in Detroit 551 cells cultured in high-glucose medium; the cell number peaked at 128.44 ± 7.67% (compared to the untreated control) at 600 µg/mL. An ethanol extract of TWK10-fermented soymilk + vaseline-treated rat model of streptozotocin-induced diabetic wounds was generated herein, and the following groups were formed herein: normal control (NC), blank control (BC), low dose group (LD, 0.24 mg/wound), intermediate dose (MD, 0.48 mg/wound), and high dose (HD, 2.40 mg/wound). On day 14 after wound infliction, the wound area in the LD, MD, and HD groups was significantly decreased to 10.2, 8.4, and 8.5% respectively (p < 0.05). Moreover, in the LD, MD, and, HD groups, tumor necrosis factor-α, interleukin 6, and matrix metalloproteinase-9 were downregulated in the wounded skin. These results show that the topical application of the ethanol extract of TWK10-fermented soymilk is beneficial for enhancing wound healing and for the closure of diabetic wounds.