UHPLC-MS/MS analysis and protective effects on neurodegenerative diseases of phenolic compounds in different parts of Diospyros kaki L. cv. Mopan.

Persimmon (Diospyros kaki L. cv. Mopan.), an important commercial crop belonging to the genus of Diospyros in the Ebenaceae family, is rich in bioactive phenolic compounds. In this study, the phenolic compounds from fruits, leaves, and calyces of persimmon were qualitatively and quantitatively determined by UPLC-Q-Exactive-Orbitrap/MS and UPLC-QqQ-MS/MS, respectively. Furthermore, the role of phenolic extract from different parts of persimmon on neuroprotective activity in vitro, through against oxidative stress and anti-neuroinflammation effect was firstly evaluated. The results showed that 75 phenolic compounds, and 3 other kinds of compounds were identified, among which 44 of phenolic compounds were quantified from different parts of persimmon. It is the first time that epicatechin-epigallocatechin, catechin-epigallocatechin, catechin-epigallocatechin (A-type), and glycoside derivatives of laricitrin were identified in persimmon extract. The dominated phenolic compounds in three parts of persimmon were significantly different. All phenolic extracts from each part of persimmon showed strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells and LPS-induced BV2 cells. The fruit extract presented the strongest activity, followed by calyx and leaf extract. The systematic knowledge on the phytochemical composition along with activity evaluation of different parts of persimmon could contribute to their targeted selection and development.

Novel l-Cysteine Incomplete Degradation Method for Preparation of Procyanidin B2-3'-O-Gallate and Exploration of its in Vitro Anti-inflammatory Activity and in Vivo Tissue Distribution.

In this study, an effective method for preparation of bioactive galloylated procyanidin B2-3'-O-gallate (B2-3'-G) was first developed by incomplete depolymerization of grape seed polymeric procyanidins (PPCs) using l-cysteine (Cys) in the presence of citric acid. The structure-activity relationship of B2-3'-G was further evaluated in vitro through establishing lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. The results suggested that the better protective effects of B2-3'-G against inflammation were attributed to its polymerization degree and the introduction of the galloyl group, compared to its four corresponding structural units. In vivo experiments demonstrated that the B2-3'-G prototype was distributed in plasma, small intestine, liver, lung, and brain. Remarkably, B2-3'-G was able to penetrate the blood-brain barrier and appeared to play an important role in improving brain health. Furthermore, a total of 18 metabolites were identified in tissues. Potential metabolic pathways, including reduction, methylation, hydration, desaturation, glucuronide conjugation, and sulfation, were suggested.

A Novel Deep Learning Model for Medical Image Segmentation with Convolutional Neural Network and Transformer.

Accurate segmentation of medical images is essential for clinical decision-making, and deep learning techniques have shown remarkable results in this area. However, existing segmentation models that combine transformer and convolutional neural networks often use skip connections in U-shaped networks, which may limit their ability to capture contextual information in medical images. To address this limitation, we propose a coordinated mobile and residual transformer UNet (MRC-TransUNet) that combines the strengths of transformer and UNet architectures. Our approach uses a lightweight MR-ViT to address the semantic gap and a reciprocal attention module to compensate for the potential loss of details. To better explore long-range contextual information, we use skip connections only in the first layer and add MR-ViT and RPA modules in the subsequent downsampling layers. In our study, we evaluated the effectiveness of our proposed method on three different medical image segmentation datasets, namely, breast, brain, and lung. Our proposed method outperformed state-of-the-art methods in terms of various evaluation metrics, including the Dice coefficient and Hausdorff distance. These results demonstrate that our proposed method can significantly improve the accuracy of medical image segmentation and has the potential for clinical applications. Illustration of the proposed MRC-TransUNet. For the input medical images, we first subject them to an intrinsic downsampling operation and then replace the original jump connection structure using MR-ViT. The output feature representations at different scales are fused by the RPA module. Finally, an upsampling operation is performed to fuse the features to restore them to the same resolution as the input image.

Wrinkling and Strengthening Behaviors in the Two-Layer-Sheet Hot-Forming-Quenching Integrated Process for an Al-Cu-Mg-Alloy Thin-Walled Curved-Surface Shell.

The thin-walled curved-surface component is an important structural element in aerospace. Wrinkling, springback and thermal distortion occur easily when forming these components. To form thin-walled components with high precision and strength, a two-layer-sheet hot-forming-quenching integrated process was proposed, in which wrinkling is prevented by thickening the upper sheet and springback is reduced by solution and die quenching. Selecting an appropriate upper sheet is crucial to suppress wrinkling and accomplish effective die quenching. The effect of the upper sheet on the wrinkling and strengthening behaviors of an Al-Cu-Mg-alloy melon-petal shell was thus studied in detail. The anti-wrinkle mechanism was analyzed through numerical simulation. The forming quality, including forming precision, deformation uniformity and strength, were further evaluated. The wrinkle gradually decreased with the increasing thickness of the upper sheet, resulting from the depressed compressive stress at the edge of the target sheet. A defect-free specimen with a smooth surface was finally formed when the thickness of the upper sheet reached three times that of the target sheet. The profile deviation was ±0.5 mm. Excellent thickness uniformity in a specimen can be obtained with a maximum thinning rate of 6%. The full strength, ranging from 455 to 466 MPa, can be obtained in all regions of the specimen, indicating that effective strengthening can be accomplished with the two-layer-sheet die quenching. The results indicated that high forming quality and full strength can be obtained in a two-layer-sheet hot-forming-quenching integrated process. This research has great potential for engineering applications using aluminum-alloy curved-surface thin-walled components.

Effects of Different Brewing Technologies on Polyphenols and Aroma Components of Black Chokeberry Wine.

The black chokeberry is a shrub of the Rosaceae family, which is characterized by strong acidity and astringency and is widely processed into wine and alcoholic beverages. However, due to the characteristics of black chokeberries, the wine brewed by traditional methods often has a strong sour taste, weak aroma, and poor sensory quality. In order to improve the sensory quality and explore the effects of different brewing technologies on polyphenols of black chokeberry wine, five brewing technologies (traditional fermentation, frozen fruit fermentation, co-fermentation, carbonic maceration, and co-carbonic maceration) were used in this study. The results showed that compared with the traditional method, the four alternative brewing technologies could reduce acidity, increase the contents of several major polyphenols, and enrich floral scents and fruity aroma, thus significantly improving the sensory qualities of black chokeberry wine. The proposed brewing technologies would be applied to the production of quality black chokeberry or other fruit wines.

Leaf Removal at Véraison and Foliar K+ Application to Beibinghong Vines Improved Berry Quality under Cold-Climate Conditions.

(1) Background: Beibinghong is a grapevine variety that is well distributed in Northeastern China due to its adaptation to extreme cold conditions and vine diseases. Nonetheless, Beibinghong wines are extremely acidic and rich in phenolic compounds. The aim of this research was to study the effects of leaf removal at véraison and foliar K+ applications on Beibinghong vines to reduce the acidity and increase their polyphenol content. (2) Methods: Beibinghong berries were harvested when they reached close to 20 °Brix, and the physicochemical parameters were determined. (3) Results: Leaf removal at véraison plus K+ foliar applications to Beibinghong vines decreased the titratable acidity and increased the total phenolic and phenolic acid contents compared with the control. Moreover, the titratable acidity in the Beibinghong berries was negatively related to their total contents of phenols, proanthocyanidins, and anthocyanins. (4) Conclusions: Leaf removal at véraison performed with foliar K+ applications to vines could be an interesting alternative for Beibinghong production under cold-climate viticulture because it allows for a decrease in the acidity and an increase in the phenolic content of the berries, without incurring the risk of sunburn.

Impact of Different Oak Chips' Aging on the Volatile Compounds and Sensory Characteristics of Vitis amurensis Wines.

In this work, different oak chips were used to age Vitis amurensis wine, and the effects on sensory properties were observed. Twenty-one different oak chips were added to a one-year-old wine made by a traditional technique. The wine was aged for 6 months before analysis by CIELab for color parameters, GC-MS for volatile compounds, and electronic tongue and a tasting panel for sensory properties. The results showed that the addition of any tested oak chip could significantly strengthen the wine's red color. Among 61 volatile compounds, alcohols presented the highest concentrations (873 to 1401 mg/L), followed by esters (568 to 1039 mg/L) and organic acids (157 to 435 mg/L), while aldehydes and volatile phenols occurred at low concentrations. Different oak species with different toasting levels could affect, to varying degrees, the concentrations of esters, alcohols, and volatile phenols, but to a lesser extent those of aldehydes. Sensory analysis by a tasting panel indicated that non- and moderately roasted oak chips gave the wines higher scores than those with heavy toasting levels. The major mouthfeel descriptors determined by electronic tongue were in good agreement with those from the tasting panel.

High-speed countercurrent chromatography as an efficient technique for large separation of plant polyphenols: A review.

Polyphenols, which are commonly found in fruits, vegetables, cereals, and legumes, are the most abundant dietary antioxidants. To date, numerous studies have demonstrated the ability of polyphenols to prevent the development of several diseases and established the corresponding structure-activity relationships. However, polyphenol standards are either not commercially available or very expensive, mainly because the structural complexity and diversity of polyphenols complicate their fractionation and isolation from plant extracts by conventional separation techniques. High-speed countercurrent chromatography (HSCCC) is based on continuous liquid-liquid partitioning, which enables one to eliminate irreversible adsorption on solid supports. This technique has been extensively used for natural product isolation and is well suited to the effective large-scale separation of polyphenols and their derivatives, achieving high purities and yields of up to several hundred milligrams per run within several hours. The present review briefly introduces briefly HSCCC technology and summarizes its applications in the separation and purification of plant polyphenols, including the recent achievements in the large-scale preparation of proanthocyanidins and anthocyanins by our laboratory.

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves.

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Corrigendum to "Macrophage membrane-coated nanocarriers Co-Modified by RVG29 and TPP improve brain neuronal mitochondria-targeting and therapeutic efficacy in Alzheimer's disease mice" [Bioactive materials 6 (2021) 529-542].

[This corrects the article DOI: 10.1016/j.bioactmat.2020.08.017.].

Panaxadiol as a major metabolite of AD-1 can significantly inhibit the proliferation and migration of breast cancer cells: In vitro and in vivo study.

Previous studies have shown that 20 (R)-25-methoxyl-dammarane-3ß, 12ß, 20 triol (AD-1) can inhibit various cancer cell lines. This study aimed to explore the effect and mechanism of AD-1 metabolite M2 (Panaxadiol; PD) on breast cancer cells of nude mice. Five AD-1 metabolites were isolated and identified using various chromatographic techniques. PD was the main component. In vitro results showed that PD could inhibit the proliferation and migration of MDA-MB-231 cells by inducing G1-phase arrest. In addition, PD down-regulated the expression of Cyclin D1, cdk2, cdk4, cdk6, P-p38, and MMP9, and up-regulated p21 and p27. In vivo results showed that PD could effectively reduce the volume, weight, and migration of breast cancer Transcriptomics analyzed 491 differentially expressed genes by GO and KEGG enrichment. RT-PCR verification confirmed that the significant down-regulation of MMP9 was consistent with transcriptomics results. In further research showed that PD regulated the protein expression of P-p38 and MMP9 in MAPK pathway. In summary, in vivo and in vitro studies showed that PD significantly inhibit the occurrence and development of breast cancer, possibly through the MAPK pathway.

Qualitative and quantitative analysis of phenolic compounds in blueberries and protective effects on hydrogen peroxide-induced cell injury.

This work was conducted to optimize an accelerated solvent extraction for ultra high performance liquid chromatography-mass spectrometry/mass spectrometry analysis of blueberry phenolic compounds. The conditions for accelerated solvent extraction were verified using response surface methodology to obtain the following optimized conditions: ethanol concentration (pH = 3), 48%; temperature, 50℃, and static cycle times, 3. Further, ultra high performance liquid chromatography with quadrupole Exactive Orbitrap mass spectrometry and ultra high performance liquid chromatography with triple-quadrupole tandem mass methods for determination of the detailed phenolic composition were developed and validated. Total of 81 phenolic compounds were identified by ultra high performance liquid chromatography with quadrupole Exactive Orbitrap mass spectrometry including 23 anthocyanins, 32 flavonols, 11 proanthocyanidins, 2 other flavonoids, and 13 phenolic acids. Fifty-one of these compounds have been simultaneously quantified by ultra high performance liquid chromatography with triple-quadrupole tandem mass including 31 anthocyanins, 8 flavonols, 6 proanthocyanidins, 2 other flavonoids, and 8 phenolic acids. Malvidin-dinhexoside has, for the first time, been detected in wild. Moreover, by verifying the protection on PC12 cells against oxidative damage, it was showed that the phenolic extracts (500 µg/mL) can improve significantly the viability (9.26-24.78%) of hydrogen peroxide-induced PC12 cells, activities of superoxide dismutase (34.59-37.90 U/mg) and glutathione peroxidase (6.87-14.42 mU/mg) and decrease the content of malonic dialdehyde (13.27-24.62 nmol/mg). Correlation analysis suggested that anthocyanins might contribute most to these activities.

The effects of linalool acupoint application therapy on sleep regulation.

The Shenque acupoint is located in the umbilicus of the human body. In the human body meridians, the Shenque acupoint can regulate body functions. The Shenque acupoint was one of the important acupuncture acupoints for the treatment of insomnia. However, the effect of linalool applied at the Shenque acupoint to improve sleep was unknown. This study explored the hypnotic and sedative effects of the main component of lavender, linalool, on the Shenque acupoint of mice and rats. The effects on the sleep latency and sleep duration were studied with the supra-threshold dose of pentobarbital sodium, and the effects on the sleep rate were studied with the sub-threshold dose of pentobarbital sodium. In order to further study the feasibility and superiority of linalool administered at the Shenque acupoint, a pharmacokinetic study was carried out. The pharmacodynamic results showed that the mice and rats treated with linalool at Shenque had the highest sleep rate, the shortest sleep latency, and the longest sleep duration compared with other groups. The T max and t 1/2 of the LS were longer than those of the LO, and had the characteristics of sustained release. The relative bioavailability of LS was 323.0 ± 31.66%. This showed that the combination of linalool and the Shenque acupoint had greater medicinal effects. This development will provide a new direction for improving sleep.

Macrophage membrane-coated nanocarriers Co-Modified by RVG29 and TPP improve brain neuronal mitochondria-targeting and therapeutic efficacy in Alzheimer's disease mice.

Neuronal mitochondrial dysfunction caused by excessive reactive oxygen species (ROS) is an early event of sporadic Alzheimer's disease (AD), and considered to be a key pathologic factor in the progression of AD. The targeted delivery of the antioxidants to mitochondria of injured neurons in brain is a promising therapeutic strategy for AD. A safe and effective drug delivery system (DDS) which is able to cross the blood-brain barrier (BBB) and target neuronal mitochondria is necessary. Recently, bioactive materials-based DDS has been widely investigated for the treatment of AD. Herein, we developed macrophage (MA) membrane-coated solid lipid nanoparticles (SLNs) by attaching rabies virus glycoprotein (RVG29) and triphenylphosphine cation (TPP) molecules to the surface of MA membrane (RVG/TPP-MASLNs) for functional antioxidant delivery to neuronal mitochondria. According to the results, MA membranes camouflaged the SLNs from being eliminated by RES-rich organs by inheriting the immunological characteristics of macrophages. The unique properties of the DDS after decoration with RVG29 on the surface was demonstrated by the ability to cross the BBB and the selective targeting to neurons. After entering the neurons in CNS, TPP further lead the DDS to mitochondria driven by electric charge. The Genistein (GS)- encapsulated DDS (RVG/TPP-MASLNs-GS) exhibited the most favorable effects on reliveing AD symptoms in vitro and in vivo by the synergies gained from the combination of MA membranes, RVG29 and TPP. These results demonstrated a promising therapeutic candidate for delaying the progression of AD via neuronal mitochondria-targeted delivery by the designed biomimetic nanosystems.

Ion channels and transporters in microglial function in physiology and brain diseases.

Microglial cells interact with all components of the central nervous system (CNS) and are increasingly recognized to play essential roles during brain development, homeostasis and disease pathologies. Functions of microglia include maintaining tissue integrity, clearing cellular debris and dead neurons through the process of phagocytosis, and providing tissue repair by releasing anti-inflammatory cytokines and neurotrophic factors. Changes of microglial ionic homeostasis (Na+, Ca2+, K+, H+, Cl-) are important for microglial activation, including proliferation, migration, cytokine release and reactive oxygen species production, etc. These are mediated by ion channels and ion transporters in microglial cells. Here, we review the current knowledge about the role of major microglial ion channels and transporters, including several types of Ca2+ channels (store-operated Ca2+ entry (SOCE) channels, transient receptor potential (TRP) channels and voltage-gated Ca2+ channels (VGCCs)) and Na+ channels (voltage-gated Na+ channels (Nav) and acid-sensing ion channels (ASICs)), K+ channels (inward rectifier K+ channels (Kir), voltage-gated K+ channels (KV) and calcium-activated K+ channels (KCa)), proton channels (voltage-gated proton channel (Hv1)), and Cl- channels (volume (or swelling)-regulated Cl- channels (VRCCs) and chloride intracellular channels (CLICs)). In addition, ion transporter proteins such as Na+/Ca2+ exchanger (NCX), Na+-K+-Cl- cotransporter (NKCC1), and Na+/H+ exchanger (NHE1) are also involved in microglial function in physiology and brain diseases. We discussed microglial activation and neuroinflammation in relation to the ion channel/transporter stimulation under brain disease conditions and therapeutic aspects of targeting microglial ion channels/transporters for neurodegenerative disease, ischemic stroke, traumatic brain injury and neuropathic pain.

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66.

Introduction: Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is important in regulating intracellular K+ and Cl- homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K+ influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb+ (K+) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb+ influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb+ influx assay showed that 10 µM BMT markedly decreased the total Rb+ influx and no additional inhibition detected at >10 µM BMT. In contrast, the maximum effects of STS66 on Rb+ influx inhibition were at 40-60 µM. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K+ influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

Neuronal mitochondria-targeted therapy for Alzheimer's disease by systemic delivery of resveratrol using dual-modified novel biomimetic nanosystems.

Reactive oxygen species (ROS)-induced neuronal mitochondrial dysfunction is a key pathologic factor in sporadic Alzheimer's disease (AD). Neuronal mitochondria have been proposed to be a promising therapeutic target for AD, especially for the failures of phase III clinical trials on conventional amyloid-ß (Aß) targeted therapy. However, the efficient intravenous delivery of therapeutic agents to neuronal mitochondria in the brain remains a major challenge due to the complicated physiological environment. Recently, biomaterials-based nanomedicine has been widely investigated for the treatment of AD. Herein, we devised a strategy for functional antioxidant delivery to neuronal mitochondria by loading antioxidants into red blood cell (RBC) membrane-coated nanostructured lipid carriers (NLC) bearing rabies virus glycoprotein (RVG29) and triphenylphosphine cation (TPP) molecules attached to the RBC membrane surface (RVG/TPP NPs@RBCm). With the advantage of suitable physicochemical properties of NLC and unique biological functions of the RBC membrane, RVG/TPP NPs@RBCm are stabilized and enabled sustained drug release, providing improved biocompatibility and long-term circulation. Under the synergistic effects of RVG29 and TPP, RVG/TPP NPs@RBCm can not only penetrate the blood-brain barrier (BBB) but also target neuron cells and further localize in the mitochondria. After encapsulating Resveratrol (RSV) as the model antioxidant, the data demonstrated that RVG/TPP-RSV NPs@RBCm can relieve AD symptoms by mitigating Aß-related mitochondrial oxidative stress both in vitro and in vivo. The memory impairment in APP/PS1 mice is significantly improved following the systemic administration of RVG/TPP-RSV NPs@RBCm. In conclusion, intravenous neuronal mitochondria-targeted dual-modified novel biomimetic nanosystems are a promising therapeutic candidate for ROS-induced mitochondrial dysfunction in AD.

Optimization of flash extraction, separation of ginsenosides, identification by HPLC-FT-ICR-MS and determination of rare ginsenosides in mountain cultivated ginseng.

In this paper, we used the flash extraction method (FEM) to extract ginsenosides from mountain cultivated ginseng (MCG), optimized the FEM process by response surface methodology (RSM), and separated 23 kinds of ginsenosides from MCG, including rare ginsenoside Rg3, 20(R/S)-Rg2, Rk3, 20(S)-Rh2, 20(R)-Rh1, F1 and Rg6. Among them, notoginsenoside R1 was isolated from MCG for the first time. Additionally, we established an HPLC-FT-ICR-MS method to accurately identify 20 ginsenosides in MCG, and quantitatively analyzed the differences in the content of rare ginsenosides in MCG and Garden-Cultivated Ginseng (CG) by HPLC-UV. The results showed that the chemical components of MCG and CG were similar, but the ginsenoside content of MCG was double that of CG. Notably, the content of ginsenoside 20 (S)-Rh2 and 20 (R)-Rh1 had the largest difference, and the content in MCG was 33 and 24 times higher than that in CG, respectively. Through quantitative analysis, we clarified the reason why the activity of MCG is stronger than that of CG, which provided a theoretical basis for clinical application and further research of MCG.

Analysis of endogenous epinephrine and norepinephrine enantiomers in rat plasma and application to a stereoselective pharmacokinetics.

Epinephrine and norepinephrine are a class of chiral endogenous catecholamines, which are known as major neurotransmitters. This work described a new LC-MS/MS method coupled with pre-column derivatization, enabling the simultaneous enantiomeric separation of epinephrine and norepinephrine in rat plasma. After protein precipitation procedure, the samples were derivatized with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester, [(S)-NIFE]. The derivatives resolved with good baseline separation on an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with mobile phase composed of methanol with 0.2% formic acid in water at a flow rate of 0.2 mL/min. Analysis was performed by multiple reaction monitoring in positive ionization mode. The linear ranges were 1.0-500 ng/mL for epinephrine enantiomers and 1.5-750 ng/mL for norepinephrine enantiomers. The lower limits of quantification for epinephrine and norepinephrine enantiomers were 1.0 and 1.5 ng/mL, respectively. The intra-day and inter-day precision were all less than 10.7% and accuracy ranged from 96.0 to 101.5%. Recoveries for all the analytes were more than 80.3%. The proposed method was successfully applied to simultaneously determine endogenous epinephrine and norepinephrine enantiomers in rat plasma. l-epinephrine and l-norepinephrine were sensitively and accurately quantified while both the d-enantiomers were not detected. Additionally, epinephrine enantiomers were analyzed for stereoselective pharmacokinetics in rats after intravenous administration of racemic epinephrine for the first time. The pharmacokinetic results indicated that the disposition of epinephrine enantiomers was stereoselective and chiral inversion did not occur in rats.