UPLC-Q-Orbitrap-MS based metabolomics and analysis of the effect of Suanzaoren Decoction on serum of chronic unpredictable mild stress depression rats / 药学学报

A UPLC-Q-Orbitrap-MS based metabolomic approach combined with biochemical assay and histopathological inspection were employed to study the intervention effects of Suanzaoren Decoction (SZRD) on chronic unpredictable mild stress (CUMS) depression rats, and to clarify the metabolic regulation pathway of SZRD. The rats were randomly divided into normal control group, CUMS model group, positive drug venlafaxine group, SZRD high (24 g·kg-1) and low (12 g·kg-1) dose groups, respectively. The CUMS model was replicated by subjecting to a variety of stimulus, such as thermal stimulation, ice water swimming, ultrasonic stimulation, tail clamping, day and night reversal, plantar electric shock and so on for rats. After oral administration of drugs for 28 days, the behavioral indexes of rats in each group were observed and the hippocampus and serum samples of rats were collected for biochemical assay and histopathological inspection. Compared with the CUMS model group, low dose and high dose SZRD groups can significantly reduce the immobility time of forced swimming (P < 0.001, P < 0.001), increase the sucrose preference rate (P < 0.01, P < 0.05), the number of crossings (P < 0.05, P < 0.01) and the number of uprights (P < 0.05, P < 0.01) in the open field test, suggesting that SZRD can significantly improve the depression-like behavior of CUMS model rats. In addition, SZRD could significantly reduce the levels of serum IL-6, IL-1β and TNF-α of CUMS model rats. A total of 21 differential metabolites in serum were identified by comparison with the data from the literature and databases. In addition, low-dose SZRD and high-dose SZRD improved the 8 and 11 perturbed potential serum biomarkers that were induced by CUMS, respectively, which related to alanine, aspartic acid and glutamic acid, tryptophan and arachidonic acid metabolism. This study provides a scientific basis for expanding the clinical indications of SZRD. This experiment was approved by the Animal Ethics Committee of Shanxi University (Approval No. SXULL2020028).

Network pharmacology strategy to investigate the pharmacological effects of Suanzaoren Decoction on insomnia

Abstract Suanzaoren Decoction (SZRD) is an ancient prescription used in the treatment of insomnia. This study aimed to investigate the components and targets of SZRD in treating insomnia. First, the compounds of five herbs in SZRD were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the putative targets for treating insomnia were obtained from DrugBank to construct the herb-compound-target- disease network. A protein-protein interaction (PPI) network was constructed in the STRING database, and then Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to predict the mechanism of action of intersection target. Finally, 30 mice were divided into five groups: control, model, and quercetin groups (100, 50, 25 mg/kg). The sleep latency and duration of pentobarbital-induced sleeping were measured. The production of interleukin-6 (IL-6) and γ-aminobutyric acid (γ-GABA) was detected by using an enzyme-linked immunosorbent assay kit (ELISA), and Gamma-aminobutyric acid type a receptor subunit alpha1 (GABRA1) was tested by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A total of 152 active ingredients, including 80 putative targets of SZRD, were obtained. The main active compounds included quercetin and kaempferol, and the key targets involved IL-6 and nitric oxide synthase 3 (NOS3). The results of pathway enrichment analysis indicated that the putative targets of SZRD mainly participated in Neuroactive ligand-receptor interaction. The experiment of P-chlorophenylalanine (PCPA)-induced insomnia model showed that quercetin obviously shortened the sleep latency and prolonged the sleep duration of the insomnia model. The production of IL-6, γ-GABA, and GABRA1 mRNA was significantly increased in mice treated with quercetin. This study predicted the active ingredients and potential targets of SZRD on insomnia on the basis of a systematic network pharmacology approach and illustrated that SZRD might exert hypnotic effects via regulating IL-6, γ-GABA, and GABRA1

Effect of Suanzaoren Decoction on molecular levels of bile acids in serum, liver, and ileum of insomnia mice / 中国中药杂志

To explore the mechanism of Suanzaoren Decoction in the treatment of insomnia from endogenous bile acid regulation, the present study investigated the hepatoprotective effect of Suanzaoren Decoction and the molecular changes of bile acids in the serum, liver, and ileum of insomnia model mice and Suanzaoren Decoction treated mice. The insomnia model in mice was established by the sleep deprivation method. After Suanzaoren Decoction(48.96 mg·kg~(-1)·d~(-1)) intervention by gavage for 7 days, the related indicators, such as water consumption, food intake, body weight, aspartate aminotransferase(AST), alanine transaminase(ALT), and total bile acid(TBA) were detected, and the pathological changes of the liver and ileum were observed. The molecular levels and distribution of 23 bile acids in the serum, liver, and ileum were analyzed by UPLC-MS/MS combined with principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). The results showed that Suanzaoren Decoction could improve the decreased water consumption and food intake, weight loss, and increased AST and ALT in the model group, and effectively reverse the injury and inflammation in the liver and ileum. The bile acids in the liver of the insomnia model mice were in the stage of decompensation, and the bile acids in the serum, liver, and ileum of the mice decreased or increased. Suanzaoren Decoction could regulate the anomaly of some bile acids back to normal. Seven bile acids including glycoursodeoxycholic acid(GUDCA), glycodesoxycholic acid(GDCA), tauro-α-MCA(T-α-MCA), α-MCA, taurodeoxycholate(TDCA), T-β-MCA, and LCA were screened out as the main discriminant components by PLS-DA. It is concluded that Suanzaoren Decoction possesses the hepatoprotective effect and bile acids could serve as the biochemical indicators to evaluate the drug efficacy in the treatment of abnormal liver functions caused by insomnia. The mechanism of Suanzao-ren Decoction in soothing the liver, resolving depression, tranquilizing the mind, and improving sleep may be related to the molecular regulation of bile acid signals.

Metabonomic study of biochemical changes in serum of PCPA-induced insomnia rats after treatment with Suanzaoren Decoction / 中国中药杂志

Suanzaoren Decoction(SZRD) is a classical formula for the clinical treatment of insomnia. This study analyzed the effect of SZRD on endogenous metabolites in insomnia rats based on metabonomics and thereby explored the anti-insomnia mechanism of SZRD. To be specific, DL-4-chlorophenylalanine(PCPA) was used to induce insomnia in rats. Then pathological changes of the liver and brain were observed and biochemical indexes such as 5-hydroxytryptamine(5-HT), dopamine(DA), glutamate(Glu), γ-aminobutyric acid(GABA), and norepinephrine(NE) in the hippocampus and prostaglandin D2(PGD2), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and IL-6 in the serum of rats were detected. On this basis, the effect of SZRD on PCPA-induced insomnia rats was preliminarily assessed. The metabolic profile of rat serum samples was further analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were combined with t-test and variable importance in projection(VIP) to identify differential metabolites, and MetaboAnalyst 5.0 was employed for pathway analysis. The results showed that SZRD could improve the pathological changes of brain and liver tissues, increase the levels of neurotransmitters 5-HT, DA, and GABA in hippocampus and the level of PGD2 in hypothalamic-pituitary-adrenal axis(HPA axis), and reduce the levels of IL-1β and TNF-α in serum of insomnia rats. Metabonomics analysis yielded 12 significantly changed potential metabolites: 5-aminovaleric acid, N-acetylvaline, L-proline, L-glutamate, L-valine, DL-norvaline, D(-)-arginine, pyroglutamic acid, 1-methylguanine, L-isoleucine, 7-ethoxy-4-methylcoumarin, and phthalic acid mono-2-ethylhexyl ester(MEHP), which were related with multiple biochemical processes including metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of arginine and proline, arginine biosynthesis, glutathione metabolism. These metabolic changes indicated that SZRD can improve the metabolism in insomnia rats by regulating amino acid metabolism.

Review of chemical constituents, pharmacological effects and clinical applications of Suanzaoren Decoction and prediction and analysis of its Q-markers / 中国中药杂志

Suanzaoren Decoction is a classic prescription for nourishing the heart and liver, nourishing blood and tranquilizing the mind. It has the functions of sedation and hypnosis, anti-anxiety, anti-depression, anti-convulsion and so on. Modern clinic is mostly used to treat different types of insomnia, depression, neurasthenia, tension headache and vertigo. In this paper, the chemical consti-tuents, pharmacological effects and clinical application of Suanzaoren Decoction are reviewed. Based on this, the quality marker(Q-marker) of Suanzaoren Decoction was predicted and analyzed according to the "five principles" of Q-marker of traditional Chinese medicine--transmission and traceability, specificity, effectiveness, measurability and compatibility environment of compound prescriptions. The results indicated that jujuboside, spinosin, ferulic acid, senkyunolide Ⅰ, sarsasapogenin, mangiferin, liquiritoside and glycyrrhizic acid were predicted and analyzed, and those can be used as Q-markers of Suanzaoren Decoction. Subsequently, the above components can be selected as indicators to control and evaluate the quality of Suanzaoren Decoction and its preparations, and establish a quality traceability system.

Effects of Suanzaoren Decoction on Learning and Memory Ability and Brain Neurotransmitters Content of Senile Insomnia Model Rats / 中国药房

OBJECTIVE:To study the effects of Suanzaoren decoction on learning and memory ability and brain neurotransmit-ters content of senile insomnia model rats. METHODS:Rats were randomly divided into normal group,model group,positive group [Estazolam tablet,2 × 10-3 g/(kg·d)] and Suanzaoren decoction low-dose,middle-dose and high-dose groups [5,10,15 g/(kg·d)],with 10 rats in each group. Except for normal group,those groups were given D-galactose ih on the back+sleep depriva-tion to induce senile insomnia model. 6 weeks after modeling,treatment groups were given relevant medicine intragastrically for one week,and normal group and model group were given normal saline intragastrically. The escape latency,the percentage of swimming time and the time of original platform crossing were observed,and the contents of Glu and GABA in cerebral tissue were determined as well as the contents of IL-1β and 5-HT in hypothalamus. RESULTS:Compared with normal group,escape la-tency of rats prolonged and the contents of IL-1β and 5-HT in hypothalamus decreased,while the percentage of swimming time of original platform quadirant,the time of original platform crossing and the contents of Glu and GABA in cerebral tissue increased (P<0.05). Compared with model group,above index of treatment groups improved significantly,with statistical significance(P<0.05). Compared with positive group,the changes of above index were more significant in Suanzaoren decoction middle-dose and high-dose groups,with statistical significance (P<0.05). CONCLUSIONS:Suanzaoren decoction could markedly enhance the learning and memory ability of senile insomnia model rats,and reduce neurotransmitters content and relieve the delayed neuronal damage which led by Glu and GABA.

Effects of Suanzaoren Decoction on Expression of NMDAR1, NMDAR2A and NMDAR2B ;RNA in Cortex and Hippocampus of Depression Model Rats / 中国中医药信息杂志

Objectives To observe the effect of Suanzaoren Decoction on the expression of NMDAR1, NMDAR2A and NMDAR2B RNA in cortex and hippocampus of depression model rats, and explore the mechanism of treating depression. Methods Ninty SD rats were randomly divided into blank group, model group, western medicine group, Suanzaoren Decoction high, medium and low dose group. Except the blank group, the other groups were replicated the rat model of depression in chronic stress. The treatment groups were given corresponding drugs by gavage. The expression of NMDAR1, NMDAR2A and NMDAR2B genes in cortex and hippocampus were detected by RT-PCR. Results Compared with the model group, the positive expression of NMDAR in cortex and hippocampus decreased in Suanzaoren Decoction high and medium dose group (P<0.01). The positive expression of NMDAR2A in cortex and hippocampus of Suanzaoren Decoction high, medium dose group and western medicine group decreased (P<0.05, P<0.01), the expression of NMDAR2A in hippocampus increased in Suanzaoren Decoction low dose group (P<0.05). The positive expression of NMDAR2B in cortex decreased in western medicine group, Suanzaoren Decoction high, medium and low dose group (P<0.01). The expression of NMDAR2B in hippocampus increased in Suanzaoren Decoction medium and low dose group (P<0.01). Conclusion Suanzaoren Decoction can play the role of treating depression by decreasing the expression of NMDAR1, NMDAR2A and NMDAR2B RNA in cortex and hippocampus.

The Effects of Suanzaoren decoction on hippocampus, cortex BDNF and TrKB geneexpression in depression rats / 国际中医中药杂志

Objective To investigate the effect of Suanzaoren Decoction on hippocampus, cortex BDNF and TrKB gene expression in depression model rats. Methods Depression rat models were established by social-isolated raise and chronic stress stimulation. Suanzaoren decoction was administrated to the models. RT-PCR was adopted to detect the expression of mRNA BDNF and TrKB genes. Results The mRNA expression of BDNF and TrKB in cortex of Suanzaoren decoction high dose group、medium dose group and clomipramine group(0.213±0.094, 0.639±0.023, 1.032±0.015, 1.089±0.014, 1.580±0.012, 1.860±0.019)were all higher than the model group(0.032±0.008, 0.001±0.000), showing a significant difference among four groups (P0.05). Conclusion Suanzaoren decoction can increase the expression of BDNF and TrKB gene, promote neuronal proliferation, and resist depression.

Determination of spinosin and ferulic acid in Suanzaoren Decoction by RP-HPLC / 中草药

Object To develop an RP-HPLC method for determination of spinosin and ferulic acid in Suanzaoren Decoction (SZRD). Methods Hypersil C 18 column was used. The mobile phase consisted of acetonitrile-water with 1% glacial acetic acid (17∶83) for spinosin and (16∶84) for ferulic acid. Detection wavelengths were set at 334 and 320 nm, respectively. The internal standard was p-hydroxybenzaldehyde for ferulic acid. Results The linear ranges for spinosin and ferulic acid were 0.960—13.44 ?g/mL (r=0.999 8) and 2.04—27.2 ?g/mL (r=0.999 7), respectively. The recoveries for them were 98.4% and 98.8%, respectively. The RSD were 2.5% and 2.8%, respectively. Conclusion This method is simple, rapids and accurate, and can be used for the determination of spinosin and ferulic acid in SZRD.

Determination of mangiferin and glycyrrhizic acid in Suanzaoren Decoction by RP-HPLC / 中成药

AIM: To develop an RP HPLC method for determination of mangiferin and glycyrrhizic acid in Suanzaoren Decoction(Semen Ziziphi Spinosae, Poria, Rhizoma Ligustici Chuanxiong, Rhizoma Anemarrhenae, Radix Glycyrrhizae). METHODS: Hypersil C 18 column was used. The mobile phase consisted of acetonitrile water(13∶87,v/v) with 1% glacial acetic acid for mangiferin and methanol acetonitrile water(25∶15∶60,v/v) with 1% acetic acid for glycyrrhizic acid, respectively. Detection wavelengths were set at 320nm and 254nm, respectively. The internal standards were vanillin for mangiferin and mebendazole for glycyrrhizic acid, respectively. The flow rates were both 0.8mL?min -1 . RESULTS: The linear ranges for mangiferin and glycyrrhizic acid were 10.72～85.76?g?mL -1 ( r =0.9998) and 0.080～0.80mg?mL -1 ( r =0.9997), respectively. The average recoveries were 97.2% with RSD 3.0% and 97.6% with RSD 2.4%, respectively. CONCLUSION: This method is simple, rapid and accurate, and can be used for the determination of mangiferin and glycyrrhizic acid in Suanzaoren decoction.