Use of Albiflorin in Treatment of Coronavirus Pneumonia

Abstract

Use of albiflorin or a pharmaceutically acceptable salt thereof or an extract or a pharmaceutical composition containing albiflorin in the preparation of a medicament for preventing or treating coronavirus pneumonia, especially novel coronavirus pneumonia, or use thereof in the preparation of a medicament for treating prolonged symptoms of novel coronavirus pneumonia, performing rehabilitation conditioning after recovery of novel coronavirus pneumonia, or alleviating a possible sequela of novel coronavirus pneumonia, the sequela being depression, anxiety, sleep disorder, pain, palpitation, asthma, intestinal function disorder, or chronic fatigue syndrome. Albiflorin has the effects of anti-coronavirus, anti-inflammatory, inflammatory storm inhibition, human microecological balance regulation, etc., can comprehensively prevent and treat coronavirus-induced pneumonia, and can be prepared into medicaments, health care products or nutrition regulators for preventing or treating coronavirus pneumonia, especially novel coronavirus pneumonia.

Claims

1.-6. (canceled)

7. A method for preventing or treating coronavirus pneumonia, comprising: administering a prophylactically or therapeutically effective amount of albiflorin or a pharmaceutically acceptable salt thereof or an extract or a pharmaceutical composition containing albiflorin to a subject in need thereof.

8. A method for treating prolonged symptoms of novel coronavirus pneumonia, performing rehabilitation conditioning after recovery of novel coronavirus pneumonia, or alleviating a possible sequela of novel coronavirus pneumonia, comprising: administering a prophylactically or therapeutically effective amount of albiflorin or a pharmaceutically acceptable salt thereof or an extract or a pharmaceutical composition containing albiflorin to a subject in need thereof, wherein the prolonged symptoms of the novel coronavirus pneumonia are pain, palpitation, asthma, consciousness disorder, or chronic fatigue; and/or the sequela is depression, anxiety, sleep disorder, pain, palpitation, asthma, intestinal function disorder or chronic fatigue syndrome.

9. The method according to claim 7, wherein the extract containing albiflorin is total glucosides of Paeonia lactiflora Pall and/or a Paeonia lactiflora Pall extract, and/or the pharmaceutical composition is a Chinese herbaceous peony and licorice preparation.

10. A medicament, a health care product or a nutrition regulator for preventing or treating coronavirus pneumonia, comprising albiflorin or a pharmaceutically acceptable salt thereof or an extract or a pharmaceutical composition containing albiflorin.

11. The method according to claim 7, wherein the coronavirus pneumonia is novel coronavirus pneumonia.

12. The method according to claim 7, wherein the albiflorin or the pharmaceutically acceptable salt thereof or the extract or pharmaceutical composition containing albiflorin is used to prevent or treat the coronavirus pneumonia by one or more of the following ways: resisting coronavirus, resisting inflammatory storm, restoring intestinal flora balance and resisting hypoxia.

13. The method according to claim 7, wherein the albiflorin or the pharmaceutically acceptable salt thereof or the extract or pharmaceutical composition containing albiflorin is used to prevent or treat the coronavirus pneumonia by one or more of the following ways: inhibiting coronavirus 3CLpro protein, promoting endogenous bile acid secretion, inhibiting sphingosine kinase SphK1 and/or SphK2, inhibiting interleukin-6 or phospholipase A2/arachidonic acid inflammatory factors, inhibiting an IDO1 inflammatory signaling pathway, regulating intestinal flora balance and promoting EPO production to resist hypoxia.

14. The method according to claim 8, wherein the extract containing albiflorin is total glucosides of Paeonia lactiflora Pall and/or a Paeonia lactiflora Pall extract, and/or the pharmaceutical composition is a Chinese herbaceous peony and licorice preparation.

15. The medicament, health care product or nutrition regulator according to claim 10, wherein the extract containing albiflorin is total glucosides of Paeonia lactiflora Pall and/or a Paeonia lactiflora Pall extract, and/or the pharmaceutical composition is a Chinese herbaceous peony and licorice preparation.

16. The medicament, health care product or nutrition regulator according to claim 10, wherein the medicament, health care product or nutrition regulator is selected from capsules, tablets, dropping pills, preparations for nasal administration or injections.

17. The medicament, health care product or nutrition regulator according to claim 10, wherein the albiflorin or the pharmaceutically acceptable salt thereof or the extract or pharmaceutical composition containing albiflorin is used to prevent or treat the coronavirus pneumonia by one or more of the following ways: resisting coronavirus, resisting inflammatory storm, restoring intestinal flora balance and resisting hypoxia.

18. The medicament, health care product or nutrition regulator according to claim 10, wherein the albiflorin or the pharmaceutically acceptable salt thereof or the extract or pharmaceutical composition containing albiflorin is used to prevent or treat the coronavirus pneumonia by one or more of the following ways: inhibiting coronavirus 3CLpro protein, promoting endogenous bile acid secretion, inhibiting sphingosine kinase SphK1 and/or SphK2, inhibiting interleukin-6 or phospholipase A2/arachidonic acid inflammatory factors, inhibiting an IDO1 inflammatory signaling pathway, regulating intestinal flora balance and promoting EPO production to resist hypoxia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 shows inhibitors of SARS-ConV;

[0059] FIG. 2 shows that albiflorin may treat COVID-19 by inhibiting 3CLpro protease;

[0060] FIG. 3 shows a binding mode of albiflorin in 3CLpro protease;

[0061] FIGS. 4A-4D show the binding modes of molecules 101-104 in 3CLpro protease, respectively, wherein FIG. 4A shows a result of docking of the molecule 101 with 6LU7, FIG. 4B shows a result of docking of the molecule 102 with 6LU7, FIG. 4C shows a result of docking of the molecule 103 with 6LU7, and FIG. 4D shows a result of docking of the molecule 104 with 6LU7;

[0062] FIG. 5 shows the changes of bile acid secretion in depression model animals;

[0063] FIG. 6 shows that albiflorin increases bile acid secretion by regulating intestinal flora;

[0064] FIG. 7 shows an effect of paeoniflorin on the secretion of IL-6 in the blood of acute stress model mice;

[0065] FIG. 8 shows an effect of albiflorin on the secretion of IL-6 in the blood of acute stress model mice;

[0066] FIG. 9 shows that albiflorin inhibits the increase of cPLA2 in chronic stress rats;

[0067] FIG. 10 shows that albiflorin resists inflammations by inhibiting cPLA2 in chronic stress rats;

[0068] FIG. 11 shows an effect of albiflorin on SphK1 and SphK2 detected by Western Blot;

[0069] FIG. 12 shows an effect of albiflorin on IDO1 detected by Western Blot;

[0070] FIG. 13 shows targeted metabolomics multiple comparison analysis (PLS-DA);

[0071] FIG. 14 shows gut population structure cluster analysis in high-throughput metagenomic sequencing after albiflorin administration; and

[0072] FIG. 15 shows that the disturbance of intestinal microbial metabolism in the rats of a model group is corrected in the albiflorin group (the Top 25 metabolites with significant changes after administration).

BEST MODE FOR CARRYING OUT THE INVENTION

[0073] The present invention will be further described below in conjunction with specific examples. However, the following examples of the present invention are only used to illustrate the present invention, but not to limit the scope of the present invention.

Example 1: Computer Molecular Docking Study of Albiflorin Inhibiting a 3CLpro Protein of 2019-nCoV

1.1 Experimental Method

[0074] 2019-nCoV, SARS-CoV, MERS-CoV, etc. are all coronaviruses, and their processes of invading a host are the same, so the corresponding drug development strategies are also similar. The coronavirus relies on the binding of a Spike protein on the surface to an angiotensin-converting enzyme 2 (ACE2) receptor on the surface of a host cell, and then enters a recipient cell. After entering the recipient cell, viral positive-sense RNA is translated into two long peptide chains by host ribosome, and the two long peptide chains are subjected to a proteolysis process, and then cut and assembled into corresponding functional proteins. This proteolysis process is mainly completed by coronavirus main protease (3CLpro) and papain-like protease (PLpro). Coronavirus RNA polymerase (RdRp) is responsible for replicating a viral RNA genome to generate new viral individuals. Therefore, four proteins of Spike, 3CLpro, PLpro and RdRp are the key enzymes of virus invasion and reproduction, and thus become the most important therapeutic targets.

[0075] At present, the sequencing of the whole genome of 2019-nCoV has been completed. According to this gene sequence, the sequences of corresponding proteins of 2019-nCoV, SARS-CoV and other viruses can be compared to find differences, thereby guiding drug development. Compared with a Spike protein of SARS-CoV, a Spike protein of 2019-nCoV has undergone major changes in some key areas, resulting in a decline in the effectiveness of drugs targeting the Spike protein of SARS-CoV for 2019-nCoV. In contrast, the three key targets of 3CLpro, RdRp and PLpro of 2019-nCoV have more than 95% sequence similarity with those of SARS-CoV. Therefore, active compounds developed against SARS-CoV may have some therapeutic effect on COVID-19.

[0076] Albiflorin is a natural product with complex activities. The inventor believes that this compound may have an inhibitory effect on 2019-nCoV, and hopes to preliminarily prove through a method of computer-aided drug design. To achieve this goal, it is first necessary to determine which target of 2019-nCoV albiflorin is most likely to have an effect on. Among the four key proteins of 2019-nCoV, only 3CLpro has completed the analysis of protein crystals, and the other three can only be subjected to a decking study through homology models, resulting in a larger error. Therefore, the inventors use a small molecule similarity method to identify the most likely targets.

[0077] Based on the above ideas, the inventors first collect 15 active compounds developed against the three key targets of 3CLpro, RdRp and PLpro of SARS-ConV from the literatures, as shown in FIG. 1. Next, the molecular structure similarity of albiflorin and these 15 SARS-ConV inhibitors is compared using molecular fingerprints in MOE software. The molecular similarity algorithm has been applied to the activity prediction of albiflorin and other natural products for many times, and has been proved to have a good accuracy rate.

[0078] Table 1 lists the top 5 SARS-ConV inhibitors with relatively high similarity to albiflorin, and it can be found that 4 of them all act on a 3Clpro protein target. Therefore, the inventors believe that if albiflorin has an inhibitory effect on 2019-nCoV, this compound most likely works by inhibiting the 3Clpro protein target, see FIG. 2.

TABLE-US-00001 TABLE 1 Five SARS-ConV inhibitors with high similarity to albiflorin Target Name Similarity 3CLpro GS376 81% 3CLpro 103 80% RdRp ATA 64% 3CLpro 101 50% 3CLpro/PLpro 302 47%

[0079] Next, the inventors study a binding mode of albiflorin and the 3CLpro protein target through molecular docking, and roughly calculate the binding free energy thereof. In order to have a comparison, the inventors use 3CLpro-101 to 104 in FIG. 1 which act on SARS-ConV, and simulate the binding mode of these four molecules with the 3CLpro protein target also by a molecular docking method and calculate the intensities, as shown in FIG. 3. A template for molecular docking is a crystal structure having a PDB number of 6LU7, which is jointly released by the Institute of Immunochemistry, ShanghaiTech University and the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, as shown in FIG. 4.

1.2 Results

[0080] From the molecular docking results, albiflorin and four other 3CLpro protein inhibitors of SARS-ConV can bind well to the 3CLpro protein of 2019-nCoV. The binding modes are relatively similar, which proves that the docking results are more reliable.

[0081] The binding free energy of 5 molecules is between −48 and −90 kCal/mol. Since this binding free energy is calculated using a molecular mechanics method in MOE software, the error is relatively large, this binding free energy has guiding significance because it is still on the order of magnitude level, and thus can be used to illustrate that these five molecules bind to the 3CLpro protein of 2019-nCoV at relatively high strength.

[0082] Due to the large number of oxygen atoms in the molecular structure, albiflorin easily interacts variously with amino acid residues in a receptor pocket. Upon the comparison of these five molecules, it can be found that Cys145, Glu166, and Gln189 are important residues in the interaction between ligands and receptors. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Comparison of albiflorin and other four ligands Binding free energy Ligands (kCal/mol) Important residues Albiflorin −90.895 His41, Gly143, Ser144, Cys145, Glu166, Gln189 101 −65.193 His163 102 −50.346 Cys145 103 −70.643 His41, His163, Glu166, Gln189, Gln192 104 −48.677 Gln189

[0083] The research results of Example 1 can prove that albiflorin is a 3CLpro protein inhibitor of 2019-nCoV with high research potential.

Example 2: Albiflorin Increases Endogenous Bile Acid Secretion by Regulating Intestinal Flora

2.1 Experimental Animals

[0084] 32 healthy male SD rats, weighing 180-220 g, are randomly divided into a blank control group (Control), a model group (CUMS), a fluoxetine group (FLX), and an albiflorin group (Albiflorin), with 8 in each group. They are reared in 2 cages, adaptively fed for a week before the experiment, had free access to water and food, and are trained with 1% sucrose water.

[0085] Except for the blank control group, all groups receive randomly designed stress stimulation, and are then administrated intragastrically with fluoxetine (10 mg/kg/d) and albiflorin (7 mg/kg/d) for 7 consecutive days in terms of 1.0 mL/100 g body weight after 29 days of stress stimulation, while the corresponding stress stimulation is continued in the course of administration. All drugs are prepared into solutions or suspensions with physiological saline before use, and dissolved by ultrasonic waves.

2.2 Collection of Metabolomic Samples

[0086] After 7 days of administration, the administration ends, and 24 hours later, behavioral tests are performed. After the tests, the animals are anesthetized and sacrificed. The plasma, hippocampal tissues, and feces of the animals are collected separately, and preserved for later use by using appropriate methods according to the experimental requirements.

2.3 Extraction of Small Molecule Metabolites from Tissues and Plasma

[0087] Hippocampus: The hippocampus of the rats is accurately weighed, added with 9 times the volume of a pre-cooled extraction solution (methanol-acetonitrile-acetone-water 30:30:30:10; V/V/V/V), homogenized by ultrasonic waves, mixed well by vortexing and stood on ice for 10-15 minutes to make the extraction solution and animal tissue powder fully react. The main purpose of this step is to lyse cell walls and precipitate macromolecular substances such as proteins and DNA, etc. After centrifuging for 10 minutes at high speed and low temperature (16000 g, 4° C.), small molecule metabolites are in supernatant in a tube. 200 μl of the supernatant is taken and placed into a new centrifuge tube and dried with nitrogen for later use.

[0088] Feces: the feces of the rats are accurately weighed, added with 9 times the volume of a pre-cooled extraction solution (methanol-acetonitrile-water (42:42:16; V/V/V), homogenized by ultrasonic waves, mixed well by vortexing and stood on ice for 10-15 minutes to make the extraction solution and the feces fully react. The main purpose is to lyse cell and precipitate macromolecular substances such as proteins and DNA, etc. After centrifuging for 10 minutes at high speed and low temperature (16000 g, 4° C.), small molecule metabolites are in supernatant in the tube. 200 μl of the supernatant is taken and placed into a new centrifuge tube and dried with nitrogen for later use.

[0089] Plasma: 100 μl of plasma is taken and transferred into a 1.5 ml centrifuge tube. 400 μl of pre-cooled extraction solution (methanol-water 50:50; V/V) is added, and mixed well by vortexing. The tube is stood on ice for 10 minutes to make the extraction solution and plasma fully react, and centrifuged at high speed (16000 g, 4° C.) for 10 minutes. Small molecule metabolites are in supernatant in the tube. 200 μl of the supernatant is taken and placed into a new centrifuge tube and dried with nitrogen for later use.

2.4 LC-MS/MS Next-Generation Targeted Metabolomic Analysis

[0090] The metabolite extract is taken, dissolved in 100 μl mobile phase, and the main metabolites in the sample are determined by LC-MS/MS (Shimadzu LC-20AD-Qtrap 5500 tandem mass spectrometer (SCIEX, USA)). Chromatographic separation conditions: chromatographic column: aPHera amino column (150×2 mm, 4 μm, Supelco, USA), mobile phase: A: 95% ultrapure water+5% acetonitrile+20 μM ammonia water, B: 100% acetonitrile; flow rate: 0.5 ml/min, column temperature: 25° C., and injection volume: 10 μl. Elution conditions: 0-3 min, 95% B; 3-6 min, 75% B; 6-7 min, 0% B; 7-12 min, 0% B, 12-15 min, 95% B. Mass spectrometry conditions: ion source: electrospray (ESI), which adopts a fast switching mode of positive and negative particles, with a switching rate of 50 ms. Ion source temperature: 500° C., gas 1: 30 psi, gas 2: 30 psi, curtain gas: 30 psi, ion spray voltage: positive: 5500V, negative: −4500V. Scanning mode: real-time multiple reaction detection mode (Scheduled MRM). There are 625 MRM ion pairs (625 major metabolites, covering 62 major metabolic pathways of organisms).

2.5 Bioinformatics Data Analysis

[0091] The obtained chromatographic peak information (Wiff file) is imported into Multiquant 3.0 (SCIEX, USA), and the obtained chromatographic peaks are area-integrated and checked manually. The correctly-checked chromatographic peak areas are imported into Excel for max/min, Z-score, and missing value analysis. The analyzed data is imported into Metaboanalyst for multivariate analysis (PLS-DA, OPLS-DA and VIP analysis), metabolic pathways analysis and correlation analysis.

2.6 The Changes of Bile Acid Secretion in Depression Model Animals

[0092] Through VIP analysis, the differences between intestinal flora metabolism of the depression model rats and the blank control group are explored. The inventors find that the intestinal flora metabolism of depression model rats is significantly lower than that of the blank control group. Among the significant Top 20 metabolites in both groups, 16 (80%) metabolites are significantly reduced (VIP>1.5) in the depression model rat group, see FIG. 5.

[0093] From the perspective of chemical classification, these metabolites that are reduced in the intestinal flora metabolism of depression rats mainly include: amino acids and vitamins (vitamin B6 (Pyridoxal), choline, L-tyrosine, citrulline and L-glutamic acid, etc.), bile acids (cholic acid and glycocholic acid)) and nucleic acid and its derivatives.

[0094] Cholic acid is a main bile acid produced by the liver using cholesterol, which is excreted into the intestine through the enterohepatic circulation, and further metabolized into various other metabolites by microorganisms in the intestine. In addition, recent studies have reported that intestine bacteria themselves can synthesize a series of sterol bile acids from metabolites in the intestine. As hormones, these bile acids are reabsorbed into the body and play a very important role in regulating fat metabolism, energy metabolism and inflammatory responses. The bile acid content in the feces of the depression model rats is significantly reduced, indicating that the intestinal bile acid reabsorption rate is increased.

2.7 The Effects of Albiflorin on Bile Acid Secretion in Depression Model Animals

[0095] VIP analysis of the effects of albiflorin administration on the intestinal flora of depression rats shows that after administration, the overall metabolism of intestinal flora is significantly improved compared with the depression group, as shown in FIG. 6. The main manifestations are the increase of bile acid content and the increase of amino acid and vitamin content. It is indicated that the normal intestinal flora function is improved and partially recovered under the action of albiflorin.

[0096] The results of this example show that the intestinal flora metabolism of the chronic stress model rats is significantly lower than that of the blank control group, especially the reabsorption of bile acids are increased and the content of amino acids are significantly reduced.

[0097] Albiflorin administration almost completely restores the normal intestinal flora metabolism, which is manifested by reducing the reabsorption of bile acids in the intestine and increasing the content of bile acids and amino acids.

Example 3: Study on Anti-Inflammatory and Immunomodulatory Effects of Albiflorin and Paeoniflorin

3.1. Materials and Methods

3.1.1 Experimental Animals and Main Reagents

[0098] Adult male ICR mice, weighing 18-22 g, are adaptively fed for one week, with normal diet and drinking water, and are randomly divided into 5 groups: a blank control group, a model group, a model-making fluoxetine group, a model-making administration group (with albiflorin, 6 doses of subgroups), and a model-making administration group (paeoniflorin, 6 doses of subgroups), with 5 animals in each group.

[0099] Paeoniflorin (a purity of 95.2%) is purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd., and albiflorin (a purity of 96.5%) is provided by Shanghai Eternal Biotechnology Co., Ltd. Albiflorin and paeoniflorin are formulated into aqueous solutions within 24 hours prior to administration. Dosages of adminstration (paeoniflorin, albiflorin) are 4 mg/kg, 8 mg/kg, 16 mg/kg, 32 mg/kg, 64 mg/kg, 128 mg/kg, respectively. All animals are housed in clean iron rearing cages with free access to water and food. A rearing room keeps good sound insulation conditions, a temperature of 18-24° C., a humidity of 50%-55%, and accepts 12 h light illumination every day. Mode of administration: administration is performed after one week of adaptive feeding, intragastric administration for 2 days, once a day, 0.5 ml each time, and experiments are started 2 hours after the second intragastric administration.

[0100] IL-6 ELISA Detection Kit, Invitrogen (Biosource), USA.

[0101] Multifunctional Microplate Analyzer, FlexStation 3, Molecular Devices, USA.

[0102] Milli-Q Ultrapure Water System, Millipore Corporation, USA.

3.1.2 Establishment of Acute Stress Model in Mice

[0103] An acute stress animal model is established by restraint braking in combination with hot and cold stimulation. The experiment is carried out between 9:00 and 15:00, and the mice in the experimental groups are put into a 50 ml plastic centrifuge tubes with ventilation at the bottom, and fixed to make them immobilized. The restrained mice are placed in a refrigerator (4° C.) for 30 min, then returned to room temperature for 10 min, placed in a ventilated oven (45° C.) for 10 min, and then kept in a restrained state for 10 min at room temperature.

3.1.3 Sampling

[0104] Blood is quickly collected from the femoral artery of the mouse after the above-mentioned model-making and anticoagulated with heparin sodium, and plasma is separated. After the animal is killed by decapitation, the whole brain is taken on ice, and the left hemisphere brain region is taken under the condition of ensuring that all the hypothalamus are obtained. The collected brain tissue samples are quickly put into liquid nitrogen for storage and reserved for the analysis of monoamine neurotransmitters and their metabolites. The separated plasma is labeled with groups and then stored in a −20° C. refrigerator for analysis of corticosterone and IL-6 content.

3.1.4 Determination of IL-6 Content in Plasma of Acute Stress Model Mice

[0105] The plasma samples stored in the −20° C. refrigerator are returned to room temperature and centrifuged at 12,000 rpm/min for 5 min, and the supernatant obtained by centrifugation is used to determine the expression level of IL-6 in the blood of acutely stressed mice by ELISA. The assay of IL-6 is performed according to the instructions of the kit.

3.2 Results

[0106] The effects of paeoniflorin and albiflorin on the content of IL-6 in the blood of acute stress model mice are as follows: after acute restraint of mice, immune cells such as peripheral lymphocytes and phagocytes in their plasma increase, the concentrations of some inflammatory cytokines such as IL-1β, IL-2, IFN-γ, TNF-α and IL-6 etc. increase, and the presence of high-concentration inflammatory cytokines existing for a long time will affect the own immune systems and induce depression under certain conditions.

[0107] In this experiment, an acute stress animal model is used to investigate the changes of IL-6 levels in mice under acute stress conditions. FIG. 7 shows that paeoniflorin has a certain inhibitory effect on the secretion of IL-6 in the blood of acute stress mice in the dose range of 4, 8, 16, 32, 64, and 128 mg/kg, and has the most significant inhibitory effect (P<0.05) at the dose of 4 mg/kg and 8 mg/kg; and FIG. 8 shows that albiflorin has a certain inhibitory effect on the secretion of IL-6 in acute stress mice in a low dose range, and has the most significant inhibitory effect (P<0.01) at the dose of 8 mg/kg, but with the increase of the administration dose, its inhibitory effect is not obvious, and the secretion of IL-6 is promoted obviously.

Example 4 Anti-Inflammatory Study of Albiflorin by Inhibiting cPLA2

4.1 Albiflorin (ALB) Inhibits the Increase of cPLA2 in Chronic Stress Rats

[0108] The inventors have established a chronic unpredictable mild stress rat model (CUMS), wherein rats under test are divided into a blank control group (Ctrl-sal), a chronic stress model group (CUMS-sal), a fluoxetine administration group (CUMS-flx) and an albiflorin administration group (CUMS-Alb).

[0109] Phospholipase A2 (cPLA2) is significantly increased in the hippocampus of chronic stress (CUMS) model rats. After 7 days of albiflorin administration (3.5 mg/day, 7 mg/day, 14 mg/day), phospholipase A2 (cPLA2) in the hippocampus of rats is significantly decreased (P<0.01), showing a significant dose dependency, see FIG. 9.

4.2 Anti-Inflammation of Albiflorin (ALB) by Inhibiting cPLA2 in Chronic Stress Rats

[0110] It is found in experiments that chronic stress (CUMS) induces a significant increase in the expression of cPLA2 in the hippocampus of model rats, resulting in an increase in inflammatory mediators PGF2α and 20-HETE. Albiflorin (ALB) administered for 7 days (7 mg/kg) can inhibit the increase of cPLA2, reduce the content of inflammatory mediators PGF2α and 20-HETE, reduce hippocampal neuroinflammation, and restore the homeostasis of membrane lipids, see FIG. 10.

Example 5: Inhibition of Albiflorin on Hippocampal Sphingosine Kinase in Chronic Stress Rats

[0111] Sphingosine kinases (SphK1, SphK2) are significantly increased in the hippocampus of chronic stress (CUMS) model rats. After 7 days of albiflorin administration (7 mg/day, 14 mg/day), sphingosine kinases (SphK1, SphK2) in the hippocampus of rats are significantly decreased (P<0.01). The results are shown in the following table.

[0112] Inhibitory effects of albiflorin on SphK1 and SphK2 in hippocampus of chronic stress rats

TABLE-US-00003 Group SphK1 SphK2 Blank control group 1.0 ± 0.007** 1.0 ± 0.007** Model group 1.1 ± 0.007 1.8 ± 0.005 Fluoxetine group 1.5 ± 0.009## 2.1 ± 0.016## Albiflorin group 3.5 1.2 ± 0.009## 2.2 ± 0.016## (mg/kg) 7 0.7 ± 0.004** 1.6 ± 0.010** 14 0.5 ± 0.001** 2.1 ± 0.015## ##means an increase compared with the model group, p < 0.01; *means a decrease compared with the model group, p < 0.05; **means a decrease compared with the model group, p < 0.01.

[0113] The effects of albiflorin on SphK1 and SphK2 are detected by Western Blot, see FIG. 11.

[0114] Conclusion: sphingosine kinases (SphK1, SphK2) serve as main rate-limiting enzymes in the synthesis of sphingosine-1-hydrochloride in cells. As can be seen from FIG. 11, the sphingosine kinases are significantly increased in the hippocampus of CUMS model rats, suggesting that chronic stress inhibits the synthesis of sphingosine-1-hydrochloride. Medium- and high-dose administration of albiflorin can significantly reduce the content of sphingosine kinases (SphK1, SphK2) in rat hippocampus, indicating that albiflorin can increase the content of sphingosine-1-hydrochloride in rat hippocampus, thereby promoting the proliferation and survival of hippocampal cells.

[0115] Albiflorin is a sphingosine kinase 2 (SphK2) inhibitor. SphK2 is a component necessary for the replication of the novel coronavirus in cells. If it is inhibited, the virus will not replicate. Therefore, the inhibitory effects of albiflorin on SphK2 can not only reduce the inflammation level, but also prevent the viral replication, and also play the functions of protecting brain nerves.

Example 6: Inhibition of Albiflorin on Overexpression of IDO1 in the Hippocampus of Chronic Stress Rats

[0116] The secretion of IDO1 is significantly increased in the hippocampus of chronic stress (CUMS) rats. After 7 days of albiflorin administration (7 mg/day, 14 mg/day), the secretion of IDO1 in the hippocampus of rats is significantly inhibited (P<0.01). The results are shown in the following table.

[0117] Inhibitory effects of albiflorin on IDO1 in hippocampus of chronic stress rats

TABLE-US-00004 Group IDO1 Blank control group 1.0 ± 0.010** Model group 1.0 ± 0.011# Fluoxetine Albiflorin group 3.5 g 1.0 ± 0.010## (mg/kg) 7 0.8 ± 0.007** 14 0.7 ± 0.007** #means an increase compared with the model group, p < 0.05; ##means an increase compared with the model group, p < 0.01; and **means a decrease compared with the model group, p < 0.01.

[0118] The effects of albiflorin on IDO1 is detected by Western Blot, see FIG. 12.

[0119] Conclusion: albiflorin has a significant inhibitory effect on overexpression of IDO1 in hippocampus of chronic stress (CUMS) rats.

Example 7: Study on Albiflorin for Restoring Intestinal Flora Balance in CUMS Rats

[0120] The inventors establish the chronic unpredictable mild stress rat model (CUMS). Using a new generation of targeted metabolomics methods, the metabolic function of the intestinal flora of stress rats is studied. PLS-DA multivariate analysis shows that there is a significant difference between the intestinal flora metabolism of rats in the depression model group (depression group) and the blank control group, see FIG. 13.

[0121] The inventors further provide treatments for the stress model rats with albiflorin for 7 consecutive days, and each treatment dose is 7 mg/kg/d. Seven days later, the applicant reassesses the functions and structures of intestinal flora of the rats by using metabolomics and 16sDNA high-throughput metagenomic sequencing technology. It is manifested through multiple comparison analysis (PLS-DA) of metabolomic data that after albiflorin administration, the intestinal flora of rats is metabolized and moved toward the blank control group, which is almost completely overlapped with the blank control group, indicating that albiflorin helps to restore normal metabolism of intestinal flora in stress rats, as shown in FIG. 13. This is consistent with the results of the flora structure analysis.

[0122] Albiflorin administration group (Alb) has similar flora structures with the blank control group (Control), both of which are clustered together without any statistical difference (P>0.05), and has a significant increase in the content of beneficial bacteria Firmicutes, especially lactic acid bacteria compared with the stress rat model group, see FIG. 14.

[0123] VIP analysis shows that after 7 days of albiflorin administration, the overall metabolism of intestinal flora is significantly improved compared with the depression group, which is mainly manifested in the increase in the content of bile acids and the content of amino acids and vitamins, see FIG. 15.