CN113813278A - New application of saikosaponin D - Google Patents

New application of saikosaponin D Download PDF

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Publication number
CN113813278A
CN113813278A CN202111323094.2A CN202111323094A CN113813278A CN 113813278 A CN113813278 A CN 113813278A CN 202111323094 A CN202111323094 A CN 202111323094A CN 113813278 A CN113813278 A CN 113813278A
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saikosaponin
dopamine
mol
antagonist
cells
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徐天瑞
秦书华
向诚
马兆斌
母茜
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Kunming University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/233—Bupleurum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

The invention discloses a new application of saikosaponin D, namely the application of saikosaponin D in preparing dopamine receptor antagonist, the invention shows that saikosaponin D can be combined with dopamine receptor D2L through molecular docking experimental results; then in vitro cell experiments show that saikosaponin D can specifically antagonize the activation of dopamine on dopamine receptor D2L on ERK phosphorylation level; the D2L antagonist provided by the invention provides a candidate compound for the development of a medicine for treating the bidirectional emotion.

Description

New application of saikosaponin D
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of saikosaponin D in preparation of a dopamine receptor antagonist.
Background
G-protein-coupled receptors (GPCRs) are a class of membrane protein receptors with 7 transmembrane helices, and currently there are about 800 members, with over 2% of the genes in the human genome encoding GPCRs. The current world drug market has 1/3 small molecule drugs as activators or antagonists of GPCRs, 34% of FDA approved drugs in the market target GPCRs, and the structural characteristics of GPCRs and the important role in signal transduction determine that GPCRs can be used as good drug targets.
Dopamine receptors in the central nervous system are important members of GPCRs. Dopamine is a monoamine neurotransmitter, is synthesized by dopaminergic neurons and stored in vesicles, acts on dopamine receptors through exocytosis and quantized release, and further participates in a plurality of physiological and pathological processes such as physical movement regulation, cardiovascular activity regulation, learning and memory, reward, psychoactive substance addiction, motivation behavior and the like through a transmembrane signal transduction process mediated by a G protein coupled receptor. ERK1/2 (extracellular regulated protein kinases) refers to extracellular regulated protein kinases, including ERK1 and ERK2, which are key kinases for conducting signals from surface receptors to the nucleus of the cell. ERK1/2 is a member of the MAPK family and is a signal transduction protein that transmits mitogen signals. It normally localizes to the cytoplasm and when activated translocates to the nucleus, regulating transcription factor activity, producing a cellular effect. The ERK1 and ERK2 pathways are the most thoroughly studied members of the ERK family, and a plurality of stimulating factors such as growth factors, cytokines, viruses, ligands of G protein coupled receptors, oncogenes and the like can activate the two pathways, while D2 is a family member of GPCRs, and recently, the ligands of the GPCRs are proved to possibly activate the signal pathway of ERK 1/2.
The medicinal part of bupleuri radix is dried root of Bupleurum scorzonerifolium or Bupleurum scorzonerifolium of Umbelliferae. Pungent and bitter in flavor and slightly cold in nature. The total saikosaponin is the main component of Chinese herbal medicine bupleurum, and the saikosaponin D has the following chemical structural formula, and has the functions of protecting liver, resisting inflammation, resisting tumor, resisting virus, resisting epilepsy and the like.
Figure 100002_DEST_PATH_IMAGE002
。
Disclosure of Invention
The invention provides a new application of saikosaponin D, namely the application of saikosaponin D in preparing dopamine receptor D2L antagonist, saikosaponin D can antagonize dopamine receptor D2L;
the saikosaponin D is used for preparing an antagonist of a dopamine receptor D2L, namely the saikosaponin D is used as an active ingredient or is compounded with other active ingredients to play a role, and can be added with one or more pharmaceutically acceptable auxiliary materials in the preparation of the dopamine receptor antagonist, wherein the auxiliary materials comprise conventional filling agents, diluents, adhesives, excipients, absorption promoters, surfactants, stabilizers and the like in the pharmaceutical field so as to improve the absorption effect of the antagonist or facilitate the administration of the antagonist, and the saikosaponin D is prepared into capsules, pills, powder, tablets, granules, oral liquid and the like.
The invention adopts a Western Blot method to confirm that the saikosaponin D has the function of antagonizing dopamine receptor D2L to phosphorylate downstream ERK 1/2; the D2L antagonist provided by the invention provides a candidate compound for the development of a medicine for treating the bidirectional emotion.
Drawings
FIG. 1 is a schematic diagram of the molecular docking 3D of saikosaponin D with dopamine D2L in example 1;
FIG. 2 is a schematic diagram of molecular docking 2D of saikosaponin D with dopamine D2L in example 1;
FIG. 3 is a VSV expression profile of the stable cell line verified in example 2;
FIG. 4 is a graph showing the results of the phosphorylation level and expression level of ERK1/2 downstream of D2L after saikosaponin D treated cells; wherein, the a picture is a Western Blot result picture, and the b picture is a column chart of the Western Blot gray scale scanning value.
Detailed Description
The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited thereto, and the methods in the examples are performed according to the conventional methods unless otherwise specified, the reagents used are commercially available reagents or prepared according to the conventional methods, and the media are commercially available products and used according to the instructions.
Example 1: computer molecular docking
In this embodiment, a molecular docking calculation method is used to assist in predicting the interaction between a ligand molecule and a target, and the specific steps are as follows:
1. preparation and optimization of Small molecules
(1) Using software ChemDraw to draw saikosaponin D, and storing in sdf format;
(2) opening ligand micromolecules on a software MOE operation interface, and processing the ligand micromolecules into a 3D structure;
(3) the structure of the ligand is simply optimized by using a Minimize function to obtain a reasonable conformation, and an mdb-format database is newly built for the optimized conformation;
2. protein receptor preparation
(1) Obtaining the three-dimensional Structure of the dopamine receptor D2L protein from the PDB database of the target protein (PDB ID:6CM 4)
(2) Introducing a protein structure into software MOE, deleting water molecules and organic solvents, only reserving protein and original substrate ligands, setting docking parameters and selecting a docking activity pocket as Ligand Atoms;
3. molecular docking
The ligand and the receptor with well defined pocket are subjected to molecular docking, and the path of the stored molecular database is input into a dialog box. The initial docking generates 50 constellations and outputs 3 docking results for reference, with the remaining parameters remaining at the default settings. The criteria for evaluation are mainly seen as S, i.e. Score, the more negative the value indicates that small molecules of this conformation bind more tightly to the ligand.
4. Butt joint result
Molecular docking verifies that the binding sites of saikosaponin D and D2L are in the same active pocket as the antagonist sulpiride, and the saikosaponin D, Ser-193, Asp-114 and Cys-118 residues form H hydrogen bond interaction by binding with Score-7.89 kcal/mol, and the results are shown in figures 1 and 2.
Example 2: western Blot technology is used for detecting influence of saikosaponin D on phosphorylation level and expression of ERK1/2 signal channel downstream of D2L
1. Construction of pcDNA5/FRT/TO-VSV-GluR5-D2L-Turquoise plasmid
(1) Construction of linearized pcDNA5/FRT/TO-VSV-D2L-Turquoise vector
According TO The principle of The clontech kit, in pcDNA5/FRT/TO-VSV-GluR5-OX 1-Turquose plasmid (using Xu T R, Ward R J, Petriani J D, et al. The orexin OX1 receptor existants predominated as a homomodimer in The basal state: potential regulation of receptor organization by body organization and antigen organization [ J]Biochemical Journal, 2011, 439(1):171. method construction in literature) was based on the design of upstream and downstream primers for linearized OX1 (upstream primer Clon-OX 1-line-F: 5 'GCGGCCGCCATGGTGAGCAAGGGCGAGGAG', the downstream primer Clon-OX 1-line-R is: 5 'CTTACCAAGGCGGTTCATTTCGATATCGGT'), obtaining a linearized vector by PCR reaction, and recovering and purifying the linearized vector; wherein the PCR reaction system (50. mu.L) is 10. mu.L of 5 XGXL buffer, 4. mu.L of dNTP, 2. mu.L of Clon-OX 1-line-F (10. mu. mol/L), 2. mu.L of Clon-OX 1-line-R (10. mu. mol/L), 1. mu.L of template DNA (pcDNA5/FRT/TO-VSV-GluR 5-OX 1-Turquose plasmid), 1. mu.L of GXL primer star GXL Enzyme, 30. mu.L of ddH2O; and (3) PCR reaction conditions: at 95 ℃ for 3 min; at 95 ℃, 10s, 60 ℃, 20s, 68 ℃, 3min, 25 cycles; 72 ℃ for 10 min;
(2) amplification of fragments of human dopamine receptor 2 (D2L) order
Extracting total RNA of human HEK293 cells by a Trizol method, synthesizing a cDNA first chain by using reverse transcriptase RevertAID RT and taking the total RNA as a template, wherein a reaction system and an operation process are as follows: taking 2 mu g of Total RNA, sequentially adding 1 mu of oligo (dT) 18Primer and DEPC water to the reaction volume of 12 mu L; after mixing, heating and denaturing at 65 ℃ for 5min, then quickly cooling on ice for 5min, then sequentially adding 4 muL of 5 × Reaction buffer, 2 muL of dNTP mix (10 nm), 1 muL of RevertAID RT and 1 muL of LRibolock RNase inhibitor, mixing uniformly and centrifuging for a short time, bathing at 42 ℃ for 1.0h, taking out, heating at 70 ℃ for 5min, terminating the Reaction, synthesizing the first strand of cDNA, and then storing at-20 ℃ for later use.
Amplifying a target gene D2L by using the synthesized first strand cDNA as a template; the sequences of the upstream primer and the downstream primer are respectively that: 5' -ATGAACCGCCTTGGTATGGATCCACTGAATCTGTCC-3' and the downstream primer are: 5'-GCCCTTGCTCACCATTCAGCAGTGGAGGATCTTCAGG-3' are provided. Amplifying a target gene by utilizing Takara GXLDNA polymerase; the reaction system (50. mu.L) was 1. mu.L of GXL Enzyme, 10. mu.L of 5 XGXL buffer, 4. mu.L of dNTP, 1. mu.L of cDNA, 1. mu.L of Clon-D2L-F (10. mu. mol/L), 1. mu.L of Clon-D2L-R (10. mu. mol/L), 32. mu.L of ddH2O; and (3) PCR reaction conditions: at 95 ℃ for 3 min; 95 ℃, 10s, 60 ℃, 20s, 68 ℃, 60s, 72 ℃, 10min, 30 cycles; 72 ℃ for 10 min; after the PCR is finished, agarose gel electrophoresis is carried out, and the target fragment is recovered and purified according to the specificity and the size of the amplified product.
(3) Construction of pcDNA5/FRT/TO-VSV-GluR5-D2L-Turquoise plasmid
The Kit used was In-Fusion HD Cloning Kit; the connection reaction system and the operation process are as follows: taking 3 mu L D2L gene fragment, 1 mu L linearized vector and 1 mu L5 XInfusion HD Enzyme premix, uniformly mixing, and treating at 37 ℃ for 15 min; treating at 50 deg.C for 15 min; standing on ice for 5 min; transferring the ligation product into escherichia coli DH5 alpha by adopting a heat shock transformation method, screening positive clones by using LB solid culture medium containing ampicillin (Amp), selecting a plurality of single colonies, shaking the colonies, identifying clones with a multi-cloning site inserted with D2L by using specific primers for amplifying D2L, selecting a plurality of single colonies, shaking the colonies, identifying clones with a multi-cloning site inserted with D2L by using specific primers for amplifying D2L, sequencing the identified clones, and finally obtaining the total-length cDNA of D2L which is 1364 bp;
the sequence obtained by sequencing is compared through NCBI BLAST, and the result shows that the D2L receptor gene is correctly connected, and no base deletion and mutation exist, thereby indicating that the pcDNA5/FRT/TO-VSV-GluR 5-D2L-Turquose plasmid is successfully constructed.
2. Screening for stable inducible expression cell lines
The HEK293 cells in good growth state are digested by 0.25% pancreatin (containing 0.1% EDTA), 1mL of culture medium is added to stop digestion, the cells are blown off from the wall, then the cells are transferred to a 1.5mL centrifuge tube and centrifuged at 800rpm for 3min, after centrifugation, the supernatant is discarded, and 1mL of culture medium is added to be mixed uniformly to prepare cell suspension. Taking out the blood counting chamber and the cover glass from the alcohol cylinder, drying,sucking 10 mu L of cell suspension liquid by using a pipette gun, slowly dripping from one side of a blood counting chamber, sucking about 10 mu L of cell suspension liquid by using the pipette gun, and slowly dripping from the other side of the blood counting chamber until a gap between a cover glass and the counting chamber is filled with liquid; the total number of cells in the four corners (16 squares per corner) was counted under a microscope in the direction of the arrow. Obtaining the required volume V = M/C of the cell suspension according to the cell density and the required total number M of the cells; the culture medium V' =10mL-V which needs to be supplemented is calculated. Uniformly mixing the V' volume culture medium and the V somatic cell suspension; taking a six-hole plate, adding the uniformly mixed cell suspension into each hole according to 2mL, and shaking the six-hole plate along the front, rear, left and right directions after adding the cell suspension to uniformly disperse cells in the hole plate; observing cell density under microscope, and placing at 37 deg.C under CO2Culturing in a constant temperature incubator.
When the cell growth state is good and the cell density reaches 60-70%, starting to perform a cell transfection experiment; putting the Opti-MEM culture medium into a 37 ℃ water bath kettle in advance for preheating; the original culture medium of the cells was aspirated by pipette, 2ml of PBS was aspirated to wash the cells and PBS was aspirated away; then, 3mL of the preheated Opti-MEM medium was aspirated into the cells, and the cells were incubated in a 37 ℃ incubator. During this time, 2 dry sterilized 1.5mL centrifuge tubes were taken to prepare solution a: 240 μ L of Opti-MEM medium +10 μ L of lipofectamine 2000/well, left for 5 min; solution b: 245 μ L of Opti-MEM medium + 0.3 μ g of plasmid (pcDNA5/FRT/TO-VSV-GluR 5-D2L-Turquose) +2.7 μ g of plasmid POG 44; then mixing the solution a and the solution b to construct a D2L stable cell line for later use; the mixture was added to 25cm with a pipette2The culture bottle is gently shaken and then is uniformly placed in a constant temperature incubator at 37 ℃; after 6-8h, fresh DMEM complete medium (containing 10% FBS) was replaced and the culture was continued.
After culturing the transfected cells for 36h, the cells were cultured from 25cm2The culture flask is completely subcultured to 175cm2The medium used during the culture IN the flask was DMEM (FLP-IN 293) + 10% FBS.
Transfected cells were at 175cm2The density in the culture bottle reaches 80 percent, and the Hygromycin B is added to screen the transfected cells. The medium was DMEM (system 100 mL: containing 10% FBS, 1% P/S, 6.6. mu.L of Blasticidin, 440. mu.L of Hygromycin B). Changing fresh culture medium every four days. After 16 days, when a cell colony is obvious on the wall of the culture flask, the cell colony can be digested by pancreatin, after centrifugation, fresh DMEM culture medium (the system is 100 mL: containing 10% FBS, 1% P/S, 6.6 muL of Blasticidin and 120 muL of Hygromycin B) is added again, and the mixture is placed in a constant temperature incubator at 37 ℃ for culture; the expression of VSV of the D2L stable cell line is verified by Western Blot experiment, VSV expression of transient transfected cells (positive control) is taken as a control, and the result shows that the stable inducible expression cell line is successfully constructed, and the result is shown in figure 3.
3. Western-Blot method for detecting effect of saikosaponin D on D2L downstream ERK channel
Paving a board: the D2L stable cell line was plated in 12-well plates in DMEM (100 mL: 10% FBS, 1% P/S, 6.6. mu.L Blasticidin, 120. mu.L Hygromycin B);
induction: when the cells grow to about 70-80% of the dish, inducing with 30ng/mL doxycycline for 24 h;
③ hungry: starvation treatment for 12h, wherein the starvation medium is DMEM (system is 100 mL: containing 1% P/S, 6.6. mu.L of Blasticidin and 120. mu.L of Hygromycin B); cell harvest was stimulated with drugs set as blank (DMSO 10)- 5mol/L), non-specific serum activated group (FBS), and agonist Dopamine hydrochloride group (Dopamine 10)-6mol/L), antagonist Sulpiride and agonist dopamine hydrochloride (Sulpiride 10)-5 mol/L + Dopamine10-6mol/L), different concentration groups of saikosaponin D (10)-5 mol/L、10-6 mol/L、10-7 mol/L、10-8 mol/L、10-9 mol/L、10-10 mol/L、10-11mol/L); wherein, the corresponding holes of the antagonist sulpiride and the group of dopamine hydrochloride and the holes of the group of saikosaponin D with different concentrations are respectively added with the corresponding antagonist sulpiride10-5Incubating M and saikosaponin D for 10min, and adding dopamine hydrochloride 10-6Stimulating for 5min by mol/L;
through Western Blot experiments, whether a D2L stable cell line is normally expressed or not is detected by using a tag antibody with VSV, and the result shows that the D2L stable cell line can cause the increase of ERK phosphorylation level under the stimulation of a specific agonist dopamine hydrochloride, so that the constructed D2L stable cell line can be further proved to be effective; compared with cells treated by agonist, the D2L stable cell line after being treated by saikosaponin D can cause phosphorylation of ERK in a certain range, and the two groups have significant difference, which indicates that saikosaponin D can antagonize D2L receptor, and the result is shown in FIG. 4.
Sequence listing
<110> university of Kunming science
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Claims (1)

1. The application of saikosaponin D in preparing dopamine receptor antagonist, wherein the saikosaponin D has the following chemical structural formula:
Figure DEST_PATH_IMAGE002
。
CN202111323094.2A 2021-11-10 2021-11-10 New application of saikosaponin D Pending CN113813278A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102283910A (en) * 2011-08-05 2011-12-21 浙江省中医药研究院 Chinese medicinal composition with anti-depression effect and preparation and preparation method thereof
CN105079016A (en) * 2014-12-01 2015-11-25 宁夏医科大学 Application of saikoside compounds in preparation of drug for treating neurodegenerative diseases
CN110179809A (en) * 2019-06-18 2019-08-30 山西大学 Saikoside B2 is preparing the application in anti-depression drug

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102283910A (en) * 2011-08-05 2011-12-21 浙江省中医药研究院 Chinese medicinal composition with anti-depression effect and preparation and preparation method thereof
CN105079016A (en) * 2014-12-01 2015-11-25 宁夏医科大学 Application of saikoside compounds in preparation of drug for treating neurodegenerative diseases
CN110179809A (en) * 2019-06-18 2019-08-30 山西大学 Saikoside B2 is preparing the application in anti-depression drug

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
LI等: "Saikosaponin D relieves unpredictable chronic mild stress induced depressive-like behavior in rats: involvement of HPA axis and hippocampal neurogenesis", PSYCHOPHARMACOLOGY, pages 3385 *
XU等: "Modulation of LPA1 receptor-mediated neuronal apoptosis by Saikosaponin-d A target involved in depression", NEUROPHARMACOLOGY, pages 150 *
张云等: "柴胡皂苷D基于PI3K/AKT/FoxO1调节神经炎症发挥抗抑郁作用", 实用药物与临床, vol. 24, no. 5, pages 395 *
李传朋等: "逍遥散及其类方与有效成分抗抑郁作用机制研究进展", 中国实验方剂学杂志, vol. 26, no. 6, pages 243 *
李妍;彭顺利;张巍;吕士杰;何哲;罗军;朱文赫;: "柴胡皂苷d对SH-SY5Y细胞生长增殖的影响", 毒理学杂志, vol. 27, no. 5, pages 353 *
李明亚: "中华医学百科全书 临床医学 麻醉学", 中国协和医科大学出版社, pages: 392 *

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