CN118078926A - Application of pharmaceutical composition in preparation of medicine for treating gouty arthritis - Google Patents

Abstract

The invention provides an application of a pharmaceutical composition in preparation of medicines for treating gouty arthritis, wherein the pharmaceutical composition consists of phellodendron bark and coix seed or extracts of the phellodendron bark and the coix seed; the extract is caffeic acid ethyl Ester (EC), sinapic Acid (SA), 4-Allylcatechol (APC). The invention discovers for the first time: the phellodendron bark and the coix seed or active ingredients thereof have synergistic effect on treating gouty arthritis and can inhibit inflammatory reaction, thereby correcting the 'immune-inflammatory' imbalance of gouty arthritis damp-heat syndrome; moreover, both of these representative active ingredients can reduce abnormal heat production by the body, thereby ameliorating energy metabolic disorders.

Description

Application of pharmaceutical composition in preparation of medicine for treating gouty arthritis

Technical Field

The invention belongs to the field of traditional Chinese medicines, and in particular relates to an application of a pharmaceutical composition in preparation of a medicine for treating gouty arthritis.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Gouty arthritis (gouty arthritis, GA) is a clinically common disease, and currently, the prevalence of gout tends to rise year by year, and the age of onset tends to be younger. The disease is mainly characterized by joint redness, pain and limited movement, is most commonly caused by first toe joints, is closely related to personal life habit, has abnormal purine metabolism and/or reduced uric acid excretion of organisms caused by long-term irregular diet, seafood eating, drinking and the like, causes rise of blood uric acid, and has high incidence and disability rate due to precipitation and deposition of monosodium urate on joints. The guidelines for gout management at the American society of rheumatology in 2020 recommended colchicine, non-steroidal anti-inflammatory drugs and glucocorticoids as drugs for the treatment of GA, and surgical excision was used if tophus was large and severely affected patient-life. Modern pharmacology proves that colchicine and benzbromarone are easy to cause liver and kidney functions to be damaged after long-term administration, other diseases are complicated, and adverse reactions of the central nervous system and gastrointestinal tracts occur in part of patients. The GA treatment of the traditional Chinese medicine has the advantages of long history, obvious curative effect, small adverse reaction and the like, and accumulates rich experience in long-term clinical practice.

The damp-heat arthralgia granule is produced by Liaoning medical nurse (group) limited company, and the approval document is national medicine standard Z20044063. The prescription is composed of rhizoma Atractylodis, caulis Lonicerae, lumbricus, fructus forsythiae, cortex Phellodendri, coicis semen, radix Saposhnikoviae, radix Cyathulae, rhizoma Dioscoreae Septemlobae, ramulus Mori, radix Stephaniae Tetrandrae, and radix Clematidis, and has effects of dispelling pathogenic wind, removing dampness, clearing heat, relieving swelling, dredging collaterals, and relieving pain. Can be used for treating damp-heat arthralgia with symptoms of red swelling, heat pain, heavy feeling, difficult walking, fever, thirst, and yellow urine. The traditional Chinese medicine composition is mainly applied to the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, cervical and lumbar pain and other bone joint diseases caused by damp-heat blockage at present. The Chinese medicine cortex phellodendri and coix seed medicine pair stored in the prescription are commonly combined in different prescriptions to treat gouty arthritis. However, there have been no reports of the treatment of the disease in the form of drug pairs.

Disclosure of Invention

In order to solve the problems, the invention provides an application of a pharmaceutical composition in preparing medicines for treating gouty arthritis. The invention discovers for the first time: the phellodendron bark and the coix seed or active ingredients thereof have synergistic effect on treating gouty arthritis and can inhibit inflammatory reaction, thereby correcting the 'immune-inflammatory' imbalance of gouty arthritis damp-heat syndrome; moreover, both of these representative active ingredients can reduce abnormal heat production by the body, thereby ameliorating energy metabolic disorders.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the invention provides an application of a pharmaceutical composition in preparing medicines for treating gouty arthritis, wherein the pharmaceutical composition consists of extracts of phellodendron bark and coix seed or both;

The extract is caffeic acid ethyl Ester (EC), sinapic Acid (SA), 4-Allylcatechol (APC).

Cortex Phellodendri is dry bark of cortex Phellodendri or cortex Phellodendri of Rutaceae. The former is called Chuan Bai Zhu and the latter is called Guan Bai, which is bitter and cold in nature and has the actions of clearing heat and drying dampness, purging fire and steaming, and removing toxin and curing sores. Has antibacterial, anticoccidial, anti-hepatitis, antiulcer, antitussive, antihypertensive, and immunity regulating effects, and can be used for treating epidemic cerebrospinal meningitis, bacillary dysentery, pneumonia, pulmonary tuberculosis, liver cirrhosis, chronic hepatitis, trichomonas vaginitis, acute conjunctivitis, chronic suppurative otitis media, chronic maxillary sinusitis, and ear eczema.

The coix seed is a dry mature seed kernel of the coix seed belonging to the Gramineae family, is a common medicament for inducing diuresis and excreting dampness, is common and frequently eaten food, has sweet, light and slightly cold nature, is rich in starch and various amino acids required by human bodies, has the effects of inducing diuresis and detumescence, strengthening spleen and eliminating dampness, relaxing tendons and removing arthralgia, clearing heat and expelling pus and the like, and is suitable for symptoms such as spleen deficiency diarrhea, heavy muscle, arthralgia, spasm and stretching of tendons, edema, beriberi, leucorrhea, lung abscess, appendicitis and the like. Has effects in tranquilizing mind, relieving fever, relieving inflammation, relieving pain, reducing blood sugar and blood calcium, and enhancing immunity.

Further, the gouty arthritis is gouty arthritis with unbalanced immunity-inflammation and energy metabolism disorder of damp-heat syndrome.

Further, the pharmaceutical composition inhibits inflammatory response and corrects immune-inflammatory imbalance of gouty arthritis damp-heat syndrome by down-regulating TLR4/PI3K/AKT/NETs signaling pathway protein expression; in addition, each component in the pharmaceutical composition can regulate abnormal expression of AMPK/PPARalpha/Mitophagy signal pathway proteins, reduce abnormal heat generation of organisms and improve energy metabolism disorder.

Further, the concentration ratio of the caffeic acid ethyl Ester (EC) to the Sinapic Acid (SA) is 0.125-4:0.125-4.

Further, the concentration ratio of the caffeic acid ethyl Ester (EC) to the Sinapic Acid (SA) is 1:2.

Further, the concentration ratio of the Ethyl Caffeate (EC) to the 4-Allylcatechol (APC) is 6.25-200:6.25-200.

In a second aspect, the invention provides application of phellodendron bark and coix seed or extracts thereof in synergistic inhibition of gouty arthritis inflammation, correction of immune-inflammation imbalance of gouty arthritis damp-heat syndrome, and reduction of abnormal heat generation of organisms, thereby improving energy metabolism disorder.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising: cortex Phellodendri and Coicis semen, or their extracts.

Further, the method further comprises the following steps: rhizoma Atractylodis, caulis Lonicerae, lumbricus, fructus forsythiae, radix Saposhnikoviae, radix Cyathulae, rhizoma Dioscoreae Septemlobae, ramulus Mori, radix Stephaniae Tetrandrae, and radix Clematidis.

In a fourth aspect of the present invention, a preparation method of a pharmaceutical composition is provided, wherein cortex phellodendri and coix seed are taken and decocted with water twice, the two extracts are combined, filtered, the filtrate is concentrated into fluid extract, 3 times of ethanol is added, the fluid extract is stood for 12 hours, the supernatant is taken and concentrated into fluid extract with a relative density of 1.33-1.35, and the fluid extract is freeze-dried into powder, thus obtaining the pharmaceutical composition.

The beneficial effects of the invention are that

(1) The invention verifies the synergistic effect of the representative active ingredients EC and SA of the damp-heat arthralgia particles at animal and cell levels, inhibits inflammatory reaction, reduces abnormal heat production of organisms, thereby correcting the 'immune-inflammatory' imbalance of damp-heat syndrome of gouty arthritis and improving energy metabolism disorder. The damp-heat arthralgia granule can correct organism 'immune-inflammation' unbalance by inhibiting NETs formation induced by neutrophil autophagy, and relieve inflammatory reaction of affected joint tissues, and induce severe pain and tissue injury; inhibit excessive activated mitochondrial autophagy in adipose tissue, prevent oxidation of fatty acid and heat production, thereby improving abnormal energy metabolism of organism, and relieving symptoms such as fever, dry mouth, and yellow urine. Thereby realizing the treatment of gouty arthritis.

(2) The invention successfully analyzes 256 in-vitro components of the damp-heat arthralgia particles by UPLC-Q-TOF-MS/MS means. By means of integrated molecular docking virtual screening, micro thermophoresis, cell experiment verification and the like, the synergistic effect of the representative active ingredient EC and SA of the damp-heat arthralgia particles is found, and inflammatory reaction is inhibited by down-regulating TLR4/PI3K/AKT/NETs signal channel protein expression, so that the 'immune-inflammatory' imbalance of damp-heat syndrome of gouty arthritis is corrected; the representative active components EC and APC of the damp-heat arthralgia granule can regulate abnormal expression of AMPK/PPARalpha/Mitophagy signaling pathway proteins, reduce abnormal heat generation of organisms, and improve energy metabolism disorder. The animal model of gouty arthritis is established to explore the molecular mechanism and action characteristics of the treatment of damp-heat arthralgia particles, and the wet-heat arthralgia particles are found to inhibit the formation of NETs induced by neutrophil autophagy by regulating and controlling a PI3K/AKT/NETs signal path, so that the immune-inflammatory imbalance of an organism is corrected, the inflammatory reaction of affected joint tissues is lightened, and severe pain and tissue injury are induced; and the AMPK/ACC signal axis can be regulated and controlled, excessive activated mitochondrial autophagy in adipose tissue is inhibited, and oxidation and heat production of fatty acid are prevented, so that abnormal metabolism of organism is improved, and symptoms such as fever, dry mouth, yellow urine and the like are relieved.

(3) The invention has simple formula, strong practicability and easy popularization.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

Fig. 1 is a view of the BPI of the internal components of the damp-heat arthralgia granule. (a) positive ion mode; and (B) negative ion mode.

FIG. 2 is a schematic representation of molecular docking of representative components EC, SA and key proteins of TLR4/NETs signaling pathways in the damp-heat arthralgia granules. (a) EC structures; (B) SA structure; (C) molecular docking diagram of PAD4 with representative component; (D) molecular docking diagram of TLR4 with representative components.

FIG. 3 binding of representative components EC and SA in the damp-heat arthralgia granules to key proteins of TLR4/NETs signaling pathway. (A) MST determination of binding force of PAD4 to EC; (B) effect of different doses of EC on PAD4 protein function; (C) effect of different doses of EC on TLR4 protein function; (D) MST measuring the binding force of PAD4 and SA; (E) effect of different doses of SA on PAD4 protein function; (F) Effect of different doses of SA on TLR4 protein function. n=3, n±s, #p <0.05, #p <0.01, #p <0.001vs Con; * P <0.05, < P <0.01, < P <0.001vs Model.

FIG. 4 combination of EC and SA components can inhibit NETs formation in cells. (a) immunofluorescent staining of the fets; (B) CitH relative fluorescence intensities; (C) Relative fluorescence intensity of MPO .Scale bar＝50μm,n＝3,n±s,#P<0.05,##P<0.01,###P<0.001vs Con;*P<0.05,**P<0.01,***P<0.001vs Model;$P<0.05,$$P<0.01,$$$P<0.001vs EC;&P<0.05,&&P<0.01,&&&P<0.001vs SA;％P<0.05,％％P<0.01,％％％P<0.001vs EC+SA.

FIG. 5 combination of EC and SA components inhibits NETs formation through TLR4/PI3K/AKT/NETs signaling pathway. (A) CitH3, p65, p-p65, PAD4, PKC, p-PKC, TLR4, PI3K, p-PI3K, AKT and p-AKT protein bands ;(B)CitH3;(C)TLR4;(D)PAD4;(E)p65;(F)p-p65;(G)p-p65/p65;(H)PKC;(I)p-PKC;(J)p-PKC/PKC;(K)PI3K;(L)p-PI3K;(M)p-PI3K/PI3K;(N)AKT;(O)p-AKT;(P)p-AKT/AKT.n＝3,n±s,#P<0.05,##P<0.01,###P<0.001vs Con;*P<0.05,**P<0.01,***P<0.001vs Model;$P<0.05,$$P<0.01,$$$P<0.001vs EC;&P<0.05,&&P<0.01,&&&P<0.001vs SA;％P<0.05,％％P<0.01,％％％P<0.001vs EC+SA.

Figure 6 is a schematic diagram of molecular docking of representative components EC, SA and AMPK/pparα signal pathway key proteins in damp-heat arthralgia granules. (A) an APC framework; (B) molecular docking diagram of pparα with representative component; (C) molecular docking diagram of AMPK with representative component.

FIG. 7 shows the binding of the representative components EC and APC to the key proteins of the AMPK/PPARα signaling pathway in the granule for damp-heat arthralgia. (A) MST assay of binding force of PPARα to EC; (B) effect of different doses of EC on pparα protein function; (C) effect of different doses of EC on AMPK protein function; (D) MST assay of pparα binding to APC; (E) effect of different doses of APC on pparα protein function; (F) Effect of different doses of APC on AMPK protein function. n=3, n±s, #p <0.05, #p <0.01, #p <0.001vs Con; * P <0.05, < P <0.01, < P <0.001vs Model.

FIG. 8EC and SA composition combination may promote lipid deposition in cells. (a) lipid deposition nile red staining; (B) Relative fluorescence intensity of lipid .Scale bar＝100μm,n＝3,n±s,#P<0.05,##P<0.01,###P<0.001vs Con;*P<0.05,**P<0.01,***P<0.001vs Model;$P<0.05,$$P<0.01,$$$P<0.001vs EC;&P<0.05,&&P<0.01,&&&P<0.001vs APC;％P<0.05,％％P<0.01,％％％P<0.001vs EC+APC.

FIG. 9EC and APC component combinations inhibit fatty acid oxidation .(A)AMPK;(B)p-AMPK;(C)p-AMPK/AMPK;(D)PPARα;(E)PGC-1α;(F)p62;(G)LC3Ⅱ;(H)Beclin1;(I)Parkin;(J)PINK1;(K)SIRT1;(L)UCP1;(M)AMPK,p-AMPK,SIRT1,PGC-1α,PPARα,PINK1,p62,Parkin,Beclin1,LC3Ⅱ and UCP1 protein bands through AMPK/PPARα/Mitophagy signaling pathway .n＝3,n±s,#P<0.05,##P<0.01,###P<0.001vs Con;*P<0.05,**P<0.01,***P<0.001vs Model;$P<0.05,$$P<0.01,$$$P<0.001vs EC;&P<0.05,&&P<0.01,&&&P<0.001vs APC;％P<0.05,％％P<0.01,％％％P<0.001vs EC+APC.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.

Example 1 Material

1.1 Laboratory animals and cells

Male SPF-grade SD rats weighing 180+ -20 g and 6-8 weeks old were purchased from Fukang Biotechnology Co., ltd., beijing, animal license SCXK (Beijing) 2020-0004. The animal center of the Chinese traditional medicine department of academy of basic theory of Chinese traditional medicine research institute feeds, models and observes. Animals were free to eat and feed water during the experiment and had normal circadian rhythms.

Human acute myeloid leukemia HL-60 cells were purchased from Pricella, cultured in 80% Iscove Modified Dulbecco Media (IMDM) medium and 20% Fetal Bovine Serum (FBS) and supplemented with 100units/ml penicillin and 100. Mu.g/ml streptomycin, the incubator environment was set at 37℃and 5% CO ₂ concentration.

Myoblast line C2C12 cells were purchased from Pricella, cultured in 90% Dulbecco's Modified Eagle Medium (DMEM) high sugar medium and 10% fetal bovine serum and added with 100units/ml penicillin and 100. Mu.g/ml streptomycin, the incubator environment was set at 37℃and 5% CO ₂ concentration.

1.2 Experimental drugs and reagents

TABLE 1 pharmaceutical and reagent for experiment

1.3 Instrumentation

Table 2 instrumentation

Example 2 Experimental methods

1. In vivo component profiling

1.1 Administration and serum collection

About 75g of damp-heat arthralgia particles are taken, and water is added to dissolve the particles to prepare a solution of about 3.75 g.mL ^-1, and the solution is taken as a rat gastric lavage solution. Rats were randomly divided into 5 groups of 6 animals, one of which was blank and the other was experimental, and the experimental groups were intragastric at 10 times the clinically equivalent dose (25 g/kg). The control group was perfused with an equal volume of physiological saline each time. After 0.5, 1,2 and 4 hours of administration, the experimental groups were anesthetized with pentobarbital, the abdominal aorta was respectively bled in blood collection tubes pre-coated with heparin sodium, centrifuged for 10min at 3 000r.min ^-1, and the supernatant was stored at-80 ℃.

1.2 Pretreatment of serum samples

Thawing serum at room temperature, mixing rat serum at each time point in equal proportion, taking 1mL of mixed serum, adding 4 times of acetonitrile, centrifuging for 10min at 2min at 12 000r/min by vortex, taking supernatant, blow-drying at room temperature by N ₂, re-dissolving residues by using 100 mu L of 70% methanol, centrifuging for 10min at 12 min at 2min by vortex, and taking supernatant as a test sample of the serum containing the drug. The same method is used for preparing the blank serum test sample.

1.3 Conditions of liquid chromatography

Waters ACQUITY UPLC HSS T ₃ chromatography column (2.1 mm. Times.100 mm,1.8 μm); the flow rate is 0.5mL/min; column temperature 40 ℃; the sample injection amount is 2 mu L; mobile phase a is 0.1% formic acid-water, B is acetonitrile; gradient elution (0-8 min,2-10% B;8-14min,10-15% B;14-20min,15-20% B;20-25min,20-40% B;25-28min,40-98% B;28-31min,98% B).

1.4 Mass Spectrometry Condition

ESI positive and negative ion mode, taper hole voltage 40V, capillary voltage 2 600V, ion source temperature 120 ℃, desolvation gas temperature 400 ℃, desolvation gas flow rate 800 L.h ^-1, taper hole back blowing 50 L.h ^-1, mass scanning range m/z 50-1 500, collision energy: low energy 4V, gradient high energy 10-30V, collision gas: high purity argon.

2 Molecular docking

The 3D structure of the representative blood-in component is downloaded through PubChem (https:// pubchem. Ncbi. Nih. Gov /) database, the crystal structure of the core receptor protein TLR4 (PDB ID:3VQ 2), PAD4 (PDB ID:5N 0M), AMPK (PDB ID:4 CFF), PPARα (PDB ID:1I 7G) is downloaded through RCSB PDB (http: www.rcsb.org /) database, the pdbqt file of the core receptor protein is obtained by means of PyMol and AutoDock Vina software, the molecular docking of the active component and the core receptor protein is further realized, and finally the docking result is visually presented by means of Discovery Studio software.

3 Micro thermophoresis

MST was used to detect binding of representative blood-entering components to the key proteins pparα, PAD 4. Representative blood components are diluted in a gradient way for 16 concentrations, and are respectively mixed with labeled PPARalpha and PAD4 proteins in an equal volume mode according to the proportion of 1:1 for 15 minutes, and the reaction system is 0.01mol/L p H7.0 PBS. The mixture was drawn by capillary tube and placed in an MST microphoresis apparatus, data were collected and exported, and data were processed using MO. Affinity Analysis v3.0.5 software (NanoTemper Technologies, germany).

4 Cell handling and grouping

The HL-60 cell line was induced to differentiate into dHL-60 (DIFFERENTIATED HL-60, dHL-60) cells, i.e., neutrophils, using complete medium containing 1.25% DMSO. Respectively adding different drugs for treatment: (1) normal control group Con without any drug treatment added; (2) Model group: no drug treatment is added; (3) EC treatment group: after 24h of 10 μM EC treatment; (4) SA treatment group: 20 mu M SA treatment for 24 hours; (5) ec+sa treatment group: 10 μM EC and 20 μM SA together for 24h; (6) Cl-amidine treatment group: 10. Mu.M Cl-amidine was treated for 24h. After 24h, 4. Mu.MA 23187 was added to stimulate cells for 4h in each of the remaining groups except for Con groups.

Myoblast C2C12 cells were cultured in DMEM containing 10% fetal bovine serum, and when the cells were polymerized to about 70%, the culture medium was changed to DMEM containing 2% horse serum to induce differentiation to grow myotubes. After C2C12 differentiation into myotubes, it was divided into (1) normal control group Con without any drug treatment; (2) Model group: cells were stimulated by addition of 500. Mu.M sodium oleate with 250. Mu MPALMITIC ACID; (3) EC treatment group: treatment with 10 μm EC for 24h after induction; (4) APC treatment group: treatment with 30. Mu.M APC for 24h after induction; (5) ec+apc treatment group: treatment with 10. Mu.M EC and 30. Mu.M APC together 24h after induction; (6) GW6471 handles group: after induction, treatment with 30. Mu.M GW6471 was carried out for 24h.

5 Cell Activity assay

HL-60 cells were seeded in 96-well plates and EC (0.125, 0.25, 0.5, 1,2, 4 μm) and SA (0.125, 0.25, 0.5, 1,2, 4 μm) were administered at different concentrations, respectively. C2C12 cells were seeded in 96-well plates and after cell attachment, EC (6.25, 12.5, 25, 50, 100, 200 μm) and APC (6.25, 12.5, 25, 50, 100, 200 μm) were administered at different concentrations, respectively. Treating for 24 hours, adding CCK-8 detection solution, and incubating for 1 hour at 37 ℃. The optical density at 450nm was measured with a microplate reader.

6 Immunoblotting

Taking out a proper amount of ankle joint tissue, cutting, adding RIPA lysate, reacting for 30min, centrifuging, collecting protein supernatant, detecting concentration, taking 30 mug protein, performing SDS-PAGE electrophoresis, transferring PVDF film, sealing at room temperature for 1.5h, incubating the film overnight at the temperature of TLR4, PKC, p-PKC, citH3 and MPO primary antibody for 4 ℃, incubating the film for 1h at the room temperature, developing by using chemiluminescence liquid, and quantitatively analyzing the relative expression condition of the protein by using Image-J software.

Immunofluorescence method 7

After the HL-60 cells were treated, the cells were fixed by adding paraformaldehyde, blocked with BSA at 37℃for 30min, and primary antibodies CitH (abcam, ab5103, 1:200) and MPO (Proteictech, 1:200) were added, respectively, overnight at 4 ℃. PBS is used for cleaning, a corresponding fluorescent secondary antibody (1:1000) is added, incubation is carried out for 1h at the temperature of 37 ℃ in the dark, DAPI is added for nuclear dyeing, anti-fluorescent quenching sealing tablet sealing is added, a laser confocal microscope is used for photographing, and an Image analysis software Image Pro Plus 6.0 is used for analyzing the result.

8 Nile red dyeing

Inoculating C2C12 cells with good growth state to a climbing slice suitable for a 24-well plate, adding different stimulators according to groups on the next day, incubating for 24 hours, discarding the supernatant, fixing 4% paraformaldehyde at room temperature, adding 10 mug/mL nile red dye into each hole, adding 0.5% Triton X-100, incubating at room temperature in a dark place for 5 minutes, then dripping an anti-DAPI-containing anti-fluorescence quenching sealing tablet, and sealing the cover glass. Observing and collecting images under a fluorescence microscope, wherein the excitation wavelength of nile red is 485nm, and the emission wavelength is 565nm; DAPI excitation wavelength 358nm and emission wavelength 461nm.

9 Data analysis and statistics

Statistical analysis was performed using SPSS 26.0, data were allAnd (3) representing. The comparison between groups is carried out by adopting single-factor analysis of variance, and P is less than 0.05, so that the difference has statistical significance.

Example 3 analysis of experimental results

1 In vivo component Spectrum identification

To screen SRBG for a representative bioactive component that ameliorates gouty arthritis, UFLC-Q-TOF-MS/MS was used to detect the blood-in profile of SRBG (fig. 1), resulting in a total of 41 chemical components identified (table 3).

TABLE 3 identification of in vivo Components of damp-heat arthralgia granules

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2EC and SA are representative drug effect substances for treating damp-heat arthralgia particles, correcting inflammatory-immune imbalance pathological links of gouty arthritis damp-heat syndrome

2.1 Potential binding of in vivo Components of the damp-heat arthralgia particles to key proteins of the TLR4/NETs signaling pathway

Molecular docking was used to evaluate the binding affinity of potential bioactive components of damp-heat arthralgia particles to key proteins in TLR4/PI 3K/AKT/nes signaling pathways associated with "inflammation-immunity" imbalance. The results show that the binding force of 21 components to these proteins is less than-5 kcal/mol (Table 4). Wherein, the binding free energy of caffeic acid ethyl ester EC (A in figure 2) in cortex Phellodendri and sinapic acid SA (B in figure 2) in Coicis semen with TLR4 and PAD4 is low. TRP347 in PAD4 protein can form hydrocarbon covalent bond with EC, intermolecular interaction force of pi-pi accumulation with SA (C in fig. 2), SER406 can form hydrocarbon covalent bond with EC and SA, so that the binding system is more stable. The intermolecular interaction force (D in figure 2) exists between PHE121 of TLR4 protein and conjugated systems in EC and SA, which is more favorable for the combination of small molecules and proteins.

TABLE 4 molecular docking score for in vivo components of damp-heat arthralgia particles and TLR4/NETs signaling pathway proteins

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2.2EC and SA binding to TLR4/NETs axis critical proteins

The MST experiment directly measures the Kd value of 16.2 mu M (A in figure 3) and the Kd value of 42.5 mu M (D in figure 3) of the PAD4 and the EC, and proves that both the EC and the SA have stronger binding capacity with the PAD4 protein. In addition, PAD4 protein promotes citrullination of histone, thereby forming CitH. In vitro cell experiments prove that the EC and SA at different doses can inhibit CitH and TLR4 proteins (B and C, E and F in figure 3) to different degrees, and the EC of 10 mu M and SA of 20 mu M have better protein inhibition effect. It is further shown that both EC and SA bind to TLR4, PAD4 proteins in vivo, thereby inhibiting TLR4 protein expression and preventing PAD 4-mediated citrullination of histone.

EC and SA may act as representative components of damp-heat arthralgia particles to correct "immune-inflammatory" imbalance by acting on TLR 4/nes signaling axes.

2.3 Combinations of EC and SA components inhibit NETs formation

The core of the traditional Chinese medicine prescription is compatibility, and in order to better represent the pharmacodynamic action of the prescription, the invention combines the representative component EC obtained in the monarch drug with the representative component SA obtained in the ministerial drug, and examines the pharmacodynamic action of the monarch drug so as to more clearly explain the prescription compatibility and the action mechanism of the damp-heat arthralgia granule.

A large number of CitH and MPO exist on the NETs, and the positions of the NETs can be positioned after co-dyeing the NETs. The immunofluorescence method detects CitH and MPO in HL-60 cells, and reflects the formation of NETs. The results show that CitH and MPO in the Model group have significantly higher fluorescence intensity, and that EC, SA and combinations thereof can effectively inhibit NETs formation in HL-60 cells after administration (P <0.001, FIG. 4), and particularly, the composition combination has stronger efficacy in inhibiting NETs formation.

2.4 Combination of EC and SA Components through TLR4/PI3K/AKT/NETs signaling pathway, correction of "inflammation-immune" imbalance in gouty arthritis damp-heat syndrome

To further verify the effects of EC and SA, the effects of EC, SA and combinations thereof on TLR4/PI 3K/AKT/nes signaling pathways were examined. As a result, EC, SA and combination drugs thereof can obviously inhibit the formation of HL-60 intracellular NETs induced by A23187, reduce the expression of CitH and PAD4 proteins (figures 5B and 5D), and the combination drugs have stronger inhibition effect on the protein expression. The invention also detects the expression of upstream and downstream proteins TLR4, P-PKC/PKC, P-PI3K/PI3K, P-AKT/AKT and P-NF- κB/NF- κB proteins formed by NETs, and the EC, SA and combined medicaments thereof can obviously inhibit the expression of TLR4, P-PKC/PKC, P-NF- κB/NF- κB proteins (C and E-J in figure 5), promote the expression of P-PI3K/PI3K, P-AKT/AKT proteins (K-P in figure 5) and have better drug effect after combination.

The combination of EC and SA components is proved to be used as a representative bioactive component for clearing heat in damp-heat arthralgia granules, and gouty arthritis inflammation-immunity imbalance is corrected through TLR4/PI3K/AKT/NETs signaling paths.

3EC and APC are representative drug effect substances for improving the pathological links of the damp-heat syndrome energy metabolism disorder of gouty arthritis by damp-heat arthralgia particles

3.1 Potential binding of the in vivo Components of the damp-heat arthralgia particles to proteins in the AMPK/PPARα Signal pathway

Molecular docking was first used to evaluate the binding affinity of potential bioactive components in damp-heat arthralgia particles to key proteins in AMPK/pparα signaling pathways associated with energy metabolism disorders. 21 components can bind to these key proteins (Table 5). Wherein, the binding energy required for the ethyl caffeate EC in the phellodendron bark to dock with 4-allylcatechol APC (A in figure 6) in the weeping forsythia and AMPK and PPARα molecules is lower (B and C in figure 6). CYS276 is a site of action for the intermolecular interactions of EC, APC and pparα forming a conjugated system, and plays an important role in the protein and small molecule binding system (B in fig. 6). In addition, VAL30, LYS45, LEU146 and EC and APC in AMPK can also form intermolecular interaction force of conjugated system, so that the protein and small molecule binding system is more stable (C in FIG. 6).

TABLE 5 molecular docking score of in vivo components of damp-heat arthralgia granules and AMPK/PPARα signal axin

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3.2EC and ACP can bind to key proteins of fatty acid oxidation

MST experiments prove that the binding force of EC and APC with PPARα is 37.8 μm and 21 μm respectively (A and D in FIG. 7), and further prove that both EC and APC have stronger binding ability with PPARα protein. In addition, in vitro cell experiments prove that the EC and the APC can inhibit the expression of the AMPK and PPARα proteins at different doses (B and C, E and F in FIG. 7), and the EC and the APC can bind with the AMPK and the PPARα proteins and perform functions in organisms.

EC and APC can act as representative components of damp-heat arthralgia particles for improving energy metabolism disorder by acting on AMPK/pparα signal axes.

3.3EC and ACP component combinations promote lipid deposition and thus inhibit fatty acid oxidation

Lipid metabolism plays a key role in energy homeostasis, inhibition of fatty acid oxidation in lipid metabolism disorders often occurring with lipid deposition. Nile red is capable of locating and quantifying intracellular lipids, and is commonly used to observe lipid deposition in cells. The results show that EC, APC and combinations thereof all significantly promote free fatty acid induced intracellular lipid deposition of C2C12 (P <0.001, fig. 8), inhibit fatty acid oxidation, and in particular, promote enhanced lipid deposition when EC is combined with APC components.

3.4 Combination of EC and ACP Components through the AMPK/PPARα/Mitophagy Signal pathway, the energy metabolism disorder of gouty arthritis due to damp-heat syndrome is improved

To further verify the effect of EC and APC, the effect of EC, APC and combination drugs thereof on AMPK/pparα/Mitophagy signaling pathways was examined. As a result, it was found that the expression of p-AMP/AMPK and PPARα proteins (A-D in FIG. 9) was significantly reduced by EC, APC and combination drugs thereof, and the inhibition effect of the combination drugs on protein expression was stronger. And the invention also detects that EC, APC and combination drugs thereof can obviously inhibit the expression of fatty acid oxidation related proteins PGC-1 alpha, SIRT1 and UCP1 protein (E, K and L in figure 9), and the drug effect after combination is even equivalent to that of positive drugs. In the early mechanism excavation, the gouty arthritis body energy metabolism disorder is closely related to the mitochondrial autophagy, and further the invention detects the mitochondrial autophagy related proteins p62, beclin1, LC3 II, parkin and PINK1, and the result shows that the EC, the APC and the combined drugs thereof can obviously reduce the expression quantity of the autophagy related proteins (F-J in figure 9) and inhibit the activation of the mitochondrial autophagy.

The EC and the APC can be used as representative bioactive components of dehumidification in damp-heat arthralgia granules, and can promote lipid synthesis through an AMPK/PPARalpha/Mitophagy signal pathway, inhibit fatty acid oxidation and further improve gouty arthritis energy metabolism disorder.

In conclusion, the synergistic effect of the representative active ingredient EC and SA of the damp-heat arthralgia granules can inhibit inflammatory reaction by down-regulating TLR4/PI3K/AKT/NETs signal pathway protein expression, thereby correcting the 'immune-inflammatory' imbalance of the damp-heat syndrome of gouty arthritis; the representative active components EC and APC of the damp-heat arthralgia granule can regulate abnormal expression of AMPK/PPARalpha/Mitophagy signaling pathway proteins, reduce abnormal heat generation of organisms, and improve energy metabolism disorder.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The application of a pharmaceutical composition in preparing medicines for treating gouty arthritis is characterized in that the pharmaceutical composition consists of extracts of phellodendron bark and coix seed or both;

The extract is caffeic acid ethyl ester, sinapic acid, 4-allyl catechol.

2. The use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of gouty arthritis, wherein said gouty arthritis is gouty arthritis with a "immune-inflammatory" imbalance of the damp-heat pattern, an energy metabolism disorder.

3. The use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of gouty arthritis, wherein the pharmaceutical composition inhibits inflammatory response and corrects "immune-inflammatory" imbalance in gouty arthritis damp-heat syndrome by down-regulating TLR4/PI 3K/AKT/nes signaling pathway protein expression; in addition, each component in the pharmaceutical composition can regulate abnormal expression of AMPK/PPARalpha/Mitophagy signal pathway proteins, reduce abnormal heat generation of organisms and improve energy metabolism disorder.

4. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of gouty arthritis, wherein the concentration ratio of ethyl caffeate to sinapic acid is 0.125-4:0.125-4.

5. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of gouty arthritis, wherein the concentration ratio of ethyl caffeate to sinapic acid is 1:2.

6. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of gouty arthritis, wherein the concentration ratio of ethyl caffeate to 4-allylcatechol is in the range of 6.25 to 200:6.25-200.

7. The application of cortex Phellodendri and Coicis semen or their extracts in synergistic inhibition of gouty arthritis inflammation, correction of gouty arthritis damp-heat syndrome of 'immune-inflammation' imbalance, and reduction of abnormal heat generation of organism, thereby improving energy metabolism disorder.

8. A pharmaceutical composition comprising: cortex Phellodendri and Coicis semen, or their extracts.

9. The pharmaceutical composition of claim 8, further comprising: rhizoma Atractylodis, caulis Lonicerae, lumbricus, fructus forsythiae, radix Saposhnikoviae, radix Cyathulae, rhizoma Dioscoreae Septemlobae, ramulus Mori, radix Stephaniae Tetrandrae, and radix Clematidis.

10. A preparation method of a medicinal composition is characterized in that cortex phellodendri and coix seed are taken and decocted twice by adding water, the two extracts are combined and filtered, filtrate is concentrated into fluid extract, 3 times of ethanol is added, standing is carried out for 12 hours, supernatant is taken and concentrated into fluid extract with the relative density of 1.33-1.35, and the fluid extract is freeze-dried into powder, thus obtaining the medicinal composition.