CN113101296B - Application of 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside in preparation of anti-inflammatory drugs - Google Patents

Abstract

The application provides an application of 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside in preparing anti-inflammatory drugs. The 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside can inhibit NO release, inhibit secretion of IL-1 beta, IL-6 and TNF-alpha, inhibit expression of iNOS and COX-2 proteins, and inhibit expression of NF-kappa B, so as to play an anti-inflammatory role, and further be used for preparing anti-inflammatory drugs. Further, the pharmaceutical composition containing 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside can also be used for preparing anti-inflammatory drugs.

Description

Application of 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside in preparation of anti-inflammatory drugs

Technical Field

The application relates to the technical field of medicines, in particular to application of 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside in preparation of anti-inflammatory medicines.

Background

Inflammation is a fundamental pathological process mainly based on a defense response caused by the stimulation of living tissues having a vascular system to various injury factors. The basic pathological changes are deterioration, exudation and hyperplasia, and the clinical manifestations of the disease are red, swelling, heat, pain and dysfunction, and the general reactions including fever, leukocytosis, hepatosplenomegaly and the like. Inflammation can be divided into acute inflammation and chronic inflammation according to duration. Acute inflammation is the rapid reaction of an organism to inflammatory factors, aims to convey leucocytes and plasma proteins to an inflammation focus, kill and eliminate the inflammatory factors and mainly generate vascular reaction and leucocyte reaction; the duration is often several days, generally not more than a month. Chronic inflammation refers to inflammation lasting for weeks or even years, with a continuous inflammatory response, tissue damage and repair that accompanies, and is mostly delayed by acute inflammation. Chronic inflammatory stimuli over a long period of time can cause a variety of chronic diseases such as obesity, cardiovascular disease, type 2 diabetes, tumors, and the like. At present, steroidal and non-steroidal anti-inflammatory drugs which are commonly used clinically cause serious adverse reactions, so that the search for anti-inflammatory drugs with good curative effects and small side effects is an urgent problem to be solved.

Natural products are a promising source of anti-inflammatory drugs. The Cimicifuga foetida is a perennial herb plant of Ranunculaceae (Ranunculaceae) Cimicifuga genus (Cimicifuga), and researches show that the Cimicifuga foetida has the effects of resisting inflammation, viruses, osteoporosis, oxidation and the like. The chemical components of the Cimicifuga foetida mainly comprise triterpenes and glycosides thereof, phenolic acids, chromones and other compounds, 23-O-acetyl cimicifugal-3-O-alpha-L-arabinoside (23-O-diacetylshengmanol-3-O-alpha-L-arabinopyranoside, DA) is firstly found in the overground part of the Cimicifuga foetida (Cimicifuga foetida L.), is one of the glycoside components in the Cimicifuga foetida, and the pharmacological activity of DA is not researched and reported at present.

Disclosure of Invention

The present inventors have found, through intensive studies, that 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside has an anti-inflammatory effect, and have completed the present application based on this.

In a first aspect of the application, there is provided the use of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside in the manufacture of an anti-inflammatory medicament.

A second aspect of the application provides a pharmaceutical composition comprising 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside.

A third aspect of the present application provides the use of a pharmaceutical composition of the second aspect of the present application in the manufacture of an anti-inflammatory medicament.

The 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside can inhibit NO release, inhibit secretion of IL-1 beta, IL-6 and TNF-alpha, inhibit expression of iNOS and COX-2 proteins, and inhibit expression of NF-kappa B, so as to play an anti-inflammatory role, and further be used for preparing anti-inflammatory drugs. Further, the pharmaceutical composition containing 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside can also be used for preparing anti-inflammatory drugs.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art that other embodiments can be obtained by using the drawings without creative efforts.

Figure 1 shows the effect of DA on RAW264.7 cell viability.

FIG. 2 shows the effect of DA on NO content released from RAW264.7 cells.

FIG. 3 shows the effect of DA on IL-1 β, IL-6 and TNF- α levels in RAW264.7 cells.

FIG. 4 shows the effect of DA on the expression of iNOS, COX-2 protein in RAW264.7 cells.

FIG. 5 shows the effect of DA on NF- κ B expression in HEK293 cells.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application are within the scope of protection of the present application.

In a first aspect of the application, there is provided the use of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside in the manufacture of an anti-inflammatory medicament.

"anti-inflammatory" as used herein includes the treatment and/or prevention of acute inflammation, and/or the treatment and/or prevention of chronic inflammation. The term "inflammation" as used herein refers to acute inflammation and/or chronic inflammation. The inventor finds in research that a plurality of inflammatory mediators and/or proinflammatory cytokines are involved in the inflammatory reaction process, and the anti-inflammatory drug disclosed by the application plays an anti-inflammatory role by regulating the secretion or expression of the inflammatory mediators and/or the proinflammatory cytokines. The inflammatory mediators and proinflammatory cytokines include, but are not limited to, NO, TNF-alpha, IL-1 beta, IL-6, and the like.

The acute inflammation mentioned in the application is the rapid inflammatory reaction of the body to the inflammatory factors, aims to transport the leucocytes and plasma proteins to the inflammation focus, kill and eliminate the inflammatory factors, and mainly generates vascular reaction and leucocyte reaction. The acute inflammation includes but is not limited to encephalitis B, viral hepatitis, acute enteritis, gouty arthritis or pericarditis, etc. Chronic inflammation as referred to in this application refers to inflammation that lasts for weeks or even years, wherein a continuous inflammatory response, tissue damage and repair response occur concomitantly. Chronic inflammation, which may be delayed by acute inflammation; also occult without acute inflammatory processes; or during the interval of repeated episodes of acute inflammation. The chronic inflammation of the application includes but is not limited to osteoarthritis, rheumatoid arthritis, colitis, ankylosing spondylitis or vasculitis, etc.

The term "treatment" has its ordinary meaning herein, and refers herein in particular to the treatment of a mammalian subject (preferably a human) already suffering from inflammation with a medicament of the present application in order to produce a therapeutic, curative, palliative, etc. effect on the disease. Similarly, the term "prevention" as used herein has its ordinary meaning and refers herein in particular to the treatment of a mammalian subject who may or is at risk of developing inflammation with a medicament of the present application in order to produce a preventing, arresting, abrogating, etc. effect on the disease.

In some embodiments of the first aspect of the present application, the 23-O-acetocimetiol-3-O- α -L-arabinoside exerts an anti-inflammatory effect by at least one of inhibiting NO release, inhibiting secretion of IL-1 β, IL-6, and TNF- α, inhibiting expression of iNOS and COX-2 proteins, or inhibiting expression of NF- κ B.

A second aspect of the application provides a pharmaceutical composition comprising 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside.

In some embodiments of the second aspect of the present application, the 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is provided in the form of a monomer, or in the form of a plant extract comprising the same. Preferably, the plant extract is cimicifugae rhizoma extract. The extraction method of cimicifuga foetida extract is reported in the prior art, and the details are not repeated herein, and those skilled in the art can obtain cimicifuga foetida extract by any existing method.

In some embodiments of the second aspect of the present application, the content of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is 1-99% based on the total weight of the pharmaceutical composition.

In some embodiments of the second aspect of the present application, the content of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is 20-80% based on the total weight of the pharmaceutical composition.

In some embodiments of the second aspect of the present application, the content of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is 40-60% based on the total weight of the pharmaceutical composition.

In some embodiments of the second aspect of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

Herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.

The "pharmaceutically acceptable carrier or excipient" useful in the pharmaceutical compositions of the present application may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. The pharmaceutically acceptable carrier or excipient described herein may be at least one selected from the group consisting of a solvent, a diluent, a dispersant, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a binder, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetening agent, an ion exchanger, a mold release agent, a coating agent, a flavoring agent, and an antioxidant.

As used herein, the term "pharmaceutical composition" has its ordinary meaning. In addition, the "pharmaceutical composition" of the present application may also be present or provided in the form of a health product, a functional food, a food additive, or the like. The pharmaceutical compositions of the present application can be prepared by conventional techniques in the pharmaceutical field, particularly in the formulation field, by obtaining the active ingredients of the raw materials of the pharmaceutical compositions of the present application by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers or excipients, and then forming the desired dosage form. The pharmaceutical composition according to the present application is a pharmaceutical formulation which may be suitable for oral, parenteral or topical, topical administration, especially for oral administration. Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.

A third aspect of the present application provides the use of a pharmaceutical composition of the second aspect of the present application in the manufacture of an anti-inflammatory medicament.

The instrument comprises: a cell culture box: likang biomedical science and technology Consortium Co., ltd; an enzyme-labeling instrument: shanghai Diken trade company, inc.; western Blotting (Western Blotting): bio-Rad, USA; a centrifuge: edengendepforf, germany; luciferase reporter assay system: promega, WI, USA.

Materials: RAW264.7 mouse macrophages: american type culture Collection (American type culture Collection)ATCC); HEK293 cells: ATCC (American ginseng). 23-O-acetocimicifugal-3-O- α -L-arabinoside (DA): the purity of the Chengdou method biological technology limited company is more than 98 percent; phosphate Buffered Saline (PBS), DMEM medium: BI Ltd; endotoxin (LPS): sigma Corp; thiazole blue (MTT, M2128): sigma company; dimethyl sulfoxide (DMSO, cell culture grade): sigma Corp; naNO ₂ And (3) standard substance: shanghai Biyuntian biotechnology, inc.; NO detection kit (S0021S): shanghai Biyuntian biotechnology, inc.; mouse IL-1 beta, IL-6, TNF-alpha enzyme linked immunosorbent assay (ELISA) kit: american R&Company D; RIPA lysate, protease/phosphatase inhibitor cocktail: kang is a century biotechnology limited; BCA Protein concentration Assay Kit (BCA Protein Assay Kit, PC 0020): beijing Solaibao science and technology, inc.; SDS-PAGE gel preparation kit: kang is a century biotechnology limited; protein Marker: thermo Scientific PageRuler ^TM Prestained Protein Ladder;10 × electrophoresis buffer, 10 × transfer solution, 20 × TBST: beijing Solaibao science and technology, inc.; immobilon-PPVDF (0.45 μm 26.5 cm. Times.3.75m roll 1/Pk): immobilon-P corporation; skim Milk powder (BD-Difco Skim Milk, 232100): BD Difco corporation; horseradish enzyme labeled secondary antibody goat anti-rabbit IgG: cell Signaling Technology, usa; iNOS, COX-2 antibody: cell Signaling Technology, usa; β -Actin antibodies: santa Cruz Biotechnology, USA; developing solution: millipore, USA; albumin Bovine serum Albumin (BSA, A8010-5): sigma Corp; PMSF solution: beijing Solaibao science and technology, inc.; NF- κ B luciferase reporter plasmid: tianjin Soromen Biotechnology Ltd; luciferase reporter plasmid pRL-TK: promega, wis., USA; polyethyleneimine (PEI): merck, germany; dexamethasone: sigma corporation, usa.

The experimental materials and methods used in the following examples are, unless otherwise specified, conventional materials and methods.

Macrophages are the major cells involved in innate immunity and are activated when invaded by foreign pathogens such as parasites, bacteria, viruses or stimulated by external signals. Lipopolysaccharide (LPS), a component of the outer wall of the cell wall of gram-negative bacteria, is an endotoxin that stimulates macrophages and induces cells to produce various inflammatory mediators and proinflammatory cytokines, such as secretion of NO, tumor Necrosis Factor (TNF) -alpha, interleukin (IL) -1 beta, IL-6, iNOS, COX-2, etc., thereby inducing an inflammatory response. RAW264.7 cells are mouse macrophage-like cell lines, LPS stimulation of RAW264.7 mouse macrophages becomes a well-known cell model for researching anti-inflammatory drugs, and LPS stimulation of RAW264.7 mouse macrophages is used as an inflammation model in the application.

Example 1 Effect of DA on the viability of RAW264.7 cells

1.1 cell culture

RAW264.7 mouse macrophage cells were cultured in DMEM medium (complete medium) containing 10% fetal bovine serum, 100U/mk penicillin, 0.1mg/ml streptomycin, and 30mg/ml L-glutamine at 37 ℃ with 5% CO ₂ The cells were placed in a constant temperature incubator and passaged every 2 days.

1.2 cell viability assay

Taking RAW264.7 cells in logarithmic growth phase, scraping with scraper, blowing off cells, adjusting cell concentration to 1 × 10 ⁴ One/ml, seeded in 96-well cell culture plates, 100. Mu.l/well. Respectively adding 50 mu L/hole LPS and 50 mu L/hole DA into each experimental group to ensure that the final concentration of LPS is 1 mu g/ml and the final concentration of DA is 2 mu mol/L, 10 mu mol/L, 25 mu mol/L, 50 mu mol/L and 100 mu mol/L respectively; model group was added 50. Mu.l/well LPS and 50. Mu.l/well complete medium to a final concentration of LPS of 1. Mu.g/ml; adding 100 mul/hole complete culture medium into the control group; blank is complete medium without cells. Culturing in a cell incubator for 24h. The supernatant in the cell plates was discarded, and 20. Mu.l of MTT solution (5 mg/ml) and 100. Mu.l of DMEM medium were added to each well, and incubation was continued for 4h. The supernatant in the cell plate was discarded, 100. Mu.l DMSO solution was added to each well, the mixture was shaken on a micro shaker for 10min, and after the crystals were completely dissolved, the absorbance (OD) of each well at 492nm was measured using a microplate reader. The measured OD values were compared to determine the effect of LPS at a final concentration of 1. Mu.g/ml on the viability of RAW264.7 cells based on the former concentration of DA. Cell viability (%) = [ (OD value of experimental group or model group-blank OD value) ÷ (OD value of control group-blank OD value)]X 100% of control groupThe cell survival rate is 100%, the cell survival rate results of each group are shown in figure 1, and the results show that LPS with the final concentration of 1 mu g/ml and DA with the final concentration of less than 100 mu mol/L added on the basis have no significant influence on the growth of RAW264.7 cells, so that the final concentration of LPS used subsequently is 1 mu g/ml, and the drug concentration is within 100 mu mol/L.

Example 2 DA inhibits NO Release in RAW264.7 cells

2.1 NaNO ₂ Standard solution preparation

Precise absorption of NaNO ₂ Adding 10 μ L standard (1 mol/L) into 90 μ L DMEM medium containing 10% fetal calf serum, penicillin 100U/mk, streptomycin 0.1mg/ml, and L-glutamine 30mg/ml, and mixing to obtain 100mmol/L NaNO ₂ The solution is prepared into 10mmol/L NaNO by the same method ₂ The solution is prepared into 100 mu mol/L NaNO by the same method ₂ The solution is diluted in sequence with the concentration as the maximum concentration to obtain NaNO with the concentration of 100, 60, 40, 20, 10, 5, 2, 1, 0 mu mol/L ₂ And (4) standard solution.

2.2 Determination of NO content

Taking RAW264.7 cells in logarithmic growth phase, scraping with scraper, centrifuging, blowing off cells, adjusting cell concentration to 1 × 10 ⁴ Each well was inoculated in a volume of 100. Mu.l/well into a 96-well cell culture plate. Respectively adding 50 mu L/hole LPS and 50 mu L/hole DA into each experimental component to ensure that the final concentration of the LPS is 1 mu g/ml and the final concentration of the DA is 25 mu mol/L, 50 mu mol/L and 100 mu mol/L respectively; model group was added with 50. Mu.l/well LPS and 50. Mu.l/well complete medium to a final concentration of LPS of 1. Mu.g/ml; adding 100 μ l/well of complete culture medium to the control group; incubate in incubator for 24h. Taking 100 μ l/well of each group of cell culture supernatant and NaNO at each concentration in step 2.1 ₂ And placing 100 mul/well of the standard solution in a 96-well plate, adding Griess Reagent I and Griess Reagent II at room temperature in sequence according to 50 mul/well, keeping out of the light, oscillating on a micro oscillator for 3min, and measuring the absorbance value of each well at the wavelength of 560nm of an enzyme marker. Determination of NaNO at the respective concentrations in step 2.1 ₂ Obtaining standard curve from the standard solution, and calculating NO of each group according to the standard curve ₂ ^- Content based on NO ₂ ^- The equivalence (1),the NO content was obtained and the results are shown in FIG. 2. As shown in FIG. 2, the amount of NO released from RAW264.7 cells was significantly increased under LPS stimulation of 1. Mu.g/ml in the model group (n =5, # # # p)<0.001, compared with the control group), indicating that LPS can obviously induce RAW264.7 cells to release NO; the NO content in the experimental group (labeled 25, 50, 100 in the figure) was significantly lower than in the model group (n =5, ^* p<0.05, ^*** p<0.001, compared with a model group), shows that DA has obvious inhibition effect on NO release of RAW264.7 cells in an activated state and has good dose dependence, and shows that DA has anti-inflammatory effect.

Example 3 DA inhibits secretion of IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells

Taking RAW264.7 macrophage in logarithmic growth phase, scraping with scraper, centrifuging for 5min, blowing off cells to obtain cell suspension, adjusting cell concentration to 1 × 10 ⁴ Cells were seeded at this concentration in 96 well cell culture plates at 100. Mu.l/well. Respectively adding 50 mu L/hole LPS and 50 mu L/hole DA into each experimental group to ensure that the final concentration of LPS is 1 mu g/ml and the final concentration of DA is 25 mu mol/L, 50 mu mol/L and 100 mu mol/L respectively; model group was supplemented with 50. Mu.l/well LPS and 50. Mu.l/well complete medium to a final concentration of LPS of 1. Mu.g/ml; adding 100 mul/well of complete culture medium into a control group; at 37 ℃ and 5% CO ₂ And (5) incubating for 24h in an incubator. The supernatant of the cell culture solution was collected, and the OD value of each well was measured at a wavelength of 450nm in an ELISA reader, according to the instructions of the ELISA kit. The amounts of IL-1. Beta., IL-6 and TNF-. Alpha.in each group were calculated according to the corresponding standard curves prepared in the kit instructions, and the results are shown in FIG. 3. As can be seen from FIG. 3, the amount of IL-1 β, IL-6 and TNF- α released from RAW264.7 cells was significantly increased under LPS stimulation in the model group (n =6, # # # p)<0.001, compared with the control group), indicating that LPS can remarkably induce RAW264.7 cells to synthesize and release IL-1 beta, IL-6 and TNF-alpha inflammatory cytokines in large quantity. Whereas the levels of IL-1 β, IL-6 and TNF- α were significantly reduced in each experimental group (labeled 25, 50, 100 in the figure) after DA treatment (n =6, ^* p<0.05, ^*** p<0.001, compared with a model group), namely the generation or release of IL-1 beta, IL-6 and TNF-alpha is obviously reduced, which shows that DA can inhibit the secretion of IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells, has good dose dependence and has better anti-inflammatory effect.

Example 4 DA inhibition of iNOS, COX-2 protein expression in RAW264.7 cells

4.1 extraction of protein sample to be tested

Taking RAW264.7 macrophage in logarithmic growth phase, scraping with scraper, centrifuging at 1000r/s for 5min, blowing off cells to obtain cell suspension, and adjusting cell concentration to 20 × 10 ⁴ Cells at this concentration were plated at 2 ml/well in 6-well cell culture plates. Respectively adding 50 mu L/hole LPS and 50 mu L/hole DA into each experimental group to ensure that the final concentration of LPS is 1 mu g/ml and the final concentration of DA is 25 mu mol/L, 50 mu mol/L and 100 mu mol/L respectively; model group was added 50. Mu.l/well LPS and 50. Mu.l/well complete medium to a final concentration of LPS of 1. Mu.g/ml; the control group was added with 100. Mu.l/well of complete medium; and (5) incubating in a constant temperature incubator for 24h. The cells were rinsed 2 times with pre-cooled PBS (2 ml/well) and then treated with lysis buffer containing 1% protease inhibitor, 1% phosphatase inhibitor and 98% RIPA, and the cell lysates were collected and transferred to centrifuge tubes. Cracking on ice for 30min, and spinning 20s every 10min in the cracking process. Centrifuging at 4 deg.C for 15min and 13000g, collecting supernatant, measuring and adjusting protein concentration to about 1.5 μ g/μ l. Adding 5 Xloading Buffer solution (Loading Buffer) according to volume, sealing, boiling in boiling water bath for 10min, subpackaging, and storing each group of protein samples to be detected at-20 ℃ for later use.

Wherein, the standard curve preparation includes: mu.l of 10. Mu.g/. Mu.l BSA stock solution was diluted with 90. Mu.l PBS solution to obtain 1. Mu.g/. Mu.l BSA. 0, 2, 4, 8, 12, 16, 18, 20. Mu.l of 1. Mu.g/. Mu.l BSA were pipetted into 96-well plates, and 20. Mu.l/well was made up with PBS, and 2 wells were repeated. Preparing BSA working solution of 1. The absorbance values measured were taken as ordinate and BSA concentration as abscissa to prepare a standard curve.

Determining and adjusting protein concentration includes: after each group of supernatant obtained by centrifugation is diluted by 20 times, the mixture is mixed evenly and added into a 96-well cell culture plate according to 20 mul/well, and 3 wells are repeated. Preparing BSA working solution of 1. The protein content of each histone extract liquid is calculated by a standard curve, and then the protein concentration is adjusted to about 1.5 mug/mul.

4.2 immunoWestern blot analysis

Preparing 10ml of 8% separation gel solution: 4.8ml of pure water, 2.7ml of a 30% acrylamide-methylenebisacrylamide solution (Acr-Bis, 29. Recovering the isopropanol in the upper layer. Preparing 4ml of 5% concentrated glue solution: 2.28ml of purified water, 30% Acr-Bis (29) 0.68ml, SDS-PAGE gel buffer concentrate (4X) 1ml, 10% APS 0.04ml, TEMED0.004ml, immediately injected into the space between the glass plates, vertically inserted with a comb, taking care to avoid mixing air bubbles. And after the concentrated gel is solidified, putting the glass plate into an electrophoresis tank, filling electrophoresis buffer solution into the electrophoresis tank, and removing the comb.

And sequentially adding 10 mu l of each group of protein samples to be detected into the loading channel, respectively adding 4 mu l of pre-dyed protein molecular weight Marker into lanes at two sides, and adding 1 Xelectrophoresis buffer solution with the same volume into blank holes. And (3) performing 90V constant voltage electrophoresis, wherein when the electrophoresis of the protein sample to be detected reaches the junction of the concentrated gel and the separation gel, the 120V constant voltage electrophoresis is changed, and the time is 1.5h. And stopping electrophoresis when the Loading Buffer electrophoresis reaches the position 0.5cm away from the rubber border. And (3) cutting off electrophoresis channels and corner cutting marks required by the separation gel according to a protein molecule Marker, and immersing the electrophoresis channels and the corner cutting marks into the membrane transferring liquid for later use.

Cutting two parts of PVDF membranes (cutting angle marks) of 5 multiplied by 8cm, sequentially soaking in methanol, water and membrane transferring liquid for 30s respectively, recovering the methanol, and keeping the membranes in the membrane transferring liquid for later use. And soaking the gel, the PVDF membrane and the filter paper in the membrane transfer buffer solution for 5min. The following sequence of placement was followed: 1 layer of sponge net, 1 layer of filter paper, gel, PVDF film, 1 layer of filter paper and 1 layer of sponge net to expel air bubbles. And (3) placing the electrode plate into a film transfer instrument, and pouring a film transfer liquid. The film is flowed for 1.5h at 300mA constant current.

And (3) sealing: the PVDF membrane is immersed in 5% skimmed milk powder of a confining liquid and incubated for 2 hours at room temperature. Primary anti-incubation: the PVDF membrane was removed, immersed in a diluted primary antibody (1. Washing the membrane: PVDF membrane immersed in 1 × TBST, placed in the shaking table vibration washing membrane 4 times, each time for 7min. And (3) secondary antibody incubation: adding horseradish enzyme labeled secondary goat anti-rabbit IgG. Secondary antibody was diluted with 1 × TBST at 1. Incubate at room temperature for 1.5h with shaking. Washing the membrane: PVDF membrane immersed in 1 × TBST, placed in the shaking table vibration washing membrane 4 times, each time for 7min.

The PVDF membrane was removed with forceps, adhered appropriately (without drying), placed in a developing solution containing 50% solution A and 50% solution B, gently shaken, and incubated for 30-60min while covering the light-shielding box. And taking out the strip, putting the strip into a gel imager, operating a gel imaging program, exposing for 1-7min, developing the result shown as a graph A of FIG. 4, and quantitatively analyzing the result of the graph A by using ImageJ, wherein the result is shown as a graph B.

As shown in FIG. 4, the protein contents of iNOS and COX-2 in the model group were significantly increased compared to the control group by using β -Actin as an internal reference correction gene (n =3, # # p)<0.01, compared to the control group), indicating that LPS can significantly increase protein expression of iNOS and COX-2; in the experimental group (25, 50, 100 in the figure) to which DA treatment was applied, the protein contents of iNOS and COX-2 were significantly reduced compared to the model group (n =3, ^** p<0.01, ^*** p<0.001, compared with the model group), indicating that DA can significantly inhibit the expression levels of iNOS and COX-2 proteins in RAW264.7 cells.

Example 5 DA inhibits expression of NF-. Kappa.B

NF-kB is used as a transcription regulator necessary for the expression of various inflammation mediators, and has a very key role in regulating inflammatory response. This example uses a luciferase reporter gene to detect the effect of DA on NF-. Kappa.B expression in HEK293 cells.

HEK293 cells were transfected with the luciferase reporter plasmid pRL-TK (10 ng/well) as a reference plasmid, and the NF-. Kappa.B luciferase reporter plasmid pGL4.32 (100 ng/well). HEK293 cells at 3X 10 ⁴ The density of individual/well was seeded in 96-well plates; tube A: mu.g pGL4.32 plasmid and 0.96. Mu.g pRL-TK reference plasmid were dissolved in 100. Mu.L serum-free DMEM medium and mixed well. And a tube B: dissolving 1 mu L of 1mg/mL Polyethyleneimine (PEI) transfection reagent in 100 mu L of serum-free DMEM medium, uniformly mixing, and standing at room temperature for 5min; c, pipe C: dissolving tube A and tube BAdding the solution into a serum-free DMEM culture medium C tube containing 9.4mL, uniformly mixing, and standing for 20min to prepare a transfection solution; 100 μ L of the transfection solution was added to each well of the 96-well plate and transfected for 24h. The transfection solution was then discarded and a drug solution was added to each well to stimulate the cells. Adding 100 μ L of serum culture medium (DMEM culture medium containing 10% fetal calf serum) into the control group; adding 100 mu L of serum culture medium containing TNF-alpha into the model group, wherein the final concentration of TNF-alpha is 20ng/mL; 100 mu.L of serum culture medium containing TNF-alpha and DA with different concentrations are respectively added into each experimental group, and the final concentration of DA is 1X 10 ^-4 、1×10 ^-5 、1×10 ^-6 mol/L, and the final concentration of TNF-alpha is 20ng/mL. Dexamethasone (DExmedetomidine, DEX) as positive control group is added into serum culture medium containing TNF-alpha and positive control drug DEX at 100 μ L to give DEX final concentration of 1 × 10 ^-6 mol/L, and the final concentration of TNF-alpha is 20ng/mL. After 6h, the luciferase activity was detected by the luciferase reporter assay system after washing, lysis and detection of each set of HEK293 cells. Calculating the expression level (RA) of the NF-. Kappa.B luciferase plasmid relative to the reference plasmid: inhibition (%) = [ RA (model group) -RA (Experimental group)]X 100%/RA (model group). The inhibition rate reflects the inhibition effect on NF-. Kappa.B expression in HEK293 cells, wherein the inhibition rate of the control group is 100%, the inhibition rate results of each group are shown in FIG. 5, and DA (10 in the figure) with different concentrations can be seen from FIG. 5 ^-4 、10 ^-5 、10 ^-6 ) The expression of NF- κ B can be inhibited (n =5, ^** p<0.01, ^*** p<0.001, compared to model group), the effect is dose-dependent; as can be seen from FIG. 5, the signal level is 1 × 10 ^-6 The inhibiting effect of DA on NF-kB is equivalent to that of dexamethasone under the concentration of mol/L and is 1 multiplied by 10 ^-4 The inhibiting effect of DA on NF-kB is obviously better than that of dexamethasone under the mol/L concentration.

In conclusion, DA can inhibit the release of NO, inhibit the secretion of inflammatory factors IL-1 beta, IL-6 and TNF-alpha, inhibit the expression of iNOS and COX-2 proteins, inhibit the expression of NF-kB and play an anti-inflammatory role, thereby being used for preparing anti-inflammatory drugs. Further, the pharmaceutical composition comprising DA can also be used for the preparation of an anti-inflammatory agent.

The above description is only for the preferred embodiment of the present application, and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application are included in the protection scope of the present application.

Claims (7)

1. Use of 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside as sole active component in preparing anti-inflammatory medicine is disclosed.
2. 2. The use of claim 1, wherein the 23-O-acetocimicinol-3-O- α -L-arabinoside exerts an anti-inflammatory effect by at least one of inhibiting NO release, inhibiting secretion of IL-1 β, IL-6 and TNF- α, inhibiting expression of iNOS and COX-2 proteins, or inhibiting expression of NF- κ B.
3. 3. Use of a pharmaceutical composition in the preparation of an anti-inflammatory medicament, wherein the only active ingredient in the pharmaceutical composition is 23-O-acetocimicifugal-3-O-alpha-L-arabinoside.
4. 4. The use according to claim 3, wherein the 23-O-acetyl cimicifugal alcohol-3-O-alpha-L-arabinoside is extracted from Cimicifuga foetida.
5. 5. The use according to claim 3, wherein the content of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is 20-80% based on the total weight of the pharmaceutical composition.
6. 6. The use according to claim 5, wherein the content of 23-O-acetocimicifugal alcohol-3-O- α -L-arabinoside is 40-60% based on the total weight of the pharmaceutical composition.
7. 7. The use of any one of claims 3-6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

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