Active substances in lycium ruthenicum murr and applications thereof

Abstract

The application discloses an active substance in Lycium ruthenicum and application thereof, relates to the technical field of natural product separation, and comprises identification of an active substance norpetanin in Lycium ruthenicum and application of the active substance norpetanin in anti-inflammation. The application obtains an active substance norpetanin for the first time, identifies the exact chemical structure of the active substance norpetanin through nuclear magnetic data, and finds that the active substance norpetanin has anti-inflammatory activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product separation, in particular to an active substance in Lycium ruthenicum and application thereof. BACKGROUND

[0002] Lycium ruthenicum is a kind of medicinal and edible plant widely planted in Qinghai Chaidamu Basin and other places in recent years, which has high nutritional and edible value and contains various nutrients such as protein, vitamins and minerals. The fruit also contains rich anthocyanin components, which have antioxidant and anti-allergy functions, and can enhance human immunity and improve sleep.

[0003] Studies have shown that Lycium ruthenicum contains various bioactive components, and its mature berries are rich in polyphenols such as anthocyanins, which have antioxidant, potential prevention and treatment of cardiovascular system diseases and other effects. Among them, polyphenols including anthocyanins and flavonoids exhibit various biological activities such as anti-inflammatory, cardiovascular protection and anti-tumor through their strong antioxidant free radical scavenging effect.

[0004] At present, the research on the active substance of Lycium ruthenicum is still relatively weak, and the exploration, separation and extraction of its active components still need further research. If the new chemical components in Lycium ruthenicum and their pharmacological effects can be studied more deeply and subtly, and the activity mechanism is discussed, more natural plant source drugs that can treat diseases safely and effectively are expected to be developed. SUMMARY

[0005] The purpose of the present application is to provide an active substance in Lycium ruthenicum and application thereof, an active substance norpetanin is obtained for the first time, the exact chemical structure of the active substance norpetanin is identified through nuclear magnetic data, and it is found that the active substance norpetanin has anti-inflammatory activity.

[0006] The technical scheme adopted by the present application is:

[0007] The compound has the structure as shown in general formula I or each optical isomer, each crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof:

[0008]

[0009] Further, the compound is selected from the compounds as shown in formula II or formula III:

[0010]

[0011] A composition comprising the compounds as shown in formula II and formula III, and the ratio of the compounds as shown in formula II and formula III in the composition is 3:1:

[0012]

[0013] Use of the compound for the preparation of a product for the treatment and / or prevention of inflammation-related diseases.

[0014] Use of the composition for the preparation of a product for the treatment and / or prevention of inflammation-related diseases.

[0015] Further, the product is a product for reducing the release amount of at least one of NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS, which are inflammation factors.

[0016] Further, the product is a product for inhibiting the expression of iNOS protein.

[0017] The pharmaceutical composition of the compound I (including cis-trans tautomers and combinations thereof) or solvates, hydrates, pharmaceutically acceptable salts or co-crystals thereof can contain pharmaceutically acceptable adjuvants.

[0018] The "pharmaceutically acceptable" in the present invention means a substance including any effective for not interfering with the biological activity of the active ingredient and being non-toxic to the host to which it is administered.

[0019] The pharmaceutically acceptable excipient is a general term for all additional materials in a drug except the main drug. The excipient should have the following properties: (1) no toxic effect on the human body, little side effect; (2) stable chemical properties, not easily affected by temperature, pH, storage time, etc.; (3) no compatibility contraindication with the main drug, does not affect the efficacy and quality inspection of the main drug; (4) does not interact with the packaging material. The excipient in the present application includes but is not limited to fillers (diluents), lubricants (glidants or anti-adhesion agents), dispersants, humectants, binders, adjusting agents, solubilizers, antioxidants, bacteriostatic agents, emulsifiers, disintegrants, etc. The binder includes syrup, gum arabic, gelatin, sorbitol, tragacanth gum, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin syrup, sugar syrup, starch paste or polyvinylpyrrolidone, etc.; the filler includes lactose, sugar powder, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricant includes micro-powder silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; the disintegrant includes starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone or microcrystalline cellulose, etc.; the humectant includes sodium dodecyl sulfate, water or alcohol, etc.; the antioxidant includes sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl phenyl acid, etc.; the bacteriostatic agent includes 0.5% phenol, 0.3% methyl phenol, 0.5% trichlorobutanol, etc.; the adjusting agent includes hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate and buffer (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; the emulsifier includes polysorbate-80, sorbitan oleate, pluronic F-68, lecithin, soybean phospholipid, etc.; the solubilizer includes tween-80, bile, glycerol, etc. The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present application with an acid or a base suitable for use as a drug. The above-mentioned acid and base are broad Lewis acids and bases. The acid suitable for forming a salt includes but is not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid and other organic acids; and aspartic acid, glutamic acid and other acidic amino acids.

[0020] The administration mode of the compound or pharmaceutical composition of the present application is not particularly limited, and the representative administration modes include but are not limited to: oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0021] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such excipients as (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) moisturizing agents, e.g., glycerol and sorbitol; (h) respiration accelerators, e.g., high-oleic acid cottonseed oil; and (i) lubricants, e.g., magnesium stearate, calcium stearate, stearic acid, glyceryl monostearate, sodium stearyl fumarate, and talc. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.

[0022] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, e.g., enteric coatings and other materials well known in the art. They can optionally contain opacifying agents, and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0023] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and the like, either with or without the addition of such

[0024] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0025] Suspensions, in addition to the active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, and the like.

[0026] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0027] Dosage forms for topical administration of a compound of this application include ointments, powders, patches, sprays, and inhalants. The active component is admixed with a carrier, which can be a sterile powder, a sterile non-fluid, or a sterile fluid.

[0028] The compound of the present application can also be used in injectable formulations. Among them, the injection is selected from liquid injection (water needle), sterile powder for injection (powder needle) or tablet for injection (refers to the tablet made by sterile operation method of medicine, which is dissolved with injection water for subcutaneous or intramuscular injection when used).

[0029] Among them, the injection powder contains at least excipients in addition to the above-mentioned compound. In the present application, the excipient is a component intentionally added to the drug, which should not have pharmacological properties in the amount used, but the excipient can help the processing, dissolution or dissolution of the drug, the delivery of the drug by the targeted drug delivery route or the stability.

[0030] The beneficial effects of the present application are:

[0031] The present application first obtains an active substance norpetanin, identifies the exact chemical structure of the active substance norpetanin through nuclear magnetic data, and finds that the active substance norpetanin has anti-inflammatory biological activity.

[0032] The numbers marked on the structural formula of the compound in the present application are only for the convenience of explaining the structure of the compound. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 norpetanin 1 H NMR chart;

[0034] Figure 2 norpetanin 13 C NMR chart;

[0035] Figure 3 High resolution mass spectrum of norpetanin;

[0036] Figure 4 HMBC chart of norpetanin;

[0037] Figure 5HSQC spectrum of norpetanin;

[0038] Figure 6 COSY spectrum of norpetanin;

[0039] Figure 7 DEPT spectrum of norpetanin;

[0040] Figure 8 UV absorption spectrum of norpetanin;

[0041] Figure 9 Effect of norpetanin at different concentrations on RAW264.7 cell viability;

[0042] Figure 10 Effect of norpetanin on RAW264.7 cell NO release amount;

[0043] Figure 11 Effect of norpetanin on RAW264.7 cell iNOS protein expression. DETAILED DESCRIPTION

[0044] The technical solutions of the present application are described below in a clear and complete manner. Obviously, the embodiments described herein are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present application.

[0045] Example 1

[0046] A separation method of a composition, comprising the following contents:

[0047] (1) 10.0 kg of dry Lycium ruthenicum Murr. fruits are weighed, and soaked in 200 L of methanol under room temperature and light shielding conditions, and extracted for 3 times, each time for 4 days, and extracted once each time, and a total of 3 times; the extract is filtered, and subjected to light shielding and reduced pressure concentration, and combined to obtain 3.643 kg of Lycium ruthenicum Murr. fruit methanol extract infusion.

[0048] (2) The methanol extract infusion prepared in step (1) is mixed with dry polyamide powder at a mass ratio of 1:1, dried in an oven at 40℃, and then ground, and passed through a 20 mesh sieve to obtain sieved powder;

[0049] (3) Take 50.00 g of the sieved powder in step (2) and load into a small medium-pressure chromatography column (26 x 100 mm) and connect a medium-pressure chromatography column (49 x 460 mm) loaded with MCI to perform dry loading; use a water / methanol / dichloromethane three-phase system to elute, 0-120 min, 100% water-100% methanol; 120-180 min, 100% methanol-100% dichloromethane; 180-210 min, 100% methanol; 210-240 min, 100% water; flow rate: 50 mL / min, detection wavelength: 210 nm; a total of three components Fr1, Fr2 and Fr3 are obtained, Fr2 is obtained at a retention time of 39-139 min, purple powder, 511.3 g, yield 15.1%.

[0050] (4) Further separate Fr2 obtained in step (3) to perform gradient elution with methanol and / or water as the mobile phase, elution conditions 0-120 min, 0-100% methanol; 120-140 min, 100% methanol; flow rate: 50 mL / min; detection wavelength: 254 nm; filler: MCI; column size: 49 x 460 mm; retention time 76-130 min to obtain Fr2-5 175.5 g, yield 34.3%.

[0051] (5) Separate Fr2-5 in step (4) and load into a C18 preparative chromatography column with a size of 21.2 x 250 mm and a 5 μm filler to perform gradient elution with water / methanol as the mobile phase: 0-60-65-90 min, 30%-42%-70%-95% methanol, flow rate: 19 mL / min; detection wavelength: 210 nm; sample volume 300 μL, retention time 19-21 min to obtain Fr2-5-3, weighing 5.2 g, yield 29.6%.

[0052] The compound of the structure shown in general formula I:

[0053]

[0054] The compound is selected from the compounds shown in formula II or formula III:

[0055]

[0056] The composition comprises compounds as shown in Formula II and Formula III, and the ratio of the compounds as shown in Formula II and Formula III in the composition is 3:1:

[0057]

[0058] The composition of the application 1 H NMR chart and 13 C NMR chart are shown in Table 1 respectively. Figure 1 and Figure 2 .

[0059] The nuclear magnetic resonance data of the active composition in Lycium ruthenicum Murr. are shown in Table 1.

[0060] Table 1 Nuclear magnetic resonance data of the composition

[0061]

[0062] Composition: yellow powder, ESI-MS m / z: 809 [M-H] - , the molecular formula of the main component is C 36 H 42 O 21 . The m / z value of the main component is 809.2140 [M-H] - , the calculated value is 809.2141, and the unsaturation degree is 19. Its nuclear magnetic resonance spectrum shows a pair of signals of different intensities, indicating the existence of tautomerism. In order to facilitate the structure analysis, the NMR signals of the main trans tautomer are preferentially used for structure elucidation. As shown in Table 1, 1 H NMR (DMSO-d6 / CF3COOD (9:1), 600 MHZ): δ H 7.53 (2H, d, J = 8.5 Hz, H-2”” / H-6””), 6.79 (2H, d, J = 8.5 Hz, H-3”” / H-5””), 7.53 (1H, d, J = 15.9 Hz, H-7””) and 6.35 (1H, d, J = 15.9 Hz, H-8””) signals show a characteristic set of trans acyl groups, δH7.53 (1H, s, H-4) shows an aromatic or olefinic proton singlet, δ H 6.54 (1H, d, J = 1.7 Hz, H-6) and 6.36 (1H, d, J = 1.7 Hz, H-8) signals show a set of 1,3,4,5-tetrasubstituted benzene ring, δ H 5.09 (1H (d), J = 7.3 Hz, H-1’) and 4.82 (1H”) signals show two β-pyranoglucoside anomeric protons, δ H 4.61 (1H, br s, H-1”) signals show α-rhamnosyl anomeric proton, δH 4.82 (1H, t-like, J = 9.6 Hz, H-4") showed an acylated methine triplet state, and the aldar-methyl doublet state was shown in the aldar region. 13 C NMR (DMSO-d6 / CF3COOD (9:1), 600 MHZ): showed 36 carbon resonances in common, including a set of trans acyl carbons, and 18 carbon signals assignable to three sugar moieties, while the remaining 9 unassigned resonances were from the aglycone nucleus. The above NMR features were generally similar to those of petanin, with the main difference being the absence of the ring B signals in the cyanidin nucleus in the present compound, which allowed us to infer that the ring B moiety should be oxidized and degraded to give the special 3,5,7-trioxy substituted coumarin nucleus. The HMBC correlations from H-4 to C-2 [δ C 157.6 (s)], C-5 [δC 154.5 (s)], and C-9 [δ C 152.1 (s)], from H-6 and H-8 to C-10 [δ C 103.1 (s)], and from H-1' to C-3 [δ C 138.0 (s)], as shown in Figure 4 , confirmed this inference. The resulting structure was further verified by careful analysis of HSQC, HMBC, and 1H, 1H-COSY correlations, as shown in Figure 1 , 4 -6. It is worth noting that the new compound was isolated as an inseparable mixture of tautomers (trans:cis ≈ 3:1). Therefore, the structure of the mixture is shown in formula II and formula III, and is named norpetanin. To our knowledge, the mixture norpetanin represents a rare class of ring B cyanidins.

[0063] The DEPT, UV absorption spectrum, and high resolution mass spectrum of norpetanin are shown in Figure 7 , 8 and 3, respectively.

[0064] Example 2

[0065] Evaluation of the anti-inflammatory effect of norpetanin:

[0066] Using the established inflammatory cell model, the effect of norpetanin on the amount of NO released by cells was determined, and the effect of norpetanin on the expression of iNOS protein was explored, and the possible mechanism of action of norpetanin in exerting anti-inflammatory effect was preliminarily discussed.

[0067] 1. Experimental materials and reagents

[0068] 1.1 Experimental materials

[0069] The active composition norpetanin of Lycium ruthenicum Murr in the embodiment 1 of the present application.

[0070] RAW264.7 mouse monocyte macrophage cells were purchased from the China Academy of Sciences Typical Culture Preservation Committee Cell Library, Catalog No. TCM13.

[0071] The test drug was prepared: the active composition norpetanin and the positive control drug dexamethasone were weighed, and a stock solution with a concentration of 100 mM was prepared using DMSO, which was stored in a refrigerator at 4°C. When used, it was diluted as required.

[0072] The materials and reagents used are shown in Table 2:

[0073] Table 2 Experimental materials and reagents

[0074]

[0075]

[0076] 2. Experimental method

[0077] 2.1 MTT method for determining cell viability

[0078] Logarithmically growing RAW264.7 cells were taken at a cell density of 5×10 4 6 / mL, 150 μL per well was inoculated in a 96-well plate, and after incubation in a cell incubator for 24 h, the DMEM medium (containing 2% FBS) containing lipopolysaccharide or the test component was replaced, and the incubation was continued for 24 h. Then 10 μL of thiazolyl blue (MTT) solution was added to each well, and the plate was incubated in a cell incubator for 4 h. Finally, the medium was discarded, and 150 μL of DMSO was added to dissolve the cells. An enzyme label instrument was used to read the absorbance value at a wavelength of 490 nm, and the cell viability was calculated according to the formula. The cell viability calculation formula is as follows:

[0079]

[0080] 2.2 Griess method for determining NO content

[0081] The Griess reagent I and II were taken out of the refrigerator and allowed to recover to room temperature for the experiment. The standard was diluted with DMEM containing 10% FBS to have concentrations of 0, 1, 2, 5, 10, 20, 40, 60, 80, and 100 μM, respectively. In a 96-well plate, 50 μL of the standard and sample solution was added to each well, and 50 μL of Griess reagent I and 50 μL of Griess reagent II were added to each well, respectively. After mixing on a shaking bed, the absorbance value at a wavelength of 540 nm was measured, and the NO content was calculated according to the standard curve.

[0082] 2.3 Western blot analysis

[0083] The experiment was divided into control group, LPS group and LPS + different concentrations of drug group, logarithmic growth period RAW264.7 cells were taken, with a density of 5x10 4 Individuals / mL were inoculated in 6-hole plates, and were incubated in a cell culture box for 24h, then were replaced with 2% FBS-containing DMEM medium, the model group was treated with LPS, the test drug group was treated with LPS + different concentrations of drug group, and was continued to be cultured for 24h, protein was extracted, and electrophoresis was performed. The specific operation method is as follows:

[0084] (1) Extraction of RAW264.7 cell protein

[0085] After the RAW264.7 cells were treated, the cells were lysed, and the cell protein was extracted. The culture solution was pumped out with a liquid pump, and PBS buffer was washed twice, and the PBS was pumped out. Cell lysis solution was added, the 6-hole plate was mixed, and was placed on ice for 10min. The cells were scraped with a cell scraper and collected into a centrifuge tube. The centrifuge tube was placed on ice for 30min. After lysis, the low-temperature centrifuge was used at 12000r / min, 4°C for 15min, and the supernatant containing protein was collected in a new EP tube.

[0086] (2) BCA method for measuring cell protein concentration

[0087] The protein concentration was determined by BCA method. First, the protein standard was prepared into a concentration of 0.5mg / mL with PBS buffer, and BCA working solution was prepared according to the ratio of A liquid:B liquid=50:1, and mixed. Three parallel sets were set up, and the protein standard solution and PBS buffer were added according to Table 3, and the standard curve was drawn. 1μL of cell-extracted protein solution sample was taken and added to a 96-hole plate, and the volume was made up to 20μL with PBS buffer, and then 200μL of BCA working solution was added, and the reaction was carried out at 37°C for 30min. The absorbance value at 562nm was measured by an enzyme label instrument, and the protein concentration of each sample was calculated according to the standard curve. The BCA method for protein quantification is shown in Table 3.

[0088] Table 3 BCA method for protein quantification

[0089]

[0090] (3) Protein denaturation

[0091] Take the diluted protein sample, add protein loading buffer and mix, and denature in a metal bath at 100°C for 15min. After the denatured protein is cooled to room temperature, it is stored in a-20°C refrigerator for standby.

[0092] (4) SDS-PAGE electrophoresis

[0093] Two clean glass plates were aligned and clamped on the gel holder, and ultrapure water was added for leak detection. After leak detection, the ultrapure water was poured out and the residual liquid between the two glass plates was absorbed with a water-absorbing paper, and the glass was prepared for pouring. As shown in Table 4, the appropriate concentration of separation gel (lower gel) was prepared according to the size of the protein molecular weight. According to the protein molecular weight to be detected in the experiment, 10% SDS-PAGE separation gel was prepared according to Table 5, and 5% SDS-PAGE concentrated gel (upper gel) was prepared according to Table 6. About 4 mL of separation gel was added between the two dry glass plates, isopropyl alcohol was added to seal the gel, and the separation gel was allowed to solidify for 1 h. The isopropyl alcohol was poured out, the residual isopropyl alcohol was absorbed with a water-absorbing paper, the concentrated gel was poured in, the comb was inserted to avoid air bubbles, and the concentrated gel was allowed to solidify. The comb was gently pulled out. The two glass plates were clamped on the electrophoresis tank, and the electrophoresis liquid was poured in to make the liquid surface above the glass plate. The protein sample was loaded, and after the loading was completed, electrophoresis was started. First, the sample was concentrated in the concentrated gel at a constant voltage of 80 V for about 30 min, the voltage was adjusted to 120 V, and the electrophoresis was continued at a constant voltage. When the loading buffer reached the bottom of the gel plate, the electrophoresis was stopped. The optimal separation range of SDS-PAGE gel is shown in Table 4; the preparation table of 10% SDS-PAGE separation gel is shown in Table 5; and the preparation table of 5% SDS-PAGE concentrated gel is shown in Table 6.

[0094] Table 4 SDS-PAGE gel optimal separation range

[0095]

[0096] Table 5 10% SDS-PAGE separation gel preparation table

[0097]

[0098] Table 6 5% SDS-PAGE concentrated gel preparation table

[0099]

[0100] (5) Transferring the membrane

[0101] The PVDF membrane was cut to the appropriate size, and a corner was cut off as a marker to distinguish the front and back after cutting the gel. The activated PVDF membrane was soaked in methanol for 1 min. The activated PVDF membrane, foam, and membrane transfer filter paper were soaked in pre-cooled membrane transfer buffer. The glass plate was pried open with a gel cutter, and the gel was cut according to the target protein molecular weight. The membrane was placed in the electrophoresis liquid, the black side of the clamp was placed below, and the foam, filter paper, gel strip, PVDF membrane, filter paper, and foam were placed in order. The clamp was placed in the membrane transfer tank with the black clamp facing the black side of the tank. The membrane liquid was poured in to immerse the clamp plate, and an ice bag was placed in a low temperature environment. The current was set to a constant current of 250 mA. The membrane transfer time was set according to the size of the protein molecular weight.

[0102] (6) Blocking

[0103] Blocking solution is 5% skim milk in lx TBST. After the transfer, take out the PVDF membrane and put it into an incubation box. Add skim milk to cover the PVDF membrane. Place it in a shaker at room temperature for 1 h. Discard the skim milk and wash the membrane with lx TBST for 10 min, 3 times.

[0104] (7) Incubation of primary antibody

[0105] Dilute the primary antibody iNOS (CST, #13120) with the antibody dilution buffer at a ratio of 1:1000. Put the PVDF membrane into the diluted primary antibody solution. Incubate the primary antibody at 4°C overnight. Discard the primary antibody solution and wash the membrane with lx TBST for 10 min, 3 times.

[0106] (8) Incubation of secondary antibody

[0107] Put the PVDF membrane into the secondary antibody solution diluted at a ratio of 1:5000. Incubate the secondary antibody at room temperature for 1 h. Discard the secondary antibody solution and wash the membrane with lx TBST for 10 min, 3 times.

[0108] (9) Development

[0109] Develop the membrane using the ECL chemiluminescence method. Mix the developing solution A and B at a ratio of 1:1. Add 100 μΐ^of the mixture to the PVDF membrane. Develop the membrane using a developing instrument and take a picture.

[0110] 3. Experimental results

[0111] 3.1 Effect of norpetanin on the activity and NO secretion of RAW264.7 cells

[0112] As shown in Table 1, the MTT method was used to detect the effect of active substances on the activity of RAW264.7 cells. The active substance norpetanin did not significantly inhibit the activity of RAW264.7 cells at 0-20 μΜ. However, it inhibited the growth of RAW264.7 cells at 50 μΜ (P < 0.05) and 100 μΜ (P < 0.01) and significantly inhibited the secretion of NO. Figure 9 10 3.2 Effect of norpetanin on the expression of iNOS protein in RAW264.7 cells

[0113] Norpetanin showed good anti-inflammatory activity in inhibiting the release of NO in the inflammation model. Further studies were conducted to investigate the anti-inflammatory mechanism. The expression level of iNOS protein in RAW264.7 cells after treatment with the compound was detected to investigate the effect of the compound on inflammation-related proteins.

[0114] As shown in Table 2, the expression of iNOS protein in RAW264.7 cells was significantly inhibited by norpetanin at 50 μΜ (P < 0.05) and 100 μΜ (P < 0.01). Figure 11 ​As shown, the expression level of iNOS in RAW264.7 cells was up-regulated after LPS stimulation, which had a very significant difference compared with the control group (P<0.01). norpetanin at a concentration of 10 μM can significantly inhibit the expression of iNOS protein (P<0.01), indicating that the compound can play an anti-inflammatory role by inhibiting the expression of iNOS protein to reduce the amount of NO release.

Claims

1. Use of a composition for the manufacture of a product for the treatment and / or prevention of inflammation, characterized in that, The composition includes the compound as shown in Formula II and Formula III, and the ratio of the compound as shown in Formula II and Formula III in the composition is 3:

1. ， 。 2. Use of a composition according to claim 1 for the manufacture of a product for the treatment and / or prevention of inflammation, characterized in that, The product is a product for reducing the release amount of at least one inflammatory factor of NO, iNOS.

3. Use of a composition according to claim 2 for the manufacture of a product for the treatment and / or prevention of inflammation, characterized in that, The product is a product for inhibiting the expression of iNOS protein.

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