A method for separating active substances in lycium ruthenicum murr.

Abstract

The application discloses a method for separating active substances in Lycium ruthenicum Murr, and relates to the technical field of natural product separation, and comprises a chromatographic separation method for separating active substances norpetanin in Lycium ruthenicum Murr, wherein the active components in the methanol extract of Lycium ruthenicum Murr are separated by using a water / methanol / dichloromethane three-phase system for elution, further separated by gradient elution of a mobile phase of methanol / water, and finally subjected to gradient elution by using a mobile phase of water / acetonitrile, so that the target active substance is obtained in a retention time of 33-35 min. The active substance norpetanin is obtained for the first time by extracting active substances from Lycium ruthenicum Murr, the exact chemical structure of the active substance norpetanin is identified by nuclear magnetic resonance data, and it is found that the active substance norpetanin has anti-inflammatory activity, hypoglycemic effect and lipid-lowering effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product separation, in particular to a separation method of active substances in Lycium ruthenicum Murr. BACKGROUND

[0002] Lycium ruthenicum Murr 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 Murr 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, anti-tumor and the like through their strong antioxidant free radical scavenging effect.

[0004] At present, the research on the active substances of Lycium ruthenicum Murr 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 Murr and their pharmacological effects can be studied more deeply and subtly, and the active mechanism is discussed, more safe and effective natural plant source drugs for treating diseases can be developed. SUMMARY

[0005] The purpose of the present application is to provide a separation method of active substances in Lycium ruthenicum Murr, which first obtains an active substance norpetanin by separating and extracting Lycium ruthenicum Murr, 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, hypoglycemic and hypolipidemic effects.

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

[0007] A separation method of active substances in Lycium ruthenicum Murr, comprising the following contents:

[0008] (1) taking Lycium ruthenicum Murr fruit methanol extract infusion;

[0009] (2) mixing the Lycium ruthenicum Murr fruit methanol extract infusion with dry polyamide powder, grinding after drying, and passing through a 20-mesh sieve to obtain a sieved powder;

[0010] (3) The sieved powder in step (2) is loaded into a small medium-pressure chromatography column, and the medium-pressure chromatography column equipped with MCI is connected to a preparative liquid chromatography for dry loading; a three-phase system of water / methanol / dichloromethane is used for elution, and three components are obtained: Fr1 with a retention time of 12-39 min, Fr2 with a retention time of 39-139 min, and Fr3 with a retention time of 139-230 min;

[0011] (4) Fr2 obtained in step (3) is subjected to gradient elution with methanol and / or water as the mobile phase, and Fr2-5 is obtained with a retention time of 76-130 min;

[0012] (5) Fr2-5 in step (4) is subjected to gradient elution with water / methanol as the mobile phase on a C18 preparative chromatography column, and Fr2-5-3 is obtained with a retention time of 19-21 min;

[0013] (6) Fr2-5-3 in step (5) is subjected to gradient elution with water / acetonitrile as the mobile phase on a C18 preparative chromatography column, and Fr2-5-3-3 is obtained with a retention time of 33-35 min, which is the target active substance.

[0014] Further, the active substance is a composition comprising the compounds as shown in Formula II and Formula III or each optical isomer, each crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof:

[0015]

[0016] Further, the ratio of the compounds as shown in Formula II and Formula III in the composition is 3:1.

[0017]

[0018] Further, the elution conditions in step (3) are as follows:

[0019] 0-120 min, 100% water to 100% methanol;

[0020] 120-180 min, 100% methanol to 100% dichloromethane;

[0021] 180-210 min, 100% methanol;

[0022] 210-240 min, 100% water;

[0023] Flow rate: 50 mL / min, detection wavelength: 210 nm.

[0024] Further, the elution conditions in step (4) are as follows:

[0025] 0-120 min, 0-100% methanol;

[0026] 120-140 min, 100% methanol;

[0027] Flow rate: 50 mL / min;

[0028] Detection wavelength: 254 nm;

[0029] Filler: MCI GEL CHP20P resin;

[0030] Column specifications: 49 x 460 mm.

[0031] Further, the elution conditions in step (5) are as follows:

[0032] 0-60-65-90 min, 30%-42%-40%-95% methanol;

[0033] Flow rate: 19 mL / min;

[0034] Detection wavelength: 210 nm;

[0035] Injection volume: 300 μL;

[0036] Filler: C18 preparative column;

[0037] Column specifications: 21.2 x 250 mm, 5 μm.

[0038] Further, the elution conditions in step (6) are as follows:

[0039] 0-10-60 min, 5%-15%-17% acetonitrile;

[0040] Flow rate: 19 mL / min;

[0041] Detection wavelength: 210 nm;

[0042] Injection volume: 500 μL;

[0043] Filler: C18 preparative column;

[0044] Column specifications: 21.2 x 250 mm, 5 μm.

[0045] The present application also provides the use of the aforementioned Fr2, Fr2-5, Fr2-5-3 or Fr2-5-3-3, composition, and pharmaceutically acceptable salts, hydrates or solvates thereof, in the preparation of a product for treating and / or preventing an inflammation-related disease.

[0046] 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 iNO.

[0047] Further, the product is a product for inhibiting the expression of iNOS protein.

[0048] The present application also provides the use of the aforementioned Fr2, Fr2-5, Fr2-5-3, composition and pharmaceutically acceptable salts, hydrates or solvates thereof in the preparation of a product for improving sugar metabolism or / and lipid metabolism.

[0049] Further, the product is a product having a lipid-lowering effect or inhibiting adipocyte differentiation.

[0050] Further, the product is a product for inhibiting the accumulation of intracellular lipid droplets and / or intracellular lipid synthesis.

[0051] According to the separation and purification route of the present application, the active substance Fr2-5-3-3 exists in the extraction fractions Fr2, Fr2-5 or Fr2-5-3, and the content increases in the above-mentioned fractions in turn. Based on the effects of compound I (including cis-trans tautomers and combinations thereof) in anti-inflammatory and improving sugar and lipid metabolism, Fr2, Fr2-5 or Fr2-5-3 containing compound I (including cis-trans tautomers and combinations thereof) also has the above-mentioned activity.

[0052] The "C18 preparative chromatography column" is a commonly used reversed-phase chromatography column, which uses C18 alkyl chains fixed on a silica gel matrix as the stationary phase to separate polar and non-polar substances. The alkyl chains of the C18 chromatography column can interact with non-polar substances in the sample, allowing them to stay on the stationary phase for a longer time and not pass through the column quickly, thus achieving separation. On the contrary, polar substances cannot interact with the alkyl chains and can pass through the column quickly. C18 chromatography columns are suitable for separating mixtures of compounds with different polarities, such as drugs, natural products and organic compounds. The separation effect is affected by multiple factors such as the properties of the stationary phase, the properties of the sample and the properties of the mobile phase.

[0053] Fine separation chromatography packing MCI GEL:

[0054] Chromatography packing for pharmaceutical analysis and preparation (MCI-GEL CHP series), MCI-GEL CHP20 / P120 series, CMG series, CHP2MG series, CHP series.

[0055] MCI GEL fine separation packing can be divided into the following series:

[0056] Ion exchange resin series:

[0057] Cation exchange resins with bonded sulfonate groups MCI-GEL SCK, CK, AFR series

[0058] Anion exchange resins with bonded quaternary ammonium groups MCI-GEL SCA, CA, CDR series.

[0059] Bioseparation resin series:

[0060] CQK, CQA ion exchange resin series for bioseparation, matrix is polyhydroxymethacrylate (HMA);

[0061] CQH hydrophobic reactive resin series for bioseparation chromatography, matrix is polyhydroxymethacrylate (HMA);

[0062] CQP series resin for size exclusion chromatography for bioseparation, polyhydroxymethacrylate (HMA).

[0063] Adsorption resin series:

[0064] MCI-GEL CHP series products for reverse chromatographic separation, matrix is polystyrene and divinyl copolymer or polymethacrylate. The MCI filler applied in the application is MCI-GEL CHP20 resin.

[0065] The beneficial effects of the present application are:

[0066] The present application first separates the active ingredients in the methanol extract of Lycium ruthenicum Murr by a chromatographic separation method: using a water / methanol / dichloromethane three-phase system for elution, finally using water / acetonitrile as the mobile phase for gradient elution, and obtaining the target active substance norpetanin with a retention time of 33-35 min. 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, hypoglycemic and lipid-lowering effects.

[0067] The numbers marked on the structural formula of the compound in the present application are only for the purpose of facilitating the description of the structure of the compound. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 Preparation chromatogram of Fr2;

[0069] Figure 2 Preparation chromatogram of Fr2-5;

[0070] Figure 3 Preparation chromatogram of Fr2-5-3;

[0071] Figure 4 Analysis chromatogram of norpetanin;

[0072] Figure 5 High resolution mass spectrum of norpetanin;

[0073] Figure 6 UV absorption spectrum of norpetanin; 1 H NMR spectrum of norpetanin;

[0074] Figure 7 C NMR spectrum of norpetanin; 13 C NMR spectrum of norpetanin;

[0075] Figure 8 HMBC spectrum of norpetanin;

[0076] Figure 9 HSQC spectrum of norpetanin;

[0077] Figure 10 COSY spectrum of norpetanin;

[0078] Figure 11 DEPT spectrum of norpetanin;

[0079] Figure 12 UV absorption spectrum of norpetanin;

[0080] Figure 13 Effect of norpetanin at different concentrations on RAW264.7 cell viability;

[0081] Figure 14 Effect of norpetanin on RAW264.7 cell NO release;

[0082] Figure 15 Effect of norpetanin on RAW264.7 cell iNOS protein expression;

[0083] Figure 16 Effect of norpetanin on 3T3-L1 adipocyte 2-NBDG uptake by FCM method;

[0084] Figure 17 Effect of norpetanin on p-PI3K and p-AKt expression in 3T3-L1 adipocytes;

[0085] Figure 18 Effect of norpetanin on 3T3-L1 cell lipid droplet accumulation (100x);

[0086] Figure 19 Effect of norpetanin on 3T3-L1 cell TG content;

[0087] Figure 20 Figure of the effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells;

[0088] Figure 21 Figure of the effect of norpetanin on the expression of proteins related to lipid metabolism in 3T3-L1 cells. DETAILED DESCRIPTION

[0089] 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, rather than all of, the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0090] Example 1

[0091] A separation method of a composition, comprising the following contents:

[0092] (1) 10.0 kg of dried Lycium ruthenicum Murr. fruits were 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, for a total of 3 times; the extract was 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.

[0093] (2) The methanol extract infusion prepared in step (1) was mixed with dry polyamide powder at a mass ratio of 1:1, dried in an oven at 40℃, and ground, and passed through a 20 mesh sieve to obtain sieved powder;

[0094] (3) 50.00 g of the sieved powder in step (2) was loaded into a small medium-pressure chromatography column (26x100 mm), and connected to a medium-pressure chromatography column (49x460 mm) loaded with MCI, and subjected to dry loading; a water / methanol / dichloromethane three-phase system was used for elution, 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; three components Fr1, Fr2 and Fr3 were obtained, as shown in Figure 1 Figure, Fr2 was obtained at a retention time of 39-139 min, in the form of purple powder, 511.3 g, with a yield of 15.1%.

[0095] (4) Further separation of Fr2 obtained in step (3) was performed by gradient elution with methanol and / or water as mobile phase, elution condition 0-120 min, 0-100% methanol; 120-140 min, 100% methanol; flow rate: 50 mL / min; detection wavelength: 254 nm; packing material: MCI GEL CHP20 resin; size: 49 x 460 mm; as shown in Figure 2 Figure 2, Fr2-5 175.5 g was obtained with a yield of 34.3% at a retention time of 76-130 min.

[0096] (5) Separation of Fr2-5 in step (4) was performed on a C18 preparative column with a size of 21.2 x 250 mm and 5 μm, by gradient elution with water / methanol as mobile phase: 0-60-65-90 min, 30%-42%-70%-95% methanol, flow rate: 19 mL / min; detection wavelength: 210 nm; injection volume 300 μL, as shown in Figure 3 Figure 3, Fr2-5-3 was obtained with a yield of 29.6% at a retention time of 19-21 min, and weighed 5.2 g.

[0097] (6) Separation of Fr2-5-3 in step (5) was performed on a C18 preparative column with a size of 21.2 x 250 mm and 5 μm, by gradient elution with water / methanol as mobile phase: 0-10-60 min, 5%-15%-17% acetonitrile; flow rate: 19 mL / min; detection wavelength: 210 nm, injection volume 500 μL; Fr2-5-3-3 was obtained at a retention time of 33-35 min, as shown in Figure 4 Figure 4, yellow powder, weighed 827 mg with a yield of 15.9%. The MS, 1 H-NMR, 13 C-NMR spectra of the target composition are shown in Figure 5 、 Figure 6 and Figure 7 .

[0098] The composition, the compound with structures as shown in general formula II and III, or each optical isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof:

[0099]

[0100] The nuclear magnetic resonance data of the active composition (Fr2-5-3-3) in Lycium ruthenicum Murr. are shown in Table 1.

[0101] Table 1 Nuclear magnetic resonance data of the composition (Fr2-5-3-3)

[0102]

[0103] Composition Fr2-5-3-3: yellow powder, ESI-MS m / z: 809 [MH] - The main component has the molecular formula C 36 H 42 O 21 The m / z value was determined to be 809.2140 [MH] by HR-ESI-MS. - The calculated value is 809.2141, with an unsaturation degree of 19. Its NMR spectrum shows paired signals of varying intensities, indicating the presence of tautomerism. For ease of structural analysis, the NMR signals of the predominantly trans-tautomers are preferentially used for structural elucidation. As shown in Table 1, 1 H NMR(DMSO-d6 / CF3COOD(9:1),600MHZ):δ H The signals at 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””) show a characteristic set of trans-p-acyl groups, and the signal at δH 7.5 3 (1H, s, H-4) shows an aromatic or alkene proton singlet state. H The signals at 6.54 (1H, d, J = 1.7 Hz, H-6) and 6.36 (1H, d, J = 1.7 Hz, H-8) indicate a set of 1,3,4,5-tetrasubstituted benzene rings, δ H Signals at 5.09 (1H(d), J = 7.3 Hz, H⁻¹') and 4.82 (1H”) indicate two β-glucanose anomeric protons, δ H The signal at 4.61 (1H, br s, H-1”) indicates an α-rhamnosyl anisoproton, δ H The signal at 4.82 (1H, t-like, J = 9.6 Hz, H-4”) shows an acylated methine triplet state, and the upper field region shows a rhamnose methyl doublet state. 13 C10 NMR (DMSO-d6 / CF3COOD(9:1), 600MHZ): shows a total of 36 carbon resonances, including a set of trans-p-acyl carbons, and 18 carbon signals attributable to the three sugar moieties, while the remaining 9 unattributed resonance carbons originate from the glycoside ligand nucleus. The above NMR characteristics are generally similar to those of petanin, the main difference being the absence of the ring B signal in the anthocyanin nucleus in this compound. This allows us to infer that the ring B moiety must have been oxidized and degraded to yield the specific 3,5,7-trioxy-substituted coumarin nucleus. From H-4 to C-2 [δ] C 157.6(s)], C-5[δC 154.5(s))] and C-9[δC 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)] of HMBC correlations, as Figure 8 shown, confirmed this deduction. The resulting structure was further verified by careful analysis of HSQC, HMBC and 1H, 1H-COSY correlations, as Figure 6 , 8 -10. 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 Fr2-5-3-3 is shown in formula II and III, and named as norpetanin. To our knowledge, the mixture norpetanin represents a rare class of cyclo-B-ring cyanidin.

[0104] The DEPT and UV absorption spectra of the active substance norpetanin are shown in Figure 11 and 12 , respectively.

[0105] Example 2

[0106] Evaluation of the anti-inflammatory effect of norpetanin:

[0107] Using the established inflammatory cell model, the effect of norpetanin on the amount of NO released by cells was determined, and the effect of the active substance norpetanin on the expression of iNOS protein was explored, and the possible mechanism of action of norpetanin in exerting anti-inflammatory effect was preliminarily explored.

[0108] 1. Experimental materials and reagents

[0109] 1.1 Experimental materials

[0110] The component Fr2-5-3-3 separated and extracted in Example 1 of the present application is the active composition norpetanin of Lycium ruthenicum Murr.

[0111] RAW264.7 mouse monocyte macrophages were purchased from the China Academy of Sciences Typical Culture Preservation Committee Cell Library, catalog number: TCM13.

[0112] Preparation of test drugs: weigh an appropriate amount of Fr2-5-3-3 and positive control drug dexamethasone, and prepare a stock solution with a concentration of 100 mM using DMSO, and store it in a 4°C refrigerator. When used, dilute it as needed.

[0113] The materials and reagents used are shown in Table 2:

[0114] Table 2 Experimental materials and reagents

[0115]

[0116]

[0117] 2. Experimental method

[0118] 2.1 MTT method for determining cell viability

[0119] Logarithmic growth RAW264.7 cells were taken at a cell density of 5x10 4 6 / mL, 150 μL per well was inoculated in a 96-well plate, after incubation in a cell incubator for 24 h, the DMEM medium containing lipopolysaccharide or test components (containing 2% FBS) was replaced, and the incubation was continued for 24 h, then 10 μL thiazolyl blue (MTT) solution was added per well, and the plate was incubated in a cell incubator for 4 h. Finally, the medium was discarded, 150 μL DMSO was added to dissolve the cells, and the absorbance value at a wavelength of 490 nm was read using an enzyme-labeled instrument. The cell viability was calculated according to the formula. The cell viability calculation formula is as follows:

[0120]

[0121] 2.2 Griess method for determining NO content

[0122] 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 per 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 shaker, the absorbance value at a wavelength of 540 nm was measured, and the NO content was calculated according to the standard curve.

[0123] 2.3 Western blot analysis

[0124] The experiment was divided into a control group, an LPS group, and an LPS+ different concentrations of drug group. Logarithmic growth RAW264.7 cells were taken at a density of 5x10 4 6 / mL, inoculated in a 6-well plate, and incubated in a cell incubator for 24 h. Then, the DMEM medium containing 2% FBS was replaced, the model group was treated with LPS, the test drug group was treated with LPS+ different concentrations of drug, and the incubation was continued for 24 h. The protein was extracted and electrophoresed. The specific operation method is as follows:

[0125] (1) Extraction of RAW264.7 cell protein

[0126] After the RAW264.7 cells are treated, the cells are lysed and cell proteins are extracted. The culture solution is pumped out using a liquid suction pump, and the cells are washed twice with PBS buffer. The cell lysate is added, the 6-well plate is shaken to mix, and the plate is placed on ice for 10 min. The cells are scraped off using a cell scraper and collected in a centrifuge tube. The centrifuge tube is placed on ice for 30 min. After the lysis is completed, a low-temperature centrifuge is used to centrifuge at 12000 r / min and 4℃ for 15 min. The supernatant containing the proteins is collected in a new EP tube.

[0127] (2) BCA method for measuring cell protein concentration

[0128] The BCA method is used to measure the protein concentration. First, the protein standard is prepared into a solution with a concentration of 0.5 mg / mL using PBS buffer. The BCA working solution is prepared according to the ratio of A liquid:B liquid = 50:1, and mixed. Three parallel samples are set up, and the protein standard solution and PBS buffer are added according to Table 3 to draw a standard curve. 1 μL of the cell-extracted protein solution sample is taken and added to a 96-well plate, and the volume is made up to 20 μL with PBS buffer. Then, 200 μL of BCA working solution is added, and the reaction is carried out at 37℃ for 30 min. The absorbance value at 562 nm is measured using an enzyme label instrument. The protein concentration of each sample is calculated according to the standard curve. The BCA method for protein quantification is shown in Table 3.

[0129] Table 3 BCA method for protein quantification

[0130]

[0131] (3) Protein denaturation

[0132] The diluted protein sample is taken, protein loading buffer is added and mixed, and the sample is denatured in a metal bath at 100℃ for 15 min. After the denatured protein is cooled to room temperature, it is stored in a refrigerator at -20℃ for standby use.

[0133] (4) SDS-PAGE electrophoresis

[0134] Two clean glass plates were aligned and clamped on a rubber frame, and super-pure water was added to check for leaks. After checking for leaks, the super-pure water was poured out and the residual liquid between the two glass plates was absorbed with a water-absorbing paper, and the glass plates were prepared for pouring glue. As shown in Table 4, the separation glue (lower glue) was prepared according to the suitable concentration of the protein molecular weight, and the 10% SDS-PAGE separation glue was prepared according to the protein molecular weight to be detected in the experiment according to Table 5, and the 5% SDS-PAGE concentrated glue (upper glue) was prepared according to Table 6. About 4 mL of separation glue was added between the two dry glass plates, isopropyl alcohol was added to seal the glue, and the separation glue 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 glue was poured in, the comb was inserted to avoid air bubbles, and the concentrated glue was allowed to solidify. The comb was gently pulled out. The two glass plates were clamped on an electrophoresis tank, and the electrophoresis liquid was poured in to make the liquid level above the glass plates. The protein sample was loaded, and after the loading was completed, electrophoresis was started. First, a constant voltage of 80 V was used to concentrate the sample in the concentrated glue for about 30 min. The voltage was adjusted to 120 V for constant voltage electrophoresis. When the loading buffer reached the bottom of the glue 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 glue is shown in Table 5; and the preparation table of 5% SDS-PAGE concentrated glue is shown in Table 6.

[0135] Table 4 SDS-PAGE gel optimal separation range

[0136]

[0137] Table 5 10% SDS-PAGE separation glue preparation table

[0138]

[0139] Table 6 5% SDS-PAGE concentrated glue preparation table

[0140]

[0141] (5) Transferring the membrane

[0142] 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 cutting knife, 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 on the bottom, and the foam, filter paper, adhesive tape, 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.

[0143] (6) Blocking

[0144] Blocking solution is 5% skim milk in 1x 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 hour. Discard the skim milk. Wash the membrane with 1x TBST for 10 minutes, 3 times.

[0145] (7) Incubate the primary antibody

[0146] Dilute the primary antibody (iNOS, CST, #13120) with the antibody dilution solution 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. Wash the membrane with 1x TBST for 10 minutes, 3 times.

[0147] (8) Incubate the secondary antibody

[0148] Put the PVDF membrane into the secondary antibody solution (horseradish peroxidase-labeled secondary antibody diluted at 1:5 000). Incubate it at room temperature for 1 hour. Discard the secondary antibody solution. Wash the membrane with 1x TBST for 10 minutes, 3 times.

[0149] (9) Develop

[0150] Develop the membrane using the ECL chemiluminescence method. Mix the developing solutions A and B at a ratio of 1:1. Add 100 μL of the mixture to the PVDF membrane. Develop the membrane using a developing instrument and take a photo.

[0151] 3. Experimental results

[0152] 3.1 Effect of norpetanin on the activity and NO secretion of RAW264.7 cells

[0153] As shown in Figure 13 and 14 , 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 μM. However, it inhibited the growth of RAW264.7 cells at 50 μM (P<0.05) and 100 μM (P<0.01), and had a significant inhibitory effect on NO secretion.

[0154] 3.2 Effect of norpetanin on the expression of iNOS protein in RAW264.7 cells

[0155] The active substance showed good anti-inflammatory activity in inhibiting the release of NO in the inflammatory 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 its effect on inflammation-related proteins. As shown in Figure 15As shown, the expression level of iNOS in RAW264.7 cells was up-regulated after LPS stimulation, and there was a significant difference compared with the control group (P < 0.01). Norpetanin at a concentration of 10 μM could significantly inhibit the expression of iNOS protein (P < 0.01), indicating that the compound could play an anti-inflammatory role by inhibiting the expression of iNOS protein and thereby reducing the release of NO.

[0156] Example 3

[0157] Effect of norpetanin on glucose metabolism:

[0158] 1. Experimental materials and instruments

[0159] 1.1 Materials and reagents

[0160] Test substance: norpetanin prepared in the laboratory.

[0161] 3T3-L1 mouse embryonic fibroblasts were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the materials and reagents used in the experiment are shown in Table 7.

[0162] Table 7 Materials and reagents

[0163]

[0164] Main experimental reagents

[0165] (1) Preparation of 2-NBDG

[0166] Dissolve 2-NBDG powder in DMSO to prepare a 10 mM stock solution, and dilute with sugar-free DMEM to a final concentration of 100 μM when used.

[0167] (2) The preparation of Western blotting working solution is as follows:

[0168] a. 10% SDS

[0169] Weigh 10 g of SDS, add 100 mL of ultrapure water, dissolve in a 50°C water bath, and store at room temperature. If precipitation occurs, it can still be used after dissolving in a water bath.

[0170] b. SDS-PAGE electrophoresis solution (5x)

[0171] Weigh 15.1 g of Tris, 94.0 g of Glycine, and 5.0 g of SDS, dissolve in ultrapure water, and dilute to 100 mL. Store at room temperature. When used, 5x electrophoresis solution can be diluted to 1x electrophoresis solution, which can be reused 2-3 times and stored at room temperature.

[0172] c. Transfer solution (10x)

[0173] Take 15.15 g of Tris, 72.0 g of glycine, and dilute to 1x transfer solution. Method: take 10x transfer solution 50 mL, add methanol 100 mL, and supplement with ultrapure water to 500 mL, i.e. 10x transfer solution: methanol: water = 1:2:7, which can be reused 3-5 times, and store in the refrigerator at 4°C.

[0174] d. TBS (10x)

[0175] Take 12.12 g of Tris and 40.03 g of NaCl, dissolve in ultrapure water, adjust pH to 7.6 with HCl (about 3.5 mL of HCl), and dilute to 500 mL, and store in the refrigerator at 4°C.

[0176] 1.2 Instruments and equipment

[0177] The main instruments and equipment used in this experiment are shown in Table 8.

[0178] Table 8 Instruments and equipment

[0179]

[0180] 2 Experimental method

[0181] 2.1 Establishment of 3T3-L1 adipocyte model

[0182] (1) In vitro culture and subculture of 3T3-L1 cells

[0183] 3T3-L1 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin mixture. The temperature of the cell culture box was 37°C, the humidity was 95%, and the CO2 concentration was 5%. Under normal culture conditions, 3T3-L1 cells need to be subcultured every 3 days. When the cell density reaches more than 85%-90%, first use a suction pump to discard the original culture medium, wash the cells with PBS in a warm bath for 1-2 times, then immediately add 850 μL of trypsin digestion solution, and gently shake the culture dish to make the digestion solution evenly cover the entire culture dish bottom. Under a microscope, when the cells are round and detached from the bottom of the dish, add fresh culture medium and repeatedly blow the cells to completely detach them from the culture dish bottom and disperse them into single cells. The cell suspension is subcultured into a new culture dish at a ratio of 1:5, and an appropriate amount of DMEM and FBS is added before being placed in the cell culture box for further culture.

[0184] (2) Freezing and recovery of 3T3-L1 cells

[0185] Freezing of cells: select the logarithmic growth phase of cells, quickly digest the cells, centrifuge the cell suspension at 1000 g for 5 min at room temperature to obtain cell pellets, add the prepared cell freezing solution (DMEM:FBS:DMSO=5:4:1), add 1.2 mL of cell suspension to each freezing tube, and mark the tube wall (cell type, cell number, number of frozen cells, and freezing time) to facilitate timely inquiry, and finally place at 4°C for 10 min, -20°C for 1 h, and -80°C overnight, and store in a liquid nitrogen tank the next day. Thawing of cells: quickly remove the frozen cell strain from the liquid nitrogen tank, quickly place it in a 37°C water bath to melt, and after the cells are completely melted, quickly transfer the cells to the culture medium for culture, and after the cells adhere (usually 6-8 h), replace the fresh culture medium.

[0186] (3) Counting of 3T3-L1 cells

[0187] The commonly used tool for cell counting is a hemocytometer, each hemocytometer has 2 H-shaped grooves, forming a counting pool with a height of 0.10 mm, and there are 9 large squares in the counting plate, and the cell counting area is the four large squares. After determining the number of cells, adjust the cell density according to the experimental requirements. Generally, the counting principle is to record the upper part and not the lower part, and to record the left part and not the right part. According to the experimental requirements, adjust the cell counting density, record the total number of cells in the four large squares, and finally calculate the number of cells according to the formula:

[0188] Number of cells = total number of cells in 4 squares / 4 x 10 4 x dilution factor

[0189] (4) Establishment of insulin resistance model

[0190] The "cocktail" method was used to induce differentiation of 3T3-L1 cells, and the induction and differentiation were carried out as follows: 3T3-L1 cells in good condition were seeded on culture plates at a plating density of 5 x 10 4 / mL, and cultured in high-glucose DMEM medium containing 10% FBS until the cell density reached about 85%-90%, then the medium was replaced, and the cells were cultured for two days after contact inhibition, then the complete culture medium was discarded, and culture medium containing 10 μg / mL insulin, 0.5 mM IBMX, and 1 μM Dex was added and cultured for 2 d (the addition of the induction solution is recorded as day 0, induction I), then the culture medium containing 10 μg / mL insulin was replaced (induction II) and cultured for 2 d, then the normal culture medium was replaced and cultured for 4 d, and the medium was replaced every other day. On the 8th day after induction and differentiation, 1 μM Dex was added to the cell solution to establish an IR model.

[0191] 2.2 Effect of norpetanin on 2-NBDG uptake by 3T3-L1 cells

[0192] 3T3-L1 preadipocytes were seeded into 12-well plates at a density of 5 x 10 4 When the cell density reached more than 80%, induction differentiation was performed, and 1 μM Dex was added to the cell solution on the 8th day after induction differentiation to establish an IR model. Normal, model, and drug treatment groups were set up, and the normal group was cultured with complete medium, while the other groups were cultured with 1 μM Dex. After 10 μM norpetanin was added to the drug treatment group for 48 h, the culture solution was removed, and the cells were washed once with DPBS, 500 μL trypsin was added at 37°C for 1 min, 2 mL DPBS was added to blow and mix evenly, and centrifugation was performed at 1000 g for 6 min. The supernatant was discarded, 1 mL sugar-free medium containing 10 μM 2-NBDG was added to each well, and incubation was performed at 37°C for 30 min. The fluorescence intensity was detected at a wavelength of 488 nm and 515-545 nm by flow cytometry.

[0193] 2.3 Effect of norpetanin on AKt phosphorylation in 3T3-L1 cells

[0194] 3T3-L1 preadipocytes were seeded into 6-well plates at a density of 5 x 10 4 When the cell density reached more than 80%, induction differentiation was performed, and 1 μM Dex was added to the cell culture solution on the 8th day after induction differentiation to establish an IR model. Normal, model, and drug treatment groups were set up, and the normal group was cultured with complete medium, while the other groups were cultured with 1 μM Dex. After 10 μM norpetanin was added to the drug treatment group for 48 h, the culture solution was removed, and the cells were collected and used for Western blotting to detect the expression levels of PI3K and AKt proteins.

[0195] 2.4 Western blot analysis

[0196] The treated cell samples were subjected to protein extraction, and the specific operation method was as follows:

[0197] (1) RIPA lysis buffer was mixed with PMSF at a ratio of 100:1 (when analyzing phosphorylated proteins, phosphatase inhibitor was added), and cell lysis buffer was prepared. The cells in the 6-well plate were washed with cold PBS for 3 times, 100 μL cell lysis buffer was added to each well, and lysis was performed on ice for 5 min. Then the cells were scraped with a cell scraper and collected into a 1.5 mL centrifuge tube. Lysis was performed again in a 4°C refrigerator for 30 min. Finally, the liver tissue homogenate and cell lysis buffer were centrifuged at 12000 g, 4°C for 15 min, and the supernatant protein was transferred to a new EP tube.

[0198] (2) BCA method for determining protein content: Take out an appropriate amount of protein sample, according to the BCA protein quantitative kit instructions, dilute the protein standard to 0.5 mg / mL with PBS buffer, mix reagent A: reagent B at a ratio of 50: 1 to prepare working solution. As shown in Table 9, add standard and PBS to the 96-well plate, 20 μL per well, generally 3 replicates for each concentration, to draw the standard curve. Add 1 μL of protein sample to the 96-well plate, add PBS to 20 μL. Add 200 μL of BCA working solution to each well and mix well. Incubate the above samples in a microplate incubator at 37°C on a shaking incubator for 30 min. Measure the absorbance value at 562 nm on a microplate reader. Calculate the protein concentration of the sample according to the standard curve. Prepare the protein sample by heating at 100°C for 10 min to denature the protein completely. Centrifuge the sample before loading to ensure uniformity.

[0199] Table 9 BCA method for protein quantification

[0200]

[0201] (3) SDS-PAGE electrophoresis: According to the molecular weight of the target protein, select the appropriate separation gel concentration (as shown in Table 10), prepare the appropriate solution system (lower gel), and let the lower gel stand at room temperature for more than 40 min until it is completely solidified. Prepare 5% concentrated gel (upper gel) and insert the appropriate comb. After the concentrated gel is solidified, place the gel plate in the electrophoresis tank, add enough electrophoresis liquid between the two plates, pull out the comb, load the protein sample and marker, and then run the gel. Electrophoresis conditions: S1 stage, 80V, 30min; S2 stage, 120V, about 60-90min, until the bromophenol blue in the buffer runs to the bottom of the separation gel.

[0202] (4) Transmembrane: Immediately after the protein electrophoresis is completed, transmembrane is carried out. According to the marker band, cut the position of the target protein according to the molecular weight. Cut the PVDF membrane to the same size as the gel, activate the cut membrane in methanol solution for 1 min, and then place it in the transmembrane liquid. According to the order of blackboard-filter-gel PVDF-filter-whiteboard, clamp the electric transfer clamp, the blackboard is against the black side of the tank, place it in the electrophoresis tank, add enough transmembrane liquid to the whole tank, and then connect the power. Place the transmembrane instrument in an ice-water mixture. The transmembrane conditions are constant current 250mA, time 90min.

[0203] Table 10 Optimal separation range of different concentrations of SDS-PAGE separation gel

[0204]

[0205]

[0206] (5) Blocking: After the end of the transfer, the PVDF membrane was quickly taken out from the transfer plate with tweezers and placed in TBST buffer for 5 min, and then immediately blocked with 5% skim milk powder in TBST at room temperature for 1 h on a shaker.

[0207] (6) Primary antibody incubation: After the end of blocking, the PVDF membrane was placed in the diluted primary antibody solution and incubated slowly on a horizontal shaker at 4°C overnight.

[0208] (7) Secondary antibody incubation: After the end of primary antibody incubation, the PVDF membrane was washed with TBST three times, 8 min each time. Then the PVDF membrane was placed in a solution containing HRP-labeled mouse or rabbit secondary antibody and incubated slowly at room temperature for 1 h.

[0209] (8) Development: After the end of secondary antibody incubation, the PVDF membrane was washed with TBST three times, 8 min each time. After washing, the mixed ECL developing solution was added to the PVDF membrane, and the gel imaging system was used for photography.

[0210] (9) Result analysis: The gray values of target proteins and internal reference proteins were quantitatively analyzed by Image J software, and the relative expression of proteins was the ratio of target proteins to internal reference proteins (the ratio of phosphorylated proteins to non-phosphorylated proteins).

[0211] 2.5 Statistical analysis

[0212] GraphPad Prism7 software was used for plotting and statistical analysis, independent sample t test was used for comparison of means between two groups, one-way ANOVA was used for comparison of means between multiple groups, statistical data were expressed as mean ± standard deviation (x ± s), * or # indicated P < 0.05, ** or ## indicated P < 0.01, and the difference was extremely statistically significant.

[0213] 3 Experimental results

[0214] 3.1 Effect of norpetanin on 2-NBDG uptake of 3T3-L1 cells

[0215] As Figure 16As shown, the uptake of glucose by 3T3-L1 adipocytes was detected using the fluorescently labeled glucose analog 2-NBDG. The fluorescent probe 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-ylamino]-2-deoxyglucose) is a 2-deoxyglucose fluorescent analog that can specifically bind to intracellular glucose, and has high sensitivity in detection. In general, the fluorescence intensity of 2-NBDG in insulin-resistant cells or organisms is very weak, at the background value, indicating that the cells or organisms have weak glucose uptake ability. The experimental results show that the glucose uptake ability of 2-NBDG in the normal group is higher than that in the insulin resistance model group, and the glucose uptake ability of the cells in the model group is weaker than that in the normal group after insulin stimulation. After the intervention of the active substance norpetanin, the uptake of 2-NBDG by adipocytes under insulin stimulation is promoted, and the active substance norpetanin has the potential to improve insulin resistance.

[0216] Note: compared with the normal group, # P < 0.5, ## P < 0.01; compared with the model group, *P < 0.5, **P < 0.01

[0217] 3.2 Effect of norpetanin on PI3K and AKt phosphorylation in 3T3-L1 cells

[0218] AKt, also known as protein kinase B (PKB), is a serine / threonine protein kinase that plays an important role in cell processes such as glucose metabolism, apoptosis, cell proliferation, cell transport, etc. In the process of glucose metabolism, activated AKt activates various enzymes, kinases and transcription factors in the insulin signaling pathway through the phosphorylation pathway, and then regulates cell function and insulin signal transmission. In general, activated AKt promotes the translocation of glucose transporter proteins from the cytoplasm to the cell membrane by activating its downstream phosphatidylinositol kinase 3 (PI3K), accelerating the absorption and utilization of glucose, thereby playing a role in insulin signal transmission. In this experiment, the effect of the active substance norpetanin in Lycium ruthenicum on the expression of p-AKt and p-PI3K in cells was detected to investigate whether it can promote the absorption and utilization of glucose by cells, enhance the insulin signaling process, and improve insulin resistance. The experimental results are shown in Figure 17 As shown, compared with the normal group, the expression levels of p-AKt and p-PI3K proteins in the model group were reduced. After treatment with the active substance norpetanin, the expression levels of p-AKt and p-PI3K in 3T3-L1 adipocytes were increased, thereby promoting the uptake of glucose by 3T3-L1 adipocytes and enhancing insulin sensitivity.

[0219] 4Summary

[0220] Insulin resistance model was established by 3T3-L1 adipocytes to study the hypoglycemic effect of active substance norpetanin in Lycium ruthenicum Murr, and FCM was used to detect the glucose uptake of adipocytes in insulin resistance state, and Western blotting was used to detect the phosphorylation level of PI3K and AKt in 3T3-L1 adipocytes. The results showed that after the intervention of active substance norpetanin in Lycium ruthenicum Murr, the uptake of 2-NBDG in adipocytes stimulated by insulin was promoted, and it had the potential to improve insulin resistance. From the results of Western blotting experiment, it can be seen that active substance norpetanin can also improve the expression level of p-PI3K and p-AKt in 3T3-L1 adipocytes.

[0221] In this study, active substance norpetanin in Lycium ruthenicum Murr can improve the expression level of p-AKt and p-PI3K in 3T3-L1 adipocytes, which suggests that active substance norpetanin in Lycium ruthenicum Murr can promote the uptake of glucose in insulin target tissues, improve the sensitivity to insulin, and has the potential to improve insulin resistance, which provides more scientific basis for the development and utilization of Lycium ruthenicum Murr to treat type II diabetes.

[0222] Example 4

[0223] Effect of norpetanin on lipid metabolism:

[0224] 1Materials and instruments

[0225] 1.1Materials and reagents

[0226] Test substance: norpetanin prepared by laboratory separation.

[0227] 3T3-L1 mouse embryonic fibroblasts were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and the materials and reagents used in the experiment are shown in Table 11.

[0228] Table 11 Materials and reagents

[0229] Materials and reagents Manufacturers 75% ethanol Tianjin Fuyu Fine Chemical Co., Ltd. PIPA lysis buffer Beyotime Biotechnology BCA Protein Assay Kit (Enhanced) Beyotime Biotechnology Protease inhibitor Beyotime Biotechnology Triglyceride Assay Kit (A110-1) Nanjing Jiancheng Bioengineering Institute DMEM Gibco PBS Corning FBS Gibco DMSO Sigma-Aldrich IBMX Sigma-Aldrich Dex Sigma-Aldrich Novolin 30R Nordmark (China) Manufacturing Co., Ltd. Polyclonal rabbit anti-FAS (#3180) Cell Signaling Technology Monoclonal rabbit anti-ACC (#3676) Cell Signaling Technology Polyclonal rabbit anti-PPARγ (#2443) Cell Signaling Technology Polyclonal rabbit anti-C / EBPα (#2295) Cell Signaling Technology Monoclonal rabbit anti-β-actin (#4970) Cell Signaling Technology

[0230] 1.2Instruments and equipment

[0231] The main instruments and equipment used in this experiment are shown in Table 12.

[0232] Table 12 Instruments and equipment

[0233] Instruments and equipment Manufacturers Haier HYCD-205 refrigerator-freezer Qingdao Haier Co., Ltd. Micro 21R high-speed refrigerated centrifuge Thermo Fisher Scientific Inc. LS-750 liquid nitrogen tank Sichuan Chengdu Jin Feng Liquid Nitrogen Container Co., Ltd. LX71 inverted microscope OLYMPUS BE-9008 microplate incubator shaker Shanghai Yanhe Instrument and Equipment Co., Ltd. Tanon 520 gel imaging system Shanghai Tian Neng Technology Co., Ltd. Multifunctional microplate reader Molecular Devices Corporation Ultrasonic cleaner Jiangsu Ding Shanhu Instrument Factory Electrophoresis apparatus Bio-Rad Company HH-2 digital constant temperature water bath Jintan City Ke Xing Instrument Factory Different size pipettes Eppendorf LDZM-80KCS vertical pressure steam sterilization pot Shanghai Shen'an Medical Instrument Factory SW-CJ-2FD double-person single-face clean bench Suzhou Purification Equipment Co., Ltd. Constant temperature carbon dioxide incubator SANYO

[0234] 2 Experimental Methods

[0235] 2.1 Establishment of 3T3-L1 adipocyte model

[0236] (1) In vitro culture and subculture of 3T3-L1 cells

[0237] 3T3-L1 cells were cultured in DMEM high glucose medium containing 10% FBS and 1% penicillin-streptomycin mixture. The temperature of the cell culture box was 37°C, the humidity was 95%, and the CO2 concentration was 5%. Under normal culture conditions, 3T3-L1 cells need to be subcultured every 3 days. When the cell density reaches more than 85% to 90%, the original culture medium is first pumped out, PBS is washed 1 to 2 times in a warm bath, then 850 μL of trypsin digestion solution is immediately added, and the culture dish is gently shaken to evenly cover the entire culture dish bottom with the digestion solution. Under a microscope, when the cells are round and detached from the bottom of the dish, fresh culture medium is added, and the cells are repeatedly blown to completely detach the cells from the bottom of the culture dish and disperse them into single cells. The cell suspension is subcultured into a new culture dish at a ratio of 1:5, and an appropriate amount of DMEM and FBS is added before being placed in the cell culture box for continued culture.

[0238] (2) Cryopreservation and recovery of 3T3-L1 cells

[0239] Cryopreservation of cells: select cells in the logarithmic growth phase, quickly digest the cells, centrifuge the cell suspension at 1000 g at room temperature for 5 min to obtain cell pellets, add the previously prepared cell cryopreservation solution (DMEM:FBS:DMSO=5:4:1), add 1.2 mL of cell suspension to each cryopreservation tube, and label the tube wall with the cell type, cell number, number of cells frozen, and freezing time to facilitate immediate inquiry. Finally, place at 4°C for 10 min, at -20°C for 1 h, and at -80°C overnight, and store in a liquid nitrogen tank the next day. Cell recovery: quickly remove the frozen cell strain from the liquid nitrogen tank, quickly melt in a 37°C water bath, and after the cells are completely melted, quickly transfer the cells to the culture medium for culture. After the cells adhere (usually 6-8 h), replace the fresh culture medium.

[0240] (3) Counting of 3T3-L1 cells

[0241] The commonly used tool for cell counting is a hemocytometer. Each hemocytometer has 2 H-shaped grooves, forming a 0.10 mm high counting pool. The counting plate has 9 large squares, and the cell counting area is the four large squares in the corners. After determining the number of cells, adjust the cell density as needed. The general counting principle is to count up but not down, and to count left but not right. Adjust the cell counting density according to the experimental requirements, record the total number of cells in the four large squares, and finally calculate the number of cells according to the formula:

[0242] Cell number = total cell number in 4 grids / 4 x 10 4 x dilution factor

[0243] (4) Induction and differentiation of 3T3-L1 cells

[0244] The 3T3-L1 cells were induced and differentiated by the "cocktail" method. The 3T3-L1 cells in good condition were inoculated on a culture plate at a plating density of 5 x 10 4 mL, and cultured in high-sugar DMEM medium containing 10% FBS until the cell density reached about 85% to 90%. After contact inhibition for two days, the complete culture medium was discarded, and a culture medium containing 10 μg / mL insulin, 0.5 mM IBMX and 1 μM Dex was added for 2 days (the time of adding the induction medium was recorded as day 0, induction I). Then, the culture medium was replaced with a culture medium containing 10 μg / mL insulin (induction II) for continuous culture for 2 days. Then, the culture medium was replaced with a normal culture medium for continuous culture for 4 days, and the medium was replaced every other day. On the 8th day of induction, more than 85% of the cells in the cells exhibited the morphology of mature adipocytes, and had fat droplets of different sizes, which were "ring-like".

[0245] 2.2 Oil red O staining

[0246] The 3T3-L1 cells in good condition were inoculated on a 6-well plate at a plating density of 5 x 10 4 mL, and cultured in high-sugar DMEM medium containing 10% FBS until the cell density reached about 85% to 90%. After contact inhibition for two days, the complete culture medium was discarded, and a culture medium containing 10 μg / mL insulin, 0.5 mM IBMX and 1 μM Dex was added for 2 days (the time of adding the induction medium was recorded as day 0, induction I). Then, the culture medium was replaced with a culture medium containing 10 μg / mL insulin (induction II) for continuous culture for 2 days. Then, the culture medium was replaced with a normal culture medium containing 10 μM norpetanin for incubation, and the medium was replaced every other day. After the induction, the cells were fixed with 4% neutral formaldehyde for 30 min. After the cells were fixed, the oil red O working solution prepared in advance was added to the cell surface, and the cells were placed in the dark for 60 min. After the staining was completed, the cells were washed with 70% ethanol, the excess dye was discarded, and the cells were washed with ultrapure water for 3 to 4 times. Finally, the cells were observed under a microscope and photographed.

[0247] 2.3 TG content determination

[0248] The 3T3-L1 cells were inoculated on a 6-well plate at a plating density of 5 x 10 4The cells were seeded in 6-well plates at a density of 1 x 105 / mL, and were induced to differentiate when the cell density reached 85% to 90%. The TG content was determined on the 8th day of induction, according to the following method.

[0249] (1) Cell pretreatment: on the 8th day of induction, the cell culture solution was removed, and the cells were washed twice with cold PBS and then digested with trypsin.

[0250] (2) Cell collection: after the cells were digested, the cells were resuspended in PBS and centrifuged at 1000g for 5 min to collect the cell precipitate.

[0251] (3) Ultrasonic disruption: the collected precipitate was added with an appropriate amount of PBS and then subjected to ultrasonic disruption (3 min).

[0252] (4) Determination: 2 μL of the cell disruption suspension was added to each well of a 96-well plate, 2 μL of distilled water was added to the blank wells, and 2 μL of the standard was added to the standard wells, followed by the addition of 200 μL of the determination solution to each well, and mixing,

[0253] After incubation at 37°C for 10 min, the absorbance was read at 510 nm. The protein concentration in the sample was determined by the BCA method, and the calibration was performed. Finally, the TG content was calculated according to the formula.

[0254] TG content = (OD sample - OD blank) / (OD calibration - OD blank) x calibration concentration (mM) / protein concentration of the sample to be tested (gprot / L)

[0255] 2.4 Western blot analysis

[0256] The cell samples obtained by the treatment were subjected to protein extraction, according to the following method:

[0257] (1) RIPA lysis buffer was mixed with PMSF at a ratio of 100:1 (if the phosphorylated protein is analyzed, a phosphatase inhibitor needs to be added), to prepare the cell lysis buffer. The cells in the 6-well plate were washed with cold PBS for 3 times, 100 μL of the cell lysis buffer was added to each well, and the cells were lysed on ice for 5 min. Then, the cells were scraped with a cell scraper and collected into a 1.5 mL centrifuge tube. The cells were again placed in a 4°C refrigerator for lysis for 30 min. Finally, the liver tissue homogenate and the cell lysis buffer were centrifuged at 12000g, 4°C for 15 min, and the supernatant protein was transferred to a new EP tube.

[0258] (2) BCA method for determining protein content: Take out an appropriate amount of protein sample, according to the BCA protein quantitative kit instructions, dilute the protein standard to 0.5 mg / mL with PBS buffer, mix reagent A: reagent B at a ratio of 50: 1 to prepare working solution. According to Table 13, add standard and PBS to the 96-well plate, 20 μL per well, generally 3 replicates for each concentration, used to draw the standard curve. Protein sample 1 μL is added to the 96-well plate, and PBS is added to 20 μL. Add 200 μL BCA working solution to each well and mix well. Incubate the above samples in a microplate incubator at 37°C on a shaking incubator for 30 min. Measure the absorbance value at 562 nm on a microplate reader. Calculate the protein concentration of the sample according to the standard curve. The prepared protein sample is heated at 100°C for 10 min to completely denature the protein. The sample is centrifuged before loading to ensure uniformity.

[0259] Table 13 BCA method for protein quantification

[0260]

[0261] (3) SDS-PAGE electrophoresis: according to the molecular weight of the target protein, select the appropriate separation gel concentration (as shown in Table 14), prepare the appropriate solution system (lower gel), and wait for the lower gel to be completely solidified at room temperature for more than 40 min, then prepare 5% concentrated gel (upper gel), and insert the appropriate comb. After the concentrated gel is solidified, place the gel plate in the electrophoresis tank, add enough electrophoresis liquid between the two plates, pull out the comb, load the protein sample and marker, and then run the gel. Electrophoresis conditions: S1 stage, 80V, 30min; S2 stage, 120V, about 60-90min, until the bromophenol blue in the buffer runs to the bottom of the separation gel.

[0262] (4) Membrane transfer: immediately after the protein electrophoresis is completed, transfer the membrane. According to the marker band, cut the target protein position according to the molecular weight. Cut the PVDF membrane to the same size as the gel, activate the cut membrane in methanol solution for 1 min, and then place it in the transfer solution. According to the order of transfer clamp blackboard-filter paper-gel PVDF-filter paper-transfer clamp whiteboard, clamp the electric transfer clamp, the blackboard is against the black side of the tank, place it in the electrophoresis tank, add enough transfer liquid to the whole tank, and then connect the power. Place the transfer instrument in an ice-water mixture. The transfer conditions are constant current 250 mA, time 90 min.

[0263] Table 14 Optimal separation range of different concentrations of SDS-PAGE separation gel

[0264]

[0265]

[0266] (5) Blocking: After the end of the transfer, the PVDF membrane was quickly taken out from the transfer plate with tweezers and placed in TBST buffer for 5 min, and then immediately blocked with 5% skim milk powder in TBST at room temperature for 1 h.

[0267] (6) Primary antibody incubation: After the end of blocking, the PVDF membrane was placed in the diluted primary antibody solution and incubated slowly on a horizontal shaker at 4°C overnight.

[0268] (7) Secondary antibody incubation: After the end of primary antibody incubation, the PVDF membrane was washed with TBST three times, 8 min each time. Then the PVDF membrane was placed in a solution containing HRP-labeled mouse or rabbit secondary antibody and incubated slowly at room temperature for 1 h.

[0269] (8) Development: After the end of secondary antibody incubation, the PVDF membrane was washed with TBST three times, 8 min each time. After washing, the mixed ECL developing solution was added to the PVDF membrane, and the gel imaging system was used for photography.

[0270] (9) Result analysis: The gray values of target proteins and internal reference proteins were quantitatively analyzed by Image J software, and the relative expression of proteins was the ratio of target proteins to internal reference proteins (the ratio of phosphorylated proteins to non-phosphorylated proteins).

[0271] 2.5 Statistical analysis

[0272] GraphPad Prism7 software was used for plotting and statistical analysis, independent sample t test was used for comparison of means between two groups, one-way ANOVA was used for comparison of means between multiple groups, statistical data were expressed as mean ± standard deviation (x ± s), * or # represented P < 0.05, which indicated that there was statistical significance, ** or ## represented P < 0.01, which indicated that the difference was extremely statistically significant.

[0273] 3 Experimental results

[0274] 3.1 Effect of norpetanin on lipid droplet accumulation in 3T3-L1 cells:

[0275] The effect of norpetanin on lipid droplet accumulation in 3T3-L1 cells was observed by oil red O staining, and the results are shown in Figure 18 There was no accumulation of lipid droplets in undifferentiated cells, and differentiated cells contained a large amount of lipid droplets. After treatment with norpetanin, intracellular lipid droplets were significantly reduced.

[0276] 3.2 Effect of norpetanin on lipid content in 3T3-L1 cells:

[0277] The results of the effect of norpetanin on TG content are shown in Figure 19As shown, TG content in differentiated cells was significantly increased compared with undifferentiated cells (P < 0.01). TG content was reduced after norpetanin treatment and there were significant differences (P < 0.01) compared with differentiated cells.

[0278] 3.3 Effect of norpetanin on the expression of lipid metabolism proteins in 3T3-L1 cells

[0279] (1) Effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells

[0280] The effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells was analyzed by Western blot, and the results are shown in Figure 20 As shown, the expression levels of PPARy and C / EBPa proteins were low in undifferentiated 3T3-L1 cells. However, the expression levels of PPARy and C / EBPa proteins were high in induced differentiated cells. Compared with the differentiation group, norpetanin treatment could reduce the protein expression levels of PPARy and C / EBPa transcription factors to some extent.

[0281] (2) Effect of norpetanin on the expression of lipid synthesis-related proteins in 3T3-L1 cells

[0282] The effect of norpetanin on the expression levels of lipid synthesis-related proteins in 3T3-L1 cells was analyzed by Western blot, and the results are shown in Figure 21 As shown, the expression levels of FAS and ACC proteins were low in undifferentiated 3T3-L1 cells. However, the expression levels of FAS and ACC proteins were significantly increased in induced differentiated cells. Compared with the differentiation group, compound treatment could significantly reduce the expression levels of FAS and ACC proteins. Based on the above results, it was found that norpetanin reduced the accumulation of intracellular lipid droplets by inhibiting the expression of adipogenic transcription factors and lipid synthesis-related proteins.

[0283] 4 Summary

[0284] The effect of norpetanin on lipid accumulation in 3T3-L1 cells was investigated at cellular level. The differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes is regulated by transcription factors and adipogenic proteins, and the cell morphology also changes, such as the final appearance of "ring-like". Norpetanin can inhibit the accumulation of lipid droplets in 3T3-L1 cells and reduce the TG content in cells. The results of this experiment also show that norpetanin can inhibit the differentiation of 3T3-L1 adipocytes by inhibiting the expression levels of transcription factors such as PPARγ and C / EBPα, and reduce the accumulation of intracellular lipid droplets. At the same time, by inhibiting the expression levels of FAS and ACC proteins, it can inhibit lipid synthesis and improve the lipid metabolism level of cells.

Claims

1. A method for isolating an active substance in Lycium ruthenicum Murr., characterized by, It comprises the following contents: (1) Take the black fruit of Lycium ruthenicum Rott. fruit methanol extract; (2) The black fruit of Lycium ruthenicum Rott. fruit methanol extract and dry polyamide powder are mixed, dried and ground, and then sieved through a 20-mesh sieve to obtain a sieved powder; (3) The sieved powder in step (2) is loaded into a small medium-pressure chromatography column, and the medium-pressure chromatography column loaded with MCI is connected to a preparative liquid chromatography for dry loading; a three-phase system of water / methanol / dichloromethane is used for elution, and three components are obtained: Fr1 with a retention time of 12-39 min, Fr2 with a retention time of 39-139 min, and Fr3 with a retention time of 139-230 min; (4) Fr2 obtained in step (3) is gradient eluted with methanol and / or water as the mobile phase, and Fr2-5 is obtained with a retention time of 76-130 min; (5) Fr2-5 in step (4) is gradient eluted with water / methanol as the mobile phase on a C18 preparative chromatography column, and Fr2-5-3 is obtained with a retention time of 19-21 min; (6) Fr2-5-3 in step (5) is gradient eluted with water / acetonitrile as the mobile phase on a C18 preparative chromatography column, and Fr2-5-3-3 is obtained with a retention time of 33-35 min, which is the target active substance; The active substance is a composition comprising compounds represented by Formula II and Formula III or each optical isomer, each crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.

2. The separation method according to claim 1, characterized in that, The ratio of the compounds represented by Formula II and Formula III in the composition is 3:

1.

3. The separation method of claim 1, wherein, The elution conditions in step (3) are as follows: 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.

4. The separation method of claim 1, wherein, The elution conditions in step (4) are as follows: 0-120 min, 0-100% methanol; 120-140 min, 100% methanol; Flow rate: 50 mL / min; Detection wavelength: 254 nm; Filler: MCI GEL CHP20P resin; Chromatography column specifications: 49x460mm.

5. The separation method of claim 1, wherein, The elution conditions in step (5) are as follows: 0-60-65-90 min, 30%-42%-40%-95% methanol; Flow rate: 19 mL / min; Detection wavelength: 210 nm; Injection volume: 300 μL; Filler: C18 preparative chromatography column; Column specifications: 21.2x250mm, 5 μm.

6. The separation method of claim 1, wherein, The elution conditions in step (6) are as follows: 0-10-60 min, 5%-15%-17% acetonitrile; Flow rate: 19 mL / min; Detection wavelength: 210 nm; Injection volume: 500 μL; Filler: C18 preparative chromatography column; Column specifications: 21.2x250mm, 5 μm.

7. Use of the Fr2, Fr2-5, Fr2-5-3 according to claim 1, the composition according to claim 2, and pharmaceutically acceptable salts, hydrates or solvates thereof, for the manufacture of a product for the treatment and / or prevention of inflammation-related diseases.

8. Use according to claim 7, characterized in that, The product is a product for reducing the release of at least one of NO, PGE2, TNF-α, IL-β, IL-6, COX-2 and iNOS.

9. Use of the Fr2, Fr2-5, Fr2-5-3 according to claim 1, the composition according to claim 2, and pharmaceutically acceptable salts, hydrates or solvates thereof, for the manufacture of a product for improving glucose metabolism or / and lipid metabolism.

10. Use according to claim 9, characterized in that, The product is a product having a lipid-lowering effect, or a product for inhibiting adipocyte differentiation.

11. Use according to claim 9, characterized in that, The product is a product for inhibiting intracellular lipid droplet accumulation and / or intracellular lipid production.

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