Preparation method and application of triterpenoid active site and triterpenoid compound

Abstract

The invention relates to a preparation method and application of a triterpenoid active site and a triterpenoid compound. The preparation method of the triterpenoid active part comprises the following steps: S1, adding an ethanol aqueous solution into fructus rosae laevigatae for reflux extraction, and concentrating to obtain an extract; s2, enabling the extract to pass through a macroporous adsorption resin column, eluting, concentrating and drying, extracting with ethyl acetate, performing chromatography, performing gradient elution with ethanol, and performing vacuum concentration to obtain a triterpenoid active part; the ethanol gradient elution process comprises the following steps: eluting by using a 0 wt% ethanol aqueous solution, a 50 wt% ethanol aqueous solution, a 75 wt% ethanol aqueous solution and a 95 wt% ethanol aqueous solution in sequence, combining eluents of the 50 wt% ethanol aqueous solution and the 75 wt% ethanol aqueous solution, and concentrating under reduced pressure. The fructus rosae laevigatae triterpenoid and the triterpenoid active part have a better effect in activity test of reducing uric acid, and are expected to become a new generation of medicines for improving and reducing uric acid.

Description

Technical Field

[0001] The present invention relates to an extract of Rosa laevigata Michx., and particularly to a preparation method and application of a triterpene active site and triterpenoid compounds. Background Art

[0002] Rosa laevigata Michx. (RLM) is an evergreen climbing shrub, which is widely distributed in southern China. The fruits of Rosa laevigata Michx. are widely used as food, such as nutritional oral liquids, fruit wines, acetic acid beverages, fruit juices, etc., and can also be used to extract brown pigments used as food additives. Traditional Chinese medicine Rosa laevigata Michx. is the dried ripe fruit of the plant Rosa laevigata Michx. of the Rosaceae family, and is a variety included in the Chinese Pharmacopoeia. It tastes sour, sweet, and astringent, is of neutral nature, and belongs to the kidney, bladder, and large intestine meridians. It has the effects of tonifying the kidney and securing essence to reduce urination, securing the metrorrhagia and leukorrhagia, and astringing the intestine to stop diarrhea. It is commonly used in symptoms such as spermatorrhea and emission, enuresis and frequent urination, metrorrhagia and leukorrhagia, chronic diarrhea and dysentery, etc. Modern research shows that Rosa laevigata Michx., which is both a medicine and a food, has various biological activities such as lipid-lowering, blood sugar-lowering, immune regulation, kidney protection, cardiovascular protection, antioxidant, anti-inflammatory, antibacterial, and antiviral effects, and has a mild effect, which makes it widely used in clinical practice and daily life.

[0003] Rosa laevigata Michx. mainly contains components such as polysaccharides, triterpenes, and flavonoids. Modern pharmacological research shows that Rosa laevigata Michx. has activities such as antioxidant, immune protection, anti-inflammatory, liver protection, kidney protection, cardiovascular protection, nerve protection, improvement of diabetes, lipid-lowering, anti-acetylcholinesterase, anti-tumor, and antibacterial effects.

[0004] The prior art CN 118697813 A discloses a traditional Chinese medicine composition, a drug, a preparation method, and a use for treating hyperuricemia. The traditional Chinese medicine composition is composed of Euryale ferox Salisb., Rosa laevigata Michx., Plantago asiatica L., Alisma orientale (Sam.) Juz., and Bupleurum chinense DC. The present invention can intervene in the early stage of hyperuricemia, has few side effects, is suitable for more people, and has many advantages.

[0005] The prior art CN 115252692 A relates to the application of a traditional Chinese medicine composition in the preparation of drugs for hyperuricemia-related diseases, belonging to the technical field of traditional Chinese medicine. The active ingredients of the traditional Chinese medicine composition are composed of the following raw materials in parts by weight: 400-800 parts of Cibotium barometz, 150-220 parts of Rosa laevigata Michx., 300-420 parts of Spatholobus suberectus Dunn, 100-200 parts of Flemingia philippinensis Merr. ex Rolfe, 300-420 parts of Kadsura coccinea (Lem.) A. C. Smith, 180-300 parts of Millettia speciosa Champ., 20-40 parts of Ligustrum lucidum Ait., 90-250 parts of Taxillus chinensis (DC.) Danser, 20-40 parts of Cuscuta chinensis Lam., 13-52 parts of Corydalis yanhusuo W. T. Wang, 13-52 parts of Zanthoxylum nitidum (Roxb.) DC., 6-24 parts of Olibanum, 10-30 parts of Myrrha. The traditional Chinese medicine composition can not only reduce the uric acid level of rats with hyperuricemia complicated with acute gouty arthritis, relieve the inflammatory reaction, but also reduce the level of inflammatory factors, inhibit the activity of XOD, promote the excretion of UA, and down-regulate the expression of related inflammatory proteins. It can be applied to the preparation of drugs for treating hyperuricemia-related diseases and has good application prospects.

[0006] However, the above prior arts all exist in the form of traditional Chinese medicine compositions, and there is no prior art research on which specific parts and active ingredients in Rosa laevigata Michx. are effective in reducing uric acid. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method of a triterpene active fraction, triterpenoids and their application in reducing uric acid.

[0008] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0009] A preparation method of a triterpene active fraction, comprising the following steps:

[0010] Step S1: Add an ethanol aqueous solution to Rosa laevigata Michx. fruit for reflux extraction, and concentrate to obtain an extract;

[0011] Step S2: Pass the extract through a macroporous adsorption resin column, elute, concentrate and dry, extract with ethyl acetate, chromatograph, elute with an ethanol gradient, and concentrate under reduced pressure to obtain a triterpene active fraction;

[0012] The process of the ethanol gradient elution is as follows: elute successively with 0wt% ethanol aqueous solution, 50wt% ethanol aqueous solution, 75wt% ethanol aqueous solution, 95wt% ethanol aqueous solution, combine the eluates of 50wt% ethanol aqueous solution and 75wt% ethanol aqueous solution, and concentrate under reduced pressure.

[0013] In one preferred embodiment, in step S1, the mass concentration of the ethanol aqueous solution is 40-60%.

[0014] In one preferred embodiment, in step S1, the mass ratio of Rosa laevigata Michx. fruit to the ethanol aqueous solution is 4:20-28.

[0015] In one preferred embodiment, in the step S1, the temperature for reflux extraction is 75 - 85 °C, the reflux time is 2 - 3 h, and the number of refluxes is 3 - 5 times.

[0016] In one preferred embodiment, in the step S1, the temperature for concentration is 60 - 70 °C, and the concentration time is 3 - 4 days.

[0017] In one preferred embodiment, in the step S2, the macroporous adsorption resin column is HP20 macroporous adsorption resin.

[0018] In one preferred embodiment, in the step S2, the elution is carried out by eluting successively with water and 80 wt% ethanol aqueous solution; the ethanol aqueous solution elution part is concentrated and dried, the dried component is dispersed with water and then extracted with ethyl acetate, the number of extractions is 8 - 12 times, the extracted solutions are combined and then subjected to chromatography.

[0019] In one preferred embodiment, in the step S2, the chromatography is carried out using a polyamide column with a mesh number of 100 - 200 meshes.

[0020] In one preferred embodiment, after the triterpene fraction is subjected to silica gel column chromatography, the TLC spots are developed, and analyzed using an ultraviolet lamp and HPLC - DAD.

[0021] Based on the same inventive concept, the present invention also claims the application of the triterpene active fraction in the preparation of a drug for reducing uric acid.

[0022] Based on the same inventive concept, the present invention also claims the application of a triterpene compound in the preparation of a drug for reducing uric acid, and the structural formula of the triterpene compound is:

[0023]

[0024] In the formula, R1 is selected from hydrogen, hydroxyl; R2 is selected from hydrogen, hydroxyl; R3 is selected from hydrogen, D - glucose; R4 is hydroxyl, and the carbon to which R4 is attached can be left - handed or right - handed.

[0025] In one preferred embodiment, when R1 and R3 are both hydrogen, R2 is not hydrogen.

[0026] In one preferred embodiment, the structural formula of the triterpene compound is:

[0027]

[0028] Based on the same inventive concept, the present invention also claims the preparation method of the triterpene compound, including the following steps:

[0029] Step A: Pass the triterpene active fraction through a silica gel column, elute with a gradient dichloromethane-methanol solution, identify the same components, and combine the same components to obtain 9 fractions, Fr.1 - 9;

[0030] Step B: Pass Fr.3 through a silica gel column, elute with a gradient dichloromethane-methanol solution to obtain 6 fractions, Fr.3.1 - 3.6; Isolate the crystals that are poorly soluble in methanol from Fr.3.4, and separate the crystals by semi-preparative liquid chromatography to obtain Compound 4 and Compound 5;

[0031] Step C: Isolate white crystals from Fr.3.3, and purify the white crystals by semi-preparative liquid chromatography to obtain Compound 3;

[0032] Step D: Crystallize Fr.5 to obtain white crystals, and purify the white crystals by semi-preparative liquid chromatography to obtain Compound 2; Purify Fr.6 by semi-preparative liquid chromatography to obtain Compound 6; Recrystallize Fr.7 with an aqueous methanol solution to obtain Compound 1.

[0033] In one preferred embodiment, in the said Step A, the gradient dichloromethane-methanol solution elution is carried out with dichloromethane-methanol solutions of different concentrations; in the dichloromethane-methanol solutions of different concentrations, the volume ratios of dichloromethane to methanol are successively 100:0, 100:1, 100:2, 100:3, 100:4, 100:5, 100:7, 100:10, 100:0.

[0034] In one preferred embodiment, in the said Step A, in the silica gel column, the mesh number of the silica gel packed in the column is 200 - 300 mesh.

[0035] In one preferred embodiment, in the said Step A, the same components are identified by the 10% sulfuric acid-ethanol color reaction method.

[0036] In one preferred embodiment, in the said Step B, the gradient dichloromethane-methanol solution elution is carried out with dichloromethane-methanol solutions of different concentrations; in the dichloromethane-methanol solutions of different concentrations, the volume ratios of dichloromethane to methanol are successively 100:1, 100:2, 100:4, 0:100.

[0037] In one preferred embodiment, in the said Step D, the volume concentration of the aqueous methanol solution is 40 - 60%.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] 1. The present invention provides a method for extracting ursane-type pentacyclic triterpene compounds 1 - 5 from Rosa laevigata Michx., and this method is simple and reproducible.

[0040] 2. The present invention has first discovered the new applications of the triterpene active fraction and the oleanane-type pentacyclic triterpene compounds 1-5, and studied their pharmacological mechanisms, which are expected to become new drugs for reducing uric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the 1 1H-NMR spectrum of Compound 1;

[0042] Figure 2 is the 13 13C-NMR spectrum of Compound 1;

[0043] Figure 3 is the 1 1H-NMR spectrum of Compound 2;

[0044] Figure 4 is the 13 13C-NMR spectrum of Compound 2;

[0045] Figure 5 is the 1 1H-NMR spectrum of Compound 3;

[0046] Figure 6 is the 13 13C-NMR spectrum of Compound 3;

[0047] Figure 7 is the 1 1H-NMR spectrum of Compound 4;

[0048] Figure 8 is the 13 13C-NMR spectrum of Compound 4;

[0049] Figure 9 is the 1 1H-NMR spectrum of Compound 5;

[0050] Figure 10 is the 13 13C-NMR spectrum of Compound 5;

[0051] Figure 11 is the 1 1H-NMR spectrum of Compound 6;

[0052] Figure 12 is the 13 13C-NMR spectrum. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0054] Example 1

[0055] 1.1 Preparation of triterpenoid active fractions: 4 kg of seedless pulp of Rosa laevigata was taken, dried and crushed, and then added to 30 L of 50% ethanol and refluxed (at 80±5°C) for 2 h. Extraction was performed four times in total. The four extracts (120 L) were combined and concentrated under reduced pressure at 60°C for 3-4 days to 5 L. Half of the concentrate (2.5 L) was taken out and vacuum dried to obtain 314.4 g of Rosa laevigata total extract. The remaining half of the concentrate was passed through HP20 macroporous adsorption resin (Beijing Green Baicao Technology Development Co., Ltd.) column chromatography, eluting with water (25 L) and 80% ethanol (40 L) in sequence. The 80% ethanol elution fraction was concentrated under reduced pressure and vacuum dried to obtain 252 g of the 80% alcohol wash fraction. The 80% alcohol portion was dispersed with 5L of water and then extracted with ethyl acetate 20 times (5L each time). The ethyl acetate extraction portion was concentrated under reduced pressure and dried to obtain 109g. The ethyl acetate extraction portion was chromatographed on a 100-200 mesh polyamide (Sinopharm Group) column and eluted with pure water (10L), 50% ethanol (20L), 75% ethanol (20L) and 95% ethanol (20L). The 50% ethanol and 75% ethanol elution portions were concentrated under reduced pressure to precipitate, the precipitate was centrifuged and dried to obtain 78.78g of the triterpene active portion.

[0056] 1.2 Study on active components of triterpenes

[0057] 1.2.1 Separation and purification:

[0058] The systematic chemical composition study of the triterpenoid active fraction was carried out using modern chromatographic separation techniques such as silica gel and Prep-HPLC. According to the characteristic UV absorption and TLC color reaction of triterpenoids, HPLC-DAD and TLC methods were used for tracking, analysis, separation and purification. 78.78 g of the triterpenoid active fraction was taken. The sample was mixed with 100 g of 80-100 mesh silica gel by silica gel column chromatography, and the column was packed with 654 g of 200-300 mesh silica gel, and gradient elution was carried out with dichloromethane:methanol system (the volume ratios of dichloromethane and methanol were 100:0 17 L, 100:1 32 L, 100:2 29 L, 100:3 18 L, 100:4 18 L, 100:5 18 L, 100:7 41 L, 100:10 10 L, 100:0 10 L). Through the UV absorption at 254 nm / 365 nm and combined with the 10% sulfuric acid-ethanol color development method to guide the fraction combination, finally 9 fractions (Fr.1-9) were combined and concentrated. 4 g of Fr.3 fraction was used, and 6 g of 80-100 mesh silica gel was added by silica gel column chromatography for sample mixing, and the column was packed with 200 g of 200-300 mesh silica gel, and gradient elution was carried out with dichloromethane:methanol system (the volume ratios of dichloromethane and methanol were 100:1 20 L, 100:2 40 L, 100:4 10 L, 0:100 5 L) to obtain 6 fractions (Fr.3.1-3.6). A part of the crystals insoluble in methanol was obtained from Fr.3.4 fraction, and compound 4 (10.33 mg, t R = 27.515 min), 5 (7.58 mg, t R = 38.171 min) were separated by semi-preparative liquid chromatography (Agilent 1260 Infinity, methanol:water = 72:18, 2 mL / min). A relatively white crystal was obtained from Fr.3.3 fraction, and compound 3 (9.25 mg, t R = 34.554 min) was purified by semi-preparative liquid chromatography (Agilent 1260 Infinity, methanol:water = 73:17, 2 mL / min). A relatively white crystal was obtained by crystallization of Fr.5 fraction, and compound 2 (1.31 g, t R = 26.339 min) was purified by semi-preparative liquid phase (Agilent 1260 Infinity, methanol:water = 65:15, 2 mL / min). Fr.6 was purified by semi-preparative liquid chromatography (Agilent 1260 Infinity, methanol:water = 65:15, 2 mL / min) to obtain compound 6. There was a main spot on the TLC plate of Fr.7 fraction, and compound 1 (7.55 g) was obtained by recrystallization with 50% methanol-water.

[0059] 1.2.2 Structure Confirmation: The structures of the compounds were confirmed by NMR, and spectroscopic characterization and systematic assignment of spectral data were carried out.

[0060] First, the absorption type of the compound and the presence of conjugated fragments were confirmed by ultraviolet absorption. Then, 1D / 2D NMR was used to determine the planar structure of the compound. The results were as follows: The 1 1H-NMR spectrum of Compound 1 was as shown in Figure 1 ; The 13 13C-NMR spectrum of Compound 1 was as shown in Figure 2 ; The 1 1H-NMR spectrum of Compound 2 was as shown in Figure 3 ; The 13 13C-NMR spectrum of Compound 2 was as shown in Figure 4 ; The 1 1H-NMR spectrum of Compound 3 was as shown in Figure 5 ; The 13 13C-NMR spectrum of Compound 3 was as shown in Figure 6 ; The 1 1H-NMR spectrum of Compound 4 was as shown in Figure 7 ; The 13 13C-NMR spectrum of Compound 4 was as shown in Figure 8 ; The 1 1H-NMR spectrum of Compound 5 was as shown in Figure 9 ; The 13 13C-NMR spectrum of Compound 5 was as shown in Figure 10 ; The 1 1H-NMR spectrum of Compound 6 was as shown in Figure 11 ; The 13 13C-NMR spectrum of Compound 6 was as shown in Figure 12 as shown.

[0061] 1.2.3 Chemical Structure

[0062] Six ursane-type pentacyclic triterpenoids (1 - 6) were isolated, and their structures are as follows:

[0063]

[0064] Example 2

[0065] 2.1 Study on the uric acid-lowering effect of the triterpene active fraction and triterpenoids

[0066] 2.1.1 Experimental method:

[0067] 64 Kunming mice (weighing 20 - 25 g, male), after 1 week of adaptive feeding, were randomly divided into 8 groups: blank control group, model control group, allopurinol group (10 mg / kg), total extract of Rosa laevigata Michx. group (310 mg / kg), triterpenoid active fraction group (80 mg / kg), compound 1 group (80 mg / kg), compound 2 group (60 mg / kg), and compound 4 group (60 mg / kg). The dosage of the total extract of Rosa laevigata Michx. group was calculated based on the extraction method. Since 78.78 g of triterpenoid active fraction was extracted from 314.4 g, when converted to the same amount, the dosage of the total extract of Rosa laevigata Michx. group was 310 mg / kg. At 9 am, except for the blank control group, mice in other groups were intragastrically administered with an adenine-containing 0.5% sodium carboxymethylcellulose (0.5% CMC-Na) suspension (adenine: 100 mg / kg) and intraperitoneally injected with a potassium oxonate suspension (potassium oxonate: 300 mg / kg), while mice in the blank control group were intragastrically administered / intraperitoneally injected with an equal dose of 0.5% CMC-Na / H2O, and the intragastric volume was 10 mL / kg. One hour later, except for the blank control group, mice in other groups were intragastrically administered with yeast extract (10 g / kg), while mice in the blank control group were intragastrically administered with an equal dose of H2O, and the intragastric volume was 10 mL / kg. Two weeks after modeling, at 2 pm, mice in each drug group were intragastrically administered with the corresponding dose of the drug, and mice in the blank control group and the model control group were intragastrically administered with an equal dose of 0.5% CMC-Na, and the drug administration continued for 28 days. On the evening of the 27th day of drug administration, the mice were fasted but allowed to drink water for 12 h. One hour after the last drug administration, the mice were weighed and then anesthetized with chloral hydrate. Blood was collected from the orbital cavity into heparin sodium anticoagulant blood collection tubes, centrifuged at 3500 rpm at 4°C for 15 min, and the plasma was aliquoted and stored in a -80°C refrigerator for later use. Plasma uric acid (uric acid test kit: Nanjing Jiancheng Bioengineering Institute, C012-2-1) and liver xanthine oxidase activity (xanthine oxidase (XOD) activity detection kit: Beijing Solarbio Science & Technology Co., Ltd., BC1095) were detected by kits. The expression levels of liver xanthine oxidase and renal uric acid transporters were detected by immunoblotting: 50.00 mg of frozen left kidney tissue / liver tissue was weighed on ice into a 2 mL centrifuge tube, and 0.5 mL of RIPA lysis buffer (protease inhibitor and phosphatase inhibitor were added in a mass ratio of 100:1, freshly prepared) was added thereto. Tissue homogenization was performed using an electric grinder, and the tissue was lysed on ice for 30 min. During this period, it was shaken several times to ensure complete lysis. After lysis, it was centrifuged at 13000 r / min at 4°C for 15 min, and the supernatant was aspirated. Protein concentration determination was performed on the lysed supernatant (BCA protein concentration determination kit: Beijing Solarbio Science & Technology Co., Ltd., BC1095PC0020). According to the measurement results, the supernatant and the lysate were quantitatively aspirated, and 5× protein loading buffer was added in proportion, mixed well, and denatured at 95°C for 10 min. After denaturation, it was mixed well and aliquoted and stored in a -80°C refrigerator for later use.Protein samples equivalent to 40 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis conditions: constant voltage (upper gel: 80 V; lower gel: 150 V). The protein bands were transferred to a polyvinylidene fluoride membrane (PVDF membrane) by the wet method. Transfer conditions: constant current of 210 mA for 2 h. Blocking was carried out with 10% skim milk at room temperature for 2 h, washed 3 times with TBST, 10 min each time, and then incubated overnight at 4 °C in the refrigerator with relevant primary antibodies (GAPDH, 1:20000, proteintech: 60004-1-Ig; β-actin, 1:5000, proteintech: 66009-1-Ig; URAT1, 1:2000, Immunoway: YN3803; GLUT9, 1:5000, proteintech: 67530-1-Ig; XOD, 1:2000, proteintech: 55156-1-AP). After completion, it was washed with TBST for 5 min, washed a total of 6 times. After washing, it was incubated with the corresponding HRP-conjugated goat anti-rabbit / mouse (proteintech: SA00001-2 and SA00001-1) on a shaker at room temperature for 1 h. After incubation, the membrane was washed with TBST for 5 min, washed a total of 6 times, and then the membrane was immersed in an enhanced chemiluminescence reagent (ECL luminescence solution, Biosharp: BL523B), and the target protein was corrected with GAPDH / β-actin.

[0068] 2.1.2 Experimental results:

[0069] As shown in Table 1, compared with the blank control group, the plasma uric acid in the model control group was significantly increased, indicating that the HUA mouse model was successfully established. Compared with the model control group, allopurinol, the total extract of Rosa laevigata Michx. and the triterpenoid active fraction could significantly reduce the plasma uric acid level in mice, indicating that the triterpenoid active fraction has the activity of reducing plasma uric acid in HUA mice and is the active fraction of Rosa laevigata Michx. in reducing uric acid. At the same time, the triterpenoid active fraction could significantly reduce the content and activity of xanthine oxidase in the liver and down-regulate the expression of renal uric acid reabsorption transporters (urate transporter 1 and glucose transporter 9), indicating that the triterpenoid active fraction can reduce uric acid production and promote uric acid excretion to reduce plasma uric acid. The total extract of Rosa laevigata Michx. could significantly reduce the activity of xanthine oxidase in the liver and down-regulate the expression of renal uric acid reabsorption transporter (urate transporter 1), and had no effect on the content of xanthine oxidase in the liver and glucose transporter 9 in the kidney. The uric acid-lowering effects of Compound 1, Compound 2 and Compound 4 were better than those of the total extract of Rosa laevigata Michx. and the triterpenoid active fraction, and the triterpenoid active fraction was better than the total extract of Rosa laevigata Michx.

[0070] Table 1 Effects of triterpenoid active fraction on plasma uric acid in hyperuricemia mice (x±s, n = 6)

[0071]

[0072]

[0073] Note: Compared with the blank group: # P < 0.05, ## P < 0.01, ### P < 0.001; Compared with the model group: * P < 0.05, ** P < 0.01, *** P < 0.001. The structural formula of compound 9 is:

[0074] 2.2 Inhibiting xanthine oxidase activity

[0075] 2.2.1 Experimental method:

[0076] The in vitro XOD activity detection system consists of a drug, XOD solution (final concentration 20 U / L), xanthine solution (final concentration 120 μM), and 2 M hydrochloric acid. Allopurinol was used as the positive drug, as shown in Table 2:

[0077] Table 2 XOD activity detection system

[0078]

[0079]

[0080] 2.2.2 Experimental results:

[0081] By measuring the inhibitory activity of XOD in vitro, the inhibition curves of the triterpene active fraction and triterpenoids on XOD were obtained, and then the inhibition rate and sample concentration were fitted to a non-linear regression to calculate the IC 50 value. The results are shown in Table 4. It can be seen that the triterpene active fraction of Rosa laevigata has a certain XOD inhibitory activity, and the IC 50 value is 1.21 ± 0.08 mg / mL. Compounds 1 - 6 showed XOD inhibitory activity. Among them, the IC 50 value of compound 2 was the lowest, at 0.76 ± 0.06 mM, and the IC 50 value of compound 4 was 0.79 ± 0.01 mM.

[0082] Table 3 IC50 values of the total extract of Rosa laevigata and its various fractions for in vitro XOD inhibition

[0083]

[0084] From the above data, it can be seen that the triterpene active fraction and triterpenoids of Rosa laevigata showed a strong effect of reducing uric acid and are expected to be developed into new drugs for reducing uric acid.

[0085] It should be noted that the above embodiments are merely examples given to clearly illustrate the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A preparation method of a triterpene active fraction, characterized in that It includes the following steps: Step S1: Add an ethanol aqueous solution to Rosa laevigata Michx. fruit for reflux extraction, and concentrate to obtain an extract. Step S2: Pass the extract through a macroporous adsorption resin column, elute, concentrate and dry, extract with ethyl acetate, chromatograph, elute with an ethanol gradient, and concentrate under reduced pressure to obtain a triterpene active fraction. The process of the ethanol gradient elution is as follows: Elute successively with 0 wt% ethanol aqueous solution, 50 wt% ethanol aqueous solution, 75 wt% ethanol aqueous solution, and 95 wt% ethanol aqueous solution, combine the eluates of 50 wt% ethanol aqueous solution and 75 wt% ethanol aqueous solution, and concentrate under reduced pressure.

2. The preparation method according to claim 1, characterized in that, In the said Step S1, the mass concentration of the ethanol aqueous solution is 40 - 60%; the mass ratio of Rosa laevigata Michx. fruit to the ethanol aqueous solution is 4:20 - 28.

3. The preparation method according to claim 1, characterized in that, In the said Step S1, the temperature of the reflux extraction is 75 - 85 °C, the reflux time is 2 - 3 h, and the number of refluxes is 3 - 5 times.

4. The preparation method according to claim 1, characterized in that, In the said Step S2, the macroporous adsorption resin column is HP20 macroporous adsorption resin; the elution is carried out by eluting successively with water and 80 wt% ethanol aqueous solution; the ethanol aqueous solution elution part is concentrated and dried, the dried component is dispersed with water and then extracted with ethyl acetate, the number of extractions is 8 - 12 times, and the combined extraction solutions are chromatographed again.

5. The triterpene active fraction prepared by the preparation method according to any one of claims 1 - 4.

6. The application of the triterpene active fraction according to claim 5 in the preparation of a drug for reducing uric acid.

7. Use of a triterpenoid compound in the preparation of a drug for reducing uric acid, characterized in that, The structural formula of the said triterpenoid compound is: In the formula, R1 is selected from hydrogen, hydroxyl; R2 is selected from hydrogen, hydroxyl; R3 is selected from hydrogen, D - glucose; R4 is hydroxyl, The carbon to which R4 is connected can be left - handed or right - handed.

8. The application according to claim 7, characterized in that The structural formula of the said triterpenoid compound is:

9. A preparation method of a triterpenoid compound, characterized in that, It includes the following steps: Step A: Pass the triterpene active fraction through a silica gel column, elute with a gradient dichloromethane - methanol solution, identify the same components, and combine the same components to obtain 9 fractions Fr.1 - 9. Step B: Pass Fr.3 through a silica gel column, elute with a gradient dichloromethane - methanol solution, to obtain 6 fractions Fr. 3.1 - 3.6; Isolate the crystals insoluble in methanol from Fr.3.4, and separate the crystals by semi - preparative liquid chromatography to obtain Compound 4 and Compound 5. Step C: Isolate white crystals from Fr.3.3, and purify the white crystals by semi - preparative liquid chromatography to obtain Compound 3. Step D: Crystallize Fr.5 to obtain white crystals, purify the white crystals by semi - preparative liquid chromatography to obtain Compound 2; Purify Fr.6 by semi - preparative liquid chromatography to obtain Compound 6; Recrystallize Fr.7 with a methanol aqueous solution to obtain Compound 1.

10. The preparation method according to claim 9, characterized in that, In the said Step A, the gradient dichloromethane - methanol solution elution is carried out by eluting with dichloromethane - methanol solutions of different concentrations; in the dichloromethane - methanol solutions of different concentrations, the volume ratios of dichloromethane to methanol are successively 100:0, 100:1, 100:2, 100:3, 100:4, 100:5, 100:7, 100:10, 100:0.

Images