Oleanolic acid as well as preparation method and application thereof

Abstract

The invention provides oleanolic acid, and a preparation method of the oleanolic acid comprises the following steps: S1, dissolving 5g of arginic acid in 500ml of dichloromethane, adding 7g of pyridinium dichlorochromate, stirring at room temperature for 18 hours, and detecting the reaction degree by thin-layer chromatography; s2, after the reaction is completed and vacuum concentration is carried out, a sample is mixed with 200-300-mesh silica gel, the mixture is volatilized to be dry and then placed on a silica gel column, column chromatography separation is carried out, petroleum ether / ethyl acetate is selected as a mobile phase, 2.87 g of oleanolic acid is obtained, the molecular formula is C30H46O3, and the yield is 57.6%. The oleanolic acid provided by the invention can be used for preparing medicines for treating oligospermia and medicines for improving dyszoospermia. The oleanolic acid provided by the invention is a natural product or an artificially synthesized product, is low in toxicity and small in side effect, and has good effects of improving dyszoospermia and promoting fertility.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a oleanonic acid, a preparation method thereof and application thereof. BACKGROUND

[0002] Oleanonic acid is a pentacyclic triterpenoid compound, and exists in different parts of traditional herbal medicines, Chinese medicines and some fruits, such as illipe nuts, olives, ginseng and licorice. The content and distribution of oleanonic acid are different due to factors such as plant species, growth environment and growth stage. Due to the extremely small content and the complex and low-yield plant extraction process, the high requirement for plant resources and the great difficulty in purification in the laboratory, the chemical synthesis method of oleanonic acid mainly includes total synthesis and semi-synthesis. The total synthesis method is to synthesize oleanonic acid from simple organic raw materials through multiple chemical reactions, and this method has more reaction steps and greater synthesis difficulty. Since oleanonic acid has chiral structure, its synthesis is very difficult, and the economic cost is high. The semi-synthesis method uses natural plant extracts as raw materials to synthesize oleanonic acid through chemical modification, and this method is relatively simple and has low cost.

[0003] Due to the difficulty in obtaining oleanonic acid, the current research on the pharmacological activity of oleanonic acid is limited. There is no report on the research of oleanonic acid in the field of reproduction. And there is no effective single drug for oligospermia patients in the current clinic. SUMMARY

[0004] To solve the above technical problems, the application provides a oleanonic acid, a preparation method thereof and application thereof. The oleanonic acid is a natural product or an artificial synthetic product, has low toxicity, small side effects, can resist oxidation and has the effect of improving sperm concentration.

[0005] To solve the above technical problems, the embodiment of the application provides a oleanonic acid, which is a crystal compound and has the following chemical formula: , The crystal compound is white powder, mp: 190 DEG C; EI-MS m / Z: 454.68.

[0006] The application further provides a preparation method of the oleanonic acid, which comprises the following steps: S1, 5g of soil angelica acid is dissolved in 500ml of dichloromethane, 7g of dichloro-chromic acid pyridine salt is added, stirring is performed at room temperature for 18h, and thin layer chromatography is used to detect the reaction degree; S2, after the reaction is completed, the sample is mixed with 200-300 mesh silica gel, dried, and then subjected to column chromatography separation on a silica gel column, and a mobile phase selected from petroleum ether / ethyl acetate (2:1) is used to obtain 2.87g of oleanonic acid with a molecular formula of C30 H 46 O3, yield 57.6%.

[0007] wherein, the preparation reaction formula is as follows: .

[0008] The application further provides an application of oleanolic acid in preparation of a drug for treating oligospermia.

[0009] Further, when the oleanolic acid is used to prepare the drug for treating oligospermia, the effective dose is in the range of 0.01 ug / mL-10 ug / ml.

[0010] The application further provides an application of oleanolic acid in preparation of a drug for improving spermatogenic disorder.

[0011] The application further provides an application of oleanolic acid in preparation of a drug for improving spermatogenic disorder.

[0012] The application has the following beneficial effects: The application provides oleanolic acid (3-oxoolean-12-ene-28-oic acid) and an application thereof in preparation of a drug for improving spermatogenic disorder. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a hydrogen spectrum of oleanolic acid in the application; Figure 2 It is a carbon spectrum of oleanolic acid in the application; Figure 3 It is a microscopic image of CASA sperm in different drug administration treatments; Figure 4 It is a statistical diagram of CASA sperm parameters in different treatment groups; Figure 5 It is a testis HE staining diagram of different drug administration groups. DETAILED DESCRIPTION

[0014] In order to make the technical problems, technical solutions and advantages of the application clearer, the following will be described in detail in combination with the drawings and specific embodiments.

[0015] The embodiment of the application provides oleanolic acid, which is a crystalline compound, and the chemical formula is as follows: , The crystal compound is white powder, mp: 190 DEG C; EI-MS m / Z: 454.68.

[0016] A preparation method of oleanolic acid, comprising the following steps: S1, dissolve 5g of the soil ligusticum acid in 500ml of dichloromethane, add 7g of dichloro chromic acid pyridine salt, stir at room temperature for 18h, and detect the reaction degree by thin layer chromatography; S2, after the reaction is completed, the sample is mixed with 200-300 mesh silica gel, dried, and then loaded on a silica gel column for column chromatography separation, and the mobile phase is selected from petroleum ether / ethyl acetate (2:1) to obtain 2.87g of oleanolic acid. 30 H 46 O3, the yield is 57.6%.

[0017] The preparation reaction formula is as follows: .

[0018] The application further provides an application of the oleanolic acid in preparation of a drug for treating oligospermia.

[0019] When the oleanolic acid is used to prepare the drug for treating oligospermia, the effective dose is in the range of 0.01ug / mL-10ug / ml.

[0020] The application further provides an application of the oleanolic acid in preparation of a drug for improving spermatogenic disorder.

[0021] When the oleanolic acid is applied, it is prepared into a medical oral preparation, a powder preparation, a granule preparation, an injection capsule preparation, a pill preparation, a targeted preparation, a controlled release preparation, a nano sustained release preparation, a liposome preparation and other preparation of a drug delivery route.

[0022] The technical scheme of the application is further described below in combination with several specific embodiments.

[0023] Example 1: preparation of oleanolic acid Dissolve 5g of the soil ligusticum acid in 500ml of dichloromethane, add 7g of dichloro chromic acid pyridine salt, stir at room temperature for 18h, and detect the reaction degree by thin layer chromatography; after the reaction is completed, the sample is mixed with 200-300 mesh silica gel, dried, and then loaded on a silica gel column for column chromatography separation, and the mobile phase is selected from petroleum ether / ethyl acetate (2:1) to obtain 2.87g of oleanolic acid. 30 H 46 O3, the yield is 57.6%.

[0024] 1H NMR (400 MHz, CDCl3) δ, 5.30 (s, 1H), 2.84 (dd, J = 12.0, 4.0 Hz,1H), 2.57 – 2.50 (m, 1H), 2.40 – 2.34 (m, 1H), 2.05 –1.85 (m, 5H), 1.82 –1.72 (m, 2H), 1.68 – 1.59(m, 3H), 1.49 – 1.31 (m, 8H), 1.28 – 1.23 (m, 4H), 1.15 (s, 3H), 1.08 (s, 3H), 1.04 (d, J =8.0, 6H), 0.98 (d, J = 4.0 Hz, 1H), 0.92 (d, J =8.0, 5H), 0.81 (s, 2H). 13 C NMR (100 MHz, CDCl3) C: 217.86, 184.04, 143.66, 122.38, 60.46,55.28, 47.46, 34.16, 33.07, 32.14,27.67, 25.85, 23.48, 22.65, 19.54, 15.03,14.22, 11.47. Example 2: The effect of oleanolic acid in improving oligoasthenospermia method: 1. After 1 week of acclimatization feeding, 48 mature 7-week-old male ICR mice were randomly divided into 8 groups: model group, blank group, low, medium and high concentration groups of angelica acid (10mg / kg, 20mg / kg and 40mg / kg), and low, medium and high concentration groups of oleanolic acid (10mg / kg, 20mg / kg and 40mg / kg), with 6 mice in each group.

[0025] 2. Cyclophosphamide was prepared into a solution with a concentration of 12 mg / mL. Except for the blank group mice, all other groups were given intraperitoneal injection at a dose of 60 mg / kg for 7 consecutive days to establish a mouse model of oligoasthenospermia.

[0026] 3, low, medium and high concentration groups of oleanolic acid (10 mg / kg, 20 mg / kg, 40 mg / kg), low, medium and high concentration groups of oleanonic acid (10 mg / kg, 20 mg / kg, 40 mg / kg) (prepared into 10% cellulose acetate sodium solvent), while the model group and the blank group are given 0.2 mL of 10% cellulose acetate sodium solvent by gavage every day, and each group is continuously gavaged for 4 weeks. After the end of the experiment, all the mice were sacrificed, the heart was taken, the serum was separated, the testis was removed and weighed, and the epididymal semen was extracted. The sperm density and sperm motility of mice were detected by CASA computer aided sperm quality analysis system. The frozen sections of mouse testis tissue were stained by HE to detect the morphological recovery of testis tissue.

[0027] Results: 1. The results of sperm count and sperm motility detection showed that compared with the model group, the difference was statistically significant (P<0.05), and compared with the model group, the administration of oleanolic acid did not show an increase in sperm parameters, and the administration of oleanonic acid significantly improved the sperm parameters, and the difference was statistically significant (P<0.05).

[0028] 2. HE staining Figure 5 The results showed that compared with the normal group, the morphology of testis tissue in the model group was obviously damaged and injured, the spermatogenesis process was disordered, and the spermatogenic cells were lost; compared with the model group, oleanolic acid did not show significant efficacy, and the morphology of testis tissue, spermatogenesis process and spermatogenic cells in the oleanonic acid administration group were obviously recovered.

[0029] Figure 1 The hydrogen spectrum of oleanonic acid in the present application is shown in the figure; Figure 2 The carbon spectrum of oleanonic acid in the present application is shown in the figure.

[0030] Figure 3 The CASA sperm microscopic images of different administration treatments are shown in the figure; the number of sperm in the oligoasthenospermia model group is significantly reduced, and oleanolic acid administration does not show obvious efficacy. The sperm concentration significantly increases with the increase of oleanonic acid concentration.

[0031] Figure 4 The CASA sperm parameter statistical graph of different treatment groups is shown in the figure; A: sperm concentration; B: sperm motility statistics. ** P<0.01 VS normal group; # P<0.05 VS oligoasthenospermia model group; ## P<0.01 VS oligoasthenospermia model group; ### P<0.001 VS oligoasthenospermia model group, n=6.

[0032] The inventors of the present application have found that oleanonic acid has obvious effects on oligospermia after long-term research. Through further research, the inventors have found that oleanonic acid has obvious therapeutic effects on white arsenic-induced oligospermia model mice in mice, and the sperm count of oligospermia mice is obviously improved after 4 weeks of continuous administration, and oleanonic acid of different concentrations has promoting effects on the sperm concentration of mice.

[0033] The present application provides a kind of oleanonic acid (3-oxoolean-12-ene-28-acid) and its application in the preparation of improving spermatogenic disorder drugs, the oleanonic acid is natural product or artificial synthesis product, it has low toxicity, side effect is small, with good improvement spermatogenic disorder, promote the effect of fertility.

[0034] The above is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled person in the art, without departing from the principles described in the present application, a number of improvements and refinements can also be made, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An oleanonic acid characterized in that, The crystal compound is white powder, and has a mp of 190 DEG C and an EI-MS m / Z of 454.

68. ， The method comprises the following steps:

2. A process for the preparation of oleanonic acid, characterized in that, S1, dissolving 5g of the aralia cordata acid in 500ml of dichloromethane, adding 7g of dichloro-chromic acid pyridine salt, stirring at room temperature for 18h, and detecting the reaction degree by thin layer chromatography; The preparation reaction formula is as follows: S2, reaction is complete, vacuum concentration, the sample is mixed with 200-300 mesh silica gel, dry and then on the silica gel column, column chromatography separation, mobile phase selected with petroleum ether / ethyl acetate, get 2.87g oleanolic acid, molecular formula is C 30 H 46 O3, yield 57.6%.

3. The method of preparing oleanonic acid according to claim 2, characterized by, 4. The use of oleanolic acid in the preparation of a drug for treating oligospermia. 。 When the oleanolic acid is used to prepare the drug for treating oligospermia, the effective dose is in the range of 0.01ug / mL-10ug / ml.

5. The use of oleanonic acid according to claim 4 for the preparation of a medicament for the treatment of oligospermia, characterized in that, 6. The use of oleanolic acid in the preparation of a drug for improving spermatogenic disorder. The oleanolic acid is prepared into a pharmaceutically acceptable oral preparation, a powder preparation, a granule preparation, an injection preparation, a capsule preparation, a pill preparation, a targeted preparation, a controlled-release preparation, a nano-sustained-release preparation, a liposome preparation and other preparations with different administration routes.

7. Use of a oleanonic acid, characterized in that, ​

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