Application of iridoid compound in medicine for resisting cerebral neuropathy degenerative diseases and preparation method of iridoid compound

Abstract

The invention provides application of iridoid compounds in drugs for resisting cerebral neuropathy degenerative diseases, and belongs to the technical field of iridoid compounds. The iridoid compound disclosed by the invention is one of acetylated loganin, loganin and loganin aglycone. The compound has a good inhibiting effect on LPS-induced BV-2 inflammatory cell NO release, is dose-dependent, possibly becomes a potential drug for resisting cerebral diseases such as cerebral ischemia and cerebral ischemia-reperfusion injury, and provides a certain reference for finding drugs for preventing and treating CIRI and other cerebral diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of iridoid compounds, and in particular to application of iridoid compounds in drugs for treating brain neuropathic diseases and a preparation method thereof. Background Art

[0002] Traditional Chinese medicine nourishes the brain by nourishing the kidneys first. The brain and kidneys have the closest relationship, because the kidneys store essence and are responsible for growth and development, and for bone and marrow production, that is, the bone marrow, spinal cord, and brain marrow. Only when the kidney qi is abundant can the brain marrow grow and develop healthily. Otherwise, if the marrow is not nourished, the person will suffer from forgetfulness, loss of consciousness, etc.

[0003] Cornus officinalis can nourish the liver and kidney, astringe and consolidate the body, and has the effects of nourishing the liver and kidney and strengthening the tendons and bones. Loganin is the main quality marker of Cornus officinalis according to the Pharmacopoeia, and its content shall not be less than 1.2%. Loganin is an O-glycoside iridoid terpenoid glycoside compound with poor stability and easy hydrolysis. Its aglycone has potential pharmacological activity and high clinical application value, but there is little research on the preparation and application of its aglycone, and it is often used in anti-tumor drugs. For example, the invention patent with publication number CN107362158A discloses the use of loganin in the preparation of anti-tumor drugs. The tumors described herein specifically relate to mammalian gastric cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, lung cancer, head and neck cancer, leukemia, malignant lymphoma, prostate cancer, breast cancer, glioma, bile duct cancer, melanoma, kidney cancer, ovarian cancer, endometrial cancer, and cervical cancer, and more specifically to human gastric cancer, human liver cancer, human esophageal cancer, human colorectal cancer, human pancreatic cancer, human lung cancer, human head and neck cancer, human leukemia, human malignant lymphoma, human prostate cancer, human breast cancer, human glioma, human bile duct cancer, human melanoma, human kidney cancer, human ovarian cancer, human endometrial cancer, and human cervical cancer. Through in vivo and in vitro experiments, it was found that loganin had a good inhibitory effect on the above-mentioned various tumors, showed good anti-tumor growth activity, and had great potential for development as an anti-tumor drug. Publication Nos.: CN113209079A, CN113209078A, CN113230246A, etc. disclose the use of loganin in the preparation of esophageal cancer prevention and treatment drugs, pancreatic cancer prevention and treatment drugs, and lung cancer prevention and treatment drugs, respectively.

[0004] It can be seen that in the prior art, loganin and its aglycones are often used in anti-tumor drugs, and there is no report on whether they can resist cerebral ischemia-reperfusion injury (CIRI). Summary of the invention

[0005] In view of this, in order to solve the technical problem that compounds such as loganin and its aglycone in the prior art have not been studied in the treatment of cerebral ischemia-reperfusion injury, the present invention provides the use of cyclopentadiene compounds in drugs for treating brain neuropathic diseases, wherein the cyclopentadiene compound is one of acetylated loganin, loganin and loganin, and has a good inhibitory effect on the release of NO from BV-2 inflammatory cells induced by LPS, and is dose-dependent, and may become a potential drug for treating brain diseases such as cerebral ischemia, cerebral ischemia-reperfusion injury and other related diseases, and provides a certain reference for finding drugs for preventing and treating brain diseases such as CIRI.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention discloses an application of an iridoid compound in a drug for treating brain neuropathic diseases. The iridoid compound comprises one of acetylated loganin, loganin and loganin.

[0008] Preferably, the concentration of the acetylated loganin is 1.56-12.5 μmol / L.

[0009] Preferably, the concentration of loganin is 3.13-50 μmol / L.

[0010] Preferably, the concentration of loganin is 6.25-100 μmol / L.

[0011] Preferably, the method for preparing acetylated loganin comprises the following steps:

[0012] Step (1), treatment of crude loganin

[0013] The crude loganin was dissolved in methanol, and the residue was removed by filtration. The filtrate was concentrated under reduced pressure and then separated and purified by silica gel column chromatography. All samples with Rf values ​​of 0.2-0.4 were concentrated to obtain a light yellow oil.

[0014] Step (2), preparation of acetylated loganin

[0015] The oil obtained in step (1) is dissolved in acetic anhydride and sodium acetate solution, refluxed, cooled to room temperature, added into water and stirred, extracted with dichloromethane and dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure, separated and purified by silica gel column chromatography, and the obtained white solid powder is acetylated loganin.

[0016] Preferably, in step (1), the developing solvents for separation and purification by silica gel column chromatography are ethyl acetate and methanol, and the volume ratio of the two is 8:1.

[0017] Preferably, in step (2), the developing solvent for separation and purification by silica gel column chromatography is ethyl acetate and petroleum ether, and the volume ratio of the two is 5:1.

[0018] Preferably, the method for preparing loganin comprises the following steps:

[0019] Step (3), adding metallic sodium to anhydrous methanol until the metallic sodium disappears, then adding acetylated loganin, and then adding a strongly acidic cation exchange resin, stirring at room temperature overnight until pH = 7;

[0020] The resin is removed by filtration, the solvent is evaporated from the filtrate, and the residue is dried to obtain pure white loganin.

[0021] Preferably, the method for preparing loganin comprises the following steps:

[0022] Step (4), sequentially adding sodium dihydrogen phosphate solution, loganin, β-glucosidase and n-butanol into a reactor, heating the reactor for reaction, adding ice water to quench the reaction after the reaction is complete, extracting with dichloromethane and drying, filtering to remove the solid, concentrating the filtrate under reduced pressure to obtain a light yellow oil, and separating and purifying by silica gel column chromatography to obtain a white solid powder, which is loganin.

[0023] Preferably, in step (4), the developing solvent for separation and purification by silica gel column chromatography is ethyl acetate and petroleum ether, and the volume ratio of the two is 1.5:1.

[0024] The present invention has the following beneficial effects compared with the prior art:

[0025] The invention provides an application of an iridoid compound in a drug for treating brain neuropathic diseases, wherein the iridoid compound is one of acetylated loganin, loganin and loganin, and has a good inhibitory effect on the NO release of BV-2 inflammatory cells induced by LPS in a dose-dependent manner, and may become a potential drug for treating brain diseases such as cerebral ischemia, cerebral ischemia-reperfusion injury and other related diseases, and provides a certain reference for finding drugs for preventing and treating brain diseases such as CIRI.

[0026] Other technical effects of the present invention will be described in the following specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The synthetic route of loganin and its aglycone is shown in the figure. DETAILED DESCRIPTION

[0028] The invention provides an application of an iridoid compound in a drug for treating brain neuropathic diseases, wherein the iridoid compound comprises one of acetylated loganin, loganin and loganin.

[0029] The invention takes loganin, a quality marker of Cornus officinalis, as a main research object, obtains pure loganin by subjecting a commercially available crude loganin product containing 10% to alcohol precipitation, column chromatography separation, acetylation and deacetylation, etc., hydrolyzes loganin with β-glucoside to obtain loganin aglycone, and uses BV-2 cells of an inflammation model induced by lipopolysaccharide (LPS) to carry out prevention and treatment of loganin and its aglycone against brain neuropathic diseases, such as cerebral ischemia-reperfusion injury and the like.

[0030] Its synthetic route is Figure 1 As shown, the details are as follows:

[0031] Step (1), treatment of crude loganin

[0032] The crude loganin was dissolved in methanol, and the residue was removed by filtration. The filtrate was concentrated under reduced pressure and then separated and purified by silica gel column chromatography. All samples with Rf values ​​of 0.2-0.4 were concentrated to obtain a light yellow oil.

[0033] The developing solvents for separation and purification by silica gel column chromatography are ethyl acetate and methanol, and the volume ratio of the two is preferably 8:1.

[0034] Step (2), preparation of acetylated loganin

[0035] The oil obtained in step (1) is dissolved in acetic anhydride and sodium acetate solution, refluxed, cooled to room temperature, added into water and stirred, extracted with dichloromethane and dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure, separated and purified by silica gel column chromatography, and the obtained white solid powder is acetylated loganin.

[0036] The developing solvents for separation and purification by silica gel column chromatography are ethyl acetate and petroleum ether, and the volume ratio of the two is 5:1.

[0037] In the present invention, the preparation method of loganin comprises the following steps:

[0038] Step (3), adding metallic sodium to anhydrous methanol until the metallic sodium disappears, then adding acetylated loganin, and then adding a strongly acidic cation exchange resin, stirring at room temperature overnight until pH = 7;

[0039] The resin is removed by filtration, the solvent is evaporated from the filtrate, and the residue is dried to obtain pure white loganin.

[0040] In the present invention, the preparation method of loganin comprises the following steps:

[0041] Step (4), sequentially adding sodium dihydrogen phosphate solution, loganin, β-glucosidase and n-butanol into a reactor, heating the reactor for reaction, adding ice water to quench the reaction after the reaction is complete, extracting with dichloromethane and drying, filtering to remove the solid, concentrating the filtrate under reduced pressure to obtain a light yellow oil, and separating and purifying by silica gel column chromatography to obtain a white solid powder, which is loganin.

[0042] The developing solvents for separation and purification by silica gel column chromatography are ethyl acetate and petroleum ether, and the volume ratio of the two is 1.5:1.

[0043] The technical solution of the present invention is described clearly and in detail below in conjunction with specific implementation methods.

[0044] 1. Preparation of products (such as Figure 1 The synthetic route shown)

[0045] 1. Processing of crude loganin

[0046] Take 10g of 10% crude loganin and dissolve it in 100mL of methanol at room temperature with stirring for 1h, then let it stand at room temperature for 1h, filter out the residue, and concentrate the filtrate under reduced pressure and separate and purify it by silica gel column chromatography. The developing solvent is ethyl acetate: methanol 8:1. All samples with Rf values ​​of 0.2-0.4 are collected and concentrated to obtain a light yellow oil, 2.2g.

[0047] 2. Preparation of Loganin

[0048] The above oily substance was dissolved in a round-bottom flask containing 30 mL of acetic anhydride and 0.15 g of sodium acetate, and directly placed in a 144°C oil bath for heating and reflux reaction for 30 min, then cooled to room temperature, added into 200 ml of water and stirred for 2 h, and then extracted with dichloromethane (100 mL*3). The combined dichloromethane was dried over anhydrous sodium sulfate for 1 h, and the filtrate was concentrated under reduced pressure after filtration. It was separated and purified by silica gel column chromatography with a developing solvent of ethyl acetate: petroleum ether in a ratio of 1:1. The white solid powder obtained after separation and purification was 1.25 g of acetylated loganin. 1H NMR(CDCl3)δ:7.27(s,1H),5.23-5.24(m,2H),5.21(s,1H),5.10-5.15(m,1H),4.99(t, J=9.6Hz,1H)4.85(d,J=8.4Hz,1H),4.30-4.32(m,1H),4.13-4.16(m,1H),3.73-3.76(m ,1H),3.70(d,J=6.0Hz,3H),3.01(d,J=5.4Hz,1H),2.22-2.27(m,2H),2.10(s,3H),2.0 4(d,J=3.0Hz,6H),2.01(s,3H),1.91(s,4H),1.81-1.84(m,1H),1.02(d,J=6.6Hz,3H).

[0049] Add 0.1 g of sodium metal to 10 mL of anhydrous methanol and stir at room temperature until the sodium metal disappears. Then add 0.5 g of acetylated loganin and continue stirring for 1 h, then add 1 g of strongly acidic cation exchange resin and stir overnight at room temperature until pH = 7.

[0050] The resin was removed by filtration, and the filtrate was evaporated on an optional evaporator to remove the solvent. The obtained residue was further dried on a vacuum pump at room temperature to obtain 0.78 g of pure white loganin.

[0051] Loganin, white powdery solid, mp = 84-85°C: 1 HNMR(D2O)δ:7.36(s,1H),5.12(d,J=4.8Hz,1H),4.96(s,2H),5.01(s,1H)4.56- 4.51(m,3H),3.89(s,1H),3.68(d,J=11.4Hz,1H),3.62(s,3H),3.41-3.44(m,1H ),3.13-3.16(m,2H),3.03(t,J=9.6Hz,1H),2.94-2.98(m,2H),2.06-2.01m,1H) ,1.82-1.86(m,1H),1.70-1.73(m,1H),1.42-1.46(m,1H),0.99(d,J=7.8Hz,3H).

[0052] 3. Preparation of Loganin

[0053] Hydrolysis of loganin: 10 mL of 0.2 mol / L sodium dihydrogen phosphate solution, 0.5 g loganin, 0.025 g β-glucosidase and 10 mL of n-butanol were added to a round-bottom flask in sequence, and the reaction system was heated at 50 degrees Celsius for 3 h. After TLC showed that the reaction was complete, 20 mL of ice water was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate for 1 h. The solid was removed by suction filtration, and the filtrate was concentrated under reduced pressure to obtain a light yellow oil, which was separated and purified by silica gel column chromatography (developing solvent: ethyl acetate: petroleum ether = 1.5:1) to obtain a white solid powder, i.e. loganin.

[0054] 1 H NMR(D2O)δ:7.37(s,1H),7.21(d,J=6.0Hz,1H),4.76(t,J=6.0Hz,1H),4.43(d,J=3.6Hz,1H),3.87(s,1H),3.61(s,3H),2.94 (q,J=9.0Hz,1H),2.05-2.09(m,1H),1.70(q,J=6.6Hz,1H),1.64(q,J=7.2Hz,1H),1.31-1.35(m,1H),0.99(d,J=7.2Hz,3H).

[0055] 2. Biologically active part

[0056] 1. BV-2 cell culture

[0057] Take a cryovial containing 1 mL of cell sample, and then put the cryovial into a water bath, set the temperature to 37°C, so that the cell sample can be quickly thawed and activated. After activation, add 4 mL of complete culture medium (10% fetal bovine serum + 1% penicillin / streptomycin mixture + 89% DMEM high glucose culture medium) to mix it with the cells thoroughly, and then place the mixed cells in a centrifuge and centrifuge at 1200 rpm for 3 minutes. After centrifugation, pour out the supernatant, then add 5 mL of complete culture medium and blow evenly, and finally set the cell growth environment to 37°C, 5% CO 2 .

[0058] 2. Effects of different drug concentrations on the activity of BV-2 cells

[0059] When BV-2 cells were in the growth phase, the cell suspension was prepared by trypsin digestion. 100 μL of the prepared cell suspension was added to a 96-well plate, and the density of inoculated cells per well was controlled to be 2×10 4 Fill the edges of the cell wells with sterile phosphate buffered saline (PBS). 2The cells were cultured in a culture incubator with a growth environment set to 5% CO 2 , cultured at 37°C for 24 hours until the cells adhered to the wall and covered the bottom of the wells of the cell plate.

[0060] Different concentrations of the iridoid terpenoid Chinese medicine monomers loganin, acetylated loganin, and loganin aglycone were added to the cell plate (8 concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μmol / L, and the 8 concentrations refer to the 8 concentrations of loganin, acetylated loganin, and loganin aglycone, respectively), and a blank control group (control) was set up. Different concentrations of acetylated loganin, loganin, and loganin aglycone were set up to treat 4 replicate wells. After 24 hours, 10 μL of CCK-8 solution was added to each well, and then the culture plate was gently tapped to mix it thoroughly. The culture environment conditions were set to 5% CO 2 , 37°C, and the treated cell plates were incubated for 1 hour. Finally, the absorbance was measured at 450nm using an ELISA reader, and the inhibition rates of acetylated loganin, loganin, and loganin aglycone on cells at different concentrations were calculated.

[0061] 3. Determination of NO Levels in BV-2 Cells

[0062] The cell seeding density per well was set to 2 × 10 4 After culturing for 24 hours, different concentrations of acetylated loganin, loganin, and loganin aglycone (8 concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μmol / L) were added to the cell plate, and a positive control group (model) and a blank control group (control) were set up. After pre-protecting the cells for 1 hour, 10 μL of lipopolysaccharide (LPS, 2 μg / mL) was added to induce a cell inflammation model.

[36] After culturing for 24 hours, 50 μL of the supernatant was aspirated, and an equal amount of Griess reagent was added to determine the NO content in the supernatant. The absorbance was measured at 550 nm using an ELISA reader, and the inhibition rates of acetylated loganin, loganin, and loganin aglycone at different concentrations on cells were calculated. The measured absolute absorbance was set as the vertical axis, and the standard concentration was set as the horizontal axis. The standard curve of NO was drawn according to the results to calculate the NO concentration of the sample.

[0063] 4. Evaluation of pharmacological activity of compounds

[0064] Table 1 Effects of acetylated loganin on LPS-induced proliferation and NO release in BV-2 cells

[0065]

[0066] The CCK-8 method was used to determine the effects of different concentrations of acetylated loganin on cell survival rate. According to Table 1, when the concentration of acetylated loganin was 25-100 μmol / L, it had a significant inhibitory effect on cell proliferation, but when it was 0.78-12.5 μmol / L, it had no significant inhibitory effect on BV-2 cells. Its LD 50 It is 58.08μmol / L.

[0067] When the concentration of acetylated loganin was 1.56-12.5 μmol / L, it had a significant inhibitory effect on the NO release level of BV-2 cells induced by LPS. Its IC 50 It was 12.44 μmol / L, indicating that acetylated loganin had good anti-inflammatory activity on BV-2 cells, indicating that the compound may have a good preventive and therapeutic effect on cerebral ischemia-reperfusion injury.

[0068] Table 2 Effects of loganin on LPS-induced proliferation and NO release in BV-2 cells

[0069]

[0070] The CCK-8 method was used to determine the effect of different concentrations of loganin on the survival rate of BV-2 cells. According to Table 2, when the concentration of loganin was 0.78-50 μmol / L, the proliferation of BV-2 cells was inhibited in a dose-dependent manner, but there was no significant inhibitory effect, indicating that loganin had very little cytotoxicity to BV-2 cells, and its LD 50 It is 488μmol / L.

[0071] When the concentration of loganin was 0.78-50 μmol / L, the compound had no inhibitory effect on cell survival rate, and at the same time, it inhibited the LPS-induced NO release level of BV-2 cells in a dose-dependent manner. At the concentration of 3.13-50 μmol / L, loganin had a significant inhibitory effect on the LPS-induced NO release level of BV-2 cells, indicating that the compound may have a good preventive and therapeutic effect on cerebral ischemia-reperfusion injury.

[0072] Table 3 Effects of loganin on LPS-induced proliferation and NO release in BV-2 cells

[0073]

[0074]

[0075] The CCK-8 method was used to determine the effect of different concentrations of loganin on cell survival rate. According to Table 3, when the concentration of loganin was 0.78-100 μmol / L, there was no significant inhibitory effect on BV-2 cells, indicating that loganin had very little cytotoxicity to BV-2 cells; when the concentration of loganin was 1.56-100 μmol / L, the compound concentration had no inhibitory effect on cell survival rate, and at the same time, it inhibited the NO release level of BV-2 cells induced by LPS in a dose-dependent manner, and at a concentration of 6.25-100 μmol / L, loganin had a significant inhibitory effect on the NO release level of BV-2 cells induced by LPS, indicating that the compound may have a certain preventive and therapeutic effect on cerebral ischemia-reperfusion injury.

[0076] In summary, in the present invention, acetylated loganin, loganin and loganin aglycone all have a good inhibitory effect on the NO release of BV-2 inflammatory cells induced by LPS in a dose-dependent manner, and may become potential drugs for treating brain diseases such as cerebral ischemia, cerebral ischemia-reperfusion injury and other related diseases, providing a certain reference for finding drugs for preventing and treating brain diseases such as CIRI.

[0077] The above are only preferred specific implementations of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and improved concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. The use of iridoid compounds in drugs for treating brain neuropathic diseases, characterized in that: The iridoid compound includes one of acetylated loganin, loganin and loganin.

2. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The concentration of the acetylated loganin is 1.56-12.5 μmol / L.

3. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The concentration of loganin is 3.13-50 μmol / L.

4. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The concentration of loganin is 6.25-100 μmol / L.

5. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The preparation method of the acetylated loganin comprises the following steps: Step (1), treatment of crude loganin The crude loganin was dissolved in methanol, and the residue was removed by filtration. The filtrate was concentrated under reduced pressure and then separated and purified by silica gel column chromatography. All samples with Rf values ​​of 0.2-0.4 were concentrated to obtain a light yellow oil. Step (2), preparation of acetylated loganin The oil obtained in step (1) is dissolved in acetic anhydride and sodium acetate solution, refluxed, cooled to room temperature, added into water and stirred, extracted with dichloromethane and dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure, separated and purified by silica gel column chromatography, and the obtained white solid powder is acetylated loganin.

6. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 5, characterized in that: In step (1), the developing solvents for separation and purification by silica gel column chromatography are ethyl acetate and methanol, and the volume ratio of the two is 8:

1.

7. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 5, characterized in that: In step (2), the developing solvent for separation and purification by silica gel column chromatography is ethyl acetate and petroleum ether, and the volume ratio of the two is 5:

1.

8. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The preparation method of loganin comprises the following steps: Step (3), adding metallic sodium to anhydrous methanol until the metallic sodium disappears, then adding acetylated loganin, and then adding a strongly acidic cation exchange resin, stirring at room temperature overnight until pH = 7; The resin is removed by filtration, the solvent is evaporated from the filtrate, and the residue is dried to obtain pure white loganin.

9. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 1, characterized in that: The preparation method of loganin comprises the following steps: Step (4), sequentially adding sodium dihydrogen phosphate solution, loganin, β-glucosidase and n-butanol into a reactor, heating the reactor for reaction, adding ice water to quench the reaction after the reaction is complete, extracting with dichloromethane and drying, filtering to remove the solid, concentrating the filtrate under reduced pressure to obtain a light yellow oil, and separating and purifying by silica gel column chromatography to obtain a white solid powder, which is loganin.

10. The use of the iridoid compound in a drug for treating brain neuropathic diseases according to claim 9, characterized in that: In step (4), the developing solvent for separation and purification by silica gel column chromatography is ethyl acetate and petroleum ether, and the volume ratio of the two is 1.5:1.

Images