CN1762967A - Enoxolone derivative, preparation method and uses - Google Patents
Enoxolone derivative, preparation method and uses Download PDFInfo
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- CN1762967A CN1762967A CN 200510099361 CN200510099361A CN1762967A CN 1762967 A CN1762967 A CN 1762967A CN 200510099361 CN200510099361 CN 200510099361 CN 200510099361 A CN200510099361 A CN 200510099361A CN 1762967 A CN1762967 A CN 1762967A
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Abstract
The present invention provides one new kind of glycyrrhetic acid derivative with high water solubility, high absorption, no irritation to blood vessel and capacity of being prepared into injection, and its preparation process, medicinal composition and preparation. The glycyrrhetic acid derivative has excellent treating effect on acute and chronic hepatitis, bacterial hepatitis, viral hepatitis, cerebral ischemia/re-perfusion damage, brain injury and myocardial ischemia/re-perfusion damage.
Description
Technical field
The present invention relates to Enoxolone derivative and preparation method thereof, purposes and preparation.
Background technology
Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.) belongs to leguminous plants, grows in temperate zone and subtropical zone, and main product is stated from the Han dynasty Shennong's Herbal in China western part as herbal medicine head commonly used.Theory of traditional Chinese medical science is thought: Radix Glycyrrhizae property is flat, flavor is sweet, has and effects such as middle emergency, moistening lung, detoxifcation, cough-relieving, qualcomm meridian, sharp qi and blood, in the traditional Chinese medicine prescription, use extremely extensive, Ancient Times in China physician's analogy be " state is old ".1932, Ruzicka determined the chemical structure of effective liquorice Potenlini, had from then on opened the prelude with modern medicine and pharmacology research Radix Glycyrrhizae.Studies show that in a large number after this, the main pharmacodynamics composition of Radix Glycyrrhizae is Potenlini and glycyrrhetinic acid.
Glycyrrhetinic acid (being called for short GA) has another name called glycyrrhetinic acid, records in the national drug standards, and its standard No. is WS-10001-(HD-1182)-2002, is white crystalline powder (C
30H
46O
6), 288～297 ℃ of fusing points, water insoluble.
Chen Xiaoguang etc. studies show that GA can suppress mice ear and ornithine decarboxylase (ODC) activity that Oleum Tiglii brings out; it is synthetic to reduce the non-program DNA that strong carcinogen benzopyrene causes simultaneously; to certain protection effect [Chen Xiaoguang is arranged by the dna damage that benzopyrene brought out; Han Rui. glycyrrhetinic acid is to damage of benzopyrene induce dna and the influence of non-program DNA synthetic. Acta Pharmaceutica Sinica; 1994,29 (10): 725-729].18 β-glycyrrhetinic acid can suppress beta-glucosidase enzyme, and (activity of β-GD) is to CCl
4The liver injury of bringing out has provide protection [Sang-Bum Shim; Nam-Jae Kim; Dong-Hyun Kim. β-Glucuronidase Inhibitory Activity and Hepatoprotective Effect of 18-GlycyrrhetinicAcid from the Rhizomes of Glycyrrhiza urelensis.Planta Medica; 2000,66 (1): 40-43].Huang Wei etc. discover: 18 β-GA and GL have the human liver cancer cell of inhibition propagation and induce its Differentiation [Huang Wei, Huang Jiqun, .18 β-glycyrrhetinic acids such as Zhang Dongfang and Potenlini are to human liver cancer cell inhibition of proliferation and induction of differentiation. Chinese Chinese materia medica science and technology, 2002,9 (2): 92-94].
Glycyrrhetinic acid is water insoluble, absorption difference, and its chemical structure is similar to Kendall compound, has the effect of Desoxycortone sample, can combine with mineralcorticoid receptor to form the effect of aldosterone sample.Because the effect of aldosterone sample is too much, influences water, electrolyte metabolism, promote water-sodium retention, row's potassium increases, thereby causes many symptoms such as the reduction of blood potassium, elevation of blood pressure and edema.
Though glycyrrhetinic acid sodium salt or sylvite that present document is reported can improve its solvability, still have sodium retention, and blood vessel irritation is arranged, and can not be prepared into injection, have limited the clinical application of glycyrrhetinic acid greatly.
The inventor has overcome the above-mentioned shortcoming of glycyrrhetinic acid by a large amount of experimental studies, provides preparation simple, convenient, good water solubility, good absorption, and to the blood vessel nonirritant, the Enoxolone derivative that toxic side effects is little has solved people and has thirsted for the difficult problem that solves for a long time.
Summary of the invention
The invention provides the Enoxolone derivative shown in the formula I,
Formula I
Wherein glycyrrhetinic acid is 18 α-glycyrrhetinic acid, 18 β-glycyrrhetinic acid or their mixture; M is basic aminoacids or organic bases, described basic aminoacids is arginine, Histidine or Methionin, organic bases is meglumine or quadrol, is preferably arginine, meglumine or quadrol, and wherein arginine can be L-arginine, D-arginine or D, L-arginine.
The preparation method of derivative shown in the formula I provided by the invention is 1: 1 glycyrrhetinic acid and basic aminoacids (or organic bases), 60-90% ethanol for add mol ratio successively in reaction flask, stirring reaction 2 hours, the evaporated under reduced pressure solvent, in residue, add ethyl acetate, grind light-yellow precipitate, filter, drying under reduced pressure, promptly.
The invention provides the application of Enoxolone derivative in preparation treatment hepatitis medicament.
The invention provides the application of Enoxolone derivative in preparation treatment acute hepatitis medicine.
The invention provides the application of Enoxolone derivative in preparation treatment chronic hepatitis medicine.
The invention provides the application of Enoxolone derivative in the bacillary hepatitis medicament of preparation treatment.
The invention provides the application of Enoxolone derivative in preparation treatment viral hepatitis medicine.
The invention provides the application of Enoxolone derivative in the medicine of preparation treatment or prevention ischemia apoplexy.
The invention provides the application of Enoxolone derivative in the medicine of preparation treatment or prevention of brain ischemia/reperfusion injury.
The invention provides the application of Enoxolone derivative in the medicine of preparation treatment or prevention of brain wound.
The invention provides the application of Enoxolone derivative in the medicine of preparation treatment or prevention myocardial ischemia.
The present invention also provides the pharmaceutical composition that contains Enoxolone derivative.This pharmaceutical composition can be prepared from method conventional on the medicine.
Enoxolone derivative of the present invention is when being used for above-mentioned arbitrary purposes, and its using dosage is 1mg～600mg/ days, and preferred dose is 1mg～250mg, and more preferably dosage range is 20～200mg.
Compound provided by the invention can exist with the form of powder injection, injection liquid, tablet, capsule, soft capsule, dripping pill or oral liquid, and preferably the form with injection liquid exists.Various formulation provided by the invention all can adopt the pharmacy ordinary method to be prepared from.
Enoxolone derivative injection liquid provided by the invention preferably adopts following method preparation: take by weighing Enoxolone derivative, be dissolved in the solubility promoter under the heating, add buffer reagent and water for injection, regulate pH to 9.0～10.0, add gac and stir, filter, divide to be filled in the ampoule, 115 ℃ of sterilizations down, promptly.
Enoxolone derivative lyophilized injectable powder provided by the invention preferably adopts following method preparation: take by weighing Enoxolone derivative, be dissolved in the solubility promoter under the heating, add buffer reagent, propping agent and water for injection, regulate pH to 9.0～10.0, add gac and stir, filter, divide to be filled in the ampoule, lyophilize, promptly.
Above-mentioned solubility promoter is one or more in propylene glycol, ethanol, tween 80, polyoxyethylenated castor oil, the Betacylcodextrin; Buffer reagent is one or more in phosphoric acid salt, citrate, ammonium chloride, the veronal; Propping agent is one or more among N.F,USP MANNITOL, sucrose, lactose, glucose, dextran, the PVP.
Enoxolone derivative tablet provided by the invention comprises Enoxolone derivative, vehicle, lubricant, and wherein vehicle is one or more in starch, pregelatinized Starch, the Microcrystalline Cellulose; Lubricant is one or more in talcum powder, the Magnesium Stearate.
Derivative preparation process provided by the invention is simple, solve the solvability of glycyrrhetinic acid in water on the one hand, improved the security of medication on the other hand, reduced pungency and angiospastic generation, solve the technical barrier of preparation injection, filled up the blank of clinical application.
The present invention further illustrates the technology of the present invention by following embodiment, but is not limited thereto.
Embodiment:
Embodiment 1: the arginic preparation of glycyrrhetinic acid
In reaction flask, add glycyrrhetinic acid 2.35g (0.005mol), L one arginine 0.87g (0.005mol), 90% ethanol (V/V) 440mL successively, stirring reaction 2 hours, the evaporated under reduced pressure solvent, in residue, add the 20mL ethyl acetate, grind light-yellow precipitate, filter drying under reduced pressure, get light yellow solid 3.0g, yield is 93%.With glycyrrhetinic acid is that reference substance carries out assay, and product content is 99.7%.
The glycyrrhetinic acid arginine shows below physicochemical characteristic:
Proterties: buff powder mp:183～186 ℃.
ESI-MS m/z：644([M-H]
+)。
Ultimate analysis % calculated value (measured value): C 67.05 (67.48), H9.38 (9.27), N8.69 (8.81).
Molecular formula: (C
36H
60N
4O
4)
UVλ
maxnm：258
IR(KBr，v
max，cm
-1)：3172(-NH
2，-OH)，1647(-C＝C-C＝O)，2949，1645，1396(-CH
3，-CH
2-)，1545(-C＝C-，-C＝N)。
1H-NMR(DMSO，δ，ppm)：0.67-1.32(s，21H，7-CH
3)，5.39(s，1H，Δ
12-H)，
13C-NMR(DMSO，δ，ppm)：38.72(C
1)，26.15(C
2)，76.56(C
3)，38.87(C
4)，54.12(C
5)，17.11(C
6)，31.43(C
7)，42.14(C
8)，61.06(C
9)，36.62(C
10)，198.99(C
11)，126.96(C12)，170.88(C
13)，44.79(C
14)，24.83(C
15)，26.03(C
16)，29.30(C
17)，48.10(C
18)，40.13(C
19)，42.87(C
20)，28.91(C
21)，37.95(C
22)，26.92(C
23)，15.92(C
24)，16.13(C
25)，18.34(C
26)，23.04(C
27)，28.08(C
28)，28.57(C
29)，173.79(C
30)，179.99(Arg-COOH)，53.86(Arg-CH-COOH)，38.56(Arg-CH-CH
2-CH
2)，32.13(Arg-CH
2-CH
2-CH
2)，43.66(Arg-CH
2-CH
2-NH-)，157.69(Arg-C＝NH)。
Embodiment 2: the arginic preparation of glycyrrhetinic acid
In reaction flask, add glycyrrhetinic acid 2.35g (0.005mol), L-arginine 0.87g (0.005mol), 85% ethanol (V/V) 440mL successively, stirring reaction 2 hours, the evaporated under reduced pressure solvent, in residue, add the 20mL ethyl acetate, grind light-yellow precipitate, filter drying under reduced pressure, get light yellow solid 3.1g, yield is 96%.With glycyrrhetinic acid is that reference substance carries out assay, and product content is 99.6%.
Embodiment 3: the preparation of glycyrrhetinic acid R-Gene 10
Glycyrrhetinic acid arginic acid salt (according to embodiment 1 preparation) 5g
Propylene glycol 150g
Sodium Citrate 10g
Water for injection adds to 1000ml
Take by weighing recipe quantity glycyrrhetinic acid arginic acid salt, be dissolved in the propylene glycol under the heating, add Sodium Citrate and water for injection, 5%NaOH transfers pH to 9.0, add 0.1% gac and stirred 30 minutes, filter, divide to be filled in the ampoule, every 1ml, 115 ℃ (sterilization is 30 minutes down, promptly.
Embodiment 4: the preparation of glycyrrhetinic acid meglumine injection
Glycyrrhetinic acid meglumine 5g
Ethanol 100g
Sodium Citrate 10g
Water for injection adds to 1000ml
Take by weighing recipe quantity glycyrrhetinic acid meglumine, be dissolved in the ethanol under the heating, adding Sodium Citrate and water for injection, 5%NaOH transfers pH to 9.0, adds 0.1% gac and stirs 30 minutes, filters, and divides to be filled in the ampoule, and every 1ml sterilizes 30 minutes down promptly for 115 ℃.
Embodiment 5: the preparation of glycyrrhetinic acid arginine lyophilized injectable powder
Glycyrrhetinic acid arginic acid salt (according to embodiment 1 preparation) 5g
Ethanol 50g
Glucose 25g
Sodium Citrate 10g
Water for injection adds to 1000ml
Take by weighing recipe quantity glycyrrhetinic acid arginic acid salt, be dissolved in the ethanol under the heating, add recipe quantity glucose, Sodium Citrate and water for injection, 5%NaOH transfers pH to 9.0～10.0, adds 0.1% gac and stirs 30 minutes, filters, divide to be filled in the ampoule, every 1ml, lyophilize is promptly.
Embodiment 6: the preparation of glycyrrhetinic acid arginine tablet
Glycyrrhetinic acid arginic acid salt (according to embodiment 1 preparation) 30g
Microcrystalline Cellulose 150g
10% starch slurry is an amount of
Magnesium Stearate 1g
Make 1000 altogether
Take by weighing the recipe quantity glycyrrhetinic acid arginic acid salt of crushing screening, add Microcrystalline Cellulose, 10% starch slurry is granulated, drying, and whole grain adds recipe quantity Magnesium Stearate mixing, and compressing tablet is promptly.
Test example 1: derivative of the present invention causes the influence of chmice acute liver injury to tetracol phenixin
1. material: Kunming mouse, 5-8 age in week, body weight 18-22g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: 200203005.The male and female dual-purpose, same sex is adopted in every batch of experiment.
Tetracol phenixin (analytical pure, Yantai three and chemical reagent company limited, lot number 990918);
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine: by method preparation provided by the invention;
Glycyrrhizic acid,diammonium salt: the lot number DG0563 of Jiangsu Zhengda Tianqing Drug Industry Co., Ltd;
ALT/GPT test kit (Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 020081);
ASP/GOT test kit (Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 020101)
Automatic clinical chemistry analyzer (Italy)
2 methods: the single sex mouse of getting qualified body weight, divide 9 groups and tail vein injection administration (0.2ml/ only): the blank group, model group (normal saline solution that contains the equivalent auxiliary material), glycyrrhetinic acid arginase i group (1mg/kg), glycyrrhetinic acid arginase i I organizes (5mg/kg), glycyrrhetinic acid arginase i II organizes (10mg/kg), glycyrrhetinic acid arginase i V organizes (20mg/kg), glycyrrhetinic acid meglumine I organizes (1mg/kg), glycyrrhetinic acid meglumine II organizes (20mg/kg), Glycyrrhizic acid,diammonium salt group (30mg/kg), 12 every group, successive administration 3 days, behind the last administration 1h with 0.2% carbon tetrachloride solution modeling, the blood sampling of fasting 16h posterior orbit, centrifugal (4000rpm, 10min), collect serum, detect ALT/GPT with medicine box, the ASP/GOT activity.Data are represented with X ± s with data, carry out statistical procedures with t check between group.
3 results: the result is as shown in table 1, with model group comparison glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine, Glycyrrhizic acid,diammonium salt obvious suppression ALT/GPT, the effect (P＜0.05) alive of ASP/GOT enzyme is arranged all, and is dosage correlation.
The anti-CCL of table 1 Enoxolone derivative
4The liver injury effect
| Group | ASP/GOT enzyme (U/L) alive | ALT/GPT enzyme (U/L) alive |
| Control group model group enoxolone arginase i group enoxolone arginase i I group enoxolone arginase i II group enoxolone arginase i V group enoxolone meglumine I group enoxolone meglumine II group diammonium glycyrrhizinate | 159.58±66.83 1228.84±151.07 905.63±61.07 * 826.81±95.01 * 719.81±65.01 ** 467±169.66 *** 897±76.09 * 477±89.66 *** 453±114.73 *** | 48.98±14.32 1120.20±126.23 899±92.82 * 827.51±158.23 * 773±129.66 ** 407±161.38 *** 867±119.62 * 443±141.18 *** 412±97.41 *** |
* compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group.
Test example 2: derivative of the present invention causes the provide protection of mouse liver injury to D-galactosamine
1, material: with test example 1 material
D-galactosamine (production of SIGMA company).
2, method: the single sex mouse of getting qualified body weight, be divided into following 7 groups at random: the blank group, model group, glycyrrhetinic acid arginase i group (5mg/kg), glycyrrhetinic acid arginase i I organizes (10mg/kg), and glycyrrhetinic acid meglumine I organizes (5mg/kg), and glycyrrhetinic acid meglumine II organizes (10mg/kg), Glycyrrhizic acid,diammonium salt group (30mg/kg), every group 12, tail vein injection administration (0.2ml/ is only), successive administration 3 days, use the D-galactosamine modeling of 150mg/kg behind the last administration 1h, the blood sampling of fasting 16h posterior orbit, centrifugal (4000rpm, 10min), collect serum, detect ALT/GPT with medicine box, the ASP/GOT activity.Data are represented with X ± s with data, carry out statistical procedures with t check between group.
3 results; As shown in table 2; relatively glycyrrhetinic acid arginase i group, II organize with model group; glycyrrhetinic acid meglumine I group, II group Glycyrrhizic acid,diammonium salt group all can significantly reduce the effect (P＜0.01) that the ALT/GPT enzyme is lived, the ASP/GOT enzyme is lived, and point out derivative of the present invention that D-galactosamine is caused mouse liver injury and have provide protection.
Table 2 Enoxolone derivative causes the provide protection of mouse liver injury to D-galactosamine
| Group | ASP/GOT enzyme (U/L) alive | ALT/GPT enzyme (U/L) alive |
| Control group model group enoxolone arginase i group enoxolone arginase i I group enoxolone meglumine I group enoxolone meglumine II group diammonium glycyrrhizinate group | 162.584±69.83 987.84±74.01 526.81±156.11 ** 345±173.38 *** 506.48±182.25 ** 321±163.59 *** 307.5±151.21 *** | 68.98±36.32 1008.84+58.07 816.36±89.23 ** 497±173.66 *** 794±109.03 ** 467±113.32 *** 438.5±110.07 *** |
* compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group.
Test example 3: derivative of the present invention is to the protective effect of rat chronic liver injury
1. material: regular grade Wistar rat, male, body weight 200-230g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106005, Shandong kinoplaszm word
IV Collagen Type VI (IV-C) ELISA measuring reagent kit (centralab of Shuguang Hospital product); Tumor necrosis factor alpha (TNF α) radioimmunoassay box (Shanghai Long March medical science company limited product).
2. method: 60 rats are divided into 6 groups at random: chronic hepatic injury model (M) group, the low dose of S of glycyrrhetinic acid arginine
1Group (iv2.5mg/kg), the heavy dose of L of glycyrrhetinic acid arginine
1Group (iv 10mg/kg), the low dose of S of glycyrrhetinic acid meglumine
2Group (iv2.5mg/kg), the heavy dose of L of glycyrrhetinic acid meglumine
2Group (iv10mg/kg), normal control (N) group, each organizes equal 10.Except that the normal control group, other each group is all used 50%CCL
4Thigh sc behind the-sweet oil 1mL/kg 2 times weekly, in totally 12 weeks, causes the rat chronic liver injury; Small dose group and heavy dose of group be iv 2.5mg/kg, 10mg/kg respectively, every day 1 time.M, N group iv equivalent physiological saline, every day 1 time.Each is organized and puts to death 6 at random in the 4th, 9,12 weeks of experiment.
Leaving and taking of sample: the anesthesia of abdominal injection 3% Sodital solution (40mg/kg), open the abdominal cavity, get blood from postcava, separation of serum ,-20 ℃ of preservations; Get liver 10% formaldehyde fixed.Serological index detects: detect with the full-automatic biochemical determinator.IV-C, TNF α detect and are all undertaken by the test kit specification sheets.Liver specimens is handled: routine paraffin wax embedding, section, HE dyeing is for conventional om observation pathological change.Statistical procedures: data are represented with x ± s, handle corresponding data with F check and t check.
3. result: with the M group relatively, the serum level of small dose group and the every serological index of heavy dose of group all obviously reduces, but reduces significantly (P＜0.01) with the heavy dose group.Small dose group: the 4th all liver lobule structural integrities, the liver cell cloudy swelling is dispersed in cavity and becomes, a small amount of cell infiltration; The 9th all portal areas enlarge, and have arc fiber to form, and the 12nd all liver lobule structure deteriorates have fiber every formation.Heavy dose of group: the 4th and 9 all liver lobule structural integrities, the liver cell marshalling, accidental cavity becomes; See that the portal area enlarges the 12nd week.As seen, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine all can suppress inflammation activity and proliferation of fibrous tissue in the hepatic tissue, thereby the rat chronic liver injury that CCl4 causes is had preventive and therapeutic effect.
Table 3 Enoxolone derivative is to the influence of rat blood serum ALT, IV-C, TNF alpha content (n=10, x ± s)
| Index | Group | Experimental period (week) | ||
| 4 | 9 | 12 | ||
| ALT(U/L) IV-C(ng/ml) TNFα(ng/ml) | N M S 1 L 1 S 2 L 2 N M S 1 L 1 S 2 L 2 N M S 1 L 1 S 2 L 2 | 78.70±20.16 272.8±48.66 221.33±53.54 139.83±49.12** 213.13±43.84* 141.38±59.52** 14.62±8.43 39.17±14.37 28.83±8.30* 17.00±7.24** 29.13±11.41 20.52±10.24* 0.74±0.27 1.74±0.48 1.18±0.28* 0.85±0.25** 1.09±0.34* 0.79±0.31** | 81.45±19.37 261.00±52.44 205.33±49.06 134.67±44.27** 198.71±85.16 124.67±47.27** 16.58±6.87 64.67±23.28 32.17±14.54* 21.33±11.74** 34.21±13.04* 23.67±9.98** 0.81±0.34 1.79±0.51 1.33±0.42* 0.81±0.28** 1.28±0.71 0.90±0.39** | 84.50±16.57 296.17±43.13 222.50±51.34 149.33±53.32** 218.50±47.21* 161.15±40.22** 15.33±6.22 91.50±25.72 51.83±24.86* 24.33±11.48** 54.33±22.34* 21.67±19.38** 0.75±0.18 1.97±0.59 1.54±0.46 0.82±0.30** 1.29±0.38* O.86±0.47** |
Compare with the M group: * P＜0.05, * * P＜0.01
Test example 4: the external anti-HBV effect of derivative of the present invention
1. material:
Cell: the Bel7402 of HBV dna clone transfection is provided by natural drug Engineering Technical Research Centre Pharmacology Lab.The HBV gene be can transcribe, translate, and HBsAg, HBeAg produced.
Medicine: by method provided by the invention preparation, be dissolved in an amount of Hank liquid, 100 ℃ of water-bath 15min sterilizations, the time spent with the DMEM doubling dilution that contains 10% calf serum to respective concentration.
Reagent and key instrument: the DMEM culture medium dry powder is produced by U.S. GBICO company: calf serum GBICO company produces:
3H-TdR is available from Chinese nuclear power research institute Isotope Research institute: HBsAg, HBeAg detection kit (the ELISA method detects, Shanghai section China product).Medical clean work station (Suzhou treating plant company product): Napco 6100 type CO
2Incubator (company of U.S. Du group product): EL-301 type enzyme-linked immunosorbent assay instrument (U.S. BIO-TEK instrument company product).LS-6500 type liquid scintillation counter (U.S. Beckman company product).
2. method
(1) the detection liver cancer cell of HBsAg, HBeAg is inoculated in 96 well culture plates with 5 * 107/ml concentration, every hole 100 μ 1, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine final concentration are (0.05,0.1,0.5,1,2mg/L), only add the cell control group that 200 μ l contain 10% calf serum DMEM nutrient solution, establish 3 multiple holes for every group.Abandon supernatant behind the adherent culture 24h, add the medicine of different concns, final volume 200 μ l.After continuing to cultivate 72h, change dressings to every hole 200 μ l.Continue to cultivate 48h, 6h before stopping gets nutrient solution supernatant 150 μ 1 packing, and-20 ℃ of storages detect HBsAg, HBeAg respectively, are undertaken by test kit specification sheets operation steps.
HBsAg, HBeAg content P/N value representation
P/N=sample A value/negative control A value
(2) after culture supernatant was got in the detection of cell proliferation, every hole added nutrient solution 130 μ l, 20 μ l
3H-TdR (9250Bq), continue to cultivate 6h after, abandon nutrient solution, with 0 25% trypsinase, 0 02%EDTA digestion, bull cell harvestor collecting cell, suction filtration on 49 type filter paper, 80 ℃ of thermostat container drying 6h.Beckman LS-6500 type liquid scintillation counter reads dpm.The result represents that with the on cell proliferation inhibiting rate inhibiting rate is calculated as follows:
Inhibiting rate=(1-experiment dpm/ contrast dpm) * 100%
Data are represented with x ± s, handle corresponding data with F check and t check.
3. result
Influence to liver cancer cell secretion HBsAg, HBeAg: shown in the table 4, derivative of the present invention (0.1,0.5,1,2mg/L) can the secretion of obvious suppression liver cancer cell HBsAg, HbeAg; Influence to hepatoma cell proliferation: shown in the table 5, derivative of the present invention (0.05,0.1,0.5,1,2mg/L) can suppress the propagation of liver cancer cell significantly.
Table 4. derivative of the present invention is secreted the influence (n=6) of HBsAg, HBeAg to liver cancer cell
| Group | Concentration (mg/l) | HBeAg | HBsAg | ||
| P/N | Prevent rate (%) | P/N | Prevent rate (%) | ||
| Control group-glycyrrhetinic acid arginine glycyrrhetinic acid meglumine | 0.05 0.1 0.5 1 2 0.05 0.1 0.5 1 2 | 9.65±0.24 9.341±0.25 8.146±0.67* 7.246±0.21* 5.921±0.36** 3.741±0.48** 9.457±0.37 7.986±0.71* 7.112±0.42* 5.817±0.54** 3.617±0.51** | 4.2 19.98 31.90 49.43 78.28 2.6 21.99 33.64 50.76 79.86 | 10.06±0.42 9.55±0.23 8.99±0.79* 7.98±0.38* 6.26±0.32** 4.59±0.27** 9.67±0.31 8.74±0.62* 7.51±0.41* 6.13±0.57** 4.37±0.35** | 6.4 13.47 26.11 47.70 68.71 4.9 16.58 32.03 49.37 71.48 |
* compare with control group: * P＜0 05, * * P＜0 01
Table 5. derivative of the present invention is to the influence (n=6) of hepatoma cell proliferation
| Group | Concentration (mg/l) | dpm | Prevent rate (%) |
| Contrast glycyrrhetinic acid arginine glycyrrhetinic acid meglumine | 0.05 0.1 0.5 1 0.05 0.1 0.5 1 | 3863.89±432.19 3240.26±235.06* 3032.96±591.79* 1704.41±208.13** 1090.23±433.04** 3314.37±217.69 3159.51±479.35* 1797.38±301.21** 1008.23±526.14** | 16.14 21.51 55.89 71.78 14.22 18.22 46.52 73.91 |
* compare with control group: *＜0 05, * * P＜0 01
Test example 5: compound Dichlorodiphenyl Acetate of the present invention brings out the influence of mouse capillary permeability
1. material
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine are provided by Shandong Province's natural drug Engineering Technical Research Centre;
Kunming mouse, age in 5-8 week, body weight 18-22g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides.The male and female dual-purpose, same sex is adopted in every batch of experiment.
2. method
Select 60 of male mice in kunming for use, fasting 16h (can't help water) before the experiment.Mouse is divided 6 groups at random, glycyrrhetinic acid arginine high dose group (iv derivative 10mg/kg), low dose group (iv derivative 1mg/kg), glycyrrhetinic acid meglumine high dose group (iv derivative 10mg/kg), low dose group (iv derivative 1mg/kg), control group is given pre-isopyknic physiological saline, and positive drug is INDOMETHACIN (10mg/kg).40min after the administration, mouse tail iv inject the blue solution 0.1mL/10g of 2% ivens, and behind the 30min, ip1% acetum 0.1mL/10g behind the 20min, takes off cervical vertebra and puts to death mouse, cuts off skin of abdomen, with 5mL normal saline flushing abdominal cavity, collects washings.In 590nm place colorimetric, measure absorbancy with 721 type spectrophotometers, relatively obtain the dyestuff seepage discharge, and carry out the t check with typical curve.
3 results: have remarkable inhibition acetic acid with model group comparison derivative and bring out the effect that vascular permeability increases, inhibiting rate is respectively 44.7% (P＜0.01) and 25.6% (P＜0.01), and high dosage is suitable with the effect of 10mg/kg INDOMETHACIN, the results are shown in Table 6.
Table 6 derivative to the influence of capillary permeability (x ± s, n=10)
| Group | Dosage (mg/kg) | Dyestuff seepage discharge (μ g)) | Inhibiting rate (%) |
| Control group glycyrrhetinic acid arginine glycyrrhetinic acid liomethacin | - 1 10 1 10 10 | 387.3±36.4 288.3±10.5** 214.1±15.6** 278.2±12.8** 220.0±17.9** 215.8±24.5** | 25.6 44.7 28.17 43.2 44.3 |
Compare * P＜0.05 with control group, * * P＜0.01,
Test example 6: the influence of compound antagonism caused by dimethylbenzene xylene small white mouse auricle edema of the present invention effect
1. material
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine are provided by Shandong Province's natural drug Engineering Technical Research Centre;
Kunming mouse, body weight 22-24g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides.The male and female dual-purpose, same sex is adopted in every batch of experiment.
2 methods
Get 70 of Kunming kind small white mouses, be divided into 7 groups at random.Glycyrrhetinic acid arginine high dose group (iv derivative 20mg/kg), middle dosage group (iv derivative 5mg/kg), low dose group (iv derivative 1mg/kg), glycyrrhetinic acid meglumine high dose group (iv derivative 20mg/kg), middle dosage group (iv derivative 5mg/kg), low dose group (iv derivative 1mg/kg), aescine (iv 2mg/kg), normal control group are given the pre-physiological saline that waits capacity.Behind the administration 1h, ear two sides, an experiment mice left side with 100% dimethylbenzene 0.02ml/ only is coated with, auris dextra is contrast, put to death mouse behind the 1h, cut two ears, prepare the mouse auricle with the 9mm punch tool along the auricle baseline, claim weight in wet base with electronic scales, with the difference of two ear weight as swelling degree (inflammation index).
3. result
All can significantly suppress the auricle edema that dimethylbenzene causes by the visible glycyrrhetinic acid arginine of table 7, glycyrrhetinic acid meglumine (1mg/kg, 5mg/kg, 20mg/kg), aescine.
Table 7 derivative p-Xylol cause the small white mouse auricle edema influence (x ± s, n=10)
| Group | Dosage (mg/kg) | Swelling degree (mg) | Inhibiting rate (%) |
| Normal control group glycyrrhetinic acid arginine glycyrrhetinic acid meglumine aescine | - 1 5 20 1 5 20 2 | 12.52±3.84 8.79±2.81* 8.03±3.05* 6.39±1.09** 8.82±3.15* 7.96±3.67* 6.47±2.07** 5.85±2.97** | 29.79 35.86 48.96 29.55 36.42 48.32 52.27 |
Compare * P＜0.05 with control group, * * P＜0.01,
Test example 7: the external bacteriostatic action test of derivative of the present invention
1 material: glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine: by the invention provides the method preparation
Streptococcus aureus, intestinal bacteria, streptococcus pneumoniae, haemolysis connect bacterium, micrococcus catarrhalis (Yantai City health and epidemic prevention station provides)
2 methods: adopt dull and stereotyped punch method respectively glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine I to be organized (0.01mg/ml), II group (0.05mg/ml), III group (0.25mg/ml) fungistatic effect detect.
3 results: the visible derivative I of table 8, II, III group all have significant fungistatic effect
Table 8 derivative bacteriostatic action
| Title | Inhibition zone size (mm) | |||||
| The glycyrrhetinic acid arginine | The glycyrrhetinic acid meglumine | |||||
| The I group | The II group | The III group | The I group | The II group | The III group | |
| Staphylococcus aureus e coli streptococcus pneumoniae haemolysis connects the bacterium micrococcus catarrhalis | 6.5 5.5 - 1.5 2 | 8 7 1.5-1 3 3.5 | 11 9 3-3.5 5.5 6 | 6.2 5.7 - 1.7 1.9 | 7.5 6.3 1.6-1 2.8 3.5 | 10.5 8.5 3-3.5 5 6.5 |
Test example 8: Enoxolone derivative is to the influence of rat local cerebral ischemia damage
1. material: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example.TCC: U.S. Sigma company product, face with preceding and be made into 4% solution with physiological saline.Laboratory animal: regular grade Wistar rat, male, body weight 280g-350g, natural drug Engineering Technical Research Centre experimentation on animals center, Shandong Province provides.Conformity certification number: No. 00106005, Shandong kinoplaszm word.
2. method: animal is divided into sham operated rats (waiting the capacity solvent) at random, model control group (waiting the capacity solvent), nimodipine group (Nim, 1.5mg/kg), glycyrrhetinic acid arginine low (1mg/kg), in (5mg/kg), high (20mg/kg) dosage group, glycyrrhetinic acid meglumine low (1mg/kg), in (5mg/kg), high (20mg/kg) dosage group, 10 every group.After the fasting 12 hours, and Chloral Hydrate (350mg/kg, i.p.) anesthesia separates right carotid, and folder closes in the neck, arteria carotis communis, external carotid artery proximal part and distal end ligation, cut off the centre.The external carotid artery free end is pulled to internal carotid artery in alignment, bolt line (selecting diameter 0.24mm nylon wire for use, length 5.0cm) is inserted into encephalic by external carotid artery, stop when meeting slight resistance, depth of penetration is about 2cm.Ligation external carotid artery opening, and open the arteria carotis communis bulldog clamp, the disinfection and stitching wound causes left side arteria cerebri media ischemia model; Sham operated rats is only carried out the separation (above experiment is all carried out at 23 ℃～25 ℃) of right carotid, internal carotid artery, external carotid artery.Each treated animal intravenous injection relative medicine of postoperative (administration volume 1ml/200g).Press document [Liu Xiaoguang, Xu Lina, a kind of rat brain medium sized artery model that can estimate thrombolysis and anti-thrombolysis after 24 hours, Acta Pharmaceutica Sinica, 1995,30:662] described method and standard is observed and the behavior disorder of record rat: (A) carry the mouse tail and observe forelimb flexing situation, stretch to ground as two forelimb symmetries, count 0 fen, the wrist flexing occurs as operation offside forelimb and count 1 fen, the elbow flexing is counted 2 fens, the shoulder inward turning is counted 3 fens, existing wrist flexing and/or elbow flexing have shoulder inward turning person again, count 4 fens.(B) animal is placed on the plane earth, push away both shoulders respectively, check resistance to side shifting.As bilateral resistance equity and strong, count 0 fen, as resistance descender when the operation offside promotes, according to decline degree difference be divided into gently, in, weigh three degree, count 1,2 and 3 fen respectively.(c) the two forelimbs of animal are put on the wire netting, observed the muscular tension of two forelimbs.Two muscle of anterior limb tension force equities and strong person count 0 fen.Count 1,2 and 3 fen according to operation offside muscular tension decline degree difference equally.(D) animal has ceaselessly to a side person of turn-taking, and counts 1 fen.According to the standard scoring, full marks are 11 minutes, and mark is high more, and expression animal behavior obstacle is serious more.
Put to death rat behind the behavior scoring, get brain, remove olfactory bulb, cerebellum and low brain stem, crownly be cut into 5, the brain sheet takes on a red color after healthy tissues is dyed with TCC (TTC) dyeing, and blocking tissue is white in color, taking a picture in dyeing back, asks the infarct size ratio with Chinese aerospace university pathological image analysis software.Data are represented with X ± s, carry out statistical procedures with t check between group.
3. result: as shown in table 9, ischemic is after 24 hours, and rat shows tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain; Give glycyrrhetinic acid arginine, the glycyrrhetinic acid meglumine of various dose, the animal behavior obstacle has alleviating in various degree, and the rat cerebral ischemia district also takes an evident turn for the better, and is dose-dependently.
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine 10mg/kg treating cerebral ischemia are better than 5mg/kg (P＜0.05), but relatively do not have significant difference (P＞0.05) with 20mg/kg.Prompting is when dosage 〉=10mg/kg, and drug effect does not continue increase.
The influence that table 9 Enoxolone derivative damages the rat local cerebral ischemia (n=10, X ± s)
| Group | Behavior disorder | Ischemic areas (%) |
| The smart 5mg/kg propylhomoserin of the sham-operation group model control group Nim group 1mg/kg enoxolone 20mg/kg 1mg/kg enoxolone 5mg/kg of Portugal methylamine 20mg/kg | 0 10.10±1.37 6.90±2.33 ** 7.90±1.24 * 7.50±3.03 * 5.34±1.28 ** 7.70±2.44 * 7.31±4.08 * 6.23±1.13 ** | 0 24.26±4.13 13.09±7.11 ** 18.21±2.73 * 16.4l±2.84 * 10.18±3.64 ** 17.06±3.20 * 15.82±3.14 * 11.08±3.01 ** |
Compare with model control group
*P＜0.05,
*P＜0.01;
Test example 9: Enoxolone derivative is to the influence of rat cerebral ischemia/reperfusion injury
1. material:
Prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example.TCC: U.S. Sigma company product, face with preceding and be made into 4% solution with physiological saline.Laboratory animal: regular grade Wistar rat, male, body weight 280g-350g, natural drug Engineering Technical Research Centre experimentation on animals center, Shandong Province provides.Conformity certification number: No. 200106005, Shandong kinoplaszm word.
2. method:
Animal is divided into sham operated rats (waiting the capacity solvent) at random, model control group (waiting the capacity solvent), nimodipine group (Nim, 1.5mg/kg), glycyrrhetinic acid arginine low (1mg/kg), in (5mg/kg), high (20mg/kg) dosage group, glycyrrhetinic acid meglumine low (1mg/kg), in (5mg/kg), high (20mg/kg) dosage group, 10 every group.After the fasting 12 hours, cause left side arteria cerebri media ischemic by experimental example 1 described method; Each treated animal intravenous injection relative medicine of postoperative 10min (administration volume 1ml/200g).Behind the ischemic 2 hours, extract nylon wire out, cause rat cerebral ischemia/reperfusion injury; After irritating 22 hours again, by carry out behavior scoring, the dyeing of brain sheet by experimental example 1 described method.Data are represented with X ± s, carry out statistical procedures with t check between group.
3. result:
As shown in table 10, after ischemic was irritated 22 hours in 2 hours again, rat showed tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain; Give glycyrrhetinic acid arginine, the glycyrrhetinic acid meglumine of various dose, the animal behavior obstacle has alleviating in various degree, and the rat cerebral ischemia district also takes an evident turn for the better, and is dose-dependently.
Table 10 Enoxolone derivative is to the influence of rat cerebral ischemia/reperfusion injury (n=10, X ± s)
| Group | Behavior disorder | Ischemic areas (%) |
| The smart 10mg/kg propylhomoserin of the sham-operation group model control group Nim group 1mg/kg enoxolone 20mg/kg 1mg/kg enoxolone 10mg/kg of Portugal methylamine 20mg/kg | 0 10.20±1.32 6.88±2.31** 7.89±1.21* 7.35±1.27** 5.31±1.11** 7.72±2.42* 7.41±3.04* 5.61±1.08** | 0 26.38±4.67 13.55±7.19** 18.56±2.77* 13.24±3.35** 10.38±3.21** 17.56±3.78* 14.34±2.12** 11.42±2.54** |
Compare with model control group
*P＜0.05,
*P＜0.01;
Test example 10: Enoxolone derivative is to the provide protection of mouse closed cerebral trauma
1. material
By preparation example preparation (one) glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection
Cleaning level kunming mouse, 18～22g, male and female half and half are provided by natural drug Engineering Technical Research Centre experimentation on animals center, Shandong Province.Conformity certification number: No. 200106003, Shandong kinoplaszm word.
2. method
Get 90 of mouse, be divided into normal control group (waiting the capacity solvent) at random, model control group (waiting the capacity solvent), glycyrrhetinic acid arginine low (2mg/kg), in (10mg/kg), high (40mg/kg) dosage group, glycyrrhetinic acid meglumine low (2mg/kg), in (10mg/kg), high (40mg/kg) dosage group, 10 every group of aescine groups (2mg/kg).Press document [Wang Chaoyun, Jiang Wanglin, the red English of intelligence, etc. baicalin is to the provide protection of brain injury. herbal medicine, 2004,35 (2): 188～190] described method causes mouse closed cerebral trauma model, and each organizes intravenously administrable, strips the mouse brain after 24 hours, the weighing weight in wet base, dried 24 hours for 70 ℃, claim dry weight, calculate brain water content; Brain water content is higher than normal value and represents the brain oedema.
3. result
As shown in table 11, the closed cerebral trauma causes the serious oedema of mouse brain, give the glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine salt of various dose after, the mouse brain oedema has alleviating in various degree, and is dose-dependently.
Table 11 Enoxolone derivative is to the influence of closed cerebral trauma (n=10, X ± s)
| Group | Dosage (mg/kg) | Brain water content (%) |
| Normal group model control group enoxolone Arginine-glycyrrhetinic acid meglumine Sodium Aescinate | - - 2 10 40 2 10 40 2 | 79.1±0.3** 82.5±1.3 81.1±0.6* 80.9±0.5* 79.4±0.4** 81.6±0.7* 80.7±0.4* 78.8±0.6** 79.1±0.4** |
Compare with model control group
*P＜0.05,
*P＜0.01.
Test example 11: Enoxolone derivative is to the influence of coronary artery ligation rat physical signs
1. material
Regular grade SD rat, male, body weight 250～300g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106005, Shandong kinoplaszm word.
Flos Carthami injection: Wanrong, Shanxi three nine-day periods after the winter solstice pharmaceutcal corporation, Ltd, lot number 040130
Given the test agent: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine salt by preparation example
Electrocardiograph: single track electrocardiograph (FX-2111), Feitian, Beijing electronic medical instruments company limited
2. method
Get 80 of male and healthy rats, be divided into 8 groups at random, model group gives isopyknic physiological saline, glycyrrhetinic acid arginine group, glycyrrhetinic acid meglumine salt are pressed 1mg/kg, 5mg/kg, the administration of 20mg/kg dosage tail vein injection respectively, Flos Carthami injection (iv 780mg/kg), administration 5min pneumoretroperitoneum is injected 20% urethane 0.65g/kg anesthesia, and back of the body position is fixing, the record normal ECG.Chest unhairing, sterilization, along the about 2cm of left mid-clavicular line longitudinal incision skin, separate the flesh layer in the 4th or the 5th intercostal passivity, open the thoracic cavity, cut off pericardium, gently press the right side thorax, extrude heart, behind ligation arteria coroaria sinistra in coronary vein place between conus arteriosus and the left auricle of heart, heart is put back to the thoracic cavity, sew up the wall of the chest rapidly, this operation was finished in 30 seconds.Electrocardiogram(ECG changes behind the record ischemic.
Data represent with X ± SD, and with model group relatively, carry out statistical procedures with the t check.
Table 12. Enoxolone derivative is to the influence of coronary artery ligation rat electrocardiogram J point
| Group | Dosage mg/kg | The J point is raised numerical value (mv) | |||||
| Initially | 30min | 60min | 90min | 180min | 240min | ||
| Model group enoxolone arginase 12 enoxolone meglumine Sofflower injection | - 1 5 20 1 5 20 780 | 0.27±0.10 0.21±0.03 * 0.20±0.05 * 0.16±0.05 * 0.22±0.06 * 0.21±0.04 * 0.17±0.06 * 0.26±0.09 | 0.26±0.04 0.20±0.04 * 0.19±0.04 * 0.16±0.05 * 0.21±0.05 * 0.20±0.07 * 0.17±0.07 * 0.23±0.06 | 0.26±0.04 0.19±0.07 * 0.18±0.08 * 0.15±0.06 ** 0.20±0.07 * 0.19±0.07 * 0.16±0.08 * 0.20±0.05 * | 0.25±0.05 0.18±0.09 * 0.18±0.09 * 0.15±0.09 ** 0.19±0.06 * 0.19±0.08 * 0.17±0.08 * 0.19±0.09 * | 0.25±0.04 0.20±0.08 0.16±0.10 * 0.17±0.06 ** 0.19±0.08 0.18±0.09 * 0.15±0.12 * 0.21±0.08 | 0.25±0.05 0.21±0.07 0.17±0.09 * 0.15±0.04 ** 0.21±0.04 0.17±0.06 * 0.15±0.08 * 0.21±0.07 |
* expression is compared * p＜0.05, * * p＜0.01 with model group
3. result
The influence that J is ordered as shown in Table 12, relatively glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine group (1mg/kg, 5mg/kg, 20mg/kg) all can significantly reduce the amplitude (p＜0.05) that the J point is raised at 30min, 60min, 90min, 180min, 240min with model group.
4. conclusion
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine have provide protection to cardiac muscle, can reduce the myocardial damage that ischemic causes.
Test example 12: Enoxolone derivative is to the influence of coronary artery ligation rat biochemical indicator
1. material
Regular grade SD rat, male, body weight 250～300g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106005, Shandong kinoplaszm word.
Flos Carthami injection: Wanrong, Shanxi three nine-day periods after the winter solstice pharmaceutcal corporation, Ltd, lot number 040130
Given the test agent: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example
Aspartate Aminotransferase: Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 020101
CK test kit: Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 090091
LDH test kit: Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 130051
Uv analyzer: 722E type visible spectrophotometer, Shanghai Spectrum Apparatus Co., Ltd.
Whizzer: table model high speed centrifuge (TGL-16G), Shanghai medical analytical instrument factory
2. method
Get 90 of male and healthy rats, be divided into 9 groups at random, sham operated rats, model group give isopyknic physiological saline, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection group are pressed 1mg, 5mg, the administration of 20mg/kg dosage tail vein injection respectively, Flos Carthami injection (iv 780mg/kg), administration 5min pneumoretroperitoneum is injected 20% urethane 0.65g/kg anesthesia, and back of the body position is fixing.Chest unhairing, sterilization, along the about 2cm of left mid-clavicular line longitudinal incision skin, separate the flesh layer in the 4th or the 5th intercostal passivity, open the thoracic cavity, cut off pericardium, gently press the right side thorax, extrude heart, behind ligation arteria coroaria sinistra in coronary vein place between conus arteriosus and the left auricle of heart, heart is put back to the thoracic cavity, sew up the wall of the chest rapidly, this operation was finished in 30 seconds.Behind the 6h with rat fixing with dissect on the plate, separate arteria carotis communis, extract blood, leave standstill 10min after, the centrifugal 15min of 4500rpm draws serum, utilizes test kit to detect CK, LDH, the AST enzyme is lived.
Data represent with X ± SD, and with model group relatively, carry out statistical procedures with the t check.
The influence that table 13 Enoxolone derivative is lived to enzyme in the coronary artery ligation rat blood
| Group | Dosage mg/kg | CK enzyme U/L alive | AST enzyme U/L alive | LDH enzyme U/L alive |
| Sham-operation group model group enoxolone Arginine-glycyrrhetinic acid meglumine Sofflower injection | - - 1 5 20 1 5 20 780 | 881.27±133.64 1213.98±96.31 ### 1211.28±155.82 1100.38±117.25 * 1016.73±194.94 * 1224.78±147.27 1104.41±93.91 * 991.57+201.2 * 1098.49±93.11 * | 80.14±22.00 150.79±23.6 ### 117.69±25.39 * 111.63±36.10 * 106.45±28.6 ** 120.08±22.14 * 115.58±29.34 * 101.31±21.49 ** 119.95±29.66 * | 186.45±43.46 420.94±27.04 ### 364.16±66.67 * 348.35±79.62 * 304.91±47.83 *** 391.42±63.34 * 363.71±53.51 * 314.87±56.78 ** 367.24±55.00 * |
* expression is compared with model group
*P＜0.05,
* *P＜0.001; # represents to compare with sham operated rats,
###P＜0.001
3. result
As shown in Table 13, this myocardial infarction and ischemia model can significantly improve the activity (p＜0.001) that CK, AST in the ischemic rat plasma, LDH enzyme live, and all can significantly reduce the activity (p＜0.05) of above-mentioned three kinds of enzymes with model group comparison glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine group.
4. conclusion
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine have provide protection to cardiac muscular tissue, can improve body effectively because of the tissue injury that myocardial ischemia causes, CK, AST in the body blood plasma, LDH enzymic activity are significantly reduced.
Test example 13: Enoxolone derivative is to the influence of coronary artery ligation rat morphological indexes
1. material
Regular grade SD rat, male, body weight 250～300g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106005, Shandong kinoplaszm word.
Flos Carthami injection: Wanrong, Shanxi three nine-day periods after the winter solstice pharmaceutcal corporation, Ltd, lot number 040130
Given the test agent: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection chlorination nitro tetrazole orchid, the chemical chemical reagent work (Shanghai) that advances, lot number 20010801 electronic balances, BP 210 S, Sartorius (Germany) by preparation example
2. method
Get 80 of male and healthy rats, be divided into 8 groups at random, model group gives isopyknic physiological saline, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine are pressed 1mg, 5mg/kg, the administration of 20mg/kg dosage tail vein injection respectively, Flos Carthami injection group (iv 780mg/kg), administration 5min pneumoretroperitoneum is injected 20% urethane 0.65g/kg anesthesia, and back of the body position is fixing.Chest unhairing, sterilization, along the about 2cm of left mid-clavicular line longitudinal incision skin, separate the flesh layer in the 4th or the 5th intercostal passivity, open the thoracic cavity, cut off pericardium, gently press the right side thorax, extrude heart, this operation was finished in 30 seconds.Behind ligation arteria coroaria sinistra in coronary vein place between conus arteriosus and the left auricle of heart, heart is put back to the thoracic cavity, sews up the wall of the chest rapidly, behind the 6h with rat fixing with dissect on the plate, open the thoracic cavity, peel off heart, use normal saline flushing, remove blood stains, reject non-cardiac muscular tissues such as blood vessel fat, along coronary sulcus excision atrium, stay ventricle, weigh.Along coronary sulcus is parallel from the apex of the heart to the heart base portion ventricle is cut into the thick myocardium sheet of 0.1cm, myocardium sheet is placed on 0.3% NBT, hatch 4min at 37 ℃, unnecessary dyestuff is removed in water flushing immediately after the dyeing, deduct the non-infarcted region cardiac muscle that each myocardium sheet is colored, the undyed infarcted region cardiac muscle of weighing utilizes following formula to calculate myocardial infarction area (%)
Data represent with X ± SD, and with model group relatively, carry out statistical procedures with the t check.
Table 14. Enoxolone derivative is to the influence of coronary artery ligation rat heart muscle infarct weight percent
| Group | Dosage (mg/kg) | Myocardial infarction weight percent (%) |
| Model group glycyrrhetinic acid arginine glycyrrhetinic acid meglumine Flos Carthami injection | - 1 5 20 1 5 20 780 | 26.12±1.71 23.64±3.62 * 22.98±3.40 * 21.79±2.77 ** 23.24±3.27 * 22.49±4.20 * 20.37±3.19 ** 22.02±2.62 * |
* expression is compared * p＜0.05, * * p＜0.01 with model group
3. result
As shown in Table 14, compare with model group, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine (1mg/kg, 5mg/kg, 20mg/kg) Flos Carthami injection (780mg/kg) all can significantly reduce myocardial infarction percentage (p＜0.05).
4. conclusion
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine have the effect of protection cardiac muscular tissue under doses, can alleviate the damage of ischemic to the myocardial cell.
Test example 14: Enoxolone derivative is to the influence of clotting time of mice
1. material
A cleaning level Kunming mouse, male, body weight 20～22g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106003, Shandong kinoplaszm word.
Given the test agent: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example.
2. method
Get 80 of male and healthy mouse, be divided into 8 groups at random, the normal control group is pressed 0.2ml/ tail vein injection saline, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine are pressed 2mg/kg, 10mg/kg, the administration of 40mg/kg dosage tail vein injection respectively, behind Flos Carthami injection group (iv 1560mg/kg) the administration 0.5h, the eye socket blood sampling, slide method is measured blood coagulation time.Data represent with X ± SD, and with model group relatively, carry out statistical procedures with the t check.
Table 15 Enoxolone derivative is to the influence of clotting time of mice
| Group | Dosage (mg/kg) | Clotting time (s) |
| Normal group glycyrrhetinic acid arginine glycyrrhetinic acid meglumine Flos Carthami injection group | - 2 10 40 2 10 40 1560 | 103±20.02 154±+26.89 ** 152.5±31.9 ** 176.6±33.40 *** 149.0±24.97 ** 157.9+29.67 ** 179.2±41.31 *** 164.3±32.7 *** |
* expression is compared * p＜0.05, * * p＜0.01 with model group
3. result:
As shown in Table 15, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine (2mg/kg, 10mg/kg, 40mg/kg), Flos Carthami injection group (iv 1560mg/kg) relatively have significant blood coagulation resisting function (p＜0.05) with model group.
4 conclusions:
Glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine be the energy significant prolongation clotting time under doses, and certain function of promoting blood circulation to disperse blood clots is arranged.
Test example 15: Enoxolone derivative is to the analgesic activity of mouse
1. material
A cleaning level Kunming mouse, male, body weight 18～22g, Shandong Province's natural drug Engineering Technical Research Centre Experimental Animal Center provides, conformity certification number: No. 200106003, Shandong kinoplaszm word.
Glycyrrhetinic acid sodium injection (Shandong Province's natural drug Engineering Technical Research Centre provides), Srm-Rhotaard (1mg/kg).
Given the test agent: prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example.
2. method
Get 140 of male and healthy mouse, be divided into 7 groups at random, the normal control group, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine group (3mg/kg, 12mg/kg), glycyrrhetinic acid sodium injection group (12mg/kg), Srm-Rhotaard group (10mg/kg), each organizes equal intraperitoneal administration.Behind the administration 30min, the equal abdominal injection 0.5% acetic acid 0.2m1/ of each mouse only.Observe interior each group of 10min and writhing response (belly indent, stretching, extension hind leg, buttocks are raised) mouse number of elements occurs, calculate each medicine analgesia percentage.
Table 16 Enoxolone derivative influences the mouse analgesic
| Group | Dosage (mg/kg) | Writhing response number (only) appears | Analgesia percentage (%) |
| Normal group glycyrrhetinic acid arginine glycyrrhetinic acid meglumine Sodium glycyrrhetinate Srm-Rhotaard | - 2 10 2 10 10 10 | 20 10 6 12 7 11 0 | 0 50 70 *** 40 65 *** 45 100 *** |
* the writhing response inhibiting rate reaches 50% when above, and just thinking has analgesic activity.
3. result:
As shown in Table 16, glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine (10mg/kg) all can obviously reduce writhing response mouse number, and more all there were significant differences (p＜0.05) with normal group.
Test example 16: the acute toxicity test of derivative of the present invention
1. material
Prepare glycyrrhetinic acid arginine, glycyrrhetinic acid meglumine injection by preparation example.Cleaning level kunming mouse, 18～22g, male and female half and half are provided by natural drug Engineering Technical Research Centre experimentation on animals center, Shandong Province.Conformity certification number: No. 200106003, Shandong kinoplaszm word.
2. method
Laboratory animal is rejected defective animal by SOP requirement one week of quarantine.Testing laboratory and receptacle term harmonization.Room temperature 18-25 ℃, humidity 40-60% filters air-supply, illumination 12 hours.Freely ingest and drink water, change the drinking-water bottle every day once.Get 50 of healthy mices, male and female half and half.Be divided into 5 groups at random, 10 every group.After weighing, by 120,90,67.5,50.6, the dosage tail vein injection administration of 35.5mg/kg.Close observation is 72 hours after the administration, the death condition of record mouse.
3. result
The arginic LD of glycyrrhetinic acid
50Be 71.52 ± 1.85mg/kg, LD
5095% the credible 63.64～80.42mg/kg that is limited to.
Glycyrrhetinic acid meglumine LD
50Be 75.68 ± 1.88mg/kg, LD
5095% the credible 66.21～86.76mg/kg that is limited to.
Claims (19)
1, a kind of glycyrrhetinic acid salt derivative, structural formula is as follows:
M is basic aminoacids or organic bases, and wherein organic bases is meglumine or quadrol.
2, glycyrrhetinic acid according to claim 1 is 18 α-glycyrrhetinic acid, 18 β-glycyrrhetinic acid or their mixture.
3, basic aminoacids according to claim 1 is arginine, Histidine or Methionin.
4, basic aminoacids according to claim 3 is an arginine.
5, the preparation method of derivative according to claim 1 is for adding glycyrrhetinic acid, basic aminoacids or organic bases, 60-90% ethanol successively in reaction flask, stirring reaction 2 hours, the evaporated under reduced pressure solvent, in residue, add ethyl acetate, grind light-yellow precipitate, filter, drying under reduced pressure, promptly.
6, the application of Enoxolone derivative according to claim 1 in the medicine of preparation treatment hepatitis.
7, the application of Enoxolone derivative according to claim 1 in the acute and chronic hepatitis medicament of preparation treatment.
8, the application in the medicine of Enoxolone derivative according to claim 1, viral hepatitis bacillary in preparation treatment.
9, the application of Enoxolone derivative according to claim 1 in the medicine of preparation treatment or prevention ischemia apoplexy.
10, the application of derivative according to claim 1 in the medicine of preparation treatment or prevention of brain ischemia/reperfusion injury.
11, the application of derivative according to claim 1 in the medicine of preparation treatment or prevention of brain wound.
12, the application of derivative according to claim 1 in the medicine of preparation treatment or prevention myocardial ischemia.
13, the pharmaceutical composition that contains the described arbitrary derivative of claim 1.
14,, it is characterized in that and to exist with the form of injection liquid, lyophilized injectable powder, tablet, capsule, soft capsule, dripping pill or oral liquid according to claim 1 or 13 described Enoxolone derivative or pharmaceutical compositions.
15, Enoxolone derivative according to claim 14 or pharmaceutical composition is characterized in that existing with the form of injection liquid.
16, Enoxolone derivative according to claim 14 or pharmaceutical composition, it is characterized in that injection liquid adopts following method preparation: take by weighing Enoxolone derivative, be dissolved in the solubility promoter under the heating, add buffer reagent and water for injection, regulate pH to 9.0～10.0, add gac and stir, filter, divide to be filled in the ampoule, 115 ℃ of sterilizations down, promptly.
17, Enoxolone derivative according to claim 14 or pharmaceutical composition, it is characterized in that lyophilized injectable powder adopts following method preparation: take by weighing Enoxolone derivative, be dissolved in the solubility promoter under the heating, add buffer reagent, propping agent and water for injection, regulate pH to 9.0～10.0, add gac and stir, filter, divide to be filled in the ampoule, lyophilize, promptly.
18,, it is characterized in that above-mentioned solubility promoter is one or more in propylene glycol, ethanol, tween 80, polyoxyethylenated castor oil, the Betacylcodextrin according to claim 16 or 17 described Enoxolone derivative or pharmaceutical compositions; Buffer reagent is one or more in phosphoric acid salt, citrate, ammonium chloride, the veronal; Propping agent is one or more among N.F,USP MANNITOL, sucrose, lactose, glucose, dextran, the PVP.
19, Enoxolone derivative according to claim 14 or pharmaceutical composition is characterized in that tablet comprises Enoxolone derivative, vehicle, lubricant, and wherein vehicle is one or more in starch, pregelatinized Starch, the Microcrystalline Cellulose; Lubricant is one or more in talcum powder, the Magnesium Stearate.
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