Glycyrrhizic acid compounds and their preparation and use in preparing drugs for alleviating nerve damage

Abstract

The present invention belongs to the field of compound medicines, and specifically relates to a compound of the glycyrrhizic acid class, which is a compound having a structural formula of Formula 1 and at least one derivative thereof in a pharmaceutically acceptable salt, ester, solvate, or crystal; Formula 1. The present invention also includes the preparation of the formula 1 and its application in the preparation of a drug for alleviating nerve damage. The present invention has shown that the new compound of Formula 1, based on the molecular structure and spatial structure characteristics, can unexpectedly exhibit excellent physiological effects of alleviating nerve damage, and can be used in the development of drugs for nerve damage.

Description

Technical field:

[0001] The invention belongs to the field of medicine, and specifically relates to a new compound capable of alleviating nerve damage. Background technology:

[0002] Neurological damage, such as the more common stroke, is a complex cascade reaction process involving a series of pathological mechanisms such as brain energy metabolism disorder, excitatory amino acid toxicity, oxidative / nitrosative stress, inflammatory response, cell apoptosis and autophagy. [1] Stroke has become one of the most serious threats to public health, and is the second leading cause of death and the third leading cause of disability worldwide, with ischemic stroke accounting for more than 70% of the total incidence. [2] .

[0003] Although research on ischemic stroke has received widespread social attention and has made some progress in preclinical studies, its transformation into new drugs is subject to many limitations. In the past 20 years, tissue plasminogen activator has remained the main drug for the treatment of ischemic stroke, but it has side effects such as a narrow treatment window, skin bleeding, and neurotoxicity. [3] Therefore, accelerating the development of new and highly effective drugs to alleviate nerve damage is of great clinical significance.

[0004] For stroke, the existing technology also has certain treatment methods. For example, the Chinese patent document with publication number CN115844885A discloses the use of a nat10 small molecule inhibitor in the preparation of a drug for treating ischemic stroke. The Chinese patent document with publication number CN116949169A discloses the use of SMEK1 in the diagnosis and treatment of ischemic stroke. The Chinese patent document with publication number CN116924457A discloses the use of modified ZnS nanoparticles in the preparation of a drug for treating ischemic stroke.

[0005] Although there are many treatment methods and strategies in the prior art, none of them involve the compounds of the present invention and reports on their application in the treatment of stroke.

[0006] [1]Wang W, Jiang B, Sun H, et al. Prevalence, Incidence, and Mortality of Stroke in China: Results from a Nationwide Population-Based Survey of 480 687Adults[J]. Circulation, 2017, 135(8):759-71.

[0007] [2]Wu S, Wu B, Liu M, et al. Stroke in China: advances and challenges inepidemiology, prevention, and management [J]. Lancet Neurol, 2019, 18(4): 394-405.

[0008] [3]Lees KR, Bluhmki E, von Kummer R, et al. Time to treatment withintravenous alteplase and outcome in stroke: an updated pooled analysis ofECASS,ATLANTIS,NINDS,and EPITHET trials[J].Lancet,2010,375(9727):1695-703. Summary of the invention:

[0009] In view of the deficiencies in the prior art, the present invention provides a glycyrrhizic acid compound, aiming to provide a new compound with excellent effect in alleviating nerve damage.

[0010] The second purpose of the present invention is to provide a method for preparing the glycyrrhizic acid compound and its application in preparing drugs for alleviating nerve damage.

[0011] The third object of the present invention is to provide a pharmaceutical composition comprising the glycyrrhizic acid compound.

[0012] A compound of the type A, which is a compound having the structural formula 1 and at least one derivative thereof of a pharmaceutically acceptable salt, ester, solvate, or crystal;

[0013]

[0014] The compound of formula 1 of the present invention can be named as: (1R, 2S, 7Z, 11S, 15S, 17R, 20R)-1, 2, 6, 6, 10, 17, 20-heptamethyl-12-oxyylidene tetracyclo [12.8.0.015, 20.02, 11] twenty-two-7, 9, 13-triene-17-carboxylic acid. The present invention shows that the new compound of formula 1, based on the molecular structure and spatial structure characteristics, can unexpectedly show excellent physiological effects of alleviating nerve damage, and can be used for the development of drugs for nerve damage.

[0015] The present invention also provides a method for preparing the glycyrrhizic acid and its derivatives, comprising mixing the raw material of formula 2, alcohol (ROH) and a sulfuric acid aqueous solution with a concentration of 30 to 45 wt.%, reacting the mixture at a temperature of 150 to 250° C., and then subjecting the mixture to alkaline hydrolysis and acidification to obtain the glycyrrhizic acid.

[0016]

[0017] The present invention allows the compound of formula 2 to react in the presence of the aqueous sulfuric acid solution and then undergoes alkaline hydrolysis, thereby unexpectedly successfully preparing the novel compound of formula 1 and helping to improve the yield and purity of the target product.

[0018] In the present invention, Formula 2 can be an existing commercial product or can be obtained based on a known method.

[0019] In the present invention, the ROH is a conventional alcohol. For example, considering the cost, the R may be a C1-C4 alkyl group, and may further be an ethyl group.

[0020] In the present invention, the control of the sulfuric acid solution in the aqueous solution of sulfuric acid is one of the keys to the desired target product with high selectivity.

[0021] Preferably, the concentration of the aqueous sulfuric acid solution (referring to the concentration of sulfuric acid at the start of the reaction) is 33-42 wt.%, more preferably 30-40 wt.%.

[0022] In the present invention, in the initial stage of the reaction, the liquid-to-solid ratio of the formula 2 and the sulfuric acid aqueous solution is above 50 ml / g, and considering the processing cost, it can be further 100-400 ml / g, and further can be 100-250 ml / g.

[0023] In the present invention, in the initial stage of the reaction, the volume ratio of alcohol to aqueous sulfuric acid solution can be 0.5 to 5:1, and can be further 1 to 3:1 in consideration of the processing cost.

[0024] In the present invention, the reaction temperature is 160-220°C.

[0025] In the present invention, the reaction time can be controlled according to a conventional in-process control detection method. Considering the preparation efficiency, it can be further 2 to 6 hours, and further can be 3 to 5 hours.

[0026] In the present invention, the alkaline hydrolysis is ester hydrolysis, that is, the ester group of C23 is hydrolyzed to form a carboxylate. The alkaline hydrolysis can be achieved by regulating based on known means and principles.

[0027] For example, in the present invention, the base used in the alkaline hydrolysis process is an alkali metal hydroxide;

[0028] The concentration of the alkali metal oxide in the initial stage of alkali hydrolysis may be 5 to 40 wt.%, and may be further 15 to 25 wt.% in consideration of cost.

[0029] In the present invention, at the initial stage of alkaline hydrolysis, the liquid-to-solid ratio is above 50 ml / g, and considering the cost, it can be further 100-300 ml / g, and further 150-250 ml / g.

[0030] In the present invention, the temperature of the alkaline hydrolysis may be 50°C or higher, further may be 80 to 110°C, and further may be 90 to 100°C.

[0031] In the present invention, there is no particular requirement for the alkaline hydrolysis time, which may be more than 2 hours. Considering the preparation efficiency, it may be further 6 to 15 hours, and further may be 9 to 13 hours.

[0032] In the present invention, the role of acidification is to acidify the carboxylate salt formed by alkaline hydrolysis. In the present invention, the acid in the acidification stage can be an inorganic strong acid, further at least one of hydrochloric acid, sulfuric acid, etc.

[0033] In the present invention, the pH at the end point of acidification may be 5 or less, and may be 3 to 4.5 in consideration of the treatment cost.

[0034] Preferably, after the acidification treatment, the crude product is redissolved and purified using an ethanol aqueous solution with a concentration of 85 to 98% by volume, preferably 90 to 95% by volume. The redissolution and purification refers to redissolving the crude product using the ethanol solution, followed by filtering and desolventizing. The above-mentioned redissolution and purification process can be repeated multiple times. There is no special requirement for the amount of the ethanol aqueous solution, as long as it can redissolve the crude product.

[0035] The present invention also provides the use of the glycyrrhizic acid compound in preparing a medicine for alleviating nerve damage.

[0036] The present invention uses in vitro PC12 cells, primary neurons and multicellular model organism Caenorhabditis elegans to conduct experiments and finds that the new compound glycyrrhizic acid can effectively alleviate neuronal hypoxia-reperfusion injury, significantly improve the survival rate of Caenorhabditis elegans after hypoxia, and enhance the vitality of the nervous system. Therefore, the new compound glycyrrhizic acid has the potential to be developed into a drug for alleviating nerve damage.

[0037] In a preferred application of the present invention, the drug for alleviating nerve damage is a drug for treating stroke, further a drug for treating cerebral stroke.

[0038] The application of the present invention is to combine the glycyrrhizic acid compound with a pharmaceutically acceptable adjuvant to prepare a pharmaceutically acceptable dosage form.

[0039] The present invention also provides a medicine for alleviating nerve damage, which comprises a pharmaceutically effective amount of a glycyrrhizic acid compound.

[0040] The medicine of the present invention further comprises pharmaceutically acceptable excipients.

[0041] The medicine of the present invention has a pharmaceutically acceptable dosage form.

[0042] Beneficial Effects

[0043] The present invention provides a novel compound of the structure of Formula 1, and the compound can unexpectedly exhibit excellent pharmacological effects of alleviating nerve damage. Description of the drawings:

[0044] Figure 1 The liquid chromatography mass spectrometry of glycyrrhizic acid. (A) Primary mass spectrum; (B) Secondary mass spectrum.

[0045] Figure 2 This is the NMR spectrum of glycyrrhizic acid. (A) 1H NMR; (B) 13C NMR; (C) HSQC; (D) HMBC. Note: 1H NMR 600MHz, 13C NMR 150MHz

[0046] Figure 3 Formula 3 ( Formula 3. ), Formula 2 and glycyrrhizic acid (Formula 1) on the repair of hypoxic injury in neurons and nematodes. (A) & (B) & (C) PC12 cell viability assay of Formula 3, Formula 2 and glycyrrhizic acid; (D) & (E) & (F) PC12 cell LDH release of Formula 3, Formula 2 and glycyrrhizic acid; (G) & (H) & (I) Primary neuron cell viability assay of Formula 3, Formula 2 and glycyrrhizic acid; (J) & (K) & (L) Primary neuron LDH release of Formula 3, Formula 2 and glycyrrhizic acid; (M) & (N) & (O) Survival rate of Formula 3, Formula 2 and glycyrrhizic acid after hypoxic injury in nematodes; (P) DAPI staining of primary neurons of glycyrrhizic acid; (Q) DAPI nuclear staining count of primary neurons of glycyrrhizic acid. Scale bar: 100μm. Data are expressed as mean ± SD (n ≥ 3), and differences among different groups were evaluated by one-way analysis of variance (ANOVA). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Specific implementation method:

[0047] 1. Preparation Example

[0048] Preparation Example 1 - Preparation of Glycyrrhizic Acid

[0049] Step (1):

[0050] Take 200 mg of formula 2, add 60 mL of ethanol and 30 mL of 40% sulfuric acid-water solution, react at 200°C for 3 hours, cool to room temperature, add 300 mL of deionized water, filter the precipitated solid, wash with deionized water 2-3 times, and dry at 70°C to obtain 126 mg of dry ethyl tetracycline (C2 in formula 1 is a compound in which carboxylic acid is esterified with ethyl).

[0051] Step (2): Ester hydrolysis-acidification

[0052] Take 100 mg of ethyl tetracycline, add 20 mL of 20% sodium hydroxide solution, hydrolyze at 100 ° C under normal pressure for 12 hours, cool to room temperature, add hydrochloric acid to acidify the pH to below 4, filter the precipitated solid, and wash with ionized water 2-3 times to remove inorganic salts. Collect the solid product, use 10 mL of 95% ethanol to redissolve and filter, collect the filtrate, dry at 70 ° C, repeat this step twice to improve the product purity, and obtain a tetracycline product with a purity of more than 98%. The total yield after ester hydrolysis, acidification and purification is 86%.

[0053] The present invention relates to a new compound (1R, 2S, 7Z, 11S, 15S, 17R, 20R)-1,2,6,6,10,17,20-heptamethyl-12-oxyylidenetetracyclo[12.8.0.015,20.02,11]twenty-two-7,9,13-triene-17-carboxylic acid, whose chemical structure, preparation method and research on alleviating nerve damage are all disclosed for the first time, and there is no possibility of providing any inspiration through other compounds. It has outstanding substantial characteristics and is expected to be used in the research and development of a new drug for alleviating nerve damage.

[0054] 1. Chemical structure analysis of tetracycline acid

[0055] pass 1 H and 13 Extensive analysis of experiments such as C, HSQC, HMBC, and HRESIMS clarified the results for glycyrrhizic acid. Figure 1 As shown, according to the HRESIMS spectrum m / z 453.3[M+H] + Its molecular formula is determined to be C 30 H 44 O3, unsaturation is 9, fragment ions are distributed at 407, 389, 241, 189 in MS2 positive ion mode. Figure 2 (A) shows that 13 In the C NMR spectrum, 30 carbon signals were observed, including 8 SP 2 Hybridized carbon (δ C 122.1, δ C 123.1, δ C 127.5, δ C136.8, δ C 139.0, δ C 168.1, carboxyl δ C 179.9 and carbonyl δ C 198.3), 5 quaternary carbon atoms (δ C 32.3, δ C 40.5, δ C 42.1, δ C 45.0, δ C 47.7), 7 methyl groups (δ C 16.2, δ C 17.2, δ C 20.3, δ C 20.7, δ C 21.1, δ C 21.6, δ C 22.3). 1 In the H NMR spectrum, the protons on the carboxyl group (δ H 12.19), δ H The signals at 5.38 (d, J = 8.4), 5.46, 5.52 (d, J = 10.2) are attributed to the four olefin protons ( Figure 2 B).

[0056] Table 1 1H and 13C NMR chemical shift assignments of tetracycline

[0057]

[0058]

[0059] Note: NMR frequency 1 H NMR 600 MHz, 13 C NMR 150MHz

[0060] In the HSQC spectrum ( Figure 2 ) in the spectral spectrum, 21 carbon signals were observed to have chemical shifts corresponding to the hydrogen spectrum, among which δ C / δ H The signals of δ16.2 / 0.68, δ17.2 / 1.13, δ20.3 / 1.07, δ20.7 / 1.26, δ21.1 / 1.17, δ21.6 / 0.91, and δ22.3 / 0.97 are attributed to the 7 methyl groups, and the relative peak areas in the hydrogen spectrum are all 3( Figure 2 C) HMBC is used to 1 H nuclei and long-range coupling 13 C nucleus associated with the effective spectrum, in the HMBC spectrum, H-30 (δ H1.17) and C-17(δ C 42.1), C-16(δ C 31.8), C-18(δ C 28.3), C-23(δ C 179.9), H-29 (δ H 0.68) and C-20(δ C 40.5), C-19(δ C 35.7) and H-25 (δ H 0.97), H-26(δ H 0.91) and C-26(δ C 21.6), C-25(δ C 22.3), C-5(δ C 26.3) and H-24 (δ H 1.07) and C-10(δ C 139.0), C-11(δ C 59.9), C-2(δ C 47.7), C-3(δ C 42.9), which is consistent with the planar structure ( Figure 2 D). Combined with 1D NMR and 2D NMR, the detailed data of glycyrrhizic acid are summarized in Table 1. Therefore, the structure of the compound is reasonably inferred to be (1R, 2S, 7Z, 11S, 15S, 17R, 20R)-1, 2, 6, 6, 10, 17, 20-heptamethyl-12-oxyylidene tetracyclo [12.8.0.015, 20.02, 11] twenty-two-7, 9, 13-triene-17-carboxylic acid, and this inferred structure is consistent with the fragment ion distribution in the MS2 experiment. Based on the above information, the structure of the new compound is determined as shown in the figure below.

[0061] Preparation Example 2:

[0062] Compared with Example 1, the difference is only that: the parameters of step 1 are changed, specifically: take 200mg formula 2, add 50mL ethanol and 40mL 35% sulfuric acid-water solution, react 5 hours at 170 ℃, cool to room temperature, add 300mL deionized water, filter the solid separated out, wash with deionized water 2-3 times, dry at 75 ℃ to obtain dry ethyl ester of tetracyclyl acid (C2 in formula 1 is the compound of carboxylic acid esterification). The yield of ethyl ester of tetracyclyl acid product is 56%.

[0063] Step (2): Ester hydrolysis-acidification

[0064] Take 100mg of ethyl tetracycline, add 15mL 25% sodium hydroxide solution, hydrolyze at 90 ℃ under normal pressure for 14 hours, cool to room temperature, add hydrochloric acid to acidify pH to below 3.5, filter the precipitated solid, and wash with ionized water 2-3 times to remove inorganic salts. Collect the solid product, use 10mL 90% ethanol to redissolve and filter, collect the filtrate, dry at 75 ℃, repeat this step twice to improve the product purity, and obtain the tetracycline product with a purity of more than 98%. The total yield after ester hydrolysis, acidification and purification is 81%.

[0065] Comparison Example 1

[0066] Compared with Example 1, the only difference is that the concentration of sulfuric acid in step 1 is changed to 20wt% sulfuric acid aqueous solution and 50wt% sulfuric acid aqueous solution respectively; and the introduced sulfuric acid solute is the same, and the other operations and parameters are the same as in Example 1. Through research, it is found that in step 1, the target ethyl ester of glycyrrhizic acid is not detected.

[0067] 2. Application Examples

[0068] Application example 1: Glycyrrhizic acid relieves nerve damage

[0069] Ischemic stroke is the most common type of clinical cerebrovascular disease, which is caused by cerebral circulation perfusion disorder, ischemia and hypoxia, resulting in localized ischemic necrosis of brain tissue and corresponding neurological deficits, accounting for about 70% of all acute cerebrovascular diseases. Promoting the repair of damaged neurons is a key entry point for drug treatment of cerebral infarction. Cell activity and lactate dehydrogenase (LDH) release are important indicators for measuring cell damage. This study selected PC12 neurons that can be passed on and primary neurons extracted from the cerebral cortex of young mice to construct a neuronal hypoxia injury model. By adding a certain amount of glycyrrhizic acid, the changes in LDH release of damaged neuronal cell activity and the toxic effects of glycyrrhizic acid on neurons were studied. At the same time, the classic model animal Caenorhabditis elegans was selected as the research object. The model has the characteristics of simple structure, easy observation and sensitive response. The experiment characterized the effect of glycyrrhizic acid on nematode hypoxic neurological injury by observing the changes in the survival rate of nematodes after hypoxic injury.

[0070] Cell viability assay

[0071] Two independent experiments were performed in this analysis, oxygen glucose deprivation (OGD) and control groups. PC12 cells (500 μL) were seeded into 48-well microplates and cultured for 24 h, and primary neurons were seeded into 48-well microplates and cultured for 12 days. For the OGD group, the culture medium was aspirated after the culture was completed and replaced with DMEM (1X) medium without glucose and placed in N 2 (94%), CO 2 (5%) and O 2For the control group, the culture medium was replaced with DMDM ​​(1X) culture medium containing 5% glucose and placed in a CO incubator for 4 h (primary neurons for 2 h). 2 (5%) incubator for 4 h (primary neurons for 2 h). After treatment, the culture medium of the control group and OGD group was removed and replaced with DMEM (1X) culture medium containing fetal bovine serum at concentrations of 0.001, 0.01, and 0.1 mmol / L of the samples (Formulas 1 to 3), 500 uL was added to each well and placed in a CO 2 After incubation in an incubator with 5% CO for 24 h, the MTT assay was used to measure absorbance at 490 nm.

[0072] Lactate dehydrogenase (LDH) assay

[0073] Each group of cells was inoculated into a 48-well microplate and placed in a CO2 (5%) incubator for culture. After the culture was completed, the oxygen glucose deprivation group (OGD) and the control group were treated according to the provided protocol, and the sugar-free medium treatment time was set to 4h (primary neurons 2h). After the treatment, the culture medium of the control group and the OGD group was removed and replaced with DMEM (1X) culture medium containing fetal bovine serum with sample (Formula 1 to Formula 3) concentrations of 0.001, 0.01, and 0.1mmol / L, respectively. 500uL was added to each well and placed in a CO2 incubator. 2 After incubation in an incubator with 5% (5%) for 24 h, 100 μL of the supernatant was aspirated from each well and transferred to a 96-well microplate, and the assay was performed using the protocol provided by the lactate dehydrogenase cytotoxicity assay kit.

[0074] Nuclear staining (DAPI) assay

[0075] Cells were seeded into 48-well microplates, and the oxygen glucose deprivation (OGD) and control groups were treated according to the provided protocol. The culture medium of the control and OGD groups was removed and replaced with DMEM (1X) culture medium containing fetal bovine serum at concentrations of 0.001, 0.01, and 0.1 mmol / L of the samples (Formulas 1 to 3), respectively. 500 uL was added to each well and placed in a CO 2 After incubation in an incubator with 5% CO for 24 h, 0.5 μL of DAPI reagent was added to each well and incubated in the dark at 37° C. for 15 min. The fluorescence intensity of each well was observed using an inverted microscope and the fluorescence intensity in different fields of view was recorded.

[0076] Nematode hypoxia injury (AS) assay:

[0077] C. elegans was cultured in a culture dish inoculated with OP50 until the nematodes grew to the L4 stage. After the culture was completed, 50 nematodes were transferred to an EP tube containing M9 solution, washed three times, and placed in a closed culture box filled with N2, and hypoxic for 72 hours at 20°C. After the treatment was completed, the nematodes were moved to OP50 culture dishes containing samples (Formula 1 to Formula 3) at concentrations of 0.05, 0.02, and 0.01 mmol / L on the surface, and cultured for another 24 hours. The state of the nematodes in each dish was observed under a microscope and the number of nematodes alive and dead was recorded.

[0078] In this study, we used different concentrations of glycyrrhizic acid (Formula 1) to treat cells under normal conditions and hypoxic conditions, and used MTT method, LDH method and DAPI staining to evaluate the repair ability of glycyrrhizic acid on nerve hypoxic injury in vitro. Figure 3 As shown in AL, in PC12 neural cells and primary neuron cell models, Formula 3, Formula 2 and glycyrrhizic acid showed no cytotoxicity, and the activity of the two cells was significantly reduced after hypoxic injury (P<0.001), while the content of LDH was significantly increased (P<0.001). Under the condition of cell hypoxia injury, we found that Formula 3 showed no obvious drug activity within the detection concentration range, and Formula 2 ( Figure 3 H&K) showed certain nerve damage repair activity on primary neurons only under higher concentration conditions. Compared with Formula 3 and Formula 2, after 24h treatment with 0.01mM and 0.1mM glycyrrhizic acid, the cell viability of PC12 cells and primary neuronal cells was significantly enhanced ( Figure 3 C&I, LDH release was significantly decreased (P<0.01) ( Figure 3 F&L). At the same time, DAPI staining results showed that after treatment with glycyrrhizic acid, the number of cell nuclei of primary neurons was significantly increased compared with the hypoxia injury group ( Figure 3 PQ). This indicates that glycyrrhizic acid can effectively alleviate the damage of nerve cells after hypoxia. Finally, we used hypoxic Caenorhabditis elegans to detect the effects of formula 3, formula 2, and glycyrrhizic acid on nerve damage. The results showed that formula 3 ( Figure 3 M) only increased the survival rate of hypoxic nematodes at high concentrations, while formula 2 ( Figure 3 N) showed no effective activity within the detected concentration range. Compared with the two, after 24h treatment with 0.01-0.05mM glycyrrhizic acid, the survival rate of nematodes damaged by hypoxia was significantly increased (P≦0.01), especially under 0.05mM glycyrrhizic acid treatment, the survival rate of nematodes increased from 64.2% to 88.8% ( Figure 3 O).

Claims

1. A tetracyclic acid compound, characterized in that: A compound having the structural formula 1; Formula 1.

2. A method for preparing the tetracyclic acid compound according to claim 1, characterized in that: The raw material of formula 2, alcohol and a sulfuric acid aqueous solution with a concentration of 30-45 wt.% are mixed and reacted at a temperature of 150-250° C., and then the glycyrrhizic acid is obtained by alkaline hydrolysis and acidification; Formula 2; The chemical formula of the alcohol is ROH, wherein R is a C1-C4 alkyl group; The base used in the alkaline hydrolysis process is an alkali metal hydroxide; The acid used in the acidification process is at least one of hydrochloric acid and sulfuric acid.

3. The method for preparing the tetracyclic acid compound according to claim 2, wherein: The concentration of the aqueous sulfuric acid solution is 33-42 wt.%.

4. The method for preparing the tetracyclic acid compound according to claim 2, wherein: The concentration of the aqueous sulfuric acid solution is 30-40wt.%.

5. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: In the initial stage of the reaction, the liquid-to-solid ratio of the formula 2 and the sulfuric acid aqueous solution is above 50 ml / g.

6. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: In the initial stage of the reaction, the liquid-to-solid ratio of the formula 2 and the sulfuric acid aqueous solution is 100-400 ml / g.

7. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: In the initial stage of the reaction, the liquid-to-solid ratio of the formula 2 and the sulfuric acid aqueous solution is 100-250 ml / g.

8. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: The volume ratio of alcohol to sulfuric acid aqueous solution is 0.5~5:

1.

9. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: The volume ratio of alcohol to sulfuric acid aqueous solution is 1~3:

1.

10. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: The reaction temperature is 160~220℃.

11. The method for preparing the tetracyclic acid compound according to claim 2, characterized in that: After acidification, the product was re-dissolved and purified using an ethanol aqueous solution with a concentration of 85-98v%.

12. Use of the glycyrrhizic acid compound according to claim 1 in preparing a drug for alleviating nerve damage.

13. The use according to claim 12, characterized in that The glycyrrhizic acid compound is combined with a pharmaceutically acceptable adjuvant to prepare a pharmaceutically acceptable dosage form.

14. The use according to claim 12, characterized in that The drug for alleviating nerve damage is a drug for treating stroke.

15. The use according to claim 14, characterized in that The medicine for alleviating nerve damage is a medicine for treating stroke.

16. A drug for alleviating nerve damage, characterized in that: It comprises a pharmaceutically effective amount of the glycyrrhizic acid compound according to claim 1.

17. The drug for alleviating nerve damage according to claim 16, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

18. The drug for alleviating nerve damage according to claim 17, characterized in that: The medicine has a pharmaceutically acceptable dosage form.

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