CN107837311B

CN107837311B - Application of Honglong analgesic tablet in preparation of nerve cell protective preparation

Info

Publication number: CN107837311B
Application number: CN201610834515.0A
Authority: CN
Inventors: 孙绪丁; 王海苹; 马宏伟; 李晗; 王红月
Original assignee: Jinhe Tibetan Medicine Shandong Health Industry Co ltd
Current assignee: Jinhe Tibetan Medicine Shandong Health Industry Co ltd
Priority date: 2016-09-20
Filing date: 2016-09-20
Publication date: 2021-03-02
Anticipated expiration: 2036-09-20
Also published as: CN107837311A

Abstract

The invention discloses an application of Honglong analgesic tablets in preparing a nerve cell protective preparation, which comprises an extract prepared by a conventional method according to the formula of the Honglong analgesic tablets, and the extract has the action mechanism of removing free radicals and reducing NO or NF-kB inflammation. The invention overcomes the technical prejudice, firstly provides the Honglong analgesic tablet and the extractive thereof with the nerve protection function, improves the survival rate of nerve cells by removing free radicals, reducing the damage of the nerve cells caused by a large amount of NO and the like, and can obviously inhibit the expression of NF-kB in the nerve cells damaged by ischemia-reperfusion so as to inhibit the damage of inflammatory reaction on the nerve cells.

Description

Application of Honglong analgesic tablet in preparation of nerve cell protective preparation

Technical Field

The invention relates to an application of Honglong analgesic tablets in preparing a nerve cell protective preparation, belonging to the technical field of medicines.

Background

The Honglong analgesic tablet is a Tibetan ancient empirical prescription and is loaded in the standard issued by the Tibetan medicine ministry, and the standard number is as follows: WS-10659(ZD-0659) -2002-2011Z, which consists of 112.0g of fossil fragments, 67.0g of India swertia herb, 67.0g of safflower, 14.0g of Chrysosporium japonicum, 67.0g of Sichuan flaveria bidentis, 14.0g of aconitum pendulum, 67.0g of aconitum tanguticum, 6.6g of bear gall powder, 13.5g of Quanhua, 13.5g of herpetospermum pedunculosum seeds, 13.5g of Marinula vesiculosa, 13.5g of lithospermum, 67.0g of Lagotis brachyporus breviflorus, 13.0g of benzoin and 35.0g of mother-of-pearl, has the effects of restoring consciousness, inducing resuscitation, dredging collaterals and relieving pain, and is used for treating migraine and angioneurotic headache caused by stasis and obstruction of brain collaterals.

The Honglong analgesic tablet has better effect on migraine and angioneurotic headache caused by stasis blocking brain collaterals in clinic, no specific pharmacological mechanism research exists in the existing literature, and the main pharmacological mechanism of the analgesic effect is presumed to be the functions of improving blood supply and resisting inflammation and relieving pain by analyzing the formula. The existing literature does not find the research of the protective effect of the Honglong analgesic tablet on nerve cells.

Disclosure of Invention

The invention aims to provide application of Honglong analgesic tablets in preparation of a nerve cell protective preparation.

The technical scheme of the invention is as follows:

application of HONGLONGZHENTONG tablet in preparing nerve cell protective preparation is provided.

Preferably, the action mechanism of the Honglong analgesic tablet on the nerve cell protective preparation is a mechanism for eliminating free radicals and reducing NO or NF-kB inflammatory reaction.

Preferably, the Honglong analgesic tablet comprises an extract prepared by a conventional method according to the formulation of the Honglong analgesic tablet.

Preferably, the extract comprises one or more of ethyl acetate extract, ethanol extract and water extract.

Wherein each extract is prepared as follows

Ethyl acetate extract: taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding ethyl acetate with the weight volume ratio of 6-10 times into the other medicinal materials except the bear gall powder and the benzoin, extracting for 2 times, each time for 2 hours, merging the ethyl acetate extracting solutions, concentrating under reduced pressure, continuously drying, adding the bear gall powder and the benzoin, and uniformly mixing to obtain an ethyl acetate extract;

ethanol extraction: taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding 70% ethanol with the weight-volume concentration of 6-10 times of the weight-volume ratio into the other medicinal materials except the bear gall powder and the benzoin for extraction for 2 times, 2 hours for the first time and 1 hour for the second time, combining the two ethanol extract solutions, filtering, concentrating under reduced pressure and continuously drying to obtain an ethanol extract;

water extract: taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding water with the weight volume ratio of 6-10 times into the other medicinal materials except the bear gall powder and the benzoin for extraction for 2 times, 2 hours for the first time and 1.5 hours for the second time, combining the two water extract solutions, filtering, concentrating under reduced pressure and continuously drying to obtain the water extract.

The relationship between the parts by weight and the parts by volume of the invention is g/ml or kg/L.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention overcomes the prejudice of the prior art and provides the Honglong analgesic tablet and the extract thereof with neuroprotective effect for the first time.

2. The neuroprotective effect of the Honglong analgesic tablet is mainly to improve the survival rate of nerve cells by eliminating free radicals, reducing the damage of nerve cells caused by a large amount of NO and the like, and can obviously inhibit the expression of NF-kB in ischemia reperfusion damaged nerve cells, thereby inhibiting the damage effect of inflammatory reaction on the nerve cells.

Detailed Description

The following experimental examples and examples are intended to further illustrate but not limit the invention.

Experimental example 1: protection effect of Honglong analgesic tablets on chemical injury of PC12 cell line caused by glutamic acid

First, experimental material

1. Samples and preparation thereof

Cell line: PC12 cell line (source: Rattus norvegicus adrenal pheochromocytoma, Katy Biotech development Co., Ltd., Nanjing);

reagent: H-DMEM cell culture medium (Hyclone, Saimer Feishale Biochemical Beijing, Inc.); fetal bovine serum (eind biold); pancreatin cell digestive juice (Biyuntian), penicillin streptomycin mixed solution double antibody, cisplatin for injection (Qilu pharmaceutical Co., Ltd.), citrate buffer solution (Beijing Zhonghua fir Jinqiao biotech Co., Ltd.), paraformaldehyde (solarbio), L-glutamic acid (Klonetech, Japan), compound Danshen drop pill (Tianjin Tianshi pharmaceutical Co., Ltd., 20131202)

The red dragon pain-relieving tablet is characterized in that each sample is called as the reference for short:

honglong analgesic tablet (Qinghai gold myrobalan Tibetan medicine, batch number 20141022)

Honglong analgesic tablet ethyl acetate extract (ester extract, self-made in example 1)

Ethanol extract of Honglong analgesic tablet (alcohol extract, self-made in example 1)

Honglong analgesic tablet aqueous extract (aqueous extract, self-made in example 1)

Honglong analgesic tablet micro powder 300 mesh (micro powder, self made in example 1)

2. Laboratory apparatus

A vertical pressure steam sterilization pot (LDZX-50FBS, Shanghai Shenan); double single-side clean bench (SW-CJ-1C, Suzhou clean); carbon dioxide incubator (BB16 μ V/BB5060 μ V, HERAE US); bench centrifuge (H3, Sigma); microplate reader (Multiskan MK3, Thermo Scientific); an electric heating constant temperature blast drying oven (101, shanghai peng shun scientific instruments ltd); inverted microscope (XDS-1B, Chongqing optical instruments factory); a water bath constant temperature oscillator (SHZ-82, medical instrument factory of gold jar city); pipettors (Gilson, Eppendorf); culture flasks (Corning); 96-well plates (Costar, USA).

Second, Experimental methods

A PC12 cytochemical injury model is prepared from glutamic acid, an absorption value is measured at 570nm by adopting a thiazole blue (MTT) method, and the repairing effect of the candidate drug on PC12 cytochemical injury is observed. SOD, NO, LDH and MDA kits are used for detecting the content of each index, and the action mechanism of each medicine is clarified.

Molding: 30mmol/l glutamic acid (this is the final concentration of the sample, dissolved in incomplete medium) acted on PC12 cells for 24 h.

Grouping: blank group, chemical injury model group, chemical injury + drug group, chemical injury + positive control drug group. Because the number of the 96-well plates is limited, two plates are adopted for simultaneous culture and detection when the sample amount is large, and each plate is respectively provided with a model group blank group and a positive control group so as to ensure the reliability and consistency of results.

Third, experimental contents and procedures

1. Cell culture

The frozen PC12 cells were recovered from the ultra-low temperature refrigerator in a culture flask and cultured in H-DMEM medium containing 10% fetal bovine serum. When the PC12 cells grew to 80% confluency, they were digested with 0.25% pancreatin cell digest (containing 0.02% EDTA), seeded into 96-well plates at 1 × 104 cells per well, and wells were zeroed without addition of cells.

2. Cell damage

After the cells in the 96-well plate grow to a monolayer, the culture medium is discarded, sterile PBS is washed for 2 times, and glutamic acid (dissolved in incomplete culture medium) with the final concentration of 30mmol/l is added into each well except for the zero adjustment group and the blank control group to be cultured in an incubator for 24 hours, so that the PC12 cell damage model is generated.

3. Medicated repair

Discarding the culture medium, washing with sterile PBS solution for 2 times, adding 100 μ l DMEM culture medium containing the screened drugs with final concentrations of 100 μ g/ml, 10 μ g/ml and 1 μ g/ml into each well of the administration group, and adding 6 wells for each drug. Taking drug-free DMEM medium as a blank control group, and adding 100 mul of serum-free DMEM medium of compound radix Salviae Miltiorrhizae dripping pills with different concentrations into the positive control group. The cells were then placed in a 5% CO2 incubator at 37 ℃ for further 24 h.

4. Index measurement

4.1 determination of cell viability by MTT method

After the medium is discarded, 100 mul of serum-free DMEM medium is added into each well, and 20 mul of MTT solution with the concentration of 5mg/ml is added into each well to react in an incubator for 4 hours in a dark place. Discarding the culture medium, adding 100 μ l DMSO, measuring OD value at 570nm with enzyme-labeling instrument, and calculating cell survival rate:

calculating the formula: cell survival (%) (drug use or model group a 570/control group a570) × 100%.

4.2 determination of the content of Nitric Oxide (NO), Lactate Dehydrogenase (LDH), superoxide dismutase (SOD), and Malondialdehyde (MDA)

Cell supernatants were collected 24 hours after dosing using sterile tubes, centrifuged for 20 minutes (2000r/min), collected carefully and assayed for LDH, NO content according to kit instructions.

And (3) discarding the supernatant 24 hours after administration, adding 2% Trion-100X, standing at 4 ℃ for 12 hours, uniformly mixing the cell suspension, centrifuging for 20 minutes (2000r/min), carefully collecting the supernatant, and determining the contents of MDA and SOD according to the operation of a kit specification.

Fourth, experimental results

All data are expressed by X +/-SD, and the significance of difference between two comparison groups of single-factor variance analysis and LSD multi-group mean is obvious when P is less than 0.05.

1. Influence of Honglong analgesic tablet on survival rate of injured PC12 cells

Table 1 results of the effect of honglong analgesic tablets on the survival rate of injured PC12 cells (n ═ 8)

Note: denotes P <0.05

The results in table 1 show that the cell survival rates of the candidate drugs with the final concentration of 1 mug/ml are all significantly different from those of the corresponding model groups in alcohol extraction, micro powder extraction, water extraction and ester extraction. In the candidate drugs with the final concentration of 10 mug/ml, the cell survival rate of the ester extraction micro powder is significantly different from that of a model group; the survival rate of the cells extracted by alcohol and water is not obviously different from that of the model group. The fat-soluble components and the Honglong analgesic tablets have stronger protective effect on in-vitro PC12 nerve cells.

2. Influence of Honglong analgesic tablet on LDH, NO, MDA and SOD content

2.1 Effect of Honglong analgesic tablets on the NO content of PC12 cells cultured in vitro

Table 2 determination of NO content in PC12 cells by each extract and serum containing drug of honglong analgesic tablets (n ═ 3)

Note: p <0.05

The results in Table 2 show that the alcohol extraction and ester extraction with final concentration of 1. mu.g/ml and 10. mu.g/ml and the water extraction with final concentration of 10. mu.g/ml are significantly different from the model group; in the serum, the blood drug has significant difference compared with the blank serum group. The results show that each extract and the serum containing the drug have great influence on the NO content of PC12 cells.

2.2 Effect of Red Dragon analgesic tablets on LDH content of PC12 cells cultured in vitro

Table 3 determination of LDH content in each extract and medicated serum of honglong analgesic tablets (n ═ 3)

Note: p <0.05

The results in Table 3 show that 1 microgram/ml, 10 microgram/ml of alcohol extraction, 10 microgram/ml of water extraction and drug-containing serum of the micropowder can obviously reduce the leakage amount of LDH in the damaged PC12 cell culture solution.

2.3 Effect of Honglong analgesic tablets on the SOD content of PC12 cells cultured in vitro

TABLE 4 determination of SOD content in each extract and serum containing drugs of HONGLONGZHENTONG tablet (n ═ 3)

Note: p <0.05

The results in Table 4 show that the ester extract of 1. mu.g/ml, the water extract of 1. mu.g/ml, the ester extract of 10. mu.g/ml and the water extract of 10. mu.g/ml are all significantly different from the model group. The ester extract and the water extract of the Honglong analgesic tablet can obviously improve the SOD concentration of damaged nerve cells and provide about oxidation resistance.

2.4 Effect of Honglong analgesic tablets on the MDA content of PC12 cells cultured in vitro

TABLE 5 content determination of each extract and drug-containing serum MDA of Honglong analgesic tablet (n ═ 3)

Note: p <0.05

The results in table 5 show that there was no significant difference between each group and the model group.

3. Research conclusion of external pharmacological test of Honglong analgesic tablet

TABLE 6 summary of the results of various preparations of HONGLONG analgesic tablet

+ indicates that the preparation or extract had a significant statistical significance (P <0.05) compared to the model group.

3.1 Effect on cell survival

The results of the cell survival rate of the PC12 determined by the MTT method show that the cell survival rate of the candidate drug with the final concentration of 1 mug/ml is significantly different from that of the corresponding model group by alcohol extraction, micropowder extraction, water extraction and ester extraction. In the candidate drug with the final concentration of 10 mug/ml, the survival rate of cells of the micro powder is significantly different from that of the corresponding model group by alcohol extraction. The positive control compound salvia miltiorrhiza dropping pill is administrated at the final concentration of 27mg/ml, and the cell survival rate is highest in each group. The other groups did not show significant difference or were significantly lower than the model control group.

3.2 Effect on SOD content

Compared with the model group, the ester extraction rate is 1 mu g/ml, the water extraction rate is 1 mu g/ml, the ester extraction rate is 10 mu g/ml and the water extraction rate is 10 mu g/ml, the significant differences exist.

3.3 Effect on MDA content

No significant difference was seen between each sample and the model group.

3.4 Effect on NO content

The final concentration of 1 mu g/ml and 10 mu g/ml are significantly different from the model group in alcohol extraction, ester extraction and 10 mu g/ml water extraction; compared with the blank serum group, the serum containing the medicine has obvious difference.

3.5 Effect on LDH content

1 microgram/ml and 10 microgram/ml of micro powder, 10 microgram/ml of alcohol extraction and 10 microgram/ml of water extraction, and the serum containing the medicine has significant differences compared with a model group.

3.6 comprehensive conclusion of Honglong analgesic tablet

The results of the MTT method cell activity screening prove that: compared with a model group, the red dragon pain relieving tablet has obvious repairing effect on PC12 cell damage caused by glutamic acid under the condition of proper concentration of alcohol extract, superfine powder and water extract. Compared with the model group, the ethyl acetate extract and the water extract of the Honglong analgesic tablet can obviously enhance the SOD activity of cells. The medicine has certain effect of eliminating free radical produced by PC12 cell damage caused by glutamic acid.

The micro powder is 1 microgram/ml and 10 microgram/ml, the water extraction is 10 microgram/ml, the alcohol extraction is 10 microgram/ml, the sample with the concentration of 10 microgram/ml, and the serum containing the medicine can reduce the leakage amount of LDH in the culture solution, which indicates that the screened medicines can reduce the damage degree of PC12 nerve cells, and have certain repairing and protecting effects on the nerve cell damage caused by glutamic acid.

The drug-containing serum of the ethyl acetate extract of the Honglong analgesic tablet can obviously reduce the NO content in a cell culture solution and reduce the damage of nerve cells caused by a large amount of NO.

Experimental example 2 protective action of Honglong analgesic tablet on nerve cells after ischemia reperfusion

1 materials and methods

1.1 materials

(1) Animal, wherein two weeks of pregnancy are pregnant two-week Kunming second-level pregnant mice provided by Shandong university animal center;

(2) the main reagent and instrument are fetal calf serum provided by Hangzhou Sijiqing company, and DMEM medium provided by GIBCO company; pancreatin is supplied by huamei corporation; rabbit anti-mouse NF-. kappa.Bp 65 polyclonal antibody (recognizing 1-286 amino acid sequences) was provided by Santa corporation, USA; fluorescein Isothiocyanate (FITC) labeled goat anti-rabbit IgG is supplied by meihua corporation; the beta-tubulin (beta-tubulin) monoclonal antibody is supplied by Progma corporation; honglong analgesic tablet is provided by gold myrobalan Tibetan medicine, and the ethanol extract and the ethyl acetate extract are prepared by the example 1; carbon dioxide incubators are available from SANYO corporation; the flow cytometer is CoulterEliteEPS type, usa.

1.2 methods

(1) Sterilizing a plurality of 25mL culture bottles together, and then coating by polylysine; preparing artificial cerebrospinal fluid, wherein the composition of the artificial cerebrospinal fluid comprises 123mmol/L NaCl, 3.75mmol/L KCl, KH2PO41.25mmol/L NaHCO326mmol/L MgCl21mmol/L CaCl22mmol/L glucose 10mmol/L and the pH value is 7.4.

(2) Cell culture, namely taking two-week pregnant Kunming second-level pregnant mice, dislocating and killing the pregnant mice, disinfecting abdomens with iodine and ethanol, dissecting skin, muscles and peritoneum of the abdomens layer by layer, taking out the fetal mice, carefully peeling amnion of fetal membranes, exposing the fetal mice, peeling meninges under a stereomicroscope, separating cortex of the lateral part of cerebral alveolus of the fetal mice, shearing brain tissues, digesting the fetal mice with trypsin containing 125mg/L (37 ℃/15min), centrifuging the fetal mice for 1000r/min and 10min, discarding supernatant, stopping digestion with DMEM of 200mL/L fetal bovine serum, diluting the fetal mice with complete culture medium (10mL/L fetal bovine serum, cyan and streptomycin 100U/mL) to the density of 7 x 10^5/mL, inoculating the fetal bovine serum, sending the fetal bovine serum into a culture flask treated by polylysine, sending the fetal mice into a 50mL/LCO2 culture box, culturing the fetal mice at 37 ℃ for 48 hours, adding cytarabine (5 mu g/mL) to inhibit the, and (3) replacing the liquid every 2d half, culturing for 10d to obtain typical nerve cells, performing immunofluorescence staining by using the beta-tubulin monoclonal antibody to show that more than 80% of the cells are positive, and culturing the cells to be pure nerve cells, and performing subsequent experimental study.

(3) Establishing an ischemia-like model according to an OGD/R (sugar and other nutrient loss) method, changing a cell culture medium into artificial cerebrospinal fluid without sugar, keeping the temperature at (30 +/-2) DEG C, and changing mixed gas into nitrogen containing 80mL/LCO 2. And (5) carrying out ischemia-like treatment for 10min, replacing the artificial cerebrospinal fluid with the culture medium, putting the artificial cerebrospinal fluid back to an incubator, and reoxygenating.

(4) Counting and detecting by a flow cytometry, namely randomly dividing primary nerve cells (1 x 10^6 bottles) inoculated in a culture bottle into 4 groups, wherein one group is an ischemia-free group, the other group is subjected to similar ischemia treatment and then reoxygenated for 1/2 hours to form an ischemia-reperfusion group, the third group and the fourth group are a treatment group I and a treatment group II, and after the similar ischemia treatment, ethanol extract of Honglong analgesic tablets (treatment group I) and ethyl acetate extract (treatment group II) are respectively added for pretreatment for 10min, wherein the two extracts are respectively diluted into a liquid containing 1mg tablets/ml by adding corresponding solvents into the extract of example 1. Each group of specimens was 4 parts. Group 4 was washed with 0.01moL/LPBS for 5min 3 times, cells were digested with 0.25% trypsin to give a single cell suspension, centrifuged and washed, then 200. mu.L of 1: 200 NF-. kappa.Bp 65 polyclonal antibody was added, the mixture was incubated at 37 ℃ for 45min, washed with 0.01moL/LPBS for 3 times, centrifuged and the supernatant was discarded, 200. mu.L of 1: 50 FITC-labeled anti-rabbit IgG was added, incubated at 4 ℃ for 45min, and washed with 0.01 moL/LPBS. Two negative control groups are additionally arranged, and one negative control group is not added with the I antibody and the II antibody and is used for correcting the fluorescence intensity baseline; another group of samples was treated with II antibody alone as a nonspecific binding control, and all samples were individually subjected to on-machine detection with 400. mu.L of 0.01mol/LPBS solution, and more than 5000 cells were counted per sample, and the results were averaged.

1.3 statistical methods

Results are expressed as x + -s, and comparisons of data between groups were performed by analysis of variance and t-test.

2 results

Flow cytometry results NF-kb/P65 were expressed as (1.38 ± 0.56)%, on a basal basis in non-ischemic neurons, with a significant increase in expression 1/2h after ischemia-reperfusion (P < 0.01). After 2 kinds of extracts of the Honglong analgesic tablets are used for pretreatment, the expression rate is remarkably reduced (P is less than 0.05), and the four groups have remarkable difference (P is less than 0.05). Among them, the ischemia-reperfusion group had a very significant difference (P <0.01) compared to treatment group ii. The ethyl acetate extract has stronger protective effect on the ischemia-reperfusion injury model.

TABLE 1 comparison of NF- κ B expression rates between groups

Note: compared with the non-ischemic group, # # P is less than 0.01; p <0.05, P <0.01 in comparison to ischemia-reperfusion group

3 small knot

NF-kB exists in brain vascular endothelial cells, nerve cells and glial cells, and the expression of the NF-kB is obviously increased after cerebral ischemia reperfusion and is closely related to an inflammation mechanism of cerebral ischemia injury. The experiment examines the intervention and treatment of the red dragon analgesic tablets on the cerebral ischemia-reperfusion injury of the mice by taking NF-kB as an index, and finds that the red dragon analgesic tablets have a remarkable reduction effect on the expression of NF-kB during the cerebral ischemia-reperfusion injury, thereby reducing the damage effect of an inflammation mechanism on the ischemia-reperfusion on nerve cells.

The following examples serve to further illustrate the invention.

Example 1 preparation of extracts from various groups of Honglong analgesic tablets

1) Preparing an ethyl acetate extract of Honglong analgesic tablets:

292g of the Honglong analgesic prescription medicinal materials and the medicinal materials taken according to the proportion are crushed. Extracting the rest materials except fel Ursi powder and Benzoinum for 2 times, adding 2250ml ethyl acetate respectively, extracting for 2h, mixing the ethyl acetate extractive solutions, concentrating under reduced pressure, drying, adding fel Ursi powder and Benzoinum, and mixing to obtain 2.11g ethyl acetate extract of HONGLONGZHENTONG tablet.

2) Preparing an ethanol extract of Honglong analgesic tablets:

292g of the Honglong analgesic prescription medicinal materials and the medicinal materials taken according to the proportion are crushed. Extracting the rest materials except fel Ursi powder and Benzoinum with 2250ml of 70% ethanol by volume for 2 times, 2 hr for the first time, 1 hr for the second time, mixing the two ethanol extractive solutions, filtering, concentrating under reduced pressure, and drying to obtain 11.78g ethanol extract of HONGLONGZHENTONG tablet.

3) Preparing an aqueous extract of Honglong analgesic tablets:

292g of the Honglong analgesic prescription medicinal materials and the medicinal materials taken according to the proportion are crushed. Adding 2820ml of water into other medicinal materials except the bear gall powder and the benzoin, extracting for 2 times, the first time for 2 hours and the second time for 1.5 hours, combining the two water extracts, filtering, concentrating under reduced pressure and continuously drying to obtain 17.84g of water extract of the Honglong analgesic tablets.

4) Honglong analgesic tablet micropowder: pulverizing HONGLONGZHENTONG tablet into fine powder.

5) Preparation of serum containing medicine:

taking ethanol extract of Honglong analgesic tablet, preparing into maximum concentration water suspension, orally feeding 5 mice with dosage of 0.0236g/g body weight, taking eyeball after 30min, taking blood without anticoagulation, centrifuging at 3000rpm for 15min, and separating serum for later use to obtain sample (blood).

Example 2, application of Honglong analgesic tablet in preparing a nerve cell protective preparation.

Example 3, application of Honglong analgesic tablet in preparing nerve cell protective agent, the action mechanism is to clear free radical, reduce NO or NF-kB inflammatory reaction mechanism.

Example 4 application of an ethyl acetate extract of Honglong analgesic tablet in preparing a nerve cell protective preparation, wherein the preparation method of the ethyl acetate extract is the same as that in example 1.

Example 5 application of ethanol extract of Honglong analgesic tablet in preparing nerve cell protective preparation, the preparation method of ethanol extract is the same as example 1.

Example 6 application of water extract of Honglong analgesic tablet in preparing nerve cell protective agent, the preparation method of the water extract is the same as example 1.

Example 7 application of a Honglong analgesic tablet extract composition in preparing a nerve cell protective preparation, the extract composition comprises an ethyl acetate extract and an ethanol extract, and the preparation method of the extract is the same as that of example 1.

Example 8, use of a Honglong analgesic tablet extract composition in the preparation of a neurocyte protective preparation, the extract composition comprising an ethyl acetate extract and a water extract, the extract preparation method being the same as in example 1.

Example 9 application of a Honglong analgesic tablet extract composition in preparing a nerve cell protective preparation, the extract composition comprises a water extract and an ethanol extract, and the preparation method of the extract is the same as that of example 1.

Example 10 application of a Honglong analgesic tablet extract composition in preparing a nerve cell protective preparation, wherein the extract composition comprises an ethyl acetate extract, an ethanol extract and an aqueous extract, and the preparation method of the extract is the same as that of example 1.

The drugs prepared in the above examples all achieved the effects described in experimental example 1 and experimental example 2.

Claims

1. The application of the Honglong analgesic tablet in preparing the nerve cell protective preparation is characterized in that the nerve cell protective preparation has the action mechanism of eliminating free radicals and reducing NO or NF-kB inflammatory reaction mechanism;

the Honglong analgesic tablet is prepared from the following components in percentage by weight: 112.0g of dragon bone, 67.0g of India swertia, 67.0g of safflower, 14.0g of Chrysosplenium, 67.0g of Sichuan yellow chrysanthemum, 14.0g of aconitum pendulum, 67.0g of aconitum tanguticum, 6.6g of bear gall powder, 13.5g of Quanhua, 13.5g of herpetospermum pedunculosum seeds, 13.5g of Maurena, 13.5g of lithospermum, 67.0g of lagotis brachypomus, 13.0g of benzoin and 35.0g of mother-of-pearl.

2. The application of the Honglong analgesic tablet in preparing the nerve cell protection preparation according to claim 1, wherein the Honglong analgesic tablet comprises an extract prepared by a conventional method according to the formulation of the Honglong analgesic tablet.

3. The use of the Honglong analgesic tablet of claim 2 in the preparation of a neuro-cytoprotective preparation, wherein the extract comprises one or more of an ethyl acetate extract, an ethanol extract, and an aqueous extract.

4. The application of the Honglong analgesic tablet in preparing the nerve cell protective preparation according to claim 3 is characterized in that the preparation method of the ethyl acetate extract is as follows:

taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding ethyl acetate with the weight volume ratio g/ml of 6-10 times into the other medicinal materials except the bear gall powder and the benzoin, extracting for 2 times, each time for 2 hours, combining the ethyl acetate extract, concentrating under reduced pressure, continuously drying, adding the bear gall powder and the benzoin, and uniformly mixing to obtain the ethyl acetate extract.

5. The application of the Honglong analgesic tablet in preparing the nerve cell protective preparation according to claim 3 is characterized in that the preparation method of the ethanol extract comprises the following steps:

taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding 70% ethanol with the volume concentration 6-10 times of the weight-volume ratio g/ml into the other medicinal materials except the bear gall powder and the benzoin, extracting for 2 times, extracting for 2 hours for the first time and 1 hour for the second time, combining the two ethanol extracting solutions, filtering, concentrating under reduced pressure and continuously drying to obtain the ethanol extract.

6. The application of the Honglong analgesic tablet in preparing the nerve cell protection preparation according to claim 3 is characterized in that the preparation method of the aqueous extract comprises the following steps:

taking the medicinal materials according to the Honglong analgesic formula and the proportion thereof, adding 6-10 times of water by weight volume ratio g/ml into the other medicinal materials except the bear gall powder and the benzoin, extracting for 2 times, 2 hours for the first time and 1.5 hours for the second time, combining the two water extract solutions, filtering, concentrating under reduced pressure and continuously drying to obtain the water extract.