阅读说明：本技术 一种对神经细胞损伤具有保护作用的组合物及其制剂和用途 (Composition with protective effect on nerve cell injury and preparation and application thereof ) 是由 张建革 詹常森 张培 周俊杰 汪飞云 姜鹏 林国强 张正光 于 2020-06-10 设计创作，主要内容包括：本发明提供一种对神经细胞损伤具有保护作用的组合物及其制剂和用途。本发明的组合物包含蟾毒配基类化合物,更具体地本发明的组合物包含华蟾酥毒基和酯蟾毒配基。本发明还提供包含本发明的组合物的药物制剂及本发明的组合物或包含本发明组合物的药物制剂在制备用于治疗由神经细胞损伤引发的疾病的药物中的用途。(The invention provides a composition with a protective effect on nerve cell injury, and a preparation and application thereof. The compositions of the invention comprise bufogenin-based compounds, more specifically the compositions of the invention comprise cinobufagin and bufogenin. The invention also provides a pharmaceutical preparation containing the composition and application of the composition or the pharmaceutical preparation containing the composition in preparing a medicament for treating diseases caused by nerve cell injury.)

1. A composition having a protective effect on nerve cell damage, which comprises a bufogenin-based compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin.

2. A composition according to claim 1, comprising cinobufagin and resibufogenin, wherein the weight ratio of cinobufagin to resibufogenin is 0-3:1, further preferably 0.9-2.5:1, still further preferably 1.0-2.0: 1.

3. The composition of claim 1 or 2, wherein the nerve cell damage is caused by hypoxia; further preferably, the disease caused by nerve cell damage is selected from the group consisting of: ischemic stroke, neonatal hypoxic ischemic disease, alzheimer's disease, and parkinson's disease.

4. The composition of any one of claims 1-3, wherein the composition is a complex.

5. A pharmaceutical formulation comprising the composition of any one of claims 1-4.

6. The pharmaceutical formulation of claim 5, wherein the composition comprises a bufogenin-like compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin;

preferably, the composition comprises cinobufagin and bufogenin, and the weight part ratio of the cinobufagin to the bufogenin is 0-3:1, more preferably 0.9-2.5:1, and still more preferably 1.0-2.0: 1.

7. The pharmaceutical preparation according to claim 5 or 6, wherein the pharmaceutical preparation may further comprise other active ingredients which may be used together or in conjunction with the composition of the invention for the protection against nerve cell damage; further preferably, the other active ingredients are selected from edaravone, nimodipine, cinepazide, gastrodine, salvianolate, vitamin B1, vitamin B6, vitamin B12 and mecobalamin;

preferably, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier.

8. Use of the composition of any one of claims 1-4 or the pharmaceutical formulation of any one of claims 5-7 in the manufacture of a medicament for treating a disease caused by nerve cell damage.

9. The use according to claim 8, wherein the disease in nerve cell damage is caused by hypoxia; the disease caused by nerve cell damage is selected from: ischemic stroke, neonatal hypoxic ischemic disease, alzheimer's disease, and parkinson's disease.

Technical Field

The invention relates to the field of medicines, in particular to a composition with a protective effect on nerve cell injury, and a preparation and application thereof.

Background

A venenum Bufonis is prepared from white serous fluid secreted by ear-back gland and skin gland of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider of Bufo siccus by processing and drying. Bufonis venenum is mainly produced in Shandong, Hebei, Jiangsu, Hunan, Zhejiang and Sichuan provinces.

According to the pharmacopoeia of 2015 edition, the venenum bufonis is sweet, pungent, warm and toxic in nature, and enters heart meridian; the functions and main indications are detoxification, pain relief, resuscitation and resuscitation, and the traditional Chinese medicine is used for treating carbuncle, deep-rooted carbuncle, furuncle, sore throat, sunstroke, coma, eruption, abdominal pain, vomiting and diarrhea. Modern pharmacological research shows that the toad venom not only can relieve pain, diminish inflammation and anaesthesia, but also has various biological activities of resisting cancer, resisting radiation, strengthening heart and the like.

Currently, there are many studies on toad venom and active ingredients in toad venom, and recent research focuses on their antitumor activity. In the existing research, bufanolide and indole alkaloid are the most studied components, and the bufanolide compounds are divided into 5 types according to different substituents on a ligand parent nucleus, wherein the bufotoxin ligand substances are important components of the bufonis venenum for exerting pharmacological activity and comprise cinobufagin and ester bufotoxin ligands. Cinobufagin is insoluble in water, has short half-life in vivo and wide distribution, has strong toxicity, and has a structure shown in formula I:

ester bufogenin is another important compound in the bufogenin class, which is almost insoluble in water, milder in toxicity compared to cinobufagin, and has the structure shown in formula II:

however, the effect of the toad venom or the main active ingredients in the toad venom on the protection of nerve cell injury is not reported in the prior art. With respect to nerve cell damage, currently known substances that may have a protective or ameliorating effect on nerve cell damage include:

gangliosides: monosialotetrahexosyl ganglioside, which is a main class of mammalian gangliosides, has been widely used in clinical applications at present.

Vitamins: (1) vitamin B1: vitamin B1 is in coenzyme form and participates in sugar catabolism in vivo, and has effect of protecting nervous system. (2) Vitamin B6: vitamin B6 is involved in the synthesis of certain neuromediators (5-hydroxytryptamine, taurine, dopamine, norepinephrine, and gamma-aminobutyric acid). (3) Vitamin B12: vitamin B12 is involved in the formation of a lipoprotein in nerve tissues, and is an essential vitamin for the functional completion of the nervous system, and thus has a function of stabilizing nerve cells. (4) Mecobalamin: the compound is easy to enter neuron organelles, participates in the synthesis of thymidine of brain cells and spinal cord neurons, promotes the utilization and the metabolism of folic acid, and promotes the synthesis of nucleic acid and protein; can promote axon transport function and axon regeneration.

Neuroprotective agents: (1) dihydropyridine calcium antagonists: comprises nimodipine, nicardipine and flunarizine, the acting receptor is L-type voltage sensitive calcium channel, the receptor is mainly located in nerve cell body, the two combine to make Ca enter into cell²⁺And nimodipine is preferentially combined with an ischemic area, so that damage to nerve cells and a blood brain barrier is obviously improved, cerebral edema is relieved, and cerebral blood flow is increased. (2) Brain protectants or free radical scavengers: such as edaravone, can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage of brain cells, vascular endothelial cells and nerve cells. (3) Calcium channel blockers, e.g. cinepazide, by blocking Ca²⁺The membrane enters into the vascular smooth muscle cells to relax the vascular smooth muscle and expand the cerebral vessels, the coronary vessels and the peripheral vessels, thereby relieving vasospasm, reducing vascular resistance and increasing blood flow. (4) Citicoline, an activator of brain metabolism, can promote the respiration of brain cells, improve brain functions, enhance the function of an ascending network structure activation system, promote awakening and reduce cerebrovascular resistance.

Natural products: (1) vinpocetine: it can block Na⁺And Ca²⁺The traditional Chinese medicine composition for treating nerve cell injury has the defects that ①, most of the traditional medicines cannot well penetrate through a blood brain barrier due to the existence of the blood brain barrier, ②, the traditional Chinese medicine composition for treating nerve cell injury has a single acting target point and cannot show a good effect when the traditional Chinese medicine composition is used for treating complex multi-target-point diseases, ③, the traditional Chinese medicine composition cannot effectively increase the treatment time window and cannot effectively increase the treatment effect④, the action range in vivo is wide, the disease area can not be locked accurately, and the potency is reduced.

Disclosure of Invention

In the process of studying the toad venom, the inventor of the present application unexpectedly found that the toad venom has a protective effect on nerve cell damage caused by hypoxia. Based on the unexpected discovery, the applicant has conducted a series of researches on toad venom from different production areas and the compatibility of different active ingredients in the toad venom, and the composition with the protective effect on nerve cell injury provided by the invention is obtained.

Accordingly, in one aspect, the present invention provides a composition having a protective effect against nerve cell damage;

in another aspect, the present invention provides a pharmaceutical formulation comprising the above composition;

in a further aspect, the present invention provides a use of the above composition or the above pharmaceutical preparation for the preparation of a medicament for treating a disease caused by nerve cell damage;

in yet another aspect, the invention provides a method of treating a disease caused by nerve cell damage. The technical scheme adopted for realizing the invention is as follows.

The invention provides a composition with a protective effect on nerve cell injury, wherein the composition comprises a bufogenin compound; or the active component of the composition is a bufogenin compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin;

preferably, the composition comprises cinobufagin and bufogenin, or the active ingredients of the composition are cinobufagin and bufogenin, wherein the weight part ratio of the cinobufagin to the bufogenin is 0-3:1, further preferably 0.9-2.5:1, further preferably 1.0-2.0: 1;

preferably, the weight part ratio of cinobufagin to bufogenin is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.4:1, 2.5:1, 3: 1;

preferably, the nerve cell damage is caused by hypoxia;

further preferably, the disease caused by nerve cell damage is selected from the group consisting of: ischemic stroke, neonatal hypoxic ischemic disease, alzheimer's disease, and parkinson's disease;

preferably, the composition is a complex.

The invention also provides a pharmaceutical preparation, which comprises the composition with the protective effect on nerve cell injury;

preferably, the composition comprises a bufogenin-like compound; or the active component of the composition is a bufogenin compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin;

preferably, the composition comprises cinobufagin and bufogenin, or the active ingredients of the composition are cinobufagin and bufogenin, wherein the weight part ratio of the cinobufagin to the bufogenin is 0-3:1, further preferably 0.9-2.5:1, further preferably 1.0-2.0: 1;

preferably, the weight part ratio of cinobufagin to bufogenin is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.4:1, 2.5:1, 3: 1;

preferably, the nerve cell damage is caused by hypoxia;

further preferably, the disease caused by nerve cell damage is selected from the group consisting of: ischemic stroke, neonatal hypoxic ischemic disease, alzheimer's disease, and parkinson's disease;

preferably, the pharmaceutical preparation may further comprise other active ingredients which may be used together or in conjunction with the composition of the present invention for protecting against nerve cell damage; further preferably, the other active ingredients are selected from edaravone, nimodipine, cinepazide, gastrodine, salvianolate, vitamin B1, vitamin B6, vitamin B12 and mecobalamin;

preferably, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier;

preferably, the composition is a complex.

The invention also provides the use of a composition of the invention or a pharmaceutical preparation of the invention in the preparation of a medicament for the prevention and/or treatment of a disease caused by nerve cell damage;

preferably, the composition comprises a bufogenin-like compound; or the active component of the composition is a bufogenin compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin;

preferably, the composition comprises cinobufagin and bufogenin, or the active ingredients of the composition are cinobufagin and bufogenin, wherein the weight part ratio of the cinobufagin to the bufogenin is 0-3:1, further preferably 0.9-2.5:1, further preferably 1.0-2.0: 1;

preferably, the weight part ratio of cinobufagin to bufogenin is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.4:1, 2.5:1, 3: 1;

preferably, the pharmaceutical formulation comprises a composition of the invention;

preferably, the pharmaceutical preparation may further comprise other active ingredients which may be used together or in conjunction with the composition of the present invention for protecting against nerve cell damage; further preferably, the other active ingredients are selected from edaravone, nimodipine, cinepazide, gastrodine, salvianolate, vitamin B1, vitamin B6, vitamin B12 and mecobalamin;

preferably, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier;

preferably, the disease in terms of nerve cell damage is caused by hypoxia; further preferably, the disease caused by nerve cell damage is selected from ischemic stroke, neonatal hypoxic-ischemic disease, alzheimer's disease and parkinson;

preferably, the composition is a complex.

The present invention also provides a method of treating a disease caused by nerve cell damage;

preferably, the method comprises the step of administering to a patient in need thereof a therapeutically effective amount of a composition of the invention or a pharmaceutical formulation of the invention;

preferably, the composition comprises a bufogenin-like compound; or the active component of the composition is a bufogenin compound;

preferably, the bufogenin-like compound is selected from cinobufagin and resibufogenin;

preferably, the composition comprises cinobufagin and bufogenin, or the active ingredients of the composition are cinobufagin and bufogenin, wherein the weight part ratio of the cinobufagin to the bufogenin is 0-3:1, further preferably 0.9-2.5:1, further preferably 1.0-2.0: 1;

preferably, the weight part ratio of cinobufagin to bufogenin is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.4:1, 2.5:1, 3: 1;

preferably, the pharmaceutical formulation comprises a composition of the invention;

preferably, the pharmaceutical preparation may further comprise other active ingredients which may be used together or in conjunction with the composition of the present invention for the protection against nerve cell damage; further preferably, the other active ingredients are selected from edaravone, nimodipine, cinepazide, gastrodine, salvianolate, vitamin B1, vitamin B6, vitamin B12 and mecobalamin;

preferably, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier;

preferably, the nerve cell damage is caused by hypoxia; further preferably, the disease caused by nerve cell damage is selected from the group consisting of: ischemic stroke, neonatal hypoxic ischemic disease, alzheimer's disease, and parkinson's disease.

The invention provides a compound type which has a protective effect on nerve cell injury, namely a compound type which mainly plays a role in protecting nerve injury in toad venom and a proportioning range thereof. Compared with the prior art, the composition can provide a new way for developing a new medicament with the nerve injury protection effect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: figure 1 shows the effect of CHS series on cell viability following hypoxia-induced PC12 cell injury; wherein the CHS-1 drug acted on hypoxic PC12 cells without significant difference (fig. 1A); the CHS-2 drug acted on hypoxic PC12 cells with no significant difference (fig. 1B); the CHS-3 drug acts on hypoxic PC-12 cells, and a significant difference exists at a concentration of 40 mug/ml (figure 1C); the CHS-4 drug acted on hypoxic PC12 cells with no significant difference in each concentration (fig. 1D); the CHS series drugs act on hypoxic PC12 cells, and at 40 mu g/ml, CHS-3 has a significant difference compared with a model group (FIG. 1E).

FIG. 2: figure 2 shows the effect of the mixture series on cell viability following hypoxia-induced PC12 cell injury; wherein the mixtures 1, 2 and 3 act on hypoxic PC12 cells, and significant differences exist at concentrations of 1. mu.g/ml, 5. mu.g/ml and 10. mu.g/ml (FIG. 2A, B, C); mixture 4 acted on hypoxic PC12 cells with no significant difference in each concentration (fig. 2D).

FIG. 3: figure 3 shows the effect of different monomers on cell viability following hypoxia-induced PC12 cell injury; wherein SHPL-1 acted on hypoxic PC12 cells, and there was a significant difference in each concentration (FIG. 3A); SHPL-2 acted on hypoxic PC12 cells, with significant differences in each concentration (FIG. 3B); SHPL-1 and SHPL-2 act on hypoxic PC12 cells, and at 1. mu.g/ml, SHPL-2 was significantly different from the model group (FIG. 3C).

FIG. 4: FIG. 4 shows a comparison of the effect of the mixture series and different monomers on cell viability after hypoxia-induced PC12 cell injury at 1. mu.g/ml; the mixture series is integrated with the results of SHPL-1 and SHPL-2 data, and the experimental results show that: at 1. mu.g/ml, there was a significant difference between mixtures 1, 2, 3 and SHPL-2 compared to the model group, and there was no significant difference between mixture 4 and SHPL-1.

FIG. 5 is a graph showing the effect of the CHS series on LDH, NO and MDA production by cells following hypoxia-induced PC12 cell injury; when CHS series traditional Chinese medicine mixture of 40 mu g/ml acts on hypoxic PC12 cells, CHS-3 can remarkably reduce the generation of LDH (figure 5A); CHS-3 was able to significantly reduce NO production (FIG. 5B); CHS series of Chinese medicinal composition can remarkably reduce MDA (figure 5C).

FIG. 6: the effects of the cinobufagin and bufogenin compositions with different proportions on the cell viability of nerve cells after hypoxia injury at the concentrations of 1 mu g/ml and 5 mu g/ml.

FIG. 7: the effect of SBP1 and SBP4 compositions at concentrations of 1 μ g/ml and 5 μ g/ml on cell viability after hypoxic injury of nerve cells in different ratios.

FIG. 8: effect of SBP1 and SBP4 monomers on cell viability following hypoxic injury of nerve cells at concentrations of 1. mu.g/ml and 5. mu.g/ml.

FIG. 9: FIG. 9A is a graph of the effect of edaravone at a concentration of 1 μ g/ml and at a concentration of 160 μ g/ml for various combinations of cinobufagin and bufogenin on LDH produced following hypoxic injury to nerve cells; FIG. 9B is a graph of the effect of edaravone at a concentration of 5 μ g/ml and a concentration of 160 μ g/ml on LDH produced following hypoxic injury to neural cells for various combinations of cinobufagin and bufogenin compositions.

FIG. 10: FIG. 10A is the effect of SBP1 and SBP4 monomers at a concentration of 1 μ g/ml and edaravone at a concentration of 160 μ g/ml on LDH produced after hypoxic injury to nerve cells, and FIG. 10B is the effect of SBP1 and SBP4 monomers at a concentration of 5 μ g/ml and edaravone at a concentration of 160 μ g/ml on LDH produced after hypoxic injury to nerve cells.

FIG. 11: FIG. 11A is a graph of the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 1 μ g/ml and edaravone at a concentration of 160 μ g/ml on LDH produced following hypoxic injury to nerve cells; FIG. 11B is a graph of the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 5 μ g/ml and edaravone at a concentration of 160 μ g/ml on LDH produced following hypoxic injury to nerve cells.

FIG. 12: FIG. 12A is a graph of the effect of edaravone at a concentration of 1 μ g/ml and at a concentration of 160 μ g/ml for various ratios of cinobufagin and bufogenin compositions on MDA produced following hypoxic injury to nerve cells; FIG. 12B is a graph of the effect of edaravone at a concentration of 5 μ g/ml and a concentration of 160 μ g/ml on MDA produced following hypoxic injury to neural cells for various ratios of cinobufagin and bufogenin compositions.

FIG. 13: FIG. 13A is the effect of SBP1 and SBP4 monomers at a concentration of 1 μ g/ml and edaravone at a concentration of 160 μ g/ml on MDA produced following hypoxic injury to nerve cells, and FIG. 13B is the effect of SBP1 and SBP4 monomers at a concentration of 5 μ g/ml and edaravone at a concentration of 160 μ g/ml on MDA produced following hypoxic injury to nerve cells.

FIG. 14: FIG. 14A is a graph showing the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 1 μ g/ml and edaravone at a concentration of 160 μ g/ml on MDA produced following hypoxic injury to nerve cells; FIG. 14B is a graph showing the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 5 μ g/ml and edaravone at a concentration of 160 μ g/ml on MDA produced following hypoxic injury to nerve cells.

FIG. 15: FIG. 15A is a graph of the effect of edaravone at a concentration of 1 μ g/ml and at a concentration of 160 μ g/ml for various ratios of cinobufagin and bufogenin compositions on NO produced following hypoxic injury to nerve cells; FIG. 15B is a graph of the effect of edaravone at a concentration of 5 μ g/ml and a concentration of 160 μ g/ml on NO produced by neuronal hypoxic injury for various combinations of cinobufagin and bufogenin.

FIG. 16A is a graph showing the effect of SBP1 and SBP4 monomers at a concentration of 1. mu.g/ml and edaravone at a concentration of 160. mu.g/ml on NO produced after hypoxic injury of nerve cells, and FIG. 16B is a graph showing the effect of SBP1 and SBP4 monomers at a concentration of 5. mu.g/ml and edaravone at a concentration of 160. mu.g/ml on NO produced after hypoxic injury of nerve cells.

FIG. 17: FIG. 17A is a graph showing the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 1 μ g/ml and edaravone at a concentration of 160 μ g/ml on NO produced following hypoxic injury to nerve cells; FIG. 17B is a graph showing the effect of different ratios of SBP1 and SBP4 compositions at a concentration of 5 μ g/ml and edaravone at a concentration of 160 μ g/ml on NO produced after hypoxic injury of nerve cells.

FIG. 18: FIG. 18A is a graph of the effect of various combinations of cinobufagin and bufogenin on the viability of neurons following hypoxic injury at a concentration of 1 μ g/ml; FIG. 18B is a graph of the effect of various combinations of cinobufagin and bufogenin on the viability of neurons following hypoxic injury at a concentration of 5 μ g/ml.

FIG. 19: FIG. 19A is a graph of the effect of various combinations of cinobufagin and bufogenin at a concentration of 1 μ g/ml on LDH production following hypoxic injury to nerve cells, and FIG. 19B is a graph of the effect of various combinations of cinobufagin and bufogenin at a concentration of 5 μ g/ml on LDH production following hypoxic injury to nerve cells.

FIG. 20: FIG. 20A is the effect of different ratios of cinobufagin and bufogenin compositions at a concentration of 1 μ g/ml on MDA produced after hypoxic injury of nerve cells, and FIG. 20B is the effect of different ratios of cinobufagin and bufogenin compositions at a concentration of 5 μ g/ml on MDA produced after hypoxic injury of nerve cells.

FIG. 21: FIG. 21A is the effect of different ratios of cinobufagin and bufogenin compositions at a concentration of 1 μ g/ml on NO produced after hypoxic injury of nerve cells, and FIG. 21B is the effect of different ratios of cinobufagin and bufogenin compositions at a concentration of 5 μ g/ml on NO produced after hypoxic injury of nerve cells.

FIG. 22: FIG. 22A shows the effect of different formulations of the composition at a concentration of 1 μ g/ml on the cell viability of the neurons after hypoxic injury, and FIG. 22B shows the effect of different formulations of the composition at a concentration of 5 μ g/ml on the cell viability of the neurons after hypoxic injury.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

1. Materials and methods

Cell lines: the PC12 cell line was purchased from Hill-Tokyo Biotech, Inc.

Sodium dithionite (Na)₂S₂O₄): national drug group, product number: 20180316, respectively;

DMEM medium: sammer Feishale Biochemical (Beijing) Ltd., cat #: 81119379, respectively;

sugar-free RPMI 1640: macgene, cat # code: 12805020.

1.1 Main laboratory instruments and Equipment

1.2 preparation of the Main solution and samples

Na₂S₂O₄Solution: the concentration is 10mM when the preparation is used.

Administration group: PC12 cells are subjected to anoxic and sugar-deficient treatment and then subjected to administration treatment with corresponding concentrations, wherein:

the preparation method of the administration groups with different concentrations is as follows: dissolving a certain amount of Chinese medicinal materials/Chinese medicinal monomer mixture/Chinese medicinal monomer/composition in a certain amount of DMSO, and diluting with RPMI culture medium containing 10% fetal calf serum to desired concentration. For example, CHS-1 with a concentration of 10. mu.g/ml is prepared by the following method: dissolving 10 μ g CHS-1 in 1 μ L LDMSO, and adding 999 μ L RPMI culture medium of 10% fetal calf serum. For example, mixture 1 at a concentration of 1. mu.g/ml can be prepared by: dissolving 1 μ g of mixture 1 in 1 μ L of LDMSO, and adding 999 μ L of RPMI culture medium containing 10% fetal calf serum.

The set dosing groups and concentrations of each dosing group were as follows:

(1) preparing Chinese medicinal material administration groups (CHS1, CHS2, CHS3 and CHS4) (mu g/ml) with different concentrations: 10. 20, 40, 80, 160, 320, 640.

(2) Preparing a traditional Chinese medicine monomer mixture administration group (mixture 1-4) with different concentrations (mu g/ml): 1.5, 10 and 20.

(3) Preparing traditional Chinese medicine monomer group administration groups (SHPL-1 and SHPL-2 monomers) with different concentrations (mu g/ml): 1.5, 10 and 20.

(4) Mixtures (μ g/ml) of SBP1 (gamabufotalin) and SBP4 (bufalin) at different concentrations;

(5) SBP1 (gamabufotalin) and SBP4 (bufalin) monomer group administration groups (mu g/ml) at different concentrations;

(6) different concentrations of Edaravone (ED) group dosing groups (μ g/ml);

(7) administration groups (μ g/ml) of different concentrations at different ratios.

Control group: PC12 cells were not subjected to hypoxic sugar deprivation treatment, nor to drug administration treatment.

Model group: PC12 cells were subjected to hypoxic-hypoglycaemic treatment, but not to drug administration.

Blank group: refers to the group without added cells.

1.3 medicinal materials and sample setting:

the toad medicinal material is provided by Shanghai and Huangyao pharmaceutical Co Ltd, and the specific information numbers are as follows (wherein, the content is weight percentage content; the content ratio is weight ratio):

wherein SHPL-1 represents cinobufagin monomer, and SHPL-2 represents resibufogenin monomer.

In addition, the following four SHPL-1 and SHPL-2 mixtures with different monomer ratios are also provided.

Wherein SHPL-1 represents cinobufagin monomer, and SHPL-2 represents resibufogenin monomer.