CN113057951B

CN113057951B - Application of biphenyl lignan compound in preparing medicine for preventing and treating amyotrophic lateral sclerosis

Info

Publication number: CN113057951B
Application number: CN202110360962.8A
Authority: CN
Inventors: 孙飏
Original assignee: Huoren Kechuang Beijing Pharmaceutical R & D Co ltd
Current assignee: Huoren Kechuang Beijing Pharmaceutical R & D Co ltd
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-06-17
Anticipated expiration: 2041-04-02
Also published as: CN113057951A

Abstract

The invention provides an application of biphenyl lignan compounds in preparing a medicament for preventing and treating amyotrophic lateral sclerosis, belonging to the technical field of medicaments. The results of the examples show that the biphenyl lignan compounds can increase the survival rate of motor neurons transfected by hSOD1-G93A dose-dependently, and the compounds have obvious protective effect on hSOD1-G93A stable ALS cell models; in addition, the biphenyl lignanoid compounds can also obviously improve the survival time of hSODl-G93A transgenic mice, reduce the expression of a spinal microglia marker Iba1 protein of the hSODl-G93A transgenic mice, and obviously increase the expression of the hSODl-G93A transgenic mice Nrf2, which suggests that the compounds can reduce neuroinflammation reaction and inhibit oxidative stress by inhibiting the activation of microglia and prolong the survival time of the hSODl-G93A transgenic mice. The biphenyl lignan compound has good prevention and treatment effects on amyotrophic lateral sclerosis.

Description

Application of biphenyl lignan compound in preparing medicine for preventing and treating amyotrophic lateral sclerosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of a biphenyl lignan compound in preparing a medicine for preventing and treating amyotrophic lateral sclerosis.

Background

Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig disease, is a severe fatal Motor Neuron Disease (MND) that selectively affects upper and lower motor neurons, and patients have a short life cycle, and mostly die from respiratory failure 3-5 years after onset. The etiology of ALS has not been elucidated, and in recent years it has been found that neurodegeneration of ALS is not limited to the motor system, but also involves sensory, speech, behavioral and other cognitive domains. In fact, a proportion of ALS patients have cognitive deficits ranging from mild cognitive impairment to overt frontotemporal dementia (FTD). Edaravone is clinically used for treating acute stroke, and is approved as an orphan drug for ALS treatment by FDA in us in 2017, which costs about $ 145524 per year and is expensive. At the same time, edaravone may also have serious adverse reactions requiring urgent treatment, such as urticaria, edema or shortness of breath, and anaphylaxis of sodium bisulfite in the drug. Sodium bisulfite may cause allergic symptoms and may be life threatening for people who are sensitive to sulfite. Furthermore, edaravone has certain functions of improving muscle strength and delaying disease progression only in early stage of ALS diseases and when symptoms are mild, so that a medicine for effectively treating ALS is searched, and clinical requirements are met.

Disclosure of Invention

In order to solve the problems, the invention provides application of biphenyl lignan compounds in preparing medicines for preventing and treating amyotrophic lateral sclerosis. The biphenyl lignan compound has obvious ALS treatment effect, and the treatment effect is superior to that of edaravone.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of biphenyl lignan compounds in preparing a medicament for preventing and treating amyotrophic lateral sclerosis.

The invention provides application of biphenyl lignan compounds in preparing a medicament for improving survival rate of motor neurons.

The invention provides an application of biphenyl lignan compounds in preparing medicines for inhibiting microglial cell activation.

The invention provides application of biphenyl lignan compounds in preparing a medicament for reducing the expression of ionic calcium binding adaptor molecule 1 protein.

The invention provides application of biphenyl lignan compounds in preparing medicines for relieving neuroinflammatory reaction.

The invention provides application of biphenyl lignan compounds in preparation of medicines for increasing expression of Nrf2 protein.

Preferably, the biphenyl lignan compound has one or more of the following structural formulas I to XIV:

preferably, the administration form of the medicament comprises injection, solid dispersion preparation, soft capsule, dripping pill, sustained release preparation or controlled release preparation.

The invention provides a composition for preventing and treating amyotrophic lateral sclerosis, the effective component of the composition is a biphenyl lignan compound in the technical scheme, and the mass percentage of the biphenyl lignan compound in the composition is 0.1-10%.

Preferably, the composition further comprises an oil phase, a surfactant, a solubilizer, an antioxidant and a complexing agent; in the composition, the mass percentage of the oil phase is 0.0-30%, the mass percentage of the surfactant is 0.0-20%, the mass percentage of the solubilizer is 1.0-40%, the mass percentage of the antioxidant is 0.1-1%, and the mass percentage of the complexing agent is 0.0-0.03%.

Preferably, the oil phase comprises one or more of long chain fatty acid glyceride, medium chain fatty acid triglyceride, oleic acid, ethyl oleate, soybean oil for injection, tricaprylin, tributyrin, corn oil for injection, and peanut oil for injection.

Preferably, the surfactant comprises one or more of egg yolk lecithin, soybean lecithin, polyethylene glycol hydroxystearate, poloxamer 188, tween 80, tween 65 and polyoxyethylene castor oil.

Preferably, the solubilizer comprises one or more of ethanol, glycerol, propylene glycol, beta-cyclodextrin, 2, 6-dimethoxy-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, sulfobutyl-gamma-cyclodextrin, methyl-beta-cyclodextrin, sulfobutyl ether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, gamma-cyclodextrin, alpha-cyclodextrin, 6- (2-glucosylamino) -beta-cyclodextrin, oligolactic acid-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxybutyl-beta-cyclodextrin, polyethylene glycol, polyglycerol ester, amino acid salt, lactose and dextran.

Preferably, the antioxidant comprises one or more of vitamin C, vitamin E, sodium sulfite and sodium metabisulfite.

Has the advantages that:

the invention provides an application of biphenyl lignan compounds in preparing a medicament for preventing and treating amyotrophic lateral sclerosis. The results of the examples show that the biphenyl lignan compound can improve the survival rate of motor neurons transfected by hSOD1-G93A in a dose-dependent manner, has better effect than edaravone, and has obvious nerve channel protection effect; meanwhile, the biphenyl lignanoid compounds can also obviously reduce the expression of spinal microglia marker Iba1 protein of the SOD1-G93A transgenic mouse, reduce neurite response and prolong the survival period of the mouse by inhibiting the activation of microglia; in addition, the biphenyl lignanoid compound can also improve the expression of Nrf2 protein, play a role in neuroprotection by inhibiting oxidative stress and prolong the survival time of mice. The biphenyl lignan compound has good effect of preventing and treating amyotrophic lateral sclerosis, and can be used for preparing medicines for preventing and treating amyotrophic lateral sclerosis.

Drawings

FIG. 1 shows the survival rate of 24h honokiol injection water injection and microemulsion injection zebra fish embryo;

FIG. 2 shows the survival rate of zebrafish embryo in 48h injection of honokiol and microemulsion;

FIG. 3 is a graph showing the comparison of the survival rates of 72h honokiol injection water injection and microemulsion injection zebra fish embryos;

FIG. 4 shows the survival rate of zebrafish embryo compared between 96h injection water injection of honokiol and microemulsion injection;

FIG. 5 is a graph showing the effect of honokiol, Magnolia triphenol B, and Magnolia aldehyde B at different concentrations on the increase in cell survival rate;

FIG. 6 is a graph showing the effect of different concentrations of edaravone on the rate of increase in cell survival;

FIG. 7 shows the effect of honokiol on the SOD activity of zebra fish embryos;

FIG. 8 shows the effect of Magnolia obovata Thunb trisphenol B on the SOD activity of zebra fish embryo;

FIG. 9 shows the effect of Magnoliac aldehyde B on SOD activity of zebra fish embryo;

FIG. 10 is a graph of the effect of honokiol on zebrafish embryo MDA viability;

FIG. 11 shows the effect of Magnolia bark trisphenol B on the activity of zebra fish embryo MDA;

FIG. 12 is a graph of the effect of Magnolia aldehyde B on the viability of zebrafish embryo MDA;

FIG. 13 is a graph of the effect of honokiol on CAT content in zebrafish embryos;

FIG. 14 shows the effect of Magnolia obovata Thunb trisphenol B on the content of CAT in zebra fish embryos;

FIG. 15 shows the effect of Magnoliac aldehyde B on the CAT content of zebra fish embryos;

FIG. 16 is a graph of the effect of honokiol on the ROS content of zebrafish embryos;

FIG. 17 is a graph of the effect of Magnolia bark triol B on the ROS content of zebra fish embryos;

FIG. 18 is a graph of the effect of Magnolia aldehyde B on the ROS content of zebrafish embryos;

FIG. 19 is a graph of the effect of honokiol on survival of hSODl-G93A transgenic mice;

FIG. 20 is a graph of the effect of Magnolia obovata phenol B on survival of hSODl-G93A transgenic mice;

FIG. 21 is a graph of the effect of Magnolia aldehyde B on survival of hSODl-G93A transgenic mice;

FIG. 22 is a graph showing the effect of honokiol on the expression of spinal microglia marker IBa1 protein in hSODl-G93A transgenic mice;

FIG. 23 shows the effect of Magnolia obovata phenol B on the expression of spinal microglia marker Iba1 protein in hSODl-G93A transgenic mice;

FIG. 24 shows the effect of Magnoliac aldehyde B on the expression of the spinal microglia marker Iba1 in hSODl-G93A transgenic mice.

FIG. 25 is a graph of the effect of honokiol on expression of Nrf2 protein in spinal cord of hSODl-G93A transgenic mice;

FIG. 26 is a graph showing the effect of Magnolia obovata phenol B on the expression of Nrf2 protein in the spinal cord of hSODl-G93A transgenic mice;

FIG. 27 is a graph of the effect of Magnolia aldehyde B on the expression of Nrf2 protein in the spinal cord of hSODl-G93A transgenic mice.

Detailed Description

The invention provides an application of biphenyl lignan compounds in preparing a medicament for preventing and treating amyotrophic lateral sclerosis. In the present invention, the biphenyl lignan compound preferably comprises one or more compounds represented by the following structural formulas I to XIV: the structural formulas of the compounds of the formulas I to XIV are shown in the summary of the invention. In the present invention, the compounds of formulae I to XIV are named as Honokiol (Honokiol), Magnolol (Magnolol), Magnolia officinalis lignin A (magnolignan A), Magnolia officinalis lignin B (magnolignan B), Magnolia officinalis lignin C (magnolignan C), Magnolia officinalis trisol B (magnoliol B), Magnolia aldehyde D (magnaldehydo D), Magnolia aldehyde B (magnaldehydo B), Magnolia aldehyde C (magnaldehydo C), Magnolia aldehyde E (magnaldehydo E), 7-O-ethyl and Magnolia officinalis triol (7-O-ethylhonokiol), 8', 9' -dihydroxy and Magnolol (8', 9' -dihydroyhonokiol), erythro-7-O-methyl and Magnolia officinalis triol (erythro-7-O-methyl-7-O-triol) in this order. In the invention, the biphenyl lignan compounds are further preferably honokiol, magnolol D and magnolia lignin C; further preferably honokiol. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound has obvious nerve channel protection effect and antioxidation effect, can obviously reduce the expression of a spinal microglia marker Iba1 protein, relieves neurite reaction by inhibiting the activation of microglia, and prolongs the survival period of mice; the biphenyl lignan compound can also improve the expression of Nrf2 protein, play a role in neuroprotection by inhibiting oxidative stress, prolong the survival time of mice, have good prevention and treatment effects on amyotrophic lateral sclerosis, and can be used for preparing medicines for preventing and treating amyotrophic lateral sclerosis.

In the present invention, the administration form of the medicament preferably includes injection, solid dispersion preparation, soft capsule, drop pill, sustained release preparation or controlled release preparation; further preferably comprises injection, soft capsule, and solid dispersion preparation; still more preferred is an injection. In the present invention, the injection further preferably comprises water injection for injection, microemulsion injection, self-emulsifying microemulsion injection; still more preferably, the injection solution is an injection solution. The microemulsion injection, the self-emulsifying microemulsion injection and the water injection for injection are prepared by adopting a microemulsion technology, so that the problem of indissolvability of biphenyl lignans can be solved, and the stability and the bioavailability are improved; the beta-cyclodextrin auxiliary material is adopted for the injection coated by the biphenyl lignans, so that the solubility and the dissolution rate of the medicine can be increased, the stability of the medicine is improved, the irritation of the medicine is reduced, and the like, the medicine is directly acted on a pathological change part by adopting an intravenous injection or intramuscular injection method, the injection has the characteristics of rapidness and high efficiency, and meanwhile, the dosage of the medicine can be greatly reduced, and the side effect of the medicine is reduced; wherein, the injection has better safety and reduces the toxic effect of the preparation on organisms.

The invention provides application of biphenyl lignan compounds in preparing a medicament for improving survival rate of motor neurons. In the present invention, the biphenyl type lignan compound preferably comprises the biphenyl type lignan compound according to the above aspect. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound has the effect of remarkably improving the survival rate of motor neurons.

The invention provides an application of biphenyl lignan compounds in preparing medicines for inhibiting microglial cell activation. In the present invention, the biphenyl type lignan compound preferably comprises the biphenyl type lignan compound according to the above aspect. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound can reduce the expression of the ionic calcium binding adaptor 1 protein and has the function of inhibiting the activation of microglia.

The invention provides application of biphenyl lignan compounds in preparing a medicament for reducing the expression of ionic calcium binding adaptor molecule 1 protein. In the present invention, the biphenyl type lignan compound preferably comprises the biphenyl type lignan compound according to the above aspect. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound has the function of reducing the expression of the ionic calcium binding adaptor molecule 1 protein.

The invention provides application of biphenyl lignan compounds in preparing medicines for relieving neuroinflammatory reaction. In the present invention, the biphenyl type lignan compound preferably comprises the biphenyl type lignan compound according to the above aspect. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound can reduce Iba1 protein expression, and has effect of relieving neuroinflammation reaction.

The invention provides application of biphenyl lignan compounds in preparation of medicines for increasing expression of Nrf2 protein. In the present invention, the biphenyl type lignan compound preferably includes the biphenyl type lignan compound according to the above aspect. The biphenyl lignan compound has no special requirement on the source, and can be prepared by a conventional preparation method of a person skilled in the art or a common commercial product. The biphenyl lignan compound has the effect of increasing the expression of Nrf2 protein.

In the present invention, the effective ingredient of the composition is the biphenyl lignan compound described in the above technical means. The biphenyl lignan compound has good effect of preventing and treating amyotrophic lateral sclerosis, and is a main effective component for treating amyotrophic lateral sclerosis in the composition. In the composition of the present invention, the content of the biphenyl lignan compound is 0.1 to 10% by mass, and more preferably 0.1 to 5% by mass.

In the composition of the present invention, the oil phase is preferably 0.0% to 30% by mass, and more preferably 5% to 25% by mass. In the present invention, the oil phase preferably comprises one or more of long chain fatty acid glyceride, medium chain fatty acid triglyceride, oleic acid, ethyl oleate, soybean oil for injection, tricaprylin, tributyrin, corn oil for injection, peanut oil for injection; further preferably one or more of medium-chain fatty acid triglyceride, soybean oil for injection, and tricaprylin; more preferably medium-chain fatty acid triglycerides. The oil phase is mainly used for solubilizing fat-soluble biphenyl lignan compounds, can promote the dispersibility of the biphenyl lignan compounds, is beneficial to absorption, reduces the decomposition of the biphenyl lignan compounds and reduces the stimulation to organisms.

In the composition of the present invention, the mass percentage of the surfactant is preferably 0.0% to 20%, and more preferably 5.0% to 15%. In the present invention, the surfactant comprises one or more of egg yolk lecithin, soybean lecithin, polyethylene glycol hydroxystearate, poloxamer 188, tween 80, tween 65 and polyoxyethylene castor oil; further preferably one or more of soybean lecithin, egg yolk lecithin, polyethylene glycol hydroxystearate; more preferably soybean lecithin. The surfactant disclosed by the invention has the main function of reducing interfacial tension to form an interfacial film and promoting the formation of microemulsion.

In the composition of the present invention, the content of the solubilizer is preferably 1.0 to 40% by mass, and more preferably 5 to 30% by mass. In the present invention, the solubilizer comprises one or more of ethanol, glycerol, propylene glycol, beta-cyclodextrin, 2, 6-dimethoxy-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, sulfobutyl-gamma-cyclodextrin, methyl-beta-cyclodextrin, sulfobutyl ether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, gamma-cyclodextrin, alpha-cyclodextrin, 6- (2-glucosylamino) -beta-cyclodextrin, oligolactic acid-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxybutyl-beta-cyclodextrin, polyethylene glycol, polyglycerol ester, amino acid salt, lactose and dextran; further preferably one or more of propylene glycol, hydroxypropyl-beta-cyclodextrin and dextran; even more preferably hydroxypropyl-beta-cyclodextrin. The solubilizer disclosed by the invention has the function of promoting the solubility of the biphenyl lignan compound in liquid.

In the composition of the present invention, the antioxidant is preferably contained in an amount of 0.1% to 1% by mass, and more preferably 0.2% to 0.5% by mass. In the present invention, the antioxidant comprises one or more of vitamin C, vitamin E, sodium sulfite and sodium metabisulfite; further preferably one or more of vitamin C, vitamin E and sodium sulfite; more preferably vitamin C. The antioxidant disclosed by the invention has the functions of preventing the oxidative hydrolysis of the biphenyl lignan compounds and improving the stability of the biphenyl lignan compounds.

In the composition of the present invention, the mass percentage of the complexing agent is preferably 0.0% to 0.10%, and more preferably 0.0% to 0.03%. In the invention, the complexing agent comprises one or more of EDTA and EDTA-2 Na; EDTA-2Na is more preferable. The complexing agent disclosed by the invention has the functions of complexing ions in a solution, reducing oxidative decomposition of the biphenyl lignan compound and improving stability.

The composition of the present invention preferably further comprises water for injection. The invention has no special requirements on the source of the water for injection, and high-purity water and ultrapure water which meet the requirements of the relevant water for injection are prepared by adopting a conventional method in the field. The water for injection is mainly used as a solvent.

In order to further illustrate the present invention, the following examples are provided to describe the usage of the biphenyl lignan compound of the present invention in the preparation of drugs for preventing and treating amyotrophic lateral sclerosis, but they should not be construed as limiting the scope of the present invention.

Example 1

An injection for preventing and treating amyotrophic lateral sclerosis comprises the following components in percentage by mass: 0.1% of honokiol, 1% of hydroxypropyl-beta-cyclodextrin, 0.05% of vitamin C and the balance of water for injection.

The preparation method comprises the following steps: 10g of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is weighed, added into 200mL of distilled water, and stirred until the mixture is completely dissolved, so as to prepare a 5% HP-beta-CD aqueous solution. Slowly adding 1.0g of honokiol into 5% HP-beta-CD aqueous solution at room temperature (25 ℃), stirring for 4h by using an electric constant-temperature magnetic stirrer until the solution is balanced, adding 0.5g of vitamin C, continuously stirring until the vitamin C is dissolved, adding sodium bicarbonate to adjust the pH value to 6.0, filtering by using a 0.45-micrometer filter membrane, adding injection water to 1000mL to obtain the magnolol injection water injection containing 1mg/mL of active ingredients, encapsulating in an ampoule, filling nitrogen, sealing by melting, and sterilizing at 121 ℃.

Examples 2 to 4

The components of the injection for preventing and treating amyotrophic lateral sclerosis in examples 2 to 4 are shown in table 1.

TABLE 1 composition of injectable solutions of examples 2 to 4

Composition (I)	Example 2	Example 3	Example 4	Function of
					Honokiol	5.0g	-	-	Main medicine
Magnolol	5.0g	-	-	Main medicine
					Magnolia officinalis aldehyde B	-	2.0g	-	Main medicine
Magnolia bark trisphenol B	-	-	5.0g	Main medicine
					Sulfobutyl-beta-cyclodextrin	100.0g	-	50.0g	Cosolvent
Hydroxypropyl-beta-cyclodextrin	-	20.0g	-	Cosolvent
					Vitamin C	1.0g	0.4g	0.5g	Antioxidant agent
Sodium bicarbonate	0.5g	0.5g	0.5g	pH regulator
					Water for injection is added to	1000mL	1000mL	1000mL

The preparation method is the same as example 1.

Example 5

A self-emulsifying microemulsion injection for preventing and treating amyotrophic lateral sclerosis comprises the following components by mass: 13.0g of honokiol, 300.0g of medium-chain triglyceride (MCT), 400.0g of polyethylene glycol-12-hydroxystearate (Solutol HS15), 200.0g of lecithin (Lipod E80) and 400.0g of absolute ethyl alcohol.

The preparation method comprises the following steps: weighing 200g of lecithin, adding 200mL of absolute ethanol, stirring for dissolving, adding 400g of polyethylene glycol stearate 15(Solutol HS15) and 400g of medium-chain triglyceride (MCT), mixing, adding absolute ethanol until the total weight of the mixed liquid is 1300g, stirring uniformly, and completely dissolving; weighing 13.0g of honokiol, adding into the solution, and stirring to completely dissolve; filtering with microporous organic filter membrane with pore diameter of 0.22 μm, packaging in ampoule, charging nitrogen, and sealing by fusing.

Examples 6 to 8

The components of the self-emulsifying microemulsion injection for preventing and treating amyotrophic lateral sclerosis in examples 6-8 are shown in Table 2.

TABLE 2 compositions of self-emulsifying microemulsion injections of examples 6-8

Composition (I)	Example 6	Example 7	Example 8	Function of
					Honokiol	5.0g	-	-	Main drug
Magnolol	5.0g	-	-	Main medicine
					Magnolia officinalis aldehyde B	-	15.0g	-	Main medicine
Magnolia bark trisphenol B	-	-	15.0g	Main medicine
					LipodE80	200.0g	200.0g	200.0g	Surface active agent
MCT	300.0	300.0g	300.0g	Cosolvent
					SolutolHS15	400.0g	400.0g	400.0g	Surface active agent
Anhydrous ethanol	90.0g	85.0g	85.0g	Cosolvent
					Total weight of	1000.0g	1000.0g	1000.0g

The preparation method is the same as example 5.

Example 9

A microemulsion injection for preventing and treating amyotrophic lateral sclerosis comprises the following components in percentage by mass: 0.4% of honokiol, 1.2% of soybean phospholipid, 0.3% of poloxamer 188(F-68), 2.2% of glycerol, 10.0% of soybean oil and 85.9% of water for injection.

The preparation method comprises the following steps: 4.0g of honokiol is precisely weighed and dissolved in 100.0g of soybean oil at 80 ℃ to be used as an oil phase. Accurately weighing 12.0g of soybean phospholipid, 3.0g of poloxamer 188(F-68), 22.0g of glycerol, adding 859g of 80 ℃ water for injection, and dispersing at 1000rpm under a heat preservation state to obtain a water phase; slowly adding the oil phase into the water phase with 80 deg.C water bath insulation under 1000rpm stirring, continuously stirring to obtain uniform primary emulsion, adjusting volume to 1000mL with water for injection, transferring the primary emulsion into a high pressure homogenizing machine, filtering the emulsion after high pressure homogenizing with 2 μm microporous membrane, subpackaging the emulsion in clean ampoules under nitrogen gas introduction, sealing, placing in a rotary hot-pressing sterilizer, slowly heating to 121 deg.C, and sterilizing for 15 min.

Example 10

A microemulsion injection for preventing and treating amyotrophic lateral sclerosis comprises the following components in percentage by mass: 0.1% of magnolol B, 1.5% of lecithin, 0.08% of vitamin E, 7.5% of soybean oil, 7.5% of medium-chain fatty glyceride, 2.25% of glycerol, 1880.5% of poloxamer and the balance of water.

The preparation method comprises the following steps: mixing 15g of lecithin, 0.8g of vitamin E, 78g of soybean oil and 75g of medium-chain fatty glyceride, heating and stirring in a water bath at 75 ℃ until the lecithin is completely dissolved, adding 10g of magnolol B, and continuously stirring until the medicine is completely dissolved to obtain an oil phase; dispersing 22.5g of glycerol, 5g of poloxamer 188 and 1g of sodium oleate in water for injection, heating to 70 ℃, and stirring until all the components are dissolved to obtain a water phase. Adding the water phase into the oil phase under high speed stirring at 12000r/min, and stirring for 10min to obtain colostrum. Diluting with water for injection to 1000mL, transferring to high pressure homogenizer, homogenizing at 25 deg.C under 900bar pressure for 10 times, filtering, bottling, charging nitrogen, sealing, and sterilizing with 121 deg.C steam for 15min to obtain 0.1% injection.

Example 11

A dripping pill for preventing and treating amyotrophic lateral sclerosis comprises the following components in percentage by mass: 25.0 percent of honokiol and PEG-600075 percent.

The preparation method comprises the following steps: heating polyethylene glycol to melt, adding precisely weighed magnolol, stirring to dissolve into transparent solution, and making into dripping pill.

Example 12

A dripping pill for preventing and treating amyotrophic lateral sclerosis comprises the following components in percentage by mass: magnolol 25.0% and PEG-2000075%.

The preparation method is the same as example 11.

Example 13

A sustained release tablet for preventing and treating amyotrophic lateral sclerosis comprises the following components by mass percent: 10.0% of magnolol, PEG-600030%, 10% of Ethyl Cellulose (EC), 25% of hydroxypropyl methyl cellulose (HPC), 20% of starch, 4% of talcum powder and 1% of magnesium stearate.

The preparation method comprises the following steps: taking mixed solvent (95% ethanol: CH)₂Cl₂1:1) of the raw materials, adding 10g of magnolol, stirring to dissolve the magnolol, adding PEG600030g, stirring to dissolve the magnolol, recovering the solvent under reduced pressure, adding 10g of EC, 25g of HPC and 20g of starch, uniformly mixing, preparing into granules, adding 4.0g of talcum powder and 1g of magnesium stearate, uniformly mixing, and tabletting to obtain the tablet.

Example 14

Observation of toxic effect of different administration doses of honokiol injection and honokiol microemulsion injection on zebra fish embryo development

1. The test method comprises the following steps:

1.1 preparation of zebra fish embryo and preparation of test solution

The adult fish were treated in a manner of male and female 1:1, placing the male and female in a mating box, and inserting a partition plate in the middle to separate the male and female. The next day, the clapboards are taken out, and the zebra fish begin to mate and lay eggs. After about 1h, the roe is collected and washed with fish culture water for later use. Under the microscope, normal-developing embryos were picked and transferred to a petri dish, approximately 3hpf (hours post fertilization) zebrafish embryos were in the blastocyst stage, and the blank control group was supplemented with E3 medium, E3 medium containing 5mM NaCl, 0.17mM KCl, 0.33mM CaCl₂，0.33mM MgSO₄. The experiment sets 10 concentrations of injection water injection of honokiol and microemulsion injection of honokiol, which are 0 mug/mL, 0.075 mug/mL, 0.106 mug/mL, 0.150 mug/mL, 0.212 mug/mL, 0.300 mug/mL, 0.423 mug/mL, 0.600 mug/mL, 0.849 mug/mL and 1.2 mug/mL respectively. Before each test with E3 preparing a solution with the required concentration by the culture solution.

1.2 Observation of toxic action of different dosages of Honokiol injectable water injection and Honokiol microemulsion injection on zebrafish embryo development

To clarify the appropriate dosing concentration and half lethal dose, a range of concentrations with mortality rates from 0 to 100% was chosen. The test is provided with 10 concentration groups which are arranged in an equal ratio series increasing by a common ratio of

Namely 0. mu.g/mL, 0.075. mu.g/mL, 0.106. mu.g/mL, 0.150. mu.g/mL, 0.212. mu.g/mL, 0.300. mu.g/mL, 0.423. mu.g/mL, 0.600. mu.g/mL, 0.849. mu.g/mL, 1.2. mu.g/mL, respectively. Morphological observation was performed on 30 embryos per dose group, zebrafish embryos of the control group and the test group were placed in the same 24-well plate, and the cumulative survival rates of 24hpf, 48hpf, 72hpf and 96hpf at which the embryos developed, and the cumulative hatchability at 72hpf and 96hpf (embryo hatchability is the ratio of the number of hatched embryos to the number of surviving embryos) were recorded. Calculating the survival rate recorded by 24hpf, 48hpf, 72hpf and 96hpf by using the SPSS software to perform regression probability analysis to obtain LD₅₀. 96hpf zebrafish embryos were observed under a stereomicroscope and embryo development and malformation were recorded, respectively. To avoid contingency and reduce errors, each experiment was repeated 3 times.

1.3 results

FIGS. 1 to 4 show the results of comparison of the survival rates of 24h, 48h, 72h and 96h of honokiol injection water injection and microemulsion injection zebra fish embryos, respectively, and Table 1 shows the survival rates of the honokiol microemulsion injection zebra fish embryos LC₅₀(half lethal concentration) results.

Analysis of magnolol microemulsion injection results: different doses of honokiol microemulsion injection infected by zebra fish embryos find that the survival rate of the highest dose group of 1.2 mug/mL at 48hpf is reduced to below 10 percent, while the survival rates of 72hpf and 96hpf are 0. The 0.4243 ug/mL and above doses tended to decrease the survival rate of zebrafish embryos, and the 0.6 ug/mL and 0.8485 ug/mL dose groups were statistically significantly different from the control group. Calculation of 24hpf, 48hpf, 72hpf and 96hpf4 time points and Using SPSSHalf lethal dose (LC) of magnolol₅₀) The results are shown in Table 3. LC of honokiol with prolonged exposure time₅₀The continuous decrease indicates that the exposure duration of the honokiol microemulsion injection has certain influence on the toxic effect, and the toxic effect is accumulated.

And (3) analyzing the results of the injection of honokiol: when zebra fish embryos are infected with different doses of honokiol injection water injections, the survival rate of the highest dose group of 1.2 mu g/mL is about 60 percent at 48hpf, and the survival rates of 72hpf and 96hpf are 40 percent. The dose of 0.849. mu.g/mL or more had a tendency to decrease the survival rate of zebrafish embryos, but no significant statistical difference was seen compared with the control group. The half lethal dose (LC) of honokiol at 24hpf, 48hpf, 72hpf and 96hpf4 time points was calculated using SPSS₅₀) The results are also shown in Table 3. LC of honokiol injection water injection along with prolonging of contamination time₅₀The exposure duration of the injection water injection does not obviously influence the toxic effect of the injection water injection, and the accumulation of the toxic effect does not occur. Comparing the injection solution of honokiol with the microemulsion injection of honokiol, the injection solution of honokiol has lower toxicity on the zebra fish embryo than the microemulsion injection of honokiol, and the safety is an important attribute for the medicine.

TABLE 3 Honokiol microemulsion injection zebrafish embryo LC₅₀(half lethal concentration) results

Example 15

Study on protective effect of honokiol, magnolol B and magnolol B on amyotrophic lateral sclerosis cell model

1. Test method

(1) Construction of NSC-34 cell line stably transfected with hSOD1-G93A (ALS cell model)

The mouse motor neuron NSC-34 cell line is purchased from Shanghai Hongshun Biotech limited. The control pEGFP and the hSOD 1G 93A-pcDNA3.1(+) -EGFP plasmid were purchased from Changsha Youbao Biotech, Inc. NSC-34 cells were cultured in DMEM medium (containing penicillin and streptomycin) containing 10% fetal bovine serum, and incubated at 37 deg.C with 5% CO₂Cultured in an incubator. And carrying out passage once every 2-3 days. Extracting plasmids from the bacterial liquid according to the operation steps of the endotoxin-free plasmid miniprep medium-volume kit, then sending the plasmids to a company for sequencing, and carrying out plasmid transfection after the sequencing is correct. NSC-34 cells were trypsinized with trypsin containing 0.25% EDTA and inoculated into 6-well plates 2mL of medium per well the day before transfection, and plasmid transfection was performed the following day when the cells reached 70% -90% confluency. The assay was performed according to the instructions of Lipofectamine 3000. After transfection for 48h, the cells in each well were digested, diluted to 2-3 cells per microscope field in a large dish, and diluted to 4 large dishes per well for screening of monoclonal cells. After the cells adhere to the wall, geneticin G418 is added for screening, the concentration is 800 mug/muL, and the culture medium containing the antibiotics is replaced once in 3-4 days. After 10 days of culture, 48 single clones of each transfected cell are picked out and cultured in a 24-well plate, the G418 concentration is changed to a maintenance concentration of 400 mug/muL, and after the cells grow in the 24-well plate for a period of time, the cells in 12 wells are selected respectively, digested and inoculated in a 6-well plate for expanded culture so as to be used for subsequent experiments.

(2) The constructed hSOD1-G93A stably transfected cells are 5 multiplied by 10⁴Is planted in a 96-well plate and is placed at 37 ℃ and 5% CO₂The incubator of (4) was cultured under the above conditions.

(3) After 24h, adding honokiol, magnolol B and magnolol B with different concentrations or edaravone 10 μ L into each hole, and culturing in an incubator.

(4) Cell viability was determined after 48 h. mu.L of MTT (tetramethylazoblue) solution was added to each well, and the mixture was incubated in an incubator for 4 hours, taken out, added with a triple solution (10% sodium dodecylsulfate, 5% isobutanol, and 0.012mol/L hydrochloric acid), incubated overnight at 37 ℃ and then measured for OD at a wavelength of 570 nm. The results are shown in tables 4 and 5, and fig. 5 and 6.

TABLE 4 survival rate of hSOD1-G93A transfected motor neurons increased by honokiol, Magnolia obovanol B and Magnolia aldehyde B

TABLE 5 Effect of survival of Edaravone hSOD1-G93A transfected motoneurons

The results in tables 4 and 5 and fig. 5 and 6 show that honokiol, magnolol B and magnolol B all increase the survival rate of hSOD1-G93A transfected motor neurons dose-dependently, and the effects are superior to edaravone, suggesting that honokiol, magnolol B and magnolol B have significant protective effects on hSOD1-G93A stable ALS cell model.

Example 16

The antioxidant effect of honokiol, Magnolia obovata phenol B and Magnolia aldehyde B on zebra fish embryo model

Influence of 1 honokiol, Magnolia triphenol B and Magnolia aldehyde B on SOD activity of zebra fish embryo

1.1 test methods

(1) The adult fish were treated in a manner of male and female 1:1, placing the male and female in a mating box, and inserting a partition plate in the middle to separate the male and female. The next day, the clapboards are taken out, and the zebra fish begin to mate and lay eggs. After about 1h, the roe is collected and washed with fish-farming water for later use. Selecting a normally developed embryo under a microscope, transferring the normally developed embryo into a culture dish, wherein about 3hpf (hours after fertilization) zebra fish embryos are in a blastocyst stage, adding E3 culture solution into a blank control group, adding 0.15 mu g/mL and 0.21 mu g/mL honokiol, magnolol B and magnolol B water injection into an administration group respectively, placing the culture dish into an artificial climate box for incubation, setting the temperature to be 28 ℃, and illuminating: dark-14 h: and (5) 10 h. Collecting tissue at 24 hr and 72 hr, freezing with liquid nitrogen, and storing in-80 refrigerator. Note that embryo development was checked every 24h and dead embryos were picked out in time.

(2) The 24h and 72h zebra fish embryo tissue is placed in a 1.5mL EP tube and 400 μ L normal saline is added, the tissue is homogenized by a grinding rod until no tissue block can be seen by naked eyes, and then the zebra fish embryo tissue is centrifuged at 2500r/min for 10min, and the supernatant is taken.

(3) Determining the SOD activity according to the operation steps of the sigma kit; MDA is measured according to the operational steps of the Nanjing construction kit; determining the activity of ROS according to the operational steps of the Nanjing built kit; and (3) performing CAT enzyme activity determination according to the operational steps of the Nanjing constructed kit. The results are shown in tables 6 to 9, FIG. 7, FIG. 18.

TABLE 6 influence of honokiol, Magnolia triphenol B on the SOD activity of zebrafish embryos (unit: U/mgprot)

Table 6 and fig. 7 to 9 show the effect of honokiol, magnolol B, and magnolol B on the SOD activity of zebrafish embryos. As can be seen from the results in Table 4 and FIGS. 7-9, on the zebrafish embryo model, honokiol, Magnolia obovata-nol B and Magnolia aldehyde B can significantly increase SOD activity. SOD (superoxide dismutase) is an important component of antioxidant enzyme in vivo, and mainly comprises two subtypes of Cu/ZnSOD and MnSOD. Superoxide dismutase catalyzes the disproportionation of superoxide anion in vivo to produce hydrogen peroxide (H)₂O₂) And oxygen (O)₂) The results show that honokiol, magnolol B and magnolol B have obvious antioxidation.

TABLE 7 influence of honokiol, Magnolia triphenol B on the vitality of zebrafish embryo MDA (unit: U/mgprot)

Table 7 and fig. 10 to 12 show the effect of honokiol, magnolol B, and magnolol B on the viability of zebrafish embryo MDA. As can be seen from the results in Table 5 and FIGS. 10 to 12, the MDA content is significantly reduced by incubating honokiol, Magnolia officinalis triphenol B and Magnolia officinalis triphenol B for 24h and 72h on the zebra fish embryo model. The active oxygen produced by the body in oxidative stress can destroy polyunsaturated fatty acids in the biological membrane, leading to lipid peroxidation to generate lipid peroxides such as Malondialdehyde (MDA), ketone group and carboxyl group. Therefore, the detection of the content of MDA can reflect the degree of lipid peroxidation in vivo and further indirectly reflect the degree of cell oxidative damage. The above results indicate that honokiol, Magnolia obovata phenol B can reduce oxidative damage to embryos by reducing lipid peroxidation.

TABLE 8 influence of honokiol, Magnolia triphenol B on the content of CAT in zebrafish embryo (unit: U/mgprot)

Table 8 and figures 13-15 show the effect of honokiol, magnolol B, and magnolol B on CAT activity of zebrafish embryos. As can be seen from the results in Table 6 and FIGS. 13-15, on the zebra fish embryo model, incubation of honokiol, magnolol B and magnolol B on the zebra fish embryo CAT content for 24h and 72h obviously improves the CAT enzyme activity. The hydroxyl radical (OH-) is the most active oxygen with chemical property, and can react with almost all organic matters in cells, such as carbohydrate, amino acid, phospholipid, nucleic acid, organic acid and the like, with high speed and strong destructive power. But it can be decomposed by catalase CAT, thereby maintaining the redox balance in the body and the homeostasis. The results show that honokiol, Magnolia obovata phenol B and Magnolia obovata phenol B can improve CAT activity and have the capability of resisting oxidative damage.

TABLE 9 influence of honokiol, Magnolia triphenol B on the ROS content of zebrafish embryos (unit: U/mgprot)

Table 9 and fig. 16-18 show the effect of honokiol, magnolol B, and magnolol B on ROS activity of zebrafish embryos. As can be seen from the results of Table 7 and FIGS. 16-18, incubation of honokiol, Magnolia officinalis triphenol B and Magnolia officinalis triphenol B for 24h and 72h on the zebra fish embryo model significantly reduced the ROS content in the administered group. Reactive Oxygen Species (ROS) are oxygen-containing products generated during the oxidative respiration of cellular mitochondria, and are involved in normal physiological activities of the body and pathological processes of many diseases such as chronic diseases and cancers. The results show that honokiol, magnolol B, and magnolol B can have an effect of resisting oxidative damage by reducing ROS.

The results provide a better basis for honokiol, magnolol B and magnolol B to be specific diseases related to oxidative damage and provide a good basis for ALS cells and animal model application.

Example 17

Research on therapeutic effect of honokiol, magnolol B and magnolol B on amyotrophic lateral sclerosis SOD1-G93A transgenic mice

1. Test materials and methods

(1) Test animals and groups

hSODl-G93A transgenic heterozygote mice [ B0048542] B6SJL-Tg (SOD1 × G93A)/J, 70 mice. Hospital BALB/C mice 10 served as normal controls. The hSODl-G93A transgenic heterozygote mice gradually have hind limb tremor or stretch weakness when in tail overhang near 80-90 days of age in days, and progressively aggravated muscle weakness, atrophy and limb paralysis until the mice cannot clear urine and feces, comb hair, climb and independently eat.

The test was divided into 8 groups, normal control group: injecting normal saline into the abdominal cavity of a littermate BALB/C mouse at the age of about 90 days, wherein n is 10; solvent control group: the hSODl-G93A transgenic mice begin to be injected with normal saline in the abdominal cavity about 90 days old, and n is 10; honokiol low dose group: the hSODl-G93A transgenic mice begin to be injected with honokiol 30 mug/kg in the abdominal cavity about 90 days old day, and n is 10; honokiol bulk dose group: the hSODl-G93A transgenic mice started to be injected with 100 mug/kg of honokiol in the abdominal cavity about 90 days of the day of age, and n is 10. Magnolol B low dose group: the hSODl-G93A transgenic mice begin to be injected with 30 mug/kg of magnolol B in the abdominal cavity about 90 days old day, and n is 10; magnolia bark trisphenol B high dose group: the hSODl-G93A transgenic mice begin intraperitoneal injection of Magnolia officinalis triphenol B100 mug/kg about 90 days old day, and n is 10. Magnolol B low dose group: the hSODl-G93A transgenic mice begin to be injected with 30 mug/kg of mangnolic aldehyde B in the abdominal cavity after being aged for about 90 days, and n is 10; magnolol B high dose group: the hSODl-G93A transgenic mice started to be injected with 100 mug/kg of magnolol B in the abdominal cavity about 90 days old day, and n is 10.

(2) Test method

1) Neurological scoring

And (3) performing nerve function scoring twice every week by adopting a 0-4 point nerve function scoring standard formulated by Vercelli and the like:

and 4, dividing: no motor dysfunction occurred; and 3, dividing: hind limb adduction or tremor occurs when the mice are suspended; and 2, dividing: obvious weakness or paralysis of hind limbs; 1 minute: dragging and walking at least one hind limb; 0 minute: the two hind limbs were completely paralyzed, and the mice could not be turned over within 30 seconds after lying on their backs. When the score of the mice reached 0 point, the mice were considered dead.

Reference: vercelli A, Mereita OM, Garbossa D, Muraca G, Mareschi K, Rustcheli D, Ferero I, Mazzini L, Madon E, Fagiol F.human sensory stem cell translation extensions Survival, improvements motor performance and development neuro migration in mouse model of anatomical translation errors Neuroplastic Dis.2008; 31(3):395-405.

2) Time to onset and survival

Neurological scoring was performed daily from the day 70 of age of the mice to assess disease progression. The mouse has a 3-4 minute neurological function score of 2 consecutive days, and is judged to have the disease if limb tremor and/or limb weakness occur. A neurological score of 1 time was considered dead.

3) Western blot analysis (Westernblot)

Tissue sample treatment:

after the hSODl-G93A transgenic mice were evaluated as dead, the spinal cords were harvested after anesthesia with chloral hydrate, frozen in liquid nitrogen, and stored in a freezer at-80 ℃.

Preparation of protein samples:

(1) spinal cord protein samples were removed from-80 ℃ and placed in EP tubes and RIPA lysate (containing RIPA 1000mL, 1m MPMSF 1mL, 1mg/mLNaF 1mL, DTT 1mL, sodium pyrophosphate 1mL, protease inhibitor 1 tablet) was added.

(2) Breaking with ultrasound on ice for 1 min.

(3) Centrifuging at 12000rpm and 4 deg.C for 15min, and collecting supernatant.

Protein quantification (BCA method):

the procedure was performed according to the instructions of the BCA protein quantification kit.

Sample preparation:

before the test, 20-40 μ g of protein solution was taken, deionized water was added to 8 μ L, 2 μ L of Loadingbuffer was added to make the total volume 10 μ L, and the mixture was boiled at 100 ℃ for 4min to denature the protein solution.

SDS-PAGE electrophoresis:

10mL of 10% separation gel is prepared, the separation gel is injected into the gap of a clean glass plate which is vertically placed, and absolute ethyl alcohol with the height of about 1cm is added to the top layer of the separation gel to cover the gel surface. Standing at room temperature for 30min, wherein a horizontal clear interface is visible between the gel and the absolute ethyl alcohol, inclining the device, if the gel surface is unchanged, the polymerization is considered to be basically completed, pouring the absolute ethyl alcohol, sucking up the liquid on the top of the gel with filter paper as much as possible, preparing 4mL of 5% concentrated gel, injecting the concentrated gel into the gap of a glass plate, immediately inserting a clean comb, vertically placing at room temperature, completing the polymerization after about 30min, pulling out the comb, and obtaining the formula of the separation gel and the concentrated gel shown in Table 10.

TABLE 10 separation and concentrate formulations

Putting the gel into a vertical electrophoresis tank, adding a proper amount of gel electrophoresis buffer solution, adding a protein sample and a marker into a comb hole, connecting an electrophoresis device with a power supply, carrying out electrophoresis in concentrated gel for 30min at a voltage of 60V, increasing the voltage to 120V after bromophenol blue enters separation gel, continuing electrophoresis until the bromophenol blue is close to the bottom of the separation gel, and cutting off the power supply. The glass plate is detached and pried open.

Film transfer:

cutting 6 pieces of filter paper with the same size as the gel block and a PVDF film (0.22 mu m), soaking the filter paper and the sponge pad in a transfer buffer solution, opening a film transfer clamp, sequentially aligning and paving the sponge pad, 3 pieces of filter paper and gel on the film transfer clamp, soaking the PVDF film in a methanol solution for about 10s, covering the film, removing bubbles layer by a glass rod, covering 3 pieces of filter paper on the film, finally covering another sponge pad, closing a clamping plate, putting the film transfer clamp into a film transfer groove (paying attention to the electrode connection position), switching on a power supply, and performing constant-current 130mA electric transfer for 1.5 h.

Antigen-antibody reaction:

after the membrane transfer is finished, putting the PVDF membrane into TBST containing 5% skimmed milk powder, shaking at room temperature for 2h, putting the PVDF membrane into an incubation box, uniformly dropwise adding a primary antibody onto the membrane, standing at 4 ℃ overnight, sucking out the primary antibody the next day, and washing the membrane with TBST at room temperature for 5min multiplied by 5 times; adding horseradish peroxidase labeled secondary antibody, shaking and incubating for 1h at room temperature, sucking out the secondary antibody, washing the membrane with TBST at room temperature for 5min × 5 times.

And (3) color development reaction:

placing the PVDF membrane on a color development tray, preparing Millipore chemiluminescence liquid (1:1), uniformly dripping the Millipore chemiluminescence liquid on the PVDF membrane, detecting chemiluminescence by an LAS-3000 gel imager, taking pictures, performing gray level analysis on protein bands in images by using Quantity One density analysis software, and quantifying target proteins: the relative content of the target protein is the gray value of the target protein band/the gray value of the internal reference protein band.

2 results of the test

(1) Prolonging survival time of hSODl-G93A transgenic mouse by using honokiol, Magnolia obovanol B and Magnolia aldehyde B

In order to determine whether honokiol, magnolol B and magnolol B can delay the disease progression of hSODl-G93A transgenic mice and whether the dose is related to the dose, the invention adopts 30 mu G/kg and 100 mu G/kg doses of honokiol, magnolol B and magnolol B for treatment. The onset time and survival time of each group are shown in Table 11, and the survival rate results at different times of each group are shown in FIGS. 19 to 21.

TABLE 11 Effect of honokiol, Magnolia obovanol B and Magnolia aldehyde B on the onset and survival of hSODl-G93A transgenic mice

Group of	Value of N	Onset time (d)	Survival time (d)
				Solvent control group	10	104.2±7.1	134.7±10.1
Honokiol 30 mug/kg	10	102±7.7	144.3±18.5
				Honokiol 100 μ g/kg	10	101.9±8.2	154.8±13.1
Magnolia officinalis triphenol B30 mu g/kg	10	100.3±4.1	140.2±15.9
				Magnolia bark triphenol B100 mu g/kg	10	106.4±2.1	151.1±9.0
Magnolia officinalis aldehyde B30 mug/kg	10	103.5±3.7	141.2±8.9
				Magnolia officinalis aldehyde B100 mu g/kg	10	101±9.8	152.1±11.6

As can be seen from the results in Table 11, the onset time of the mice in the solvent control group is 104.2 + -7.1 days, the mean onset time of the mice in the honokiol treatment group of 30 μ g/kg and 100 μ g/kg is 102 + -7.7 days and 101.9 + -8.2 days, the mean onset time of the mice in the magnolol treatment group of 30 μ g/kg and 100 μ g/kg is 100.3 + -4.1 days and 106.4 + -2.1 days, and the mean onset time of the mice in the magnolol treatment group of 30 μ g/kg and 100 μ g/kg is 103.5 + -3.7 days and 101 + -9.8 days. There were no statistical differences (P >0.05) compared between groups. The mean survival time of the mice in the treated group, which was 30. mu.g/kg, was 9 days longer than that of the solvent control mice. The survival time of the treatment group of 100 mu g/kg of honokiol is 154.8 +/-13.1 days, which is increased by 20 days (P < 0.01). The mean survival time of mice in the treatment group of 30 mu g/kg of the magnolol B is prolonged by 5 days compared with that of the solvent control mice. The survival time of the treatment group of the magnolol B100 mu g/kg is 151.1 +/-9.0 days, and 16d is increased (P is less than 0.01). The mean survival time of the mice in the magnolol B treatment group with the concentration of 30 mu g/kg is prolonged by 6 days compared with that of the mice in the solvent control group. The survival time of the treatment group of the magnolol B with the concentration of 100 mu g/kg is 152.1 +/-11.6 days, and 17 days are increased (P < 0.01). The suggestion shows that honokiol, magnolol B and officinal B have obvious therapeutic effect on ALS.

FIGS. 19-21 are graphs showing the effect of honokiol, Magnolia triphenol B and Magnolia aldehyde B on survival of hSODl-G93A transgenic mice. The results of fig. 19-21 also show that honokiol, pyrogallol B, and magnolol B significantly improve survival of the hSODl-G93A transgenic mice at the same survival time.

(2) Improvement of activation of hSODl-G93A transgenic mouse spinal microglia by honokiol, Magnolia officinalis triphenol B and Magnolia officinalis aldehyde B

Activation and proliferation of microglia are important features of the SOD1 mutation-mediated process of degeneration of spinal motor neurons. The invention examines the expression of the ionized calcium binding adaptor molecule 1(Iba1) protein in the spinal cord of hSODl-G93A transgenic mice by WesternBlot, and the detection results are shown in FIGS. 22 to 24. The results in fig. 22 to fig. 24 show that honokiol, magnolol B and magnolol B can reduce the expression of the spinal microglia marker Iba1 protein of the hSODl-G93A transgenic mice, which suggests that honokiol, magnolol B and magnolol B can alleviate neuroinflammatory reaction by inhibiting microglia activation, thereby prolonging the survival of the mice.

(3) Honokiol, Magnolia obovata phenol B and Magnolia aldehyde B enhance expression of hSODl-G93A transgenic mouse spinal cord Nrf2 protein

The cells contain a plurality of endogenous antioxidases which participate in maintaining redox balance. The production of antioxidant enzymes by these cells can be induced by regulation at the transcriptional level when ROS are elevated in the cellular environment. The body mainly regulates the transcription expression of more than 200 downstream antioxidant genes by activating transcription factor nuclear factor E2 related factor 2(Nrf 2). Increased markers of oxidative stress around degenerated motor neurons were observed in ALS patients and in rodent models of ALS. The expression of Nrf2 protein in spinal cord of hSODl-G93A transgenic mice is investigated by Western Blot, and the detection results are shown in FIGS. 25 to 27. The results in fig. 25 to 27 show that honokiol, magnolol B, and magnolol B can significantly increase the expression of hSODl-G93A transgenic mouse Nrf2, suggesting that honokiol, magnolol B, and magnolol B exert neuroprotective effect by inhibiting oxidative stress, and prolong the survival time of mice.

The results of the above examples show that the biphenyl lignan compounds and the derivatives thereof have good effects of preventing and treating amyotrophic lateral sclerosis, and can be used for preparing medicines for preventing and treating amyotrophic lateral sclerosis.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. The application of honokiol in preparing medicine for preventing and treating amyotrophic lateral sclerosis;

the honokiol is the only active ingredient of the medicament;

the dosage form of the medicine is injection;

the preparation method of the injection water injection comprises the following steps: weighing 10g of hydroxypropyl-beta-cyclodextrin, adding 200mL of distilled water, stirring until the hydroxypropyl-beta-cyclodextrin is completely dissolved to prepare a 5% HP-beta-CD aqueous solution, slowly adding 1.0g of honokiol into the 5% HP-beta-CD aqueous solution at room temperature, stirring for 4h by using an electric constant-temperature magnetic stirrer until the solution is balanced, adding 0.5g of vitamin C, continuously stirring until the honokiol is dissolved, adding sodium bicarbonate to adjust the pH value to 6.0, filtering by using a 0.45 mu m filter membrane, and adding injection water to 1000mL to obtain the magnolol injection water injection containing 1mg/mL of active ingredient.

2. The use of claim 1, wherein the prevention or treatment of amyotrophic lateral sclerosis is achieved by increasing survival of motor neurons.

3. The use of claim 1, wherein the prevention or treatment of amyotrophic lateral sclerosis is achieved by inhibiting microglial activation.

4. The use of claim 1, wherein the prevention or treatment of amyotrophic lateral sclerosis is achieved by reducing ionic calcium binding adaptor molecule 1 protein expression.

5. The use according to claim 1, wherein the prevention or treatment of amyotrophic lateral sclerosis is achieved by reducing neuroinflammatory response.

6. The use of claim 1, wherein the prevention and treatment of amyotrophic lateral sclerosis is achieved by increasing Nrf2 protein expression.