CN111658630B

CN111658630B - Application of rosmarinic acid C in preparation of medicine for preventing and/or treating epilepsy

Info

Publication number: CN111658630B
Application number: CN202010706800.0A
Authority: CN
Inventors: 徐仁爱; 邵圆圆; 叶雪梅
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2021-03-23
Anticipated expiration: 2040-07-21
Also published as: CN111658630A

Abstract

The invention relates to application of a therapeutically effective amount of rhodizonic acid C or pharmaceutically acceptable salt or hydrate thereof in preparing a medicament for preventing and/or treating epilepsy in a subject in need thereof, wherein the rhodizonic acid C can restore abnormal electroencephalogram during epileptic seizure, reduce epileptic seizure rate and seizure level, inhibit activity of hippocampal excitatory neurotransmitter receptors NMDA, stimulate activity of hippocampal inhibitory neurotransmitter receptors GABA, reduce excitability of cerebral cortex, further inhibit formation of epilepsy and play an anti-epileptic role.

Description

Application of rosmarinic acid C in preparation of medicine for preventing and/or treating epilepsy

Technical Field

The invention relates to the technical field of medicines, in particular to application of rosmarinic acid C in preparing a medicine for treating epilepsy.

Background

Epilepsy (epilepsy) is a chronic disease in which neurons in the brain suddenly discharge abnormally, resulting in transient cerebral dysfunction. Status Epilepticus (SE) refers to the condition in which epileptic seizures occur frequently in a short period of time, resulting in persistent coma during seizures. After SE, the incidence rate of chronic encephalopathy, encephalatrophy and focal neurosis is obviously increased, the CA1 area of the hippocampus of a patient has typical neuron necrosis and deletion, but the brain injury mechanism is not clarified yet, and an effective treatment means is also lacking clinically.

At present, the number of patients with epilepsy is increasing at home and abroad, and statistics shows that: epileptic patients account for approximately 0.5% to 1% of the world population. Despite the continuous and intensive research on epilepsy, the pathogenesis of epilepsy is still poorly understood, and the currently used drugs can only partially relieve the condition of epilepsy patients, and the effective rate of the drugs for the developed grand-mal patients is only 60-70%.

In recent years, with the continuous and intensive research on pathophysiological changes caused by epileptic seizures and neuronal discharge diffusion mechanisms, it was found that a decrease in the concentration of gamma-aminobutyric acid (GABA) or a decrease in receptors is an important cause of epileptic seizures. Animal experiment research shows that the GABA level in the frontal cortex of the mouse is increased, reduced and increased again along with the prolonging of time during the epileptic seizure. GABA is present only in the central nervous system and is an important inhibitory transmitter of the central nervous system. The brain tissue has limited utilization of the hydatidiform mole and the substrate thereof during the epileptic seizure, the reduction of the utilization of oxygen can cause the reduction of the amount of glutamic acid and GABA, the inhibition of the postsynaptic membrane of the neuron is weakened, and the excessive excitation of the neuron is stimulated or the epileptic seizure is caused. Recent studies have shown that there is also a reduction in GABA receptors in the brain of patients with partial SE, which can also produce neuronal hyperexcitability.

In addition, neuronal damage caused by the N-methyl-D-aspartate receptor (GCSE) may be caused by an excitatory neuronal overage. Glutamate, the most common excitatory neurotransmitter, is highly specific for the N-methyl-D-aspartate (NMDA) receptor and regulates neuronal hyperexcitation in GCSE, which regulates neuronal damage. In normal excitatory synaptic neurotransmission, glutamate is released from axon terminals and binds to glutamate receptors on the postsynaptic membrane, and when it binds to non-NMDA ionotropic receptors, the receptor channels open, and so on. Glutamate also binds to NMDA receptors, but since Mg2+ blocks the receptor channel opening, there is no effect under normal conditions, but when the depolarization state persists longer or stronger, Mg2+ is released from the channel opening and Ca2+ flows into the cell. NMDA receptor channels, by their very nature, allow prolonged Ca2+ influx and lead to prolonged depolarization times. NMDA receptor channel activation can modulate neuronal damage through the following mechanisms: (1) leading to an extended depolarization time of the cells, opening more NMDA receptor channels, and activating other types of voltage-dependent receptor channels, leading to SE; (2) leading to the accumulation of intracellular Ca2+, activating many intracellular pathological processes; (3) the accumulation of intracellular ions may also regulate neuronal damage by osmotic pressure or some other mechanism; (4) intracellular Ca2+ may also activate some cascade amplification reactions by activating early genes, etc., leading to apoptosis. Apoptosis can occur over a longer period of time than neuronal death and can persist after depolarization is complete, a mechanism that is applicable only where NMDA receptors accumulate, such as the hippocampus and other limbic systems, which may explain why neuronal loss occurs selectively in the hippocampal region. Injury and long-term changes in the limbic system can lead to subsequent seizures.

Propofol is a GABA agonist and is used clinically to treat Status Epilepticus (SE), and clinical studies have demonstrated that continuous infusion of propofol is the currently preferred method of treating status epilepticus. The inventor of the present invention found that the monomeric compound, rhodizonic acid C, extracted from rose leaves (Rosa rugosa Thunb.) has an anti-epileptic effect similar to that of propofol in the process of studying rose extract. The rhodizonic acid C has strong affinity to GABA receptors, has obvious GABA agonism, can antagonize NMDA receptors, regulates the release of Mg2+ from the channel port and the Ca2+ influx of a slow Ca2+ channel. Has protective effect on hippocampal neurons, reduces excitability of cerebral cortex, inhibits epileptogenesis, and exerts antiepileptic effect.

Disclosure of Invention

The object of the present invention is to provide the use of a therapeutically effective amount of rosmarinic acid C or a pharmaceutically acceptable salt or hydrate thereof for the manufacture of a medicament for the prevention and/or treatment of epilepsy in a subject in need thereof.

Further, the epilepsy refers to epileptic seizures, including grand mal seizures (generalized tonic clonic seizures), petit mal seizures (absence seizures), simple partial seizures, complex partial seizures (psychomotor seizures), and autonomic seizures (episodic seizures).

Further, the rosmarinic acid C can restore abnormal electroencephalogram during the epileptic seizure, and obviously reduce the epileptic seizure level.

Furthermore, the rosmarinic acid C inhibits the activity of hippocampal excitatory neurotransmitter receptor NMDA, excites the activity of hippocampal inhibitory neurotransmitter receptor GABA, reduces the excitability of cerebral cortex, further inhibits the formation of epilepsy and plays a role in resisting the epilepsy.

Further, the drug contains, as an active ingredient, from about 0.1mg to 500mg, preferably about 50 to 100mg of rosmarinic acid C.

Further, the drug may be administered at a dosage of about 0.01-20.0 mg/kg/day, preferably about 0.1-10 mg/kg/day, more preferably about 0.5-5 mg/kg/day, more preferably about 1.0-5.0 mg/kg/day.

Further, the medicament further comprises a second active ingredient selected from propofol, clobazam, clonazepam, levetiracetam, topiramate, valproic acid, phenytoin, carbamazepine, lamotrigine and rufinamide.

Further, the medicine also comprises a pharmaceutically acceptable carrier or excipient.

Further, the medicament can be prepared into any conventional medicament dosage form.

Preferably, the dosage form of the present invention includes the form of tablets, capsules, granules, suspensions, syrups, solutions, transdermal patches, emulsions, liposomes, injections, lyophilized powder injections.

Drawings

FIG. 1 structural formula of Murashiic acid C.

FIG. 2 Effect of Murashioic acid C on the electroencephalogram of the cortex of rats in the epilepsy model of pentaerythrine. Wherein, A: electroencephalograms of saline control groups; b: electroencephalograms of the pentaerythrine model control group; c: electroencephalogram of the rosmarinic acid C treatment group.

Detailed Description

Terms and noun explanations

Rose acid C

Murasonic acid C (1,2,3,3a,4,5,8,8 a-octahydro-5, 8,8 a-trihydroxy-3 a-methyl-1- (1-methylethyl) -, [1R- (1 alpha, 3a alpha, 5 beta, 8a beta) -6-azulene-1-carboxylic acid]) Is a natural carotenoid monomer compound extracted from rose leaves (Rosa rugosa Thunb.) by extracting rose leaves with methanol, dissolving the solvent evaporated extract in ethyl acetate, and dissolving in ethyl acetate5% NaHCO for ester₃Washed, acidified to pH 3.0 with 5N HCl, and extracted again with ethyl acetate to give an acidic extract. The acidic extract was chromatographed sequentially on silica gel column (Si02, n-hexane fixed). Eluting with a mixed solvent of methanol and dichloromethane containing a small amount of formic acid to obtain eluates of different fractions, concentrating the eluates, and separating in a fraction of dichloromethane/methanol/formic acid (400: 60: 1.5) to obtain the rosolic acid C.

The term "epilepsy" or "seizure" refers to a patient experiencing one to many seizure disorders. Suitable examples include, but are not limited to, grand mal seizures (generalized tonic clonic seizures), petit mal seizures (absence seizures), simple partial seizures, complex partial seizures (psychomotor seizures), and autonomic seizures (episodic seizures). Seizure as a complication of epilepsy, essential tremor, restless leg syndrome, and the like.

The term "therapeutically effective amount" means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

The term "composition" as used herein includes a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The rosmarinic acid C of the present invention may exist in enantiomeric or diastereomeric forms. It is to be understood that all such isomers and mixtures thereof are included within the scope of the present invention. In addition, the rosmarinic acid C of the present invention may be polymorphic forms, and such polymorphic forms are included within the scope of the present invention. Furthermore, the rosmarinic acid C of the present invention forms a solvate with water (i.e., hydrate) or common organic solvents, and such a solvate is also included in the scope of the present invention.

Prodrugs of rosmarinic acid C of the present invention are also included within the scope of the invention. Typically, such prodrugs are functional derivatives of the rhodizonic acid C of the present invention and are readily converted to rhodizonic acid C in vivo.

The salts of rhodizonic acid C of the present invention are referred to as "pharmaceutically acceptable salts" which are non-toxic. Suitable pharmaceutically acceptable salts of rosolic acid C of the present invention include acid addition salts which may be prepared, for example, by mixing a solution of a compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, when the rhodizonic acid C of the present invention contains a carboxyl group-containing moiety, suitable pharmaceutically acceptable salts include alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; salts with suitable organic ligands, for example quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium ethylenediaminetetraacetate, camphorsulfonate, carbonate, chloride, clavulanate, citrate, dihydrochloride, ethylenediaminetetraacetate, edisylate, laurylsulfonate (etonate), ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycolylaspartate, hexylresorcinol formate, N' -bis (dehydroabietyl) ethylenediamine (hydrabamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothioglycolate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, ethylsulfate, ethylnonacetate, ethylolate, Galactarates, naphthalenesulfonates, nitrates, ammonium N-methylglucamine salts, oleates, pamoates, palmitates, pantothenate, phosphates/diphosphates, polygalacturonates, salicylates, stearates, sulfates, basic acetates, succinates, tannates, tartrates, 8-chlorotheyl salt, tosylates, triethyliodides and valerates.

The invention also includes pharmaceutical compositions comprising one or more of rosmarinic acid C and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions containing one or more of the compounds of the present invention may be prepared by mixing the compound rhodizonic acid C with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the desired route of administration (e.g., oral, parenteral). Thus, for liquid oral preparations (e.g., suspensions, elixirs and solutions), suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral formulations, such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Solid oral formulations may also be coated with substances such as sugars, or enteric coatings, to modulate the primary site of absorption. For parenteral administration, the carrier will usually consist of sterile water, and other ingredients may be added to increase solubility or shelf life. Injectable suspensions or solutions may also be prepared using aqueous carriers and appropriate additives.

To prepare the pharmaceutical compositions of this invention, one or more of the compounds of the invention, rhodizonic acid C, as the active ingredient, is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the desired route of administration (e.g. oral, parenteral such as intramuscular). In preparing the compositions for oral dosage form, any of the usual media may be employed. Thus, for liquid oral preparations (e.g., suspensions, elixirs and solutions), suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral formulations such as powders, capsules, caplets, gel capsules (gelcaps) and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease of administration, tablets and capsules are the most preferred oral unit dosage form in which case solid pharmaceutical carriers are obviously employed. Tablets may be sugar-coated or enteric-coated, if desired, by standard techniques. For parenteral compositions, the carrier will usually comprise sterile water, although other ingredients may be included for purposes of improving solubility or for preservation, etc. Suspensions for injection may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be used.

The pharmaceutical composition of the present invention per dosage unit (e.g., one tablet, one capsule, one pack of powder, one injection, etc.) will contain an amount of the active ingredient necessary to administer the effective dose as described above. Each dosage unit of the pharmaceutical composition of the present invention will contain from about 0.1 to about 500mg, preferably from about 50 to about 100mg, of rosmarinic acid C, and may be administered in a dosage of from about 0.01 to about 20.0 mg/kg/day, preferably from about 0.1 to about 10 mg/kg/day, more preferably from about 0.5 to about 5 mg/kg/day, and even more preferably from about 1.0 to about 5.0 mg/kg/day. However, the dosage may vary depending on the needs of the patient, the severity of the condition being treated, and the compound being used.

Preferably these compositions are in unit dosage forms, such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered dose aerosols or liquid sprays, drops, ampoules, autoinjectors or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition may be in a dosage form suitable for once weekly or monthly administration; for example, insoluble salts of the active compound (e.g., caprate) may be used to provide a depot formulation for intramuscular injection. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutically acceptable carrier (e.g., conventional tablet ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums) and other pharmaceutically acceptable diluents (e.g., water) to produce a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a pharmaceutically acceptable salt thereof. Where it is stated that these preformulation compositions are homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition is readily divided into equivalent dosage forms such as tablets, pills and capsules. This solid preformulation composition is then divided into unit dosage forms of the type described above containing from 0.1mg to about 500mg of the active ingredient of the invention, rosolic acid C. Tablets or pills of the novel compositions may be coated or otherwise formulated to provide a sustained action dosage form. For example, a tablet or pill can comprise an inner dosage component and an outer dosage component, the latter encapsulating the former. The two components may be separated by an enteric layer which serves to prevent disintegration in the stomach, allow the inner component to pass intact into the duodenum, or delay release. A number of materials may be used as such enteric layers or coatings, such materials including various polymeric acids, such as shellac, cetyl alcohol and cellulose acetate.

The novel compositions of this invention may be incorporated in liquid forms for oral or injectable administration, including aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils (e.g., cottonseed oil, sesame oil, coconut oil, or peanut oil), as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums, such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.

For use in the treatment of epilepsy and related disorders described herein, pharmaceutical compositions comprising rosolic acid C, as defined herein, and a pharmaceutically acceptable carrier may also be used. The pharmaceutical composition may contain about 0.1mg to 500mg, preferably about 50 to 100mg, of rosolic acid C and may be formulated in any form suitable for the chosen mode of administration. Carriers include the necessary inert pharmaceutical excipients including, but not limited to, binders, suspending agents, lubricants, flavoring agents, sweetening agents, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid dosage forms such as pills, tablets, caplets, capsules (each dosage form including immediate release, timed release and sustained release formulations), granules and powders; and liquid dosage forms such as solutions, syrups, elixirs, emulsions and suspensions. Dosage forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

The compounds of the invention may be administered in a single daily dose, or the total daily dose may be administered in 2,3 or 4 divided doses throughout the day. In addition, the compounds of the present invention may be administered in intranasal dosage forms by topical application of suitable intranasal vehicles or transdermal patches well known to those of ordinary skill in the art. Of course, for administration in the form of a transdermal delivery system, the dosage administration will be continuous rather than intermittent throughout the dosage regimen.

For example, for oral administration in the form of tablets or capsules, the active pharmaceutical ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired, suitable binders, lubricants, disintegrating agents and coloring agents may also be added to the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

Liquid dosage forms may include suitable flavoring suspending or dispersing agents, for example, synthetic and natural gums (e.g., tragacanth, acacia, methylcellulose, and the like). For parenteral administration, sterile suspensions and solutions are required. When intravenous administration is desired, isotonic formulations, which typically contain suitable preservatives, are employed.

The compounds of the present invention may be administered in any of the compositions described above, as long as treatment of epilepsy or related disorders is desired, according to a dosage regimen established in the art.

The daily dosage for an adult may vary from about 0.01mg to about 1000mg per day. For oral administration, the compositions are preferably tablets containing 0.01mg, 0.05mg, 0.1mg, 0.5mg, 1.0mg, 2.5mg, 5.0mg, 10.0mg, 15.0mg, 25.0mg, 50.0mg, 100mg, 150mg, 200mg, 250mg, 500mg and 1000mg of the active ingredient rosmarinic acid C, the dose being adjusted to the patient to be treated according to the symptoms. A pharmaceutically effective amount is a dosage level that generally provides from about 0.01mg/kg to about 20mg/kg of body weight per day. A preferred range is from about 0.5mg/kg to about 10.0mg/kg body weight per day, most preferably from about 1.0mg/kg to about 5.0mg/kg body weight per day. The compounds of the present invention may be administered on a regimen of 1-4 times per day.

Optimal dosages will be readily determined by those skilled in the art and will vary with the particular compound employed, the mode of administration, the size of the formulation, the mode of administration and the course of the disease. In addition, the dosage may be adjusted taking into account various factors specific to the particular patient being treated, including the age, weight, diet, and time of administration of the patient.

It will be appreciated by those skilled in the art that the ability of a test compound to treat or prevent a particular condition may be predicted, both in vivo and in vitro, using suitable, known, accepted cellular and/or animal models.

Advantageous effects

The rosmarinic acid C can restore abnormal electroencephalogram during the epileptic seizure period, and obviously reduce the epileptic seizure frequency and seizure level. Inhibiting activity of hippocampal excitatory neurotransmitter receptor NMDA, stimulating activity of hippocampal inhibitory neurotransmitter receptor GABA, reducing excitability of cerebral cortex, inhibiting formation of epilepsy, and resisting epilepsy.

Detailed Description

The present invention is described in more detail below to facilitate an understanding of the present invention.

Effect of rhodizonic acid C on pentaerythrityl epilepsy model rats.

Epilepsy is characterized by paroxysmal abnormal discharges of neurons leading to sudden, recurrent and transient central nervous system dysfunction, and increased neuronal excitation and hypersynchronized discharges may be the basic conditions for the development of epilepsy. The PTZ-fired model of classical epileptogenic agents is considered to be one of the ideal animal models to mimic human epilepsy. The PTZ is injected into abdominal cavity or cerebral cortex to induce animal epilepsy model, and the PTZ has no special neurotoxicity and is a common model for researching epilepsy attack and neuron damage.

(I) procedure of experiment

1. Experimental reagent and instrument

Penetradecyltetrazol (PTZ) was purchased from Sigma, USA; the rosolic acid C is prepared by laboratories; propofol is a product of the national rui company, llc; EEG-4418K electroencephalograph, available from Hitachi, Japan. Pentobarbital sodium was purchased from chemical reagent works in Harbin; dental burs, stereotaxic instruments (japan); silver needle electrode, guide electrode, biological function experiment system, purchased from union bio-technology corporation of Gongtai; NMDA ζ 1 goat anti-mouse polyclonal antibody and GABAAR α 1 rabbit anti-mouse polyclonal antibody were purchased from Santa Cruz; the SP immunohistochemical staining kit is purchased from Beijing Zhongshan bioengineering, Inc.

2. Experimental animals and groups

50 Wistar rats weighing 150-220 g are immediately divided into 5 groups, namely a blank control group, an epilepsy model group, a rhodizonic acid C group, a propofol positive control group and a rhodizonic acid C + propofol combined drug group, wherein each group comprises 10 rats.

Epilepsy model group: PTZ intraperitoneal injection and normal saline intragastric administration;

blank control group: injecting normal saline into abdominal cavity and irrigating stomach with normal saline;

drug treatment group: PTZ intraperitoneal injection + gavage with rosolic acid C;

positive control group: PTZ intraperitoneal injection + propofol intraperitoneal injection;

combination treatment group: PTZ intraperitoneal injection, propofol intraperitoneal injection and rosmarinic acid C intragastric administration.

The intraperitoneal injection dosage of PTZ or normal saline is 35mg/kg, and the concentration is 10 g/L; the intraperitoneal injection dose of the propofol is 50 mg/kg; the dose of the drug or the normal saline for gastric lavage is 150mg/kg, the concentration is 30g/L, and the drug or the normal saline is fasted for 6 hours, 1 time/day and 28 days continuously before gastric lavage.

3. Observation of behavioral changes in rats

Rats were observed for 60 minutes after each injection and recorded seizure status, with seizure scoring criteria: grade 0, no behavioral seizure; class I, rhythmic nodding or head twitching; grade II, clonic chewing; class III, the head twitching plus the clonic tic of the forelimbs; level IV, kangaroo posture, upright upper body; grade V, fall; grade VI, tonic convulsions. Rats showing 5 consecutive convulsions above grade II were rated to reach the fire point criteria.

4. Electroencephalogram recording

2 rats were taken from each group and electroencephalograms of animals in each group were recorded using a biological function experiment system. Injecting 0.4% sodium pentobarbital (40mg/kg) into abdominal cavity of rat, keeping rat in general anesthesia state after 10min, and fixing rat prone position on stereotaxic apparatus to be tested. A2 cm long incision was cut at the center of the rat calvaria, and the periosteum was peeled off. And (4) positioning electrodes, namely placing needle electrodes with the diameter of 0.5mm in the right cerebral cortex and the hippocampus. Frontal cortex electrode 2.0mm anterior bregma, 2.0mm lateral to midline, and 0.5mm under dura mater. The hippocampal electrode is 3.8mm behind bregma, 2.0mm beside midline and 2.6mm under dura mater. Drilling with a dental drill, placing a guide electrode, and fixing all the electrodes with the dental desquamation powder. The electrodes are connected with (1) the left hippocampus between the left scalps; (2) right hippocampus between right scalp; scaling, 2mm to 50 μ V; the paper speed is 30 mm/s. Gain 5000, time constant 5s, filtering 10 Hz. The test was started after the rats were awake and recorded 1 time every 10 min.

5. Immunohistochemical examination

After the last observation of the rest 8 rats in each group is finished, the heart is exposed, a puncture needle is inserted into the left ventricle under direct vision and the right auricle is cut, the rats are quickly perfused by cold physiological saline 100ml, then 0.1mol/L phosphate buffer solution of 40g/L paraformaldehyde is perfused 500ml, the total perfusion time is 1h, the cranium is opened, the brains are taken out, the rats are placed in the perfusion solution for overnight at 4 ℃, then the rats are placed in 200g/L sucrose for 12h, and then the rats are dehydrated, soaked in wax, embedded and sliced conventionally.

Dewaxing paraffin sections conventionally, placing the paraffin sections in 0.01mol/L citric acid buffer salt for microwave repair for 10min, dropwise adding 50 microliters of 1g/L Triton solution to each section, incubating for 10min at 37 ℃, washing for 3min x 3 times by PBS, dropwise adding animal serum for sealing, incubating for 15min at 37 ℃, dropwise adding NMDA zeta 1 goat anti-mouse polyclonal antibody (1:100) or GABA alpha 1 rabbit anti-mouse polyclonal antibody 1(1:3000) for overnight incubation at 4 ℃, washing with PBS for 5min x 3 times, dropwise adding biological cable labeled goat anti-rabbit IgG, incubating for 20min at 37 ℃, washing for 3min x 3 times by PBS, dropwise adding horseradish enzyme labeled streptavidin working solution, incubating for 20min at 37 ℃, washing for 3min x 3 times by PBS, DAB color development, gradient ethanol dehydration, xylene transparency and neutral gum sealing.

The average absorbance and percentage of positive cells were determined for rat hippocampal NMDA ζ 1 and GABAAR α 1 positive-reacting cells.

6. Statistical treatment

SPSS statistical software processes data, counts data and compares rate with chi-square test, and compares rank data with rank test.

(II) results

1. The change of seizure rate and ignition rate of epileptic rats caused by PTZ ignition of each group.

The attack rate of rats with epileptic attacks above II after intraperitoneal injection is calculated, the highest attack rate is 100% in a model group, no epileptic attack exists in a blank group, the attack rate of a rhodizonic acid C treatment group, a propofol treatment group and a combined drug group is obviously lower than that of the model group, wherein the three groups have significant difference (P <0.05) compared with the model group at the 14 days and the 28 days, and the combined drug group, the rhodizonic acid C treatment group and the propofol treatment group also have significant difference (P <0.05, P < 0.01). At the end of 28 days, the ignition rate of the model group reached 100%, the ignition rate of the blank group was 0, while the ignition rates of the rosc acid treatment group, the propofol treatment group and the combination treatment group were lower than those of the model group and significantly different from the model group (P <0.05), and the combination treatment group also significantly different from the rosc acid treatment group and the propofol treatment group (P <0.05) (see table 1).

TABLE 1 Effect of Rose acid C on seizure and ignition rates in rats

P <0.05 compared to model group, P <0.05 compared to model group; p <0.05 compared to positive control group, P <0.05 compared to positive control group; delta is compared with the C-type rhodizonic acid group, P is less than 0.05, Delta is compared with the C-type rhodizonic acid group, P is less than 0.01.

2. Effect of rhodizonic acid C on brain waves in rats.

The electroencephalogram of each group of rats is shown in fig. 2. The electroencephalogram of the hippocampus of the A group of rats is mainly based on basic waves and has no obvious rhythmicity; the rats in the model group are mainly spike waves, sharp waves and spike-slow composite waves which are scattered epilepsy waves, and a large amount of epilepsy discharges occur; the spike wave, the spike wave and the spike-slow wave of the rats in the treatment group are obviously reduced, and the spike-slow wave is basically expressed as alpha wave and beta wave. The result shows that the rosmarinic acid C can inhibit PTZ from igniting epileptic discharge of rats and reduce epileptic seizure.

3. Effect of Rose acid C on rat hippocampal excitatory neurotransmitter receptor NMDA and inhibitory neuroreceptor GABA

The results show that compared with the rats in the normal control group, the rats in the PTZ ignition epilepsy-induced model group have increased hippocampal NMDA zeta 1 expression and reduced GABAAR alpha 1 expression, which indicates that activation of hippocampal NMDA zeta 1 and inhibition of GABAAR alpha 1 are closely related to epileptic seizure. Compared with the rats in the model group, the rats in the rosmarinic acid C treatment group have obviously reduced NMDA zeta 1 expression and obviously increased GABAAR alpha 1 expression while reducing the epileptic seizure and the ignition rate, and the results are obviously different (P < 0.05). The rosmarinic acid C can inhibit the activity of hippocampal excitatory neurotransmitter receptor NMDA and stimulate the activity of inhibitory neurotransmitter receptor GABA, thereby reducing the excitability of cerebral cortex and inhibiting epileptic seizure. The results are shown in tables 2 and 3.

Table 2 average absorbance and percentage of positive cells in rat hippocampal NMDA ζ 1 positive reaction cells (X ± S, n ═ 8)

Group of	Average absorbance	Percentage of positive cells (%)
			Blank group	0.1491±0.0068*	36.59±0.54*
Treatment group	0.1532±0.0085*	38.18±0.28*
			Model set	0.2985±0.0088	50.00±0.36

P <0.05 compared to model group

Table 3 average absorbance and percentage of positive cells in rat hippocampal GABAAR α 1 positive reaction cells (X ± S, n ═ 8)

Group of	Average absorbance	Percentage of positive cells (%)
			Blank group	0.2035±0.0084*	60.13±1.54*
Treatment group	0.1847±0.0065*	58.18±2.25*
			Model set	0.1298±0.0037	40.06±1.96

P <0.05 compared to model group

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. Use of a therapeutically effective amount of rosmarinic acid C or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing and/or treating epilepsy in a subject in need thereof.

2. Use according to claim 1, characterized in that said epilepsy is a seizure.

3. Use according to claim 1, the epilepsy being selected from the group consisting of grand mal seizures (generalized tonic clonic seizures), petit mal seizures (absence seizures), simple partial seizures and complex partial seizures (psychomotor seizures).

4. Use according to any one of claims 1 to 3, characterized in that the rosmarinic acid C is capable of restoring abnormal electroencephalogram during the duration of epileptic seizures, reducing the rate and the level of epileptic seizures.

5. The use according to any one of claims 1 to 3, wherein the rosmarinic acid C inhibits the activity of hippocampal excitatory neurotransmitter receptor NMDA, agonizes the activity of hippocampal inhibitory neurotransmitter receptor GABA, decreases cerebral cortex excitability, inhibits the formation of epilepsy, and exerts an antiepileptic effect.

6. Use according to any one of claims 1 to 3, characterized in that the medicament contains, as active ingredient, 0.1mg to 500mg of C rosmarinic acid.

7. Use according to claim 6, wherein the content of rosolic acid C is 50-100 mg.

8. Use according to any one of claims 1 to 3, wherein the medicament is to be administered in a dose of 0.01 to 20.0 mg/kg/day.

9. Use according to claim 8, said medicament being administered in a dose of 0.1-10 mg/kg/day.

10. Use according to claim 9, said medicament being administered in a dose of 0.5-5 mg/kg/day.

11. Use according to claim 10, said medicament being administered at a dose of 1.0-5.0 mg/kg/day.

12. Use according to any one of claims 1 to 3, wherein the medicament further comprises a second active ingredient selected from propofol, clobazam, clonazepam, levetiracetam, topiramate, valproic acid, phenytoin, carbamazepine, lamotrigine and rufinamide.

13. The use according to any one of claims 1 to 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.

14. Use according to any one of claims 1 to 3, wherein the medicament is formulated in any conventional pharmaceutical dosage form.

15. Use according to claim 14, characterized in that the dosage form is selected from the form of tablets, capsules, granules, suspensions, syrups, solutions, transdermal patches, emulsions, liposomes, injections, lyophilized powder injections.