CN115624543B - Medicine for treating migraine, pharmaceutical composition, preparation method and pharmaceutical use thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to a medicine for treating migraine, a medicine composition, a preparation method and a pharmaceutical use thereof, and further relates to a medicine for treating migraine caused by dysfunction of the trigeminal ganglion, and a medicine for treating migraine caused by migraine. Drugs for pathological damage of midbrain tissue. The medicine of the present invention contains one or a combination of paeonol and glycyrrhizic acid, which can significantly improve the behavioral manifestations of migraine, and at the same time, treat migraine caused by trigeminal ganglion dysfunction and midbrain tissue caused by migraine. Pathological damage has a good therapeutic and improvement effect. Compared with traditional non-steroidal anti-inflammatory drugs, triptans and CGRP receptor antagonists, the therapeutic effect is equivalent or better, and it has the advantages of lower cost and fewer side effects, and can play the role of "treating both symptoms and root causes". Effect.

Description

Medicine for treating migraine, pharmaceutical composition, preparation method and pharmaceutical use thereof

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a medicine for treating migraine, a pharmaceutical composition, a preparation method and a pharmaceutical use thereof, and further relates to a medicine for treating migraine caused by abnormal function of the trigeminal ganglion and/or a migraine anti-inflammatory drug. Drugs for pathological damage to brain tissue.

Background technique

Migraine is a common episodic headache disorder characterized by symptoms of gastrointestinal and autonomic changes, mainly unilateral, bilateral, or generalized headache, lasting 4 to 72 hours, accompanied by nausea, vomiting, misophonia Or photophobia etc. Migraine attacks are frequent, easy to repeat, severe pain, and seriously affect normal life, work or study. It is classified by the World Health Organization (WHO) as the sixth disabling disease and the third common disease in humans. In addition, migraine can also lead to impaired cognitive function, increase the overall risk of stroke and other cerebrovascular diseases, and often coexist with sleep disorders, depression and anxiety. Recent studies have shown that migraine has more than 1 billion patients worldwide, with an annual prevalence rate of 15%, while the annual prevalence rate of young adults in my country is about 14.3%, and the ratio of male to female is close to 1:3. Therefore, migraine has caused a huge economic burden for our country. According to the 2011 census, the total cost of migraine is estimated to be 331.7 billion RMB per year. Studies have shown that migraine causes 45.1 million years lived with disability (YLDs), accounting for 5.6% of the global disease burden, higher than the sum of all other neurological diseases.

In terms of drug treatment, non-steroidal anti-inflammatory drugs, triptans, and CGRP receptor antagonists are usually used internationally. However, these drugs are costly and have multiple adverse reactions or serious adverse events. Too many contraindications also prevent it from being widely used in patients with migraine. For example, ibuprofen can cause adverse reactions in multiple systems such as the gastrointestinal tract, cardiovascular system, and endocrine system, and even severe cardiovascular disease and liver disease. People with digestive tract diseases, cardiovascular diseases, and allergic constitution should be cautious. The most common side effects of zolmitriptan are paresthesia, throat, jaw and chest pain, chest pressure, nausea and insomnia, and cardiovascular discomfort. Patients with high-risk family history of diabetes, high cholesterol and coronary artery disease should be cautious. use.

Therefore, there is an urgent need for new drugs for the treatment of migraine in the prior art.

Contents of the invention

One object of the present invention is to provide a medicament that can effectively relieve and treat migraine in vertebrates, especially humans.

Another object of the present invention is to provide a medicine for improving or treating pathological damage of migraine to midbrain tissue.

Another object of the present invention is to provide a drug capable of up-regulating the content of 5-HT in cerebrospinal fluid and/or down-regulating the content of HIS in cerebrospinal fluid.

Another object of the present invention is to provide a drug for improving or treating migraine caused by trigeminal ganglion dysfunction, especially by intervening in the TRPV1/CGRP signaling pathway, inhibiting the high expression of pro-inflammatory factors IL-1β and TNF-α, Promote the high expression of anti-inflammatory factor IL-10, thereby improving migraine.

Another object of the present invention is to provide the application of paeonol and/or glycyrrhizic acid in the preparation of the aforementioned medicine, and the preparation method of the medicine.

Paeonol referred to in the present invention is an organic compound, the chemical name is 2'-hydroxy-4'-methoxyacetophenone, another name is paeonol, paeonol, and the chemical formula is C ₉ H ₁₀ O ₃ , is an active ingredient from the dry root bark of the Ranunculaceae plant peony or the dry root extract of the Ranunculaceae plant Paeoniae officinalis.

Glycyrrhizic acid referred to in the present invention is an organic compound with a chemical formula of C ₄₂ H ₆₂ O ₁₆ , and is an active ingredient extracted from licorice, a leguminous plant of the genus Glycyrrhizae.

In one embodiment, the medicine of the present invention can treat or improve the pathological damage of the midbrain tissue caused by migraine. The pathological damage of the midbrain tissue in the present invention is not particularly limited, as long as it is caused or caused by migraine All encompassed, the midbrain histopathological damage includes, inter alia, reduction of neuronal cell axes and macules and/or neuronal degenerative folds.

In one embodiment, the medicament of the present invention is capable of modulating the levels of biomarkers associated with migraine. In a specific embodiment, the drug can up-regulate the content of 5-HT in cerebrospinal fluid, and/or down-regulate the content of HIS in cerebrospinal fluid. Long-term low levels of 5-HT in the brain can enhance the sensitivity of nociceptors and lead to recurrent migraine; histamine can induce vascular pulsation migraine in normal people in a dose-dependent manner; both are biomarkers of migraine .

In general, the content ranges of the above biomarkers are as follows:

Under normal conditions (i.e. Con group): 5-HT, 19.83±2.19ng/mL; HIS, 4.63±0.99 ng/mL;

In the model group (i.e. the Mod group): 5-HT, 10.28±0.71 ^** ; HIS, 13.12±0.50 ^** (compared with the Con group, ** indicates p<0.01).

The inventors of this patent further found that migraine is not accompanied by organic lesions of the trigeminal ganglion, but is associated with dysfunction of the trigeminal ganglion. Based on this creative discovery, the present invention creatively proposes the application of paeonol, glycyrrhizic acid or the combination of the two in the preparation of drugs for treating or improving trigeminal ganglion dysfunction.

In one embodiment, the drug for treating migraine caused by trigeminal ganglion dysfunction has a down-regulation effect on TRPV1/CGRP gene transcription and/or protein expression, and can down-regulate the expression of pro-inflammatory factors IL-1β and TNF-α, and / Or up-regulate the expression of anti-inflammatory factor IL-10. The occurrence of migraine is closely related to TRPV1/CGRP. Noxious substances such as pro-inflammatory factors can induce the activation of TRPV1 in neurons, enhance the sensitivity of nociceptors, increase the release of CGRP in the local meninges, dilate meningeal blood vessels, and accelerate blood flow. Pain information transmission increases, and further stimulates the release of pro-inflammatory factors, thereby inducing migraine.

In general, the relative transcript level ranges of the above genes are as follows:

Under normal conditions (i.e. Con group): TRPV1 mRNA, 0.61±0.37; CGRP mRNA, 0.95±0.79;

In the model group (i.e. Mod group): TRPV1 mRNA, 1.57±0.41 ^** ; CGRP mRNA, 5.95±0.24 ^** (compared with Con group, ** indicates P <0.01).

In general, the ranges of relative protein expression and transcription level of the above genes are as follows:

Under normal conditions (i.e. Con group): TRPV1, 0.77±0.21; CGRP, 0.83±0.31;

In the model group (i.e. the Mod group): TRPV1, 1.53±0.49 ^** ; CGRP, 1.70±0.55 ^** (compared with the Con group, ** indicates P <0.01).

Generally speaking, the content ranges of the above-mentioned inflammatory factors are as follows:

Under normal conditions (i.e. Con group): IL-1β, 18.98±1.68pg/mL; TNF-α, 182.34±29.36pg/mL; IL-10, 19.66±5.91pg/mL;

In the model group (i.e. Mod group): IL-1β, 34.88±5.10 ^** pg/mL; TNF-α, 322.05±27.96 ^** pg/mL; IL-10, 31.23±6.74 ^** pg/mL (with Compared with Con group, ** indicates P <0.01).

In one embodiment, the medicament of the present invention includes only paeonol as an active ingredient, or only glycyrrhizic acid as an active ingredient, or a combination of both paeonol and glycyrrhizic acid as active ingredients.

In one embodiment, the medicament of the present invention is composed of paeonol and glycyrrhizic acid or a combination of both, and pharmaceutically acceptable excipients. Wherein, the pharmaceutical excipient is selected from one of conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants in the pharmaceutical field. one or more species.

In one embodiment, in the medicine of the present invention, the dosage of paeonol is 50~500 mg·kg ^-1 ·d ^-1 , preferably 100~400 mg·kg ^-1 ·d ^-1 , more preferably 400 mg·kg ^-1 d ^-1 .

In one embodiment, in the medicine of the present invention, the dosage of glycyrrhizic acid is 10~300 mg·kg ^-1 ·d ^-1 , preferably 50~200 mg·kg ^-1 ·d ^-1 , more preferably 200 mg·kg -1 ·d ^{-1 . 1} ·d ^-1 .

In one embodiment, the medicament of the present invention includes a combination of paeonol and glycyrrhizic acid, and the mass ratio of the two is paeonol: glycyrrhizic acid=0.2~5, preferably 0.5~2, more preferably 2.

For the medicine of the present invention, its administration form or dosage form is not particularly limited, and any form known in the prior art can be used. For example, oral preparations, injection preparations or external preparations can be used.

The subject of administration of the aforementioned drugs may be any vertebrate, especially human.

The invention further relates to processes for the preparation of medicaments. The medicine is used for treating migraine, and treating migraine caused by abnormal function of trigeminal ganglion, and/or treating pathological damage of migraine to midbrain tissue; the method includes using one or both of paeonol and glycyrrhizic acid The step of using a combination of species as a raw material preferably includes using both paeonol and glycyrrhizic acid at the same time. The so-called "simultaneously" means that the aforementioned method of the present invention includes both the operation of adding paeonol and the operation of adding glycyrrhizic acid, and these two operations can be completed in one step or in multiple steps.

The present invention further relates to the application of the combination of one or both of paeonol and glycyrrhizic acid in the preparation of medicines, and the medicines have one or more of the following effects:

Treating or improving migraine's pathological damage to midbrain tissue, which includes reduction of neuron cell axis and spots and/or neuron degeneration folds;

Treat or improve trigeminal ganglion dysfunction;

Up-regulate the content of 5-HT in cerebrospinal fluid, and/or down-regulate the content of HIS in cerebrospinal fluid;

Down-regulation of TRPV1 and/or CGRP gene transcription or protein expression;

Down-regulate the expression of pro-inflammatory factor IL-1β and TNF-α, and/or up-regulate the expression of anti-inflammatory factor IL-10.

The beneficial effects of the present invention are: based on the active ingredients of natural plant extracts, a medicine for treating migraine is provided, which can have a significant therapeutic effect on migraine, and is different from traditional non-steroidal anti-inflammatory drugs, triptans and CGRP receptor antagonists have equivalent or better effects, and have the advantages of lower cost and fewer side effects. Further discovered the pathological basis of the midbrain and neurology related to migraine, and based on this discovery, a corresponding therapeutic drug was proposed, which contained one or a combination of paeonol and glycyrrhizic acid, and the drug could significantly Treating or improving migraine caused by abnormal function of trigeminal ganglion, treating or improving pathological damage to midbrain tissue caused by migraine, has the effect of "treating both symptoms and root causes".

Description of drawings

Figure 1: Periorbital hyperalgesia in rats with migraine after intragastric administration (in the figure: compared with the Con group, ^** P <0.01; compared with the Mod group, ^f P <0.05, ^a P <0.01; compared with the Pos group Compared with group, ^# P <0.05 , ^## P <0.01; compared with HD, HZ, ^b P <0.05; compared with DG, GG, ^e P <0.05; compared with DG, ^r P <0.050);

Figure 2: Behavioral changes of migraine rats after intragastric administration (in the figure: compared with the Con group, ^* P <0.01, ^** P <0.01; compared with the Mod group, ^f P <0.05, ^a P <0.01 ; compared with Pos group, ^# P <0.05 , ^## P <0.01; compared with HD, HZ, ^b P <0.05; compared with HD, ^j P <0.05);

Figure 3: HE staining of the midbrain of rats with migraine (400×) (in the figure: rats in Con group, Pos group and other treatment groups can see more neurons with normal morphology; Mod group has different degrees of damaged neurons, It is manifested as a shuttle shape with reduced spots and deep dyeing;

Figure 4: Expression levels of biomarkers 5-HT and HIS in migraine rats (in the figure: Note: Compared with Con group, ** P <0.01; compared with Mod group, ^f P <0.05, ^a P <0.01; compared with Pos group, ^# P <0.05 , ^## P <0.01; compared with HD, HZ, ^b P <0.05; compared with DD, DZ, ^c P <0.05; compared with GD, GZ, ^dP <0.05; compared with DG and GG, ^eP < 0.05 .);

Figure 5: HE staining of trigeminal ganglion in migraine rats (100×);

Figure 6: Transcript levels of TRPV1 and CGRP genes in the trigeminal ganglia of rats with migraine (in the figure: compared with the Con group, ^** P <0.01; compared with the Mod group, ^f P <0.05, ^a P <0.01; compared with the HZ group ^kP <0.05 compared with DD; ^zP <0.05 compared with DD; ^rP < 0.05 compared with DG ) ;

Figure 7: Expression levels of TRPV1 and CGRP proteins in the trigeminal ganglia of migraine rats (in the figure: compared with the Con group, ^** P <0.01; compared with the Mod group, ^f P <0.05, ^a P <0.01);

Figure 8: Expression levels of plasma pro-inflammatory factors IL-1β, TNF-α and anti-inflammatory factor IL-10 in migraine rats (in the figure: compared with Con group, ^** P <0.01; compared with Mod group, ^fP <0.05, ^aP <^0.01; kP < 0.05 compared with HZ; zP < 0.05 compared with DD; ^{rP <} 0.05 compared with DG).

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Preparation example

(experimental animals)

Seventy-two SPF-grade male SD rats, weighing (200±10) g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. [Certificate No.: SCXK (Beijing) 2021-0011]. The animals were kept in the barrier environment animal room of Beijing University of Traditional Chinese Medicine, with a temperature of (22±1)°C, a humidity of (65±5)%, and a 12-h day and night cycle light environment. They were fed with common feed and had free access to food and water. The animal experiment protocol has been approved by the Ethics Committee of Beijing University of Traditional Chinese Medicine.

(model preparation and grouping)

After 1 week of adaptive feeding, 72 SD male rats were randomly divided into a blank group (Con) with 6 rats and a model group with 66 rats according to their body weight.

Referring to the internationally recognized modeling method, the rats in the model group were subcutaneously injected with nitroglycerin 10 mg·kg ^-1 ·d ^-1 on the back of the neck, once every other day, for a total of 5 times to establish a migraine model.

Rats in the Con group were subcutaneously injected with 0.9% normal saline 20 μL·kg ^-1 ·d ^-1 on the back of the neck, and the injection method and frequency were the same as those in the model group.

Modeling criteria: within 30 minutes frequent head scratching, decreased orbital pain threshold, reduced activity, curling up, etc., and the above-mentioned symptoms peaked at about 60 minutes, and the model was considered successful.

66 model rats were randomly divided into model group (Mod), positive drug group (Pos), paeonol and glycyrrhizic acid combined low, middle and high dose group (HD, HZ, HG), paeonol low, medium and high dose group according to body weight. groups (DD, DZ, DG), glycyrrhizinic acid low, medium and high dose groups (GD, GZ, GG), 6 rats in each group.

(reagents and instruments)

Glycyrrhizic acid (purity ≥95%), paeonol (purity ≥98%) (Shanghai Yuanye Biotechnology Co., Ltd., batch numbers: N09GS166788, X21A11S111651); zolmitriptan tablets (Wante Pharmaceutical Co., Ltd., China , National Pharmaceutical Accreditation H20061077), as a positive drug group drug; Nitroglycerin Injection (Beijing Yimin Pharmaceutical Co., Ltd., China, National Pharmaceutical Accreditation H11020289).

(method of administration)

After grouping, the rats in each group were given the corresponding drugs for continuous gavage for 7 days. Paeonol, glycyrrhizic acid and zolmitriptan tablets were dissolved in 3% DMSO by volume and 1% Tween 80 saline by volume, respectively, and mixed well. The rats in the Con group were given an equal volume of normal saline 10mL·kg-1·d-1 every day; the rats in the Mod group were given 10 mL·kg-1·d-1 of normal saline with a volume percentage of 3% DMSO and a volume percentage of 1% Tween 80; Rats in the Pos group were given 0.25 mg·kg-1·d-1 zolmitriptan tablets 10 mL·kg-1·d-1; the administration volume of each treatment group was 10 mL·kg-1·d-1, Dosage is given in Table 1.

Table 1 Dosage of each treatment group

(Behavioral observation and detection of periorbital pain threshold)

The orbital pain threshold of the rats in each group was detected after the 2d, 4d, and 6d of administration, and the behavioral changes of the rats were observed after the 1d, 3d, and 5d.

Behavioral observation: within 1 hour after administration, observe and record the number of head scratches (including the total number of head scratches of front paws and hind paws) of rats in each group, and count as one head scratching for several consecutive head scratchings without pause.

Periorbital pain threshold detection: Periorbital pain threshold detection was performed with a Von Frey Hairs tactile measurement kit 1 hour after administration. At the beginning of the test, start from the smallest fiber size (0.008g, corresponding to a log value of 1.65) to stimulate the skin around the orbit of the rat, and gradually increase to the largest fiber size (300g, corresponding to a log value of 6.65), until a positive reaction occurs, and record The log value corresponding to the fiber filament was used as the periorbital pain threshold. Positive reaction standard: each fiber was stimulated 5 times, and there were 3 or more positive reactions, such as the rat shrank/turned his head, pulled the fiber with his forepaws, and made a sound.

(Specimen collection and indicator detection)

The materials were collected on the seventh day after administration. Rats in each group were fasted for 12 hours, and the rats were anesthetized by intraperitoneal injection of 50 mg·kg ^-1 of 1% pentobarbital sodium. Connect a disposable blood collection needle with a 1ml syringe, penetrate the ventricle horizontally through the foramen magnum, slowly extract 100-150 μL of cerebrospinal fluid, and use ELISA to detect the content of 5-HT and HIS in the cerebrospinal fluid of rats; then take blood from the abdominal aorta and separate plasma , ELISA was used to detect the contents of IL-1β, TNF-α, and IL-10 in rat plasma; the rats were sacrificed, the heart was perfused with 120-180ml of 4°C normal saline, and the midbrain and trigeminal ganglion were quickly isolated; the midbrain and 1/2 2 The trigeminal ganglion is soaked and fixed in 4% paraformaldehyde solution (for example: take 4g of paraformaldehyde, add pure water to dissolve, and make the volume to 100ml) for 3 days, and routinely dehydrate, paraffin-embedded, sectioned, and HE stained The remaining 1/2 of the trigeminal ganglion was placed in a -80°C refrigerator, and the gene transcription and protein expression levels of TRPV1 and CGRP in the rat trigeminal ganglion were detected by qRT-PCR and Western Blot. The primer sequences are shown in Table 2.

Table 2 Primer sequences

(statistical methods)

The data are represented by x±s, analyzed by SPSS 23.0 software, and drawn by GraphPad Prism 8.0 software. When the data of each group conformed to the normal distribution, the statistical analysis was carried out by One-way ANOVA method; if the variances were homogeneous, the least significant difference method (LSD) was used to compare the differences between groups; if the variances were not homogeneous, the differences between groups were compared by Games- Howell's method. When the data in each group did not conform to the normal distribution, non-parametric tests were used for comparison between groups. P <0.05 was considered statistically significant.

Example 1: Intervention on Migraine Behavior

See Figure 1. Compared with the Con group, the orbital pain threshold of the Mod group decreased significantly on the 2nd, 4th, and 6th day of intragastric administration in migraine-forming rats ( P <0.01); compared with the Mod group, the periorbital pain threshold of the Pos group and each treatment group The hyperalgesic response was improved to varying degrees ( P <0.05 or P <0.01).

On the second day of intragastric administration, the orbital pain threshold of the HG group, DD group, and GD group was significantly higher than that of the Pos group ( P <0.05 or P <0.01); on the fourth day of intragastric administration, the orbital pain threshold of the HG group was significantly higher than that of the Pos group ( P <0.05). On the 2nd day and the 4th day of gavage, the periorbital pain threshold of HG group was significantly higher than that of HD group and HZ group ( P <0.05), but there was no significant difference between DG group and GG group and their corresponding low and middle dose groups. And on the first day of intragastric administration, the periorbital pain threshold of HG group was higher than that of DG group, and on the fourth day of intragastric administration, the threshold of HG group was higher than that of GG and DG groups ( P <0.05). On the 6th day of intragastric administration, the degree of improvement in each treatment group was relatively similar, with no significant difference.

See Figure 2. Compared with the Con group, the number of head scratching in the Mod group was significantly increased on the 1st, 3rd, and 5th day of intragastric administration in migraine-forming rats ( P <0.05 or P <0.01); compared with the Mod group, the Pos group and each treatment The head scratching times of the two groups were improved to varying degrees ( P <0.05 or P <0.01). Compared with Pos group, on the 5th day of gavage, the number of head scratching in HG group, DG group, and GG group was significantly lower than that of Pos group ( P <0.05); on the first day of gavage, the number of head scratching in HG was significantly lower than that in HD and HZ ( P <0.05). Paeonol and glycyrrhizic acid high-dose treatment group had no significant difference in behavioral improvement compared with low and middle-dose groups, but there was a better improvement trend. The degree of improvement in the three high-dose groups was similar, and on day 1 and day 5 of gavage, HG had a better improvement trend.

Animal experiments have proved that paeonol, glycyrrhizic acid and the combination of the two have a good effect on improving the periorbital pain threshold and behavior of migraine rats, and can effectively relieve and treat the symptoms of migraine. Since the genome sequences of rats and humans are highly similar, it can be considered that it also has the same or similar effect on the treatment of human migraine.

in conclusion:

The combined use of paeonol and glycyrrhizic acid has a better effect on improving the periorbital pain threshold; its effect is equal to or better than that of the positive drug group.

At high doses (paeonol 400mg·kg ^-1 ·d ^-1 , glycyrrhizic acid 200mg·kg ^-1 ·d ^-1 ), there is a better tendency to improve migraine behavior, and Dan The combination of corticosterol and glycyrrhizic acid is better.

Example 2: Intervention on Pathological Damage of Midbrain Histopathology Caused by Migraine

See Figure 3, the midbrain tissue, neuron cells, and tissue structure of the rats in the Con group were normal; the neuron cell axes and spots in the midbrain tissue of the rats in the Mod group were reduced, and many neurons were degenerated, shrunken, and deeply stained; the Pos group and each The pathological damage of the midbrain tissue of the rats in the administration group was improved to varying degrees.

Conclusion: Paeonol, glycyrrhizic acid and their combination can improve the pathological damage of midbrain tissue caused by migraine.

Example 3: Intervention on migraine biomarkers

See Figure 4, after 7 days of treatment, compared with the Con group, the 5-HT of the cerebrospinal fluid of the rats in the Mod group decreased significantly, while the HIS increased significantly ( P <0.01); compared with the Mod group, the Pos group, paeonol, glycyrrhizic acid And the combination of the two has a callback effect ( P <0.01), and each treatment group has different degrees of improvement ( P <0.05 or P <0.01). The up-regulation effect of paeonol, glycyrrhizic acid and combination groups on 5-HT was significantly higher in the high-dose group and middle-dose group than in the Pos group ( P <0.05 or P <0.01). The improving effects of paeonol, glycyrrhizic acid and their combination groups on 5-HT and HIS tended to increase with the increase of doses, and the improvement effects of high doses were significantly higher than those of corresponding low and middle doses ( P <0.05). For HIS, the adjustment effect of HG group was better than that of DG group ( P <0.05), and it had a better regulation trend than GG group.

in conclusion:

Paeonol, glycyrrhizic acid and the combination of the two have obvious regulatory effects on cerebrospinal fluid biomarkers, which can improve the levels of 5-HT and HIS in cerebrospinal fluid, thereby alleviating and treating migraine.

Judging from the changes of cerebrospinal fluid biomarkers, the high-dose group (paeonol 400mg·kg ^-1 ·d ^-1 , glycyrrhizic acid 200mg·kg ^-1 ·d ^-1 ) had a better regulatory effect; HIS results showed that paeonol The combination of the two with glycyrrhizic acid is better than a single one.

Example 4: Intervention of Trigeminal Ganglion Dysfunction in Migraine

See Figure 5, the rats in the Con group, the Mod group, the Pos group, and the treatment groups had no obvious morphological differences, the distribution of neurons and nerve fibers was normal, the anatomical structure was complete, and no obvious organic lesions were seen, thus proving that migraine is a trigeminal disease. Caused by abnormalities in ganglion function.

See Figure 6, qRT-PCR results showed that compared with the Con group, the transcript levels of TRPV1 and CGRP in the trigeminal ganglion of rats in the Mod group were significantly increased ( P <0.01); compared with the Mod group, the Pos group, paeonol , glycyrrhizic acid and the combination of the two all have down-regulation effects ( P <0.05 or P <0.01).

See Figure 7, the results of Western Blot showed that, taking β-Actin as the protein expression level reference, compared with the Con group, the protein expression levels of TRPV1 and CGRP in the trigeminal ganglion of the rats in the Mod group were significantly increased ( P <0.01); Compared with the group, the Pos group, paeonol, glycyrrhizic acid and the combination of the two all had a down-regulation effect ( P <0.05 or P <0.01).

See Figure 8, ELISA results showed that compared with the Con group, the plasma IL-1β, TNF-α, and IL-10 in the Mod group increased significantly ( P <0.01); compared with the Mod group, the Pos group, paeonol, licorice Acid and the combination of the two had down-regulation effects on IL-1β and TNF-α ( P <0.05 or P <0.01), but up-regulation effects on IL-10 ( P <0.05 or P <0.01). Paeonol, glycyrrhizic acid and the combination group had a tendency to increase the down-regulation of IL-1β and TNF-α and the up-regulation of IL-10 with the increase of dose. Compared with DG group and GG group, HG group has a better tendency to improve IL-1β and TNF-α. Among them, the regulating effect of HG group on TNF-α was significantly better than that of DG group ( P <0.05).

in conclusion:

Paeonol, glycyrrhizic acid and their combination can down-regulate the gene transcription and protein expression levels of TRPV1 and CGRP in migraine rats, down-regulate the expression of plasma pro-inflammatory factors IL-1β, TNF-α and up-regulate the expression of anti-inflammatory factor IL-10 level. The high dose group (paeonol 400mg·kg ^-1 ·d ^-1 , glycyrrhizic acid 200mg·kg ^-1 ·d ^-1 ) had better effect.

Compared with DG group and GG group, HG group has a better tendency to improve IL-1β and TNF-α. Among them, the regulating effect of HG group on TNF-α was significantly better than that of DG group ( P <0.05).

Embodiment 5: pharmaceutical dosage form and preparation method

The present invention adopts the administration method through the gastrointestinal tract. In actual operation, the administration method can be improved, and it is not limited to the intravenous injection administration method, the subcutaneous injection administration method, the nasal mucosa administration method and the like.

What the present invention adopts is the liquid dosage form that is used for oral administration, in actual operation, can improve dosage form, not limited to liquid dosage form, solid dosage form and semisolid dosage form etc., as capsule, pill, tablet, ointment, patch, injection, drop Pills, sprays, etc.

Regardless of the administration method or dosage form, it only affects the absorption site, speed and time of the drug, and it needs to enter the systemic circulation to exert its effect, all based on the exact research results that paeonol and glycyrrhizic acid have therapeutic effects on migraine. Therefore, different administration methods or dosage forms are within the protection scope of the present invention.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (6)

1. The combination of paeonol and glycyrrhizic acid is used as the only active ingredient in the preparation of a medicine for treating or improving nitroglycerin-induced migraine, and the medicine is composed of paeonol, glycyrrhizic acid, and pharmaceutically acceptable auxiliary materials. Composition, the mass ratio of described paeonol and described glycyrrhizic acid is paeonol: glycyrrhizic acid=2. 2. application as claimed in claim 1, is characterized in that, described medicine can: Treat or improve migraine caused by abnormal function of trigeminal ganglion; Treating or improving pathological damage of midbrain tissue caused by migraine, the pathological damage of midbrain tissue includes reduction of neuron cell axis and spots and/or degeneration of neuron folds; Up-regulate the content of 5-HT in cerebrospinal fluid, and/or down-regulate the content of HIS in cerebrospinal fluid; Downregulation of TRPV1/CGRP gene transcription or protein expression; Down-regulate the expression of pro-inflammatory factor IL-1β and TNF-α, and/or up-regulate the expression of anti-inflammatory factor IL-10. 3. The application according to claim 1, characterized in that the dosage of paeonol is 100-400 mg·kg ^-1 ·d ^-1 . 4. The application according to claim 1, characterized in that the dosage of the glycyrrhizic acid is 50-200 mg·kg ^-1 ·d ^-1 . 5. The application according to claim 1, wherein the medicine is an oral preparation, an injection preparation or an external preparation. 6. The application according to claim 1, characterized in that, the administration object of the drug is a vertebrate.

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