CN111920835A - Application of medicinal preparation in preparing medicament for treating senile dementia - Google Patents

Abstract

The invention relates to an application of a medicinal preparation in preparing a medicament for treating senile dementia, wherein the medicinal preparation comprises the following raw material medicaments in part by weight: the invention aims to provide a new clinical treatment application of a medicament for treating senile dementia, expand the clinical application indication range of the medicament and provide a new medicament selection for senile dementia patients, wherein the ginkgo leaf extract is 2-4 parts, and the honey ring powder is 80-120 parts.

Description

Application of medicinal preparation in preparing medicament for treating senile dementia

Technical Field

The invention relates to an application of a medicinal preparation in preparing a medicament for treating senile dementia, belonging to the field of new clinical application of medicaments.

Background

Senile dementia can be classified into Alzheimer's Disease (AD) dementia, vascular dementia and the coexistence of them. However, AD is more common clinically, and most of brain dysfunction caused by organic injury of the brain degrades and intelligently damages the abilities of memory, understanding, judgment and control and the like, thereby seriously affecting the daily life of patients. The prevalence rate of the senile dementia in China is about 2% -5%, but the senile dementia tends to gradually increase with the age. At present, the annual new onset is about 1 percent, and the death rate of the senile dementia is the 4 th death rate of common diseases, and is only second to cardiovascular and cerebrovascular diseases, tumors and cerebral apoplexy. With the further development of the aging of the social population structure in China, the proportion of the elderly is getting larger, so that the prevention and treatment of the senile dementia disease become a serious social problem.

Based on the existing pathogenesis of the senile dementia, different approaches of drug treatment are proposed. In the 70 s the field was mainly focused on the relationship of acetylcholine to AD, while in the 80 s the effects of cholinesterase inhibitors on AD were mainly studied, and in the 90 s acetylcholine receptor agonists were implicated, including estrogens, anti-inflammatory analgesics, antioxidants and free radical scavengers, drugs inhibiting amyloid deposition, and calcium antagonists. But due to AD at the onset, the degree of dementia, impairment of brain function, genetic factors and differences in responses that exist between individuals. At present, the medicines for treating senile dementia comprise tacrine, donepezil, galanthamine, huperzine A, metabolism promoting medicines, antioxidants, amyloid protein resistance and the like, but the medicines can generate larger toxic and side effects of liver and kidney after being taken for a long time. At present, no low-toxicity and effective specific medicine exists clinically. And the traditional Chinese medicine has obvious advantages in treating the diseases.

The pharmaceutical preparation is prepared by taking ginkgo honey ring as a composing prescription raw material, wherein the oral solution preparation is a core variety of an qiqigong & ltu & gt Tianyin pharmaceutical company, and the approved literature numbers of the pharmaceutical preparation are as follows: the national drug standard H20013079, the national drug standard number executed today is: WS 1-XG-004-2001. The medicine is prepared from folium Ginkgo extract and Armillaria mellea, and can be used for treating coronary heart disease, angina pectoris, ischemic cerebral blood diseases, and relieving heart and cerebral ischemia. The product has effects in dilating coronary artery and cerebral vessels, increasing coronary blood flow and cerebral blood flow, improving microcirculation of cardiac and cerebral tissues, inhibiting platelet aggregation, and resisting thrombosis. Since the product comes into the market, the product has obvious and reliable clinical curative effect and is well received by the general patients. The applicant has made layout 3 patent applications, wherein the patent numbers are respectively: 200610078022.5, 201721437087.4, 201930114171.0 and the subject matter of the above patent protection: the preparation method of the ginkgo honey-prepared Armillaria mellea oral preparation, the improvement of medicine production equipment, the external packing box of the medicine and the like. Subsequently, the applicant conducted a systematic search and grooming of the prior art documents to which the pharmaceutical preparation relates, which found that the diseases in which the drug is clinically applied involved: coronary heart disease, angina pectoris, acute cerebral infarction, sudden deafness patients, hypertension, acute coronary syndrome, coronary atherosclerotic heart disease, thoracic obstruction and cardialgia, tympanitis, ischemic stroke, senile hypertension, multiple cerebral infarction, etc., and the treatment diseases mostly belong to the cardiovascular and cerebrovascular field. In a large number of clinical use processes, the product is unexpectedly found to have remarkable treatment effects on senile dementia patients besides treating cardiovascular and cerebrovascular diseases. Therefore, the inventor develops the pharmaceutical research of the prescription composition and various oral dosage forms based on the oral liquid dosage form so as to expand the clinical treatment range of the variety.

Disclosure of Invention

The invention aims to provide a new application of a medicinal preparation, and particularly relates to an application of the medicinal preparation in preparing a medicament for treating senile dementia.

The application of a medicinal preparation in preparing a medicament for treating senile dementia comprises the following raw material medicaments in part by weight: 2-4 parts of ginkgo leaf extract and 80-120 parts of honey ring powder.

Preferably, the pharmaceutical preparation comprises the following raw material medicines in parts by weight: 3 parts of ginkgo leaf extract and 100 parts of honey ring powder.

The preparation method of the pharmaceutical preparation comprises the following steps:

(1) and (3) extracting:

putting the honey-soaked doughnut powder into a bag decocting bag, placing the bag decocting bag into an extraction tank, adding water into the extraction tank for extracting for 1-3 times, and collecting an extracting solution;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) and (4) uniformly mixing the honey ring powder extract prepared in the step (4) and the ginkgo leaf extract to obtain a mixed extract, and adding pharmaceutically common auxiliary materials to prepare various oral preparations.

The dosage form of the pharmaceutical preparation can be prepared into oral liquid, granules, capsules, tablets and dropping pills.

Oral liquid preparation: mixing the obtained total mixture (semen Ginkgo extract and Armillaria mellea), appropriate antiseptic (such as benzoic acid, parabens, sorbic acid, etc.), cosolvent (sorbitol ester-80 or sodium dodecyl sulfate, etc.), and sweetener (sucrose, fructose, starch sugar, sugar alcohol, oligofructose, isomaltulose, aspartame or aspartame, etc. as medicinal adjuvants).

And (3) tablet preparation: mixing the obtained total mixture (semen Ginkgo extract and Armillaria mellea), appropriate amount of filler (lactose, microcrystalline cellulose, mannitol, starch, dextrin, etc.), binder (starch, dextrin, carboxymethyl cellulose, etc.), disintegrant (such as carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricant (pulvis Talci, magnesium stearate, etc.), mixing, granulating, and making into tablet.

Granules: mixing the obtained total mixture (semen Ginkgo extract and Armillaria mellea) with appropriate amount of dextrin and sucrose, granulating, drying, and making into granule.

And (3) capsule preparation: adding appropriate adjuvant into the obtained total mixture (semen Ginkgo extract and Armillaria mellea), granulating, and making into hard capsule.

Dripping pills: mixing the obtained total mixture (semen Ginkgo extract and Armillaria mellea), melting with proper matrix (the adjuvants can be selected from stearic acid, PEG4000, PEG6000, sodium stearate, poloxamer, glycerol, gelatin, etc.), stirring, making into dripping pill.

Senile dementia is a chronic brain hypofunction disease, and belongs to the categories of dull disease, amnesia, insanity and the like in traditional Chinese medicine. The etiology and pathogenesis of the disease are as follows: the disease is located in the brain, and the marrow is the cause of brain loss, leading to the loss of the brain spirit. Its root cause is related to the dysfunction of the heart, spleen, kidney and other viscera. The disease is characterized by deficiency in origin, marked excess, and intermingled deficiency and excess. Senile dementia has no other than phlegm, deficiency and stasis, and most patients have malnutrition of brain and marrow due to dysfunction of viscera, insufficiency of qi and blood circulation, and inability of kidney essence to generate essence and blood, or stagnation of qi, blood and phlegm leading to obstruction and resuscitation. In the elderly, the deficiency of the kidney-qi, essence and blood, brain and sea are caused by deficiency of the liver and kidney, and the absence of the primary cause of the mental retardation is dull. Or dysfunction of the zang-fu organs, disorder of qi and blood circulation, phlegm turbidity in the body and blood stasis which cannot be removed in time due to deficiency or disturbance of the zang-fu organs, which may turn into turbid-toxin after long-term heat transformation. When blood stasis and toxicity damage brain collaterals, it can cause obstruction of brain orifices and mental disorder, resulting in dementia.

The medicine is widely applied to patients with cardiovascular and cerebrovascular diseases clinically, but the clinical symptoms of dizziness, amnesia, expression apathy and the like of the patients with senile dementia are obviously relieved after the patients taking the oral preparation of the medicine are discovered accidentally. According to the principle of traditional Chinese medicine treatment and the formula compatibility of medicines, further attempts are made to explain the theoretical basis of the traditional Chinese medicine treatment for treating the senile dementia by the medicines.

The folium ginkgo extract is selected from folium ginkgo extract in the formula, wherein the folium ginkgo extract has a characteristic taste, and is sweet, bitter and astringent in taste. They enter heart and lung meridians. Has effects of promoting blood circulation, removing blood stasis, dredging collaterals and relieving pain. Can make qi and blood pass, eliminate blood stasis in the body of a patient, smooth blood circulation can make food essence slightly distributed in the brain, and gradually eliminate the dementia symptom. The research finds that the effective components of the ginkgo biloba extract not only contain flavonoids such as kaempferol, quercetin, rutin, and quercitrin, but also contain components such as bilobalide A, bilobalide C, catechin terpene lactones, and bilobalide. The modern pharmacological research shows that the composition has the effects of dredging and improving cerebral circulation on cerebral blood flow, regulating cerebral lipid metabolism and protecting brain cells. In the recipe, honeydew powder is also added, which is sweet and mild in nature and taste. Meridian tropism and liver meridian tropism. The Armillaria mellea powder contains various active ingredients, sesquiterpene compounds, purine compounds, various essential amino acids, Armillaria mellea polysaccharide, etc. Has effects in regulating qi and blood, tonifying kidney, and promoting salivation. And can invigorate spleen and kidney, and promote the generation of qi and blood to nourish brain and marrow. Can nourish blood and vessels and pass body fluid, so as to nourish viscera and tranquilize mind, nourish brain and sea, refresh mind, and relieve amnesia and insomnia. The long-term use of the medicine is discovered accidentally, and the medicine has excellent clinical curative effect on treating senile dementia and other symptoms.

In order to illustrate the clinical treatment effect of the medicine on senile dementia, the questionnaire mode is adopted for investigation, and the investigation is based on the marketed oral dosage form, and the situation is illustrated as follows:

a certain patient, female, is 68 years old, has cardiovascular and cerebrovascular diseases for more than 2 years, is accompanied by senile dementia, and has amnesia, anxiety, hypomnesis, reaction retardation, lethargy, lusterless complexion, cognitive function reduction, motor proficiency reduction, and slight symptoms such as three-drop four, unclear thinking and the like, which cause the gradual reduction of the daily life capacity of the patient. Patients often take oxazepam, alprazolam and other medicaments, but the effect is not ideal after long-term administration, and the patients often have the side effects of somnolence, dizziness, hypodynamia and the like. The medicine (ginkgo honey-ring oral liquid) is taken by a patient to treat the cardiovascular and cerebrovascular diseases for many years, and the taking mode is as follows: the medicine is orally taken for 3 times a day by 10ml once, after 6 months of medicine treatment, the family members of patients complain that the symptoms of forgetfulness, anxiety, lethargy and the like are obviously improved when the cardiovascular and cerebrovascular diseases are stably controlled, and the memory of the patients is gradually recovered after the patients continue to take the medicine for 6 months. Compared with the prior art, the thinking judgment and the analysis capability of the medicine are obviously improved, and all symptoms of the medicine gradually disappear.

The medicine preparation has the beneficial effects that:

in order to verify the pharmacological effect of the drug in treating chronic senile dementia and design experiments of senile dementia rat learning memory and hippocampal monoamine neurotransmitters, the inventor records the capability of rats in an EL (rat escape latency) detection model of rats 1d, 3d and 5d through a rat Morris water maze experiment. Pharmacological experiment results show that the drug group can obviously shorten the Escape Latency (EL) of rats, so that the drug can improve the learning and memory abilities of the rats with the senile dementia. The drug group can also obviously increase the contents of norepinephrine, dopamine and 5-hydroxytryptamine in the hippocampus of rats, which also reveals that the drug (ginkgo biloba armillaria oral solution) can improve the learning and memory abilities of the senile dementia rats.

The medicinal preparation has influence on the brain protection effect of the rat focal cerebral ischemia reperfusion injury, and the test result shows that the medicament has reduced activity on rat TNOS (nitric oxide synthase) and has significant difference compared with a model group; the pharmaceutical preparation can achieve the level of NO (nitric oxide), has significant difference compared with a model group, and has insignificant influence on the activity of NOS (nitric oxide synthase).

According to the experimental results of the influence of the composition on neurotransmitters in brain tissues, the composition can remarkably increase the level of NE (epinephrine) in the brain of a rat, and the levels of 5-HT (5-hydroxytryptamine) and 5-HIAA (5-oxindole acetic acid) are not obviously changed; the NE level in the brain is remarkably increased, and the 5-HIAA level is remarkably reduced. Studies have shown that learning and memory ability is closely related to the levels of monoamine neurotransmitters in the brain and hippocampus. NE, and 5-HT belong to the same central monoamine neurotransmitters, which play an extremely important role in regulating learning and memory behavior, and the decrease of the content thereof can cause the decline of learning and memory functions. The ginkgo leaf extract and the honey ring powder contained in the medicine have synergistic effect, promote the repair of damaged brain cells and neurons, improve the learning and memory capacity of senile dementia rats and improve monoamine neurotransmitter metabolism in hippocampus.

The pharmaceutical preparation can also reduce the area of the cerebral infarction area of a rat, and the brain infarction area of the rat occasionally has the swelling of endothelial cells, nerve cells and astrocytes, has inflammatory cell infiltration phenomenon, and has better lesion degree improvement. The experimental effect is the pharmacological basis for treating the senile dementia, and provides a theoretical basis for developing the clinical treatment of the senile dementia by the medicine in the later stage.

The present invention is further illustrated by the following exemplary embodiments in order that the practice of the invention may be more fully understood. The beneficial therapeutic effects of the drug are demonstrated below and by pharmacodynamics.

Drawings

The accompanying drawings, which are described herein and form a part of the specification, illustrate embodiments of the present invention and, together with the description, serve to explain the present invention and not to limit the scope of the invention, wherein the medicament is a total mixture of extracts from 3 parts of ginkgo biloba leaf extract and 100 parts of Armillaria mellea powder (hereinafter referred to as the total mixture of extracts from the formulation of the materials).

FIG. 1-Effect of the drug of the invention on serum TNOS in MCAO rats;

FIG. 2-Effect of the drug of the invention on serum iNOS in MCAO rats;

FIG. 3-Effect of the agents of the invention on MCAO rat serum NO;

FIG. 4-Effect of the drug of the invention on NE in MCAO rat brain tissue;

FIG. 5-Effect of the drug of the invention on MCAO rat brain tissue 5-HT and 5-HIAA;

FIG. 6-results of HE staining of MCAO rat brain tissue with the drug of the present invention, wherein: a-sham operation group, B-model group, C-gold groups, D-ginkgo biloba extract group, E-gastrodia elata armillaria mellea group, F-gastrodia elata armillaria powder group, G-ginkgo biloba armillaria oral solution high-dose group and H-ginkgo biloba armillaria oral solution low-dose group. HE staining, 40X.

Detailed Description

In order to better understand the invention, the following examples are presented to illustrate its new use in the pharmaceutical field. The following experiments are intended to illustrate the invention and not to limit it.

EXAMPLE 1 preparation of oral liquid solution of pharmaceutical preparation

(1) And (3) extracting:

putting 100Kg of honeydew ring powder into a bag-decocting bag, wherein the filling amount is 1/5-1/2 of the bag-decocting bag, and adding water into a multifunctional extraction tank.

(2) And concentrating:

mixing the extracting solutions, filtering, concentrating the filtrate until the relative density is 1.14-1.15, and obtaining the ointment.

(3) And alcohol precipitation:

pumping the cooled extract into an alcohol precipitation tank, accurately adding ethanol while stirring to make the ethanol content of the liquid medicine reach 75%, refrigerating, and standing.

(4) And ethanol recovery:

and extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, recovering ethanol, and concentrating to a relative density of 1.15-1.17.

(5) And liquid preparation:

a. preparation of ginkgo leaf extract solution

Dissolving cosolvent sodium dodecyl sulfate 4Kg in pure water to 75Kg, heating to 80 deg.C, slowly adding weighed folium Ginkgo extract 3Kg while stirring, and stirring at constant temperature to completely dissolve to obtain folium Ginkgo extract solution.

b. Preparation of sodium benzoate solution

Adding 400L of purified water into a diluting preparation tank, slowly adding 4kg of sodium benzoate, and stirring until the sodium benzoate is completely dissolved for later use.

c. Operation of liquid preparation

Adding 180kg of extract into a concentration tank, diluting with pure water to 500L, stirring, heating, stirring for 30 min, standing, filtering while hot, pumping the filtrate into a dilution tank, adding 6kg of solution containing folium Ginkgo extract and 10kg of stevioside or aspartame, stirring while adding, supplementing purified water to 1500L, and standing. Slowly adding purified water to 2000L, and stirring to obtain oral liquid.

EXAMPLE 2 preparation of capsules

1) And (3) extracting:

putting 110Kg of honey ring powder into a bag decocting bag, putting the bag decocting bag into an extraction tank, adding water into the extraction tank for extracting for 3 times, and collecting an extracting solution;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) mixing the components

And then 4Kg of ginkgo leaf extract is taken, and after being uniformly mixed, a proper amount of starch and calcium hydrophosphate are added, and the mixture is granulated and encapsulated to prepare hard capsules.

EXAMPLE 3 preparation of dropping pills

(1) And (3) extracting:

putting 120Kg of honey ring powder into a bag decocting bag, putting the bag decocting bag into an extraction tank, adding water into the extraction tank for extracting for 1-3 times, and collecting the extracting solution;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) dropping method

Adding PEG4000 and PEG6000 into the extract obtained from the step (4) and 2Kg of folium Ginkgo extract, heating and stirring, dripping, and making into dripping pill.

EXAMPLE 4 preparation of granules

1) And (3) extracting:

putting 80Kg of honey ring powder into a bag decocting bag, placing the bag decocting bag into an extraction tank, adding water into the extraction tank for extracting for 1-3 times, and collecting the extracting solution;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) and granulating

Taking the extract prepared from the honeydew powder (4) and 4Kg of ginkgo biloba extract, adding a proper amount of dextrin, sucrose and lactose, mixing uniformly, granulating, drying, and preparing into granules.

EXAMPLE 5 preparation of tablets

1) And (3) extracting:

putting 90Kg of honeydew ring powder into a bag decocting bag, placing the bag decocting bag in an extraction tank, adding water and extracting for 1-3 times, and collecting the extract;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) and tableting

And (3) taking the extract prepared from the honeydew powder (4) and 3Kg of ginkgo leaf extract, uniformly mixing, adding a proper amount of sodium carboxymethyl starch and magnesium stearate, uniformly mixing, granulating, and pressing into tablets.

Example 6 pharmacological Activity test of the drug of the present invention

1. Influence on learning and memory of senile dementia rats and hippocampal monoamine neurotransmitters

1.1 sources of Material

1.1.1 Experimental animals

Selecting 40 healthy adult SD rats with SPF grade, weight (220 +/-20) g and half of male and female, carrying out the experiment in 5-6 months in 2018.

1.1.2 Main instruments and reagents

The Morris water maze; an enzyme linked immunosorbent assay (ELISA) detector; 25-35 of Abeta; the medicine is provided by ginkgo honey ring oral solution (qiqiqiu Tianyin pharmaceutical Co., Ltd.); naofukang piracetam tablets; NE test kit, dopamine DA test kit, 5-HT test kit.

1.2 Experimental methods

1.2.1 model preparation and administration

After being raised for 1 week in accordance with standard environment adaptability, each experimental rat is randomly divided into a blank control group, a model group, a positive drug control group and a drug group of the invention, 4 groups are counted, 10 rats in each group are anesthetized by intraperitoneal injection of chloral hydrate (0.3ml/100g) with the drug concentration of 10%, the rats are fixed on a brain stereotaxic apparatus, a Paxinos rat brain stereotaxic map is referred, the distance from the Bergman point to the back is about 3.4mm, the distance from the midline is about 2.1mm, bone drills are used for drilling through bilateral skull of the rats, a micro-injector is used for vertically inserting needles for 4-5mm under the guidance of the brain stereotaxic apparatus, the model group and the drug administration group are slow, 4 mul of A beta 25-35 solution injected into bilateral hippocampal tissues is slow (2 mul on each side), and the control group is injected with equal volume of sterile physiological saline. The positive control medicine of brain rehabilitation group and the medicine group of the invention are respectively administered to the total mixture of extracts of the raw material medicine formula of brain rehabilitation (piracetam tablets) and ginkgo honey ring on the next day after operation, and are diluted by adding a proper amount of normal saline for preparation, and then are irrigated by stomach, the administration dosage is respectively 200mg/kg and 300mg/kg, and the air-white control group and the model group are administered with 2ml/100g of normal saline every day for irrigating for 1 time/day and continuously for 20 days. The body weight was recorded once a week for each group of animals and the dose was adjusted. (the clinical dose of human is Xmg/Kg, converted into the dose of rats: (Xmg/Kg × 70Kg × 0.018/200g is 6.3 Xmg/Kg), and the equivalent dose of rats is 6.3 times of that of human body by the conversion of the formula, the clinical administration dose of the marketed oral dosage form of the drug of the invention in the application of treating cardiovascular and cerebrovascular diseases is about 300mg/Kg by the conversion of the administration dose of rats, and the conversion of the administration dose of the positive control drug Naokang is the same as the same).

1.2.2 detection of escape latency in rats

After the rat learning and memory behavior test medication is finished, the learning and memory capacity of the rat is detected by recording EL (rat escape latency) of 1d, 3d and 5d of the rat by adopting a Morris water maze test method.

1.2.3 detection of NE, DA, 5-HT content in Hippocampus tissue of rat

After the Morris water maze test is carried out on a rat, the rat is cut off and killed, the brain is rapidly taken out on a frozen operating table, cerebral hemispheres on two sides are separated, a hippocampal tissue is taken out, an ELISA method is adopted according to the requirements of a kit, the learning and memory capacity of the rat is measured, and the content of Noradrenaline (NE), Dopamine (DA) and 5-hydroxytryptamine (5-HT) in the hippocampal tissue is detected. The administration dosage of the experimental animal is converted by referring to an equivalent dosage ratio method converted from the surface areas of human and animal intermediates.

1.3 statistical treatment

Through the positioning navigation test data result in the designed Morris water maze measurement, the variance analysis of repeated measurement data is carried out by adopting SPSS18.0, and the single-factor variance analysis is adopted by other experimental indexes. All experimental data are expressed in x ± s, and differences of P <0.05 are statistically significant.

1.4 Experimental results:

table 1-comparison of rat EL for each experimental group (x ± s, n ═ 10)

Note: compared with the blank control group, the composition of the composition,^■■P＜0.01，^■P＜0.05。

from the experimental results shown in table 1, the model group rats EL were significantly prolonged, and the Escape Latency (EL) of the rats on days 3 and 5 of the positive drug brain rehabilitation group and the drug group of the present invention was significantly shortened compared to the blank control group, and the differences thereof were statistically significant (P < 0.01). The difference of EL between the brain rehabilitation group and the drug group of the invention at different time is not statistically significant (P is more than 0.05).

TABLE 2 Effect on NE, DA, 5-HT of hippocampus in rats with Alzheimer's disease (x. + -.s, n 10. mu.g/g)

Note: comparing with a blank control group; compared with the blank control group, the composition of the composition,^■■P＜0.01，^■P＜0.05。，

as can be seen from the results shown in Table 2, compared with the control group, NE, DA and 5-HT in the hippocampus of the model group are obviously reduced, and the difference has statistical significance (P < 0.01); NE, DA and 5-HT in the positive control brain rehabilitation group and the drug group of the invention are obviously increased, and the difference has statistical significance (P <0.05 or P < 0.01); compared with the drug group of the invention, the positive control Naokukang group has no statistical significance on the difference when comparing the contents of norepinephrine, dopamine and 5-hydroxytryptamine in rat hippocampus (P is more than 0.05).

Example 7 brain protective Effect on rat focal cerebral ischemia reperfusion injury

1. Materials and instruments

1.1 drugs and reagents

1.1.1 test samples

(1) Ginkgo honey-ring oral solution, 10 ml/piece, batch number: 160312; (2) ginkgo biloba leaf extract, batch number: 160402; (3) gastrodia elata armillaria mellea powder, batch number: 20151027, respectively; (4) gold multi (ginkgo biloba extract tablet), 40 mg/tablet, produced by the german wilama schobenberg pharmaceutical factory, batch number: 5260613, respectively; (5) rhizoma gastrodiae armillaria mellea tablet, 0.25 g/tablet, produced by Jiangsu Shenhua pharmaceutical Co., Ltd, batch number: 20160602. (1) all the medicines are provided by Qiqin Ganjin pharmacy Co., Ltd, and are stored in a dry way; (4) and (5) is a commercially available medicine.

1.1.2 Chlorination-2, 3,5-Triphenyltetrazolium Chloride (TTC), national drug group chemical reagent company, 0.2mol/L PBS (pH 7.2) to 1.2%, low temperature and light-proof storage.

1.1.3 chloral hydrate, national pharmaceutical group chemical Co., Ltd., batch No. 20131027.

1.1.4. The kit comprises: SOD, NOS, GSH-PX, MDA, NO kit, provided by Nanjing institute of bioengineering, and having the batch numbers of: 20161216, 20161216, 20161214, 20161217, 20161216.

1.2 Experimental Equipment

1.2.1 Nylon wire, 2432-100, Beijing Saedong Biotechnology Ltd.

1.2.2 multimedia color pathology image-text analysis system (MPIAS-500).

1.2.3 ultrasonication apparatus, model VCX150PB, SONICS, USA.

1.2.4 ultracentrifuge (HITACHI 55P-72, Japan).

1.2.5 syringe type microporous membrane filter: water system (0.22 μm), Tianjin Tengda filter plant product.

1.3 animals SD rat, male, 220-240 g, commercially available.

2 method of experiment

2.1 test grouping

The surgery model group comprises 18mg/kg of gold, 9mg/kg of ginkgo biloba extract (silver extract), 600mg/kg of gastrodia elata armillaria mellea tablet, 300mg/kg of gastrodia elata armillaria powder (armillaria mellea powder), 618mg/kg of ginkgo biloba armillaria oral solution (high dosage of silver honey) and 309mg/kg of ginkgo biloba armillaria oral solution (low dosage of silver honey). Total 8 groups, animals were randomly assigned to each group.

2.2 model building

A4% chloral hydrate anesthetized rat (1ml/100g) is adopted, the body temperature of the animal is maintained at 37 ℃ by a constant-temperature electric blanket, the animal is fixed in a supine position, skin and subcutaneous tissues are cut along the midline of the neck, a right Common Carotid Artery (CCA), an Internal Carotid Artery (ICA) and an External Carotid Artery (ECA) are separated, the ECA and the CCA are ligated, the ICA distal end is clamped, a V-shaped incision is made at the bifurcation of the ECA and the ICA, a nylon wire plug with the diameter of 0.24mm and the diameter of 0.24mm is inserted, the nylon wire plug is heated into a smooth spherical shape, an artery clamp is loosened, a nylon wire is continuously inserted until the nylon wire is slightly withdrawn after slight resistance, the insertion depth of the wire plug is about 20 +/-2 mm, the head end of the wire penetrates through the middle cerebral artery starting part, about 1cm is left outside the nylon wire, the incision of. After 1.5h, gently pulling the thread head to a slight resistance, indicating that the head end of the thread is pulled out of the internal carotid artery, realizing reperfusion, and cutting off the outer section of the thread-inserting skin. Indication of model success: after the animals are anesthetized and conscious, the Horner's sign of ischemia and the hemiplegia mainly treating forelimbs on the contralateral side appear, the animals without the symptoms are discarded, and the rest animals are returned to cages for feeding and kept at the body temperature.

2.3 administration of drugs

Each test drug was administered via the duodenum after insertion of a wire plug (ischemia). Except for the ginkgo biloba armillaria oral solution 618mg/kg group, each administration group was administered with the test drug at a volume of 3mL/kg, and the sham operation and model group was administered with NS at a volume of 3 mL/kg.

2.4 detection index

2.4.1 serum TNOS, iNOS and NO level measurements were performed as described in the instructions.

2.4.2 detection of levels of monoamine neurotransmitters in the brain

A certain amount of brain tissue was weighed and 1ml of working solution (0.15mol/L HCLO4) was added per 100mg of brain. Homogenizing in ice bath (11000rpm/min) for 20s, ultracentrifuging at 0-4 deg.C (14000rpm/min) for 20min, collecting supernatant, filtering with 0.22 μm (water system) syringe type microporous membrane filter, packaging, storing in refrigerator at-80 deg.C, and measuring the concentration the next day.

The high-efficiency liquid phase mobile phase composition adopted in the content determination comprises: 90mmol/L of sodium dihydrogen phosphate, 50mmol/L of citric acid, 1.7mmol/L of sodium octyl sulfonate and 0.05 mu mol/L of ethylene diamine tetraacetic acid tetrasodium salt (EDTA). The solution was filtered through a 0.22 μm aqueous membrane, and ACN (10%) was added to the filtrate. Flow rate of mobile phase was 0.6mL/min, working electrode 1: -150mV, working electrode 2: 450mV, working electrode 3: 500mV, working electrode 4: 550 mV.

2.4.3 pathological examination

And (3) observing morphological indexes such as a cerebral tissue infarction focus, a neuron structure, lesion degree, microglial cell proliferation degree and the like of the rat by adopting HE staining under a light mirror.

2.5 statistical analysis

Experimental animals which died, were in the subarachnoid hemorrhage and were not ischemic were excluded, and each group of data was expressed as mean ± SD. Except for the behavioral data, statistics were performed using the SPSS16.0 for windows statistics software package, with one-way ANOVA. The neurological scores were counted in the Kruskal-wallis test and Mann-Whitney test in the SPSS16.0 for windows statistics software package nonparametric test.

3 results of the experiment

3.1 Effect on serum TNOS, iNOS, NO

After ischemia reperfusion, iNOS activity is increased and NO level is increased in the model group, and the significant difference (P < 0.05-0.01) is obtained compared with that in a sham operation group; after administration, the iNOS activity in the serum of rats with 18mg/kg more control drug gold is reduced, and the iNOS activity has significant difference (P < 0.05-0.01) compared with that of a model group; however, the pharmacological action of the ginkgo biloba extract group with the dosage of 9mg/kg and the gastrodia elata armillaria mellea tablet group with the dosage of 600mg/kg is weaker, and the indexes are improved. The activity of TNOS in the gastrodia elata armillaria mellea powder 300mg/kg group is reduced, and the significant difference (P is less than 0.05-0.01) is obtained compared with that in the model group; the ginkgo biloba armillaria oral solution can be used for treating the NO level, has significant difference (P is less than 0.05-0.01) compared with a model group, and has NO significant influence on the iNOS activity. The results are shown in Table 3, and can be referred to in the description of the figures from FIGS. 1 to 3.

Table 3-effects on MCAO rat serum TNOS, iNOS, NO (n ═ 10,

)

compared with the group of the pseudo-operation,^■■P<0.01，^■P<0.05; in comparison with the set of models,^**P<0.01，^*P<0.05; compared with the silver extract group,^△△P<0.01，△P<0.05, compared with the halimasch tablet group, a-solidup-P<0.01，▲P<0.05。

3.2 Effect on neurotransmitters in brain tissue

The experiment shows that when the cerebral ischemia-reperfusion injury of rats occurs for 24 hours, the central NE level is reduced, the 5-HT and 5-HIAA level are increased, and the differences are very obvious compared with a sham operation group (the P is less than 0.01); compared with the model group, the levels of NE in the brain of the 18 mg/kg-more gold group, the armillaria tablet 600mg/kg group and the ginkgo and armillaria oral solution 309mg/kg group are remarkably increased (the average P is less than 0.01), and the levels of 5-HT and 5-HIAA are not obviously changed; the level of NE in the brain of the ginkgo biloba extract group with the concentration of 9mg/kg is remarkably increased (P <0.01), and the level of 5-HIAA is remarkably reduced (P < 0.05); the NE of 618mg/kg group of ginkgo and honey looper oral solution is not changed obviously, and the levels of 5-HT and 5-HIAA are reduced greatly (the P is less than 0.01). The results are shown in Table 4, FIG. 4, and FIG. 5.

Table 4 effect on neurotransmitters in MCAO rats brain (n 10,

)

compared with the group of the pseudo-operation,^■■P<0.01，^■P<0.05; in comparison with the set of models,^**P<0.01，^*P<0.05; compared with the silver extract group,^△△P<0.01，^△P<0.05, compared with the Armillaria mellea slice group,^▲▲P<0.01，^▲P<0.05。

3.3 pathological examination

The brain tissue of the experimental rat is subjected to HE staining by a dehydrated embedded section, and observation under a microscope shows that the cortex, medullary neuron cells and glial cells of the brain tissue of the rat in the sham operation group are orderly arranged, the nucleus is large, the chromatin is uniformly distributed, the nucleolus is clear, and pathological changes such as degeneration, necrosis and the like are not seen. The rat brain tissue of the model group has large peduncle area, is liquification necrosis (softening range), shows a blank structure nearly vanishing due to brain tissue edema, is in a screen-shaped structure, reduces neuron cells, can show a plurality of microglial cell hyperplasia in the necrotic area, and can show a plurality of neutrophil infiltration at necrotic edges. The area of the brain infarct area of rats of other administration groups is reduced, the swelling of endothelial cells, nerve cells and astrocytes is occasionally seen, the inflammatory cell infiltration phenomenon is caused, the best lesion degree is 309mg/kg of ginkgo honey ring oral solution and 618mg/kg of ginkgo honey ring oral solution, and the pathological examination result can be shown in figure 6.

In conclusion, the new clinical treatment application of the medicinal preparation for treating the senile dementia enlarges the clinical application indication range of the medicament. In the later period, the company develops the effectiveness and the safety of the medicine for treating the motor cognition risk syndrome and develops multi-center, random and double-blind clinical tests of senile dementia patients. The development of the new application of the medicine provides a new medicine selection for treating the senile dementia and has good market application prospect.

Claims (4)

1. The application of the medicinal preparation in preparing the medicament for treating the senile dementia is characterized in that the medicinal preparation comprises the following raw material medicaments in part by weight: 2-4 parts of ginkgo leaf extract and 80-120 parts of honey ring powder.

2. The use of claim 1, wherein the pharmaceutical preparation comprises the following raw material herbs in parts by weight: 3 parts of ginkgo leaf extract and 100 parts of honey ring powder.

3. The use of claim 1, wherein the pharmaceutical formulation is prepared by a method comprising:

(1) and (3) extracting:

putting the honey-soaked doughnut powder into a bag decocting bag, placing the bag decocting bag into an extraction tank, adding water into the extraction tank for extracting for 1-3 times, and collecting an extracting solution;

(2) and concentrating:

combining the extracting solutions obtained in the step (1), filtering, concentrating the filtrate to the relative density of 1.10-1.16, and collecting the paste;

(3) and alcohol precipitation:

pumping the extract obtained in the step (2) into an alcohol precipitation tank, adding ethanol, stirring to ensure that the alcohol content of the liquid medicine reaches 60-80%, refrigerating, and standing;

(4) and ethanol recovery:

extracting supernatant after alcohol precipitation into a vacuum concentrator for concentration, simultaneously recovering ethanol, and concentrating to obtain a honeydew powder extract with the relative density of 1.15-1.17;

(5) and (4) uniformly mixing the honey ring powder extract prepared in the step (4) and the ginkgo leaf extract to obtain a mixed extract, and adding pharmaceutically common auxiliary materials to prepare various oral preparations.

4. The use of claim 1, wherein the pharmaceutical preparation is in the form of oral liquid, granule, capsule, tablet, or drop pill.

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