A pharmaceutical composition, its preparation method and application

Abstract

The application provides a kind of pharmaceutical composition and its preparation method and application, it is related to medical technical field.The pharmaceutical composition of the application includes components: rehderin D, tortoise shell glue, berberine hydrochloride, beta-sitosterol, salt know mother, paeoniflorin and peony phenol;Wherein, radix rehmanniae preparata is mixed with methanol solution, heated extraction, extract liquid passes chromatographic column, finally eluted with methanol aqueous solution to obtain rehderin D;Take salt huang bai chop and mix with acid ethanol solution and soak, heated extraction is carried out ultrasonic treatment, filtrate is recovered ethanol under reduced pressure, to obtain berberine hydrochloride.The tortoise shell glue of the application is crushed to particle size 80-200 mesh, mixed with salt know mother extract and other components in the pharmaceutical composition, the obtained pharmaceutical composition can be applied to prepare menopausal syndrome drug, and / or treatment of nervous system disease drug, can significantly relieve menopausal syndrome symptoms, and has certain effect on the treatment of Parkinson, Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition, a preparation method and an application thereof. Background Art

[0002] Menopausal syndrome falls under the category of "perimenopause syndromes" in Traditional Chinese Medicine. These syndromes refer to the symptoms women experience around the time of menopause, along with menstrual irregularities or menopause, such as paroxysmal hot flashes and sweats, restless feeling, irritability, mood swings, dizziness, tinnitus, palpitations, insomnia, facial and limb swelling, joint pain, osteoporosis, back pain, or a crawling sensation on the skin. These symptoms often vary in severity and duration, from just a few months to years.

[0003] Western medicine believes that menopausal symptoms are primarily caused by a series of clinical manifestations resulting from a decrease in estrogen levels. Currently, Western medicine treatments for menopausal symptoms primarily use estrogen and progesterone to increase or restore estrogen levels in the body to normal, thereby alleviating menopausal symptoms. However, simply increasing estrogen levels in menopausal women does not effectively eliminate the impact of the various symptoms brought on by menopausal physiological changes on women's physical and mental health.

[0004] According to Chinese medicine, the etiology and pathogenesis of menopausal syndrome are attributed to the kidney. Before and after menopause, kidney qi gradually declines, the body will be exhausted, the Chong and Ren meridians will be deficient, and the essence and blood will be insufficient, resulting in imbalance of yin and yang, and organ dysfunction and menopausal syndrome. Traditional treatment prescription Guanhuangmu granules, whose Chinese medicinal ingredients are prepared rehmannia root, tortoise shell glue, salt Guan Huangbai, salt Anemarrhena and white peony root, have the effect of tonifying the liver and kidney, nourishing yin and reducing internal heat. It is used for female menopausal syndrome (pre-menopausal syndrome) and TCM syndrome differentiation belongs to liver and kidney yin deficiency syndrome, with symptoms such as hot flashes and sweating, dizziness, tinnitus, soreness of waist and knees or heel pain, insomnia and frequent dreams, impatience and irritability. Chinese invention patent CN101879269A discloses a medicine for treating liver and kidney deficiency syndrome, nourishing yin and reducing internal heat, and a method for preparing the medicine. The medicine is prepared from five Chinese medicinal herbs: prepared rehmannia root, tortoise shell glue, salt Guan Huangbai, salt Anemarrhena and white peony root. The preparation method comprises the following steps: pulverize tortoise shell glue into a fine powder and set aside; soak Phellodendron chinense in 3 times the amount of 1% phosphoric acid for 4 hours, then decoct it twice with Rehmannia glutinosa, Anemarrhena asphodeloides, and White Peony Root in 12 times the amount of water, each time for 1.5 hours. The decoctions are combined and filtered. When the filtrate is concentrated to 1.10-1.15 (at 50°C-60°C), ethanol is added to adjust the alcohol content to 70%, and the mixture is allowed to stand overnight. The ethanol in the supernatant is recovered until the alcohol taste is eliminated. The tortoise shell glue powder is added to the medicinal solution and dissolved, and the medicinal solution is further concentrated to an extract with a relative density of 1.32-1.35 (at 50°C-60°C), which is then prepared into granules.

[0005] Chinese invention patent CN103948778A discloses a medicine comprising 10-30 parts of Rehmannia root, 5-10 parts of Bupleurum root, 1-10 parts of tortoise shell glue, 10-20 parts of Phellodendron chinense, 10-20 parts of Anemarrhena rhizome, 1-5 parts of Cyperus rotundus, 10-30 parts of White Peony Root, 10-20 parts of Leonurus japonicus, 2-3 parts of Cinnamon Twig, 1-10 parts of Zedoariae rhizome, 10-30 parts of Cornus officinalis, 1-5 parts of Carthamus tinctorius, 25-35 parts of White Poria cocos, 5-10 parts of Myrrh, 1-5 parts of Angelica sinensis, 1-5 parts of Dipsacus asper, 1-5 parts of Corydalis yanhusuo, 10-20 parts of Cuscuta seed and 10-30 parts of Schizonepeta tenuifolia. Chinese invention patent CN103393927A discloses a medicine prepared by mixing Phellodendron amurense, Anemarrhena asphodeloides, Alisma orientalis, Poria cocos, Ligustrum lucidum fruit, Ophiopogon japonicus, Schisandra chinensis, and Angelica sinensis in a certain weight ratio. The medicine has the functions of nourishing yin and moistening the lungs, clearing the heart and relieving restlessness, replenishing and harmonizing blood, regulating menstruation and relieving pain, nourishing yin and reducing fire. It is effective in treating menopausal syndrome quickly, has good efficacy, and has a high cure rate.

[0006] Although in recent years, extensive research has been conducted on the composition, pharmacological effects, and applications of traditional Chinese medicines at home and abroad, and a large number of drugs for the treatment of menopausal syndrome have been developed, their therapeutic effects still have room for improvement. Therefore, we further studied the traditional formula Guanhuangmu Granules to explore the significant role of the synergistic enhancement of technical effects brought about by different traditional Chinese medicine ingredients. It is of great significance to develop a pharmaceutical composition that significantly tonifies the liver and kidneys, nourishes yin and reduces internal heat, and improves menopausal syndrome. In addition, the present invention also found that the traditional Chinese medicine composition has a neuroprotective effect and can treat neurological diseases such as Parkinson's disease and Alzheimer's disease. Summary of the Invention

[0007] The present invention addresses the problems existing in the prior art and provides a pharmaceutical composition, a preparation method and an application thereof; further in-depth research is conducted based on the synergistic enhancement of traditional Chinese medicine components and specific active ingredients, as well as the preparation method, to prepare a pharmaceutical composition that significantly treats menopausal syndrome and has therapeutic effects on neurological diseases such as Parkinson's disease and Alzheimer's disease.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] First, the present invention provides a pharmaceutical composition comprising the following components: rehmannia glutinosa, tortoise shell glue, berberine hydrochloride, β-sitosterol, anemarrhena asphodeloides, paeoniflorin and paeonol.

[0010] Rehmannia root is a commonly used Chinese medicinal material with the effects of nourishing blood and yin, replenishing essence and filling marrow; Rehmannia root includes a large number of active ingredients, such as rehmannia glycoside D, which has the effects of nourishing yin and replenishing blood; rehmannia glycoside D may inhibit the activation of mouse microglia N9 cells by adjusting the release of inflammatory factors and inhibiting the transformation of microglia from M2 to M1, thereby improving neuronal inflammation.

[0011] Yanguan Huangbo has the effects of nourishing yin and reducing fire, clearing away heat and dampness, purging fire and removing steaming, detoxifying and treating sores; Yanguan Huangbo contains a variety of effective ingredients, among which berberine hydrochloride helps to improve women's body composition, regulate hormone levels, and to a certain extent effectively treat polycystic ovary syndrome; β-sitosterol has estrogenic activity, which can keep the ovaries younger, and also has anti-diabetic, lipid-lowering, anti-cancer, anti-arthritis and liver protection effects.

[0012] White peony root nourishes blood and regulates menstruation, calms liver yang, restrains yin and stops sweating, softens the liver and relieves pain. It is rich in paeoniflorin, paeonol, and β-sitosterol. Paeoniflorin has been shown to improve cognitive function, provide sedation, and offer anti-inflammatory, analgesic, and antispasmodic effects. It can effectively regulate levels of abnormally phosphorylated tau protein and excessive β-amyloid protein deposition in Alzheimer's disease animals and cell models, restore the balance of neurotransmitters and neuroreceptors such as acetylcholine and nerve growth factor, reduce tissue inflammation and oxidative stress, and mitigate cell apoptosis. Paeonol has central nervous system inhibitory effects such as sedation, cooling, antipyretic, analgesic, and antispasmodic, as well as anti-atherosclerotic, diuretic, and anti-ulcer properties, alleviating menopausal symptoms. Paeonol has also been shown to improve learning and memory in Alzheimer's disease animals.

[0013] Preferably, the pharmaceutical composition comprises the following components in parts by weight: 0.5-3 parts of rehmannia glutinosa, 12-40 parts of tortoise shell glue, 1.5-6 parts of berberine hydrochloride, 1-4 parts of β-sitosterol, 80-150 parts of anemarrhena asphodeloides, 2-5 parts of paeoniflorin and 0.5-2 parts of paeonol.

[0014] Further preferably, the pharmaceutical composition comprises the following components, in parts by weight: 1.5-2.5 parts of rehmannia glutinosa, 24-36 parts of tortoise shell glue, 2-3.5 parts of berberine hydrochloride, 1.8-3 parts of β-sitosterol, 90-110 parts of anemarrhena asphodeloides, 3-4.2 parts of paeoniflorin and 0.8-1.2 parts of paeonol.

[0015] More preferably, the pharmaceutical composition comprises the following components in parts by weight: 2 parts of rehmannia glutinosa, 35 parts of tortoise shell glue, 3 parts of berberine hydrochloride, 2.4 parts of β-sitosterol, 100 parts of anemarrhena asphodeloides, 4 parts of paeoniflorin and 1 part of paeonol.

[0016] Preferably, the preparation method of rehmannia glutinosa glycoside D is as follows: rehmannia root prepared is mixed with 5-20 times the volume amount of a methanol solution with a concentration of 20-75%, subjected to multiple heating and reflux extractions, filtered, the filtrate is recovered, passed through a D101 macroporous adsorption resin chromatography column, and then eluted with a methanol aqueous solution to obtain rehmannia glutinosa glycoside D.

[0017] Further preferably, the preparation method of the rehmannia glutinosa glycoside D is: mixing Rehmannia glutinosa with 8-15 times the volume amount of a methanol solution with a concentration of 50-65%, extracting 1-3 times under reflux heating at 55-70°C, each time for 1-3 hours, filtering, recovering the filtrate, passing it through a D101 macroporous adsorption resin chromatography column, and then eluting with a 20-50% methanol aqueous solution, collecting the eluate, and recovering the methanol under reduced pressure to obtain rehmannia glutinosa glycoside D.

[0018] More preferably, the preparation method of the rehmannia glutinosa glycoside D is as follows: Rehmannia glutinosa glutinosa is mixed with 10 times the volume concentration of 60% methanol solution, extracted twice by reflux heating at 70°C, each time for 2 hours, filtered, recovered the filtrate, passed through a D101 macroporous adsorption resin chromatography column, and then eluted with 40% methanol aqueous solution, collected the eluate, and recovered the methanol under reduced pressure to obtain rehmannia glutinosa glycoside D.

[0019] Preferably, the preparation method of berberine hydrochloride is: chop the salt-guan Huangbai, mix it with 5-20 times the amount of acidic ethanol solution, and soak it for 3-5 hours, perform heating-assisted ultrasonic extraction 1-3 times, each extraction for 0.5-3 hours, filter, reduce the pressure of the filtrate to recover ethanol, add sodium chloride for salting out, and obtain berberine hydrochloride.

[0020] More preferably, the preparation method of berberine hydrochloride is as follows: take salt-off cork and chop into 0.5-1.5cm 3 After that, the mixture is mixed with 8-15 times the amount of acidic ethanol solution and soaked for 3-5 hours, and then subjected to heating-assisted ultrasonic extraction at 40-50°C for 2-3 times, each extraction for 1.5-2.5 hours. The filtrate is decompressed to recover ethanol, and sodium chloride is added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution is 40-60%; the added acid is phosphoric acid, and the volume content of the acid in the acidic ethanol solution is 0.5-3%.

[0021] More preferably, the preparation method of berberine hydrochloride is as follows: take salt-off cork and chop 1.0cm 3 Afterwards, the mixture was mixed with 12 times the amount of acidic ethanol solution and soaked for 4 hours, and then subjected to ultrasonic extraction assisted by heating at 45°C for 2 hours each time. The filtrate was decompressed to recover the ethanol, and sodium chloride was added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution was 50%; the acid added was phosphoric acid, and the volume content of the acid in the acidic ethanol solution was 1%.

[0022] Then, the present invention provides a method for preparing the above-mentioned pharmaceutical composition, comprising the steps of:

[0023] (1) crushing the tortoise shell glue and grinding it into a fine powder to obtain tortoise shell glue powder;

[0024] (2) decocting the Anemarrhena Rhizoma, concentrating the decoction, mixing it with ethanol, recovering the supernatant, and removing the ethanol from the supernatant to obtain the Anemarrhena Rhizoma extract;

[0025] (3) Mixing tortoise shell glue powder, salt anemarrhena extract, rehmannia glutinosa glycoside D, berberine hydrochloride, β-sitosterol, paeoniflorin and paeonol to obtain a pharmaceutical composition.

[0026] Preferably, in step (1), the particle size of the tortoise shell glue powder is 80-200 mesh; further preferably, the particle size of the tortoise shell glue powder is 100-150 mesh; even further preferably, the particle size of the tortoise shell glue powder is 100-120 mesh.

[0027] Preferably, in step (2), the decoction of the salt anemarrhena is specifically as follows: the salt anemarrhena is mixed with 6-15 times the amount of water and decocted for 1-3 hours; the residue is filtered and repeatedly decocted 1-2 times; the decoctions are combined and concentrated to a relative density of 1.10-1.15 (50-60° C.) to obtain the salt anemarrhena concentrate.

[0028] Further preferably, the decocting of salt Anemarrhena asphodeloides is specifically as follows: mixing salt Anemarrhena asphodeloides with 10 times the amount of water and decocting for 2 hours; filtering the residue and repeating the decocting once; combining the decoctions and concentrating to a relative density of 1.10-1.15 (50-60° C.) to obtain salt Anemarrhena asphodeloides concentrate.

[0029] Preferably, in step (2), the mixing with ethanol is specifically as follows: adding ethanol to the salt Anemarrhena concentrate to an ethanol concentration of 55-70%, stirring evenly and then standing for 8-16 hours, recovering the supernatant, and concentrating the supernatant under reduced pressure to remove ethanol to obtain the salt Anemarrhena extract.

[0030] Further preferably, the mixing with ethanol is specifically as follows: adding ethanol to the salt Anemarrhena concentrate to an ethanol concentration of 65%, stirring evenly and then standing for 12 hours, recovering the supernatant, and concentrating the supernatant under reduced pressure at 40-50°C to remove ethanol to obtain the salt Anemarrhena extract.

[0031] Preferably, in step (3), after mixing, the pharmaceutical composition is dried; the drying is not limited to freeze drying, vacuum drying, and spray drying.

[0032] Finally, the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of drugs for treating menopausal syndrome and / or drugs for treating nervous system diseases.

[0033] Preferably, the nervous system diseases include Alzheimer's disease and Parkinson's disease.

[0034] Preferably, the dosage form of the drug can be any pharmaceutically acceptable dosage form, including but not limited to tablets, capsules, granules, mixtures, and pills.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The pharmaceutical composition of the present invention is based on the original Huangmu granules, and the traditional Chinese medicine ingredients and active ingredients of the formula are creatively compounded. The ingredients work synergistically with each other, which can significantly alleviate the symptoms of menopausal syndrome, has a good effect of improving the content of related hormones and monoamine neurotransmitters in menopausal model rats, and has a significant calming and sleep-inducing effect.

[0037] 2. The present invention also conducted research on new indications for the prepared pharmaceutical composition and found that it has certain effects on the treatment of neurological diseases such as Alzheimer's disease and Parkinson's disease, improves the new object recognition ability of Alzheimer's disease model animals, significantly reduces neuroinflammation, and broadens the application of the pharmaceutical composition.

[0038] 3. The present invention comprehensively utilizes various traditional Chinese medicines and active ingredients. The ingredients of the pharmaceutical composition are clear and reasonably matched. Each ingredient is combined with a specific extraction method. The resulting pharmaceutical composition has high activity and has significant effects on alleviating menopausal syndrome symptoms and treating nervous system diseases. DETAILED DESCRIPTION

[0039] The following non-limiting examples can make those of ordinary skill in the art understand the present invention more comprehensively, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and they should also fall within the scope of protection of the present invention. When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those of ordinary skill in the art to which the present invention belongs.

[0040] The present invention is further described below in the form of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels. In the following examples, the β-sitosterol (CAS No. 83-46-5) was purchased from Aladdin with the item number S434260; the paeoniflorin (CAS No. 23180-57-6) was purchased from Aladdin with the item number P101691; and the paeonol (CAS No. 552-41-0) was purchased from Med Chem Express with the catalog number HY-N0159. For the above components, products from different manufacturers do not have a significant effect on the effects.

[0041] Examples 1-5

[0042] A pharmaceutical composition, the components and weight proportions of which are shown in Table 1:

[0043] Table 1

[0044] parts by weight Example 1 Example 2 Example 3 Example 4 Example 5 Rehmannia glutinosa D 2 1.5 2.5 0.5 3 Tortoise Shell Glue 35 24 36 12 40 Berberine hydrochloride 3 2 3.5 1.5 6 β-Sitosterol 2.4 1.8 3 1 4 Salt Anemarrhena 100 90 110 80 150 Paeoniflorin 4 3 4.2 2 5 Paeony 1 0.8 1.2 0.5 2

[0045] In Examples 1-4, the preparation method of the rehmannia glutinosa glycoside D is as follows: Rehmannia glutinosa ...

[0046] In Example 1-3, the berberine hydrochloride is prepared by: taking salt-cleared Phellodendron chinense and chopping it into 1.0 cm 3 Afterwards, the mixture was mixed with 12 times the amount of acidic ethanol solution and soaked for 4 hours, and then subjected to heating at 45°C with the assistance of ultrasonic extraction twice, each extraction for 2 hours. The filtrate was decompressed to recover the ethanol, and sodium chloride was added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution was 50%; the acid added was phosphoric acid, and the volume content of phosphoric acid in the acidic ethanol solution was 1%.

[0047] In Example 4, the berberine hydrochloride is prepared by: taking salt-cleared Phellodendron chinense and chopping it into 0.5 cm 3 Afterwards, the mixture was mixed with 8 times the amount of acidic ethanol solution and soaked for 4 hours, and then subjected to heating-assisted ultrasonic extraction at 40°C for 2 times, each extraction for 2 hours. The filtrate was decompressed to recover the ethanol, and sodium chloride was added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution was 40%; the added acid was phosphoric acid, and the volume content of phosphoric acid in the acidic ethanol solution was 3%.

[0048] In Example 5, the preparation method of the rehmannia glutinosa glycoside D is as follows: Rehmannia glutinosa ...

[0049] In Example 5, the berberine hydrochloride is prepared by: taking salt-cleared Phellodendron chinense and chopping it into 1.5cm pieces 3 Afterwards, the mixture was mixed with 15 times the amount of acidic ethanol solution and soaked for 4 hours, and then subjected to heating at 50°C with ultrasonic extraction for 2 hours each time. The filtrate was decompressed to recover the ethanol, and sodium chloride was added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution was 60%; the added acid was phosphoric acid, and the volume content of phosphoric acid in the acidic ethanol solution was 0.5%.

[0050] The preparation method of the pharmaceutical composition of Examples 1-5 comprises the steps of:

[0051] (1) crushing the tortoise shell glue and grinding it into a fine powder to obtain a tortoise shell glue powder with a particle size of 100-120 mesh;

[0052] (2) Mixing the salt anemarrhena with 10 times the amount of water, decocting for 2 hours; filtering the residue and repeating the decoction once; combining the decoctions and concentrating to a relative density of 1.10-1.15 (50-60°C) to obtain the salt anemarrhena concentrate;

[0053] The Anemarrhena Rhizoma concentrate was added with ethanol to a concentration of 65%, stirred evenly, and then allowed to stand for 12 hours. The supernatant was recovered and concentrated under reduced pressure at 40-50° C. to remove ethanol and obtain the Anemarrhena Rhizoma extract.

[0054] (3) Mix the tortoise shell glue powder, the salt anemarrhena extract, rehmannia glutinosa glycoside D, berberine hydrochloride, β-sitosterol, paeoniflorin and paeonol, and freeze-dry to obtain a pharmaceutical composition.

[0055] Comparative Example 1

[0056] The difference from Example 1 is that β-sitosterol is replaced by quercetin in the pharmaceutical composition, and the rest is the same as Example 1.

[0057] Comparative Example 2

[0058] The difference from Example 1 is that in the pharmaceutical composition, paeoniflorin is replaced by rehmannia glutinosa D. That is, the weight portion of rehmannia glutinosa D is 6 parts, and the weight portion of paeoniflorin is 0 parts. The rest is the same as Example 1.

[0059] Comparative Example 3

[0060] The difference from Example 1 is that rehmannia glutinosa D is replaced by paeoniflorin. That is, the weight portion of rehmannia glutinosa D is 0 parts and the weight portion of paeoniflorin is 6 parts. The rest is the same as Example 1.

[0061] Comparative Example 4

[0062] The difference from Example 1 is that the preparation method of berberine hydrochloride is different, specifically the heating extraction and ultrasonic treatment are different: Take salt-off Phellodendron chinense and chop 1.0cm 3 Afterwards, the mixture was mixed with 12 times the amount of acidic ethanol solution and soaked for 4 hours, and then ultrasonically treated at 45°C for 1 hour; then heated and extracted at 45°C for 1 hour; the filtrate was decompressed to recover ethanol, and sodium chloride was added for salting out to obtain berberine hydrochloride; the volume concentration of the ethanol solution was 50%; the added acid was phosphoric acid, and the volume content of phosphoric acid in the acidic ethanol solution was 1%.

[0063] The rest are the same as in Example 1.

[0064] Comparative Example 5

[0065] The difference from Example 1 is that the preparation method of rehmannia glutinosa D is different, specifically the concentration of methanol is different. The methanol used in this comparative example is pure methanol: Rehmannia glutinosa is mixed with 10 times the amount of methanol, heated under reflux at 70°C and extracted twice, each time for 2 hours, filtered, and the filtrate is recovered and passed through a D101 macroporous adsorption resin chromatography column, and then eluted with a 40% methanol aqueous solution. The eluate is collected and the methanol is recovered under reduced pressure to obtain rehmannia glutinosa D.

[0066] The rest are the same as in Example 1.

[0067] Comparative Example 6

[0068] The pharmaceutical composition differs from Example 1 in that the weight ratios are different. The components, by weight, include: 2 parts of rehmannia glutinosa glycoside D, 65 parts of tortoise shell glue, 1 part of berberine hydrochloride, 2.4 parts of β-sitosterol, 74.5 parts of anemarrhena asphodeloides, 1.5 parts of paeoniflorin, and 1 part of paeonol. The remaining ingredients are the same as in Example 1.

[0069] Comparative Example 7

[0070] The difference from Example 1 is that the preparation method of the pharmaceutical composition is different, specifically the particle size of the tortoise shell glue powder is different. The particle size range of the tortoise shell glue powder used in this comparative example is 50-60 mesh. All other steps are the same as in Example 1.

[0071] Comparative Example 8

[0072] The difference from Example 1 is that the preparation method of the pharmaceutical composition is different, specifically: the salt Anemarrhena Rhizoma is not decocted in water, but directly extracted with ethanol, comprising the steps of:

[0073] (1) Same as Example 1;

[0074] (2) Mixing the salt anemarrhena with 10 times the amount of 65% ethanol, and reflux extraction for 2 hours; filtering the residue and repeating the reflux extraction once; combining the decoctions and concentrating to a relative density of 1.10-1.15 (50-60° C.) to obtain a salt anemarrhena concentrate; concentrating the salt anemarrhena concentrate under reduced pressure at 40-50° C. to remove the ethanol, and obtaining a salt anemarrhena extract;

[0075] (3) Same as Example 1.

[0076] Comparative experiment 1: Effects of related hormones and monoamine neurotransmitter levels in menopausal model rats

[0077] 1. Experimental drugs:

[0078] The pharmaceutical composition described in the Examples and Comparative Examples has a low dose of 0.3 g / kg / d, a medium dose of 0.6 g / kg / d, and a high dose of 1.2 g / kg / d.

[0079] 2. Experimental Animals: Female SD rats weighing 200-220 g were fed for 3 days of adaptive feeding. The model group and experimental group were then ovariectomized (OVX) to establish a menopausal rat model. After the model rats were successfully established, they were randomly divided into a model group (gavage with normal saline), a low-dose group in Example 1, a medium-dose group in Example 1, a high-dose group in Example 1, medium-dose groups in Examples 2-5, and medium-dose groups in Comparative Examples 1-8. Rats that were not subjected to model establishment were set as a blank control group (gavage with normal saline). 10 rats were included in each group.

[0080] 3. Experimental Methods: Each group of rats received the drug by gavage once daily at the appropriate dose for 6 consecutive weeks. Twenty-four hours after the last dose, rats were decapitated and blood was collected. Serum was centrifuged at 4000 rpm for 15 minutes and serum was prepared. Serum levels of relevant hormones and monoamine transmitters were determined by radioimmunoassay. The results were statistically analyzed to compare differences between groups. The results of the relevant hormone and monoamine transmitter assays are shown in Tables 2.1 and 2.2.

[0081] Table 2.1

[0082]

[0083]

[0084] In Table 2.1, the model group was compared with the blank control group. * P<0.05, ** P<0.01; Comparison between each drug-treated group and the model group, & P<0.05, && P<0.01; each drug administration group is compared with the dosage in Example 1, # P<0.05, ## P<0.01.

[0085] Table 2.2

[0086]

[0087] In Table 2.2, the model group was compared with the blank control group. * P<0.05, ** P<0.01; compared with the embodiment group and the model group, & P<0.05, && P < 0.01; each comparative example group was compared with the dosage group in Example 1, # P<0.05, ## P<0.01.

[0088] As can be seen from Tables 2.1 and 2.2, the pharmaceutical composition of the present invention significantly increased the serum E2 estradiol level, decreased the LH gonadotropin level, increased the P progesterone level, and decreased the serum NA norepinephrine level in menopausal rats. The remaining comparative groups showed varying degrees of improvement, but the effects were significantly lower than those achieved by the present invention.

[0089] Comparative experiment 2: sedation-hypnosis test

[0090] 1. Experimental drugs:

[0091] The pharmaceutical composition described in the Examples and Comparative Examples has a low dose of 0.3 g / kg / d, a medium dose of 0.6 g / kg / d, and a high dose of 1.2 g / kg / d.

[0092] 2. Experimental Animals: Quarantine-qualified female ICR mice weighing 20-22 g were fed for one week of adaptive feeding. The model group and experimental group were then ovariectomized (OVX) to establish a menopausal mouse model. After the menopausal model mice were successfully established, they were randomly divided into a model group (gavage with normal saline), a low-dose group in Example 1, a medium-dose group in Example 1, a high-dose group in Example 1, medium-dose groups in Examples 2-5, and medium-dose groups in Comparative Examples 1-8. Mice that were not subjected to model establishment were set as a blank control group (gavage with normal saline). 10 mice were included in each group.

[0093] 3. Experimental Methods: Each group of mice was administered the drug once daily according to the dosage for 7 consecutive days. One hour after the last administration, each group of mice received an intraperitoneal injection of a subthreshold dose of 25 mg / kg of sodium pentobarbital. Loss of righting reflex for more than 1 minute was used as the criterion for falling asleep. The number of sleeping mice in each group was recorded, and the statistical differences between the groups were compared. After all mice recovered, 48 hours later, a subthreshold dose of 40 mg / kg of sodium pentobarbital was injected intraperitoneally again. The time from the completion of sodium pentobarbital injection to the loss of righting reflex was defined as the sleep onset latency, and the time from the loss of righting reflex to recovery was defined as the sleep duration. The sleep latency and sleep time of each mouse were recorded, and the statistical differences between the groups were compared. The test results are shown in Tables 3.1 and 3.2.

[0094] Table 3.1

[0095]

[0096] In Table 3.1, the model group was compared with the blank control group. * P<0.05, ** P<0.01; Comparison between each drug-treated group and the model group, & P<0.05, && P<0.01; each drug administration group is compared with the dosage in Example 1, #P<0.05, ## P<0.01.

[0097] Table 3.2

[0098] Sleep only, only Sleep incidence, % Sleep latency, s Sleep time, min Blank control group 7 70％ 270±26.4 46.5±3.66 Model Group 2 20％ <![CDATA[451±22.9 ** ]]> <![CDATA[18.3±2.12 ** ]]> Dose group in Example 1 7 70％ <![CDATA[288±23.8 && ]]> <![CDATA[38.9±2.61 && ]]> Comparative Example 1 3 30％ <![CDATA[417±28.0 ## ]]> <![CDATA[23.8±2.92 ## ]]> Comparative Example 2 2 20％ <![CDATA[406±22.5 ## ]]> <![CDATA[27.0±2.07 ## ]]> Comparative Example 3 2 20％ <![CDATA[382±30.6 ## ]]> <![CDATA[28.5±2.90 ## <!-- 8 -->]]> Comparative Example 4 4 40％ <![CDATA[346±25.3 ## ]]> <![CDATA[32.7±2.63 ## ]]> Comparative Example 5 4 40％ <![CDATA[368±24.8 ## ]]> <![CDATA[31.0±1.99 ## ]]> Comparative Example 6 2 20％ <![CDATA[423±27.3 ## ]]> <![CDATA[21.8±2.67 ## ]]> Comparative Example 7 4 40％ <![CDATA[349±33.7 ## ]]> <![CDATA[33.9±3.45 ## ]]> Comparative Example 8 3 30％ <![CDATA[370±30.6 ## ]]> <![CDATA[30.4±2.59 ## ]]>

[0099] In Table 3.2, the model group was compared with the blank control group. * P<0.05, ** P<0.01; compared with the embodiment group and the model group, & P<0.05, && P < 0.01; each comparative example group was compared with the dosage group in Example 1, # P<0.05, ## P<0.01.

[0100] As can be seen from Tables 3.1 and 3.2, the pharmaceutical composition of the present invention can significantly improve the sleep quality of mice and has better sedative and tranquilizing effects. The other comparative groups showed varying degrees of improvement, but the effects were significantly lower than the technical effects of the examples of the present invention.

[0101] Comparative experiment 3: novel object recognition ability and neuroinflammation inhibition detection

[0102] 1. Experimental drugs: the pharmaceutical compositions described in the Examples and Comparative Examples, low dose 0.4 g / kg / d, medium dose 0.8 g / kg / d, high dose 1.6 g / kg / d.

[0103] 2. Experimental Animals: Sprague-Dawley rats, half male and half female, weighing (220 ± 20) g. Animals were housed in an SPF environment at a room temperature of 18-23°C and a relative humidity of 45%-55%, with free access to water and food, and acclimated for 5 days. All animal experiments were approved by the Animal Ethics Committee of the experimental unit.

[0104] The AD model was established in SD rats: scopolamine was administered intraperitoneally at a dose of 3 mg / kg / day, while the normal control group received an equal amount of saline subcutaneously for 9 consecutive days. After successful model establishment, the rats were divided into groups and given the drug orally for 3 weeks.

[0105] AD model SD rats were randomly divided according to body weight into the following groups: an Alzheimer's disease AD model group (oral administration of normal saline), a medium-dose group in Example 1, medium-dose groups in Examples 1-3, and medium-dose groups in Comparative Examples 1-8. Rats not undergoing model establishment were set up as a blank control group (oral administration of normal saline). Each group consisted of 10 rats.

[0106] 3. Experimental methods

[0107] 3.1. Novel object recognition: The novel object recognition experiment was tested in an opaque plastic chamber (40 cm × 40 cm × 50 cm). A camera installed on the wall directly above the test chamber was used to record the test process for offline analysis. 24 hours before the test, the rats were placed in the test room to adapt to the test environment, and all rats were placed in the plastic chamber for 5 minutes to adapt. The test began 24 hours later. Two identical objects A and B were placed at the left and right ends of one side wall, 10 cm away from the wall. Each rat stayed in the plastic chamber for 5 minutes, and the rat's exploration of the two objects was recorded. The test was conducted again 24 hours later. One of the two identical objects, object B, was replaced with a different object C. The rat was also placed in the plastic chamber for 5 minutes, and the rat's exploration behavior was recorded.

[0108] The novel object recognition experiment primarily tests rats' ability to discriminate between familiar and novel objects, including their ability to remember old objects and become familiar with new ones. This experiment assesses the animals' cognitive memory abilities by behaviorally measuring the length of time they explore familiar and new objects. Rats with poor cognitive abilities will explore the new and old objects equally; rats with normal cognitive abilities will explore the new object longer than the old one.

[0109] The formula for calculating the cognitive index is: cognitive index (index, %) = new object / (new object + old object) × 100%.

[0110] 3.2 Elisa test

[0111] After the animal behavior test, the levels of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the rat cerebral cortex and β-amyloid peptide-42 (Aβ42) in the hippocampus were detected according to the Elisa instructions.

[0112] The test results are shown in Table 4.

[0113] Table 4

[0114]

[0115]

[0116] In Table 4, the model group was compared with the blank control group. * P<0.05, ** P<0.01; compared with the embodiment group and the model group, & P<0.05, && P < 0.01; each comparative example group was compared with the dosage group in Example 1, # P<0.05, ##P<0.01.

[0117] As can be seen from Table 4, the pharmaceutical composition of the present invention can significantly improve the new object recognition ability of AD model rats, and at the same time has a significant reducing effect on the content of neuroinflammation IL-1β, TNF-α and Aβ42. The remaining comparative groups also have improvement trends to varying degrees, but the effects are significantly lower than the technical effects of the embodiments of the present invention. Therefore, it can be concluded that the pharmaceutical composition of the present invention has a significant effect on the treatment of neurological diseases such as Alzheimer's disease and Parkinson's disease. Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A pharmaceutical composition for treating menopausal syndrome and / or Alzheimer's disease, characterized in that: The invention is composed of the following components in parts by weight: 0.5-3 parts of rehmannia glutinosa glycoside D, 12-40 parts of tortoise shell glue, 1.5-6 parts of berberine hydrochloride, 1-4 parts of β-sitosterol, 80-150 parts of anemarrhena asphodeloides, 2-5 parts of paeoniflorin and 0.5-2 parts of paeonol.

2. The pharmaceutical composition according to claim 1, characterized in that The invention is composed of the following components in parts by weight: 1.5-2.5 parts of rehmannia glutinosa, 24-36 parts of tortoise shell glue, 2-3.5 parts of berberine hydrochloride, 1.8-3 parts of β-sitosterol, 90-110 parts of anemarrhena asphodeloides, 3-4.2 parts of paeoniflorin and 0.8-1.2 parts of paeonol.

3. The pharmaceutical composition according to claim 2, characterized in that The invention is composed of the following components in parts by weight: 2 parts of rehmannia glutinosa, 35 parts of tortoise shell glue, 3 parts of berberine hydrochloride, 2.4 parts of β-sitosterol, 100 parts of anemarrhena asphodeloides, 4 parts of paeoniflorin and 1 part of paeonol.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The preparation method of rehmannia glutinosa glycoside D comprises the following steps: mixing prepared rehmannia root with 5-20 times the volume of a methanol solution with a concentration of 20-75%, performing multiple heating and reflux extractions, filtering, recovering the filtrate, passing the filtrate through a D101 macroporous adsorption resin chromatography column, and then eluting with a methanol-water solution to obtain rehmannia glutinosa glycoside D.

5. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The preparation method of berberine hydrochloride comprises the following steps: chopping salt-guan Huangbai, mixing the mixture with 5-20 times the amount of acidic ethanol solution, and soaking the mixture for 3-5 hours; performing heating-assisted ultrasonic extraction for 1-3 times, each extraction lasting 0.5-3 hours; filtering, reducing the pressure of the filtrate to recover ethanol, and adding sodium chloride for salting out to obtain berberine hydrochloride.

6. The method for preparing the pharmaceutical composition according to any one of claims 1 to 5, characterized in that: Including steps: (1) crushing the tortoise shell glue and grinding it into fine powder to obtain tortoise shell glue powder; (2) decocting the Anemarrhena Rhizoma, concentrating the decoction, mixing it with ethanol, recovering the supernatant, and removing the ethanol from the supernatant to obtain the Anemarrhena Rhizoma extract; (3) Mixing tortoise shell glue powder, salt anemarrhena extract, rehmannia glutinosa glycoside D, berberine hydrochloride, β-sitosterol, paeoniflorin and paeonol to obtain a pharmaceutical composition.

7. The preparation method according to claim 6, characterized in that In step (1), the particle size of the tortoise shell glue powder is 80-200 mesh.

8. The preparation method according to claim 7, characterized in that In step (1), the particle size of the tortoise shell glue powder is 100-150 mesh.

9. The preparation method according to claim 7, characterized in that In step (1), the particle size of the tortoise shell glue powder is 100-120 mesh.

10. The preparation method according to claim 6, characterized in that Step (2) is specifically as follows: after mixing the salt anemarrhena with 6-15 times the amount of water, the mixture is decocted for 1-3 hours; after filtering, the medicinal residue is repeatedly decocted for 1-2 times; the decoctions are combined and concentrated to a relative density of 1.10-1.15 to obtain a salt anemarrhena concentrate; ethanol is added to the salt anemarrhena concentrate to an ethanol concentration of 55-70%, the mixture is stirred evenly, and the mixture is allowed to stand for 8-16 hours, the supernatant is recovered, and the supernatant is concentrated under reduced pressure to remove ethanol to obtain a salt anemarrhena extract.

11. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug for treating menopausal syndrome and / or a drug for treating Alzheimer's disease.

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