WO2015074494A1 - 人参皂苷Rg3在制备用于预防或/和治疗痴呆病症药物中的应用及药物 - Google Patents

人参皂苷Rg3在制备用于预防或/和治疗痴呆病症药物中的应用及药物 Download PDF

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WO2015074494A1
WO2015074494A1 PCT/CN2014/090475 CN2014090475W WO2015074494A1 WO 2015074494 A1 WO2015074494 A1 WO 2015074494A1 CN 2014090475 W CN2014090475 W CN 2014090475W WO 2015074494 A1 WO2015074494 A1 WO 2015074494A1
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ginsenoside
brain
group
oil
dementia
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PCT/CN2014/090475
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English (en)
French (fr)
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富力
王凯乾
付强
刘正贤
鲁岐
盖鑫
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富力
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Priority to KR1020167016598A priority Critical patent/KR101863739B1/ko
Priority to RU2016124765A priority patent/RU2016124765A/ru
Priority to EP14864431.3A priority patent/EP3072517A4/en
Priority to AU2014352441A priority patent/AU2014352441B2/en
Priority to US15/036,494 priority patent/US10238676B2/en
Priority to CA2931066A priority patent/CA2931066A1/en
Priority to JP2016531692A priority patent/JP2017501975A/ja
Publication of WO2015074494A1 publication Critical patent/WO2015074494A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/236—Ligusticum (licorice-root)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates to a ginsenoside Rg3 external preparation, a preparation method thereof and use thereof, and belongs to the field of medicine.
  • Vascular dementia is a variety of dementia caused by intracranial hemorrhage, multiple infarction, arteriosclerotic encephalopathy, acute craniocerebral trauma, intracranial space-occupying lesions, alcoholism, brain nutrient deficiencies, and metabolic endocrine diseases. disease.
  • senile dementia Regardless of the cause of senile dementia, early symptoms are forgetfulness, computing power and language ability decline, the second stage is, severe memory loss, lost direction and ignorance, the third stage is, serious thinking ability disorder The orientation is blurred. Therefore, research on prevention and treatment of senile dementia firstly prevents memory and mental decline, and ginsenoside Rg3 has an obvious effect of improving memory and delaying aging, which is also one of the objects of the present invention.
  • acetylcholine Ach
  • ChoAT presynaptic specific cholinergic marker choline acetyltransferase
  • ginsenoside Rg3 can It is also the second object of the present invention to effectively activate the function of the brain stem network up-regulation system, promote the synthesis of acetylcholine, increase the blood supply to the brain, and improve the microcirculation, so that it can be used for the treatment and prevention of senile dementia.
  • the drugs currently used in the prevention and treatment of dementia are mainly donepezil, rivastigmine, galantamine, piracetam, aniracetam, Vinpocetine, nicergoline, dihydroergotoxine, huperaine, ginkgo leaf agent, brain protein hydrolysate, cell Citicoline, cholinesterase inhibitor, xanthine, non-steroidal anti-inflammatory drugs, estrogen Etc., these drugs have a certain mitigation effect, but the corresponding side reactions are very large, limiting their widespread use.
  • Tim Jingyi marrow prescription drugs such as Rehmannia, Hawthorn, Polygonatum, turtle shell, Ejiao, medlar, antler, etc.
  • Tim Jingyi marrow prescription drugs such as Rehmannia, Hawthorn, Polygonatum, turtle shell, Ejiao, medlar, antler, etc.
  • ginsenoside Rg3 an active ingredient extracted by the natural plant of the invention, has high purity (80% or more), and the curative effect is remarkable and unique, and the prepared preparation is convenient to use, and the patient can adjust the dosage according to the self-reaction, and the effect is mild after transdermal absorption. lasting.
  • Ginseng is a perennial herb of the genus Araliaceae.
  • the ancient Chinese pharmacy book on the Chinese medicine "Shen Nong's Herbal Classic” lists ginseng as the top grade, saying that it "mainly fills the five internal organs, the spirit of the spirit, the soul of the soul, the horror, the eyesight, the happy puzzle , long service has the effect of light body extension.”
  • ginseng contains a chemical called ginsenoside, which has obvious effects on regulating the central nervous system, strengthening heart, anti-fatigue, regulating substance metabolism, etc., so it is used to treat the nervous system.
  • Many diseases of the cardiovascular system, endocrine system and reproductive system have a good therapeutic effect.
  • the results show that it has an effect on both the excitation and inhibition of neural processes, but mainly strengthens the excitatory process of the cerebral cortex. Since it acts on the inhibition process at the same time, the inhibition tends to concentrate, and the differentiation is accelerated and more complete.
  • Ginseng regulates nerve function and restores the neural processes that cause tension.
  • ginseng extract has an antagonistic effect on the memory loss caused by anisodine and pentobarbital sodium, and can also improve the memory consolidation disorder caused by cycloheximide and sodium nitrite and the memory caused by 40% ethanol. Reproduce the defect.
  • Intraperitoneal injection of ginseng stem saponins 200mg/kg, 100mg/kg, 50mg/kg can significantly inhibit the action of anisodine and improve the memory of mice, increase the RNA in the brain, but no DNA or protein content. Significant impact.
  • Ginseng also has a significant effect on cerebral blood flow and brain energy metabolism.
  • Ginseng preparation can increase the glucose uptake of rabbit brain, while reducing the ratio of lactic acid, pyruvic acid and lactic acid/pyruvate, and can change the utilization of glucose from anaerobic metabolic pathway to aerobic metabolism.
  • Ginseng also increases free inorganic phosphorus in the cerebral cortex by 25%.
  • Ginseng fruit saponin can improve brain oxygen uptake ability.
  • Ginseng total saponins and ginseng root saponins have protective effects on cerebral ischemia/reperfusion injury. In short, ginseng can make animal brain more rational use of energy substance glucose, oxidation capacity, and synthesize more ATP for activities such as learning and memory.
  • Ginsenoside Rg3 is a rare saponin of ginseng. It contains only 3 parts per 100,000 red ginseng in 5 years. It is the most essential ingredient in ginseng. Ginsenoside Rg3 has an improvement and prevention effect on many diseases, and has good curative effect on common diseases of middle and old age, cardiovascular and cerebrovascular diseases, coronary heart disease, limb weakness, incompetence of legs and feet, and memory loss.
  • Ginsenoside Rg3 is a ginseng diol tetracyclic triterpenoid saponin, which is found in practical applications due to ginsenoside Rg3 molecule Larger amount (785.02), its fat solubility and water solubility are poor, so that its oral absorption percentage is about 10.28%, resulting in poor absorption of the oral preparation, animal pharmacokinetic test results show that about 80% of the original drug It is not absorbed and excreted in the feces, and the drug cannot be fully utilized in the body, which affects the full effect of its clinical efficacy.
  • the ginsenoside Rg3 is made into a topical preparation, which is absorbed through the skin of the neck, the mucous membrane of the ear, directly enters the lymphatic system, and is absorbed through the blood-brain barrier to achieve therapeutic effects on the cranial nervous system, thereby obtaining better clinical efficacy, and Further confirmed by pharmacological experiments and clinical applications.
  • borneol could not induce the expression of ICAM-1 on the brain microvessel EC of normal rats, but it could significantly reduce the adhesion of white blood cells and EC during brain injury, and protect the EC.
  • the opening of the blood-brain barrier has nothing to do with ICAM-1.
  • the borneol has a certain protective effect on the brain tissue of rats with traumatic brain injury.
  • Ge Chaoli et al. divided the rats into 10,13 mL ⁇ kg-12 groups of borneol paraffin oil solution. According to the sampling time points, each group was further divided into 1, 2, 4, 8, 24, 48 hours, and the control group was given the same. A quantity of paraffin oil solution.
  • the rats in the two groups were sacrificed at the above time points and observed by transmission electron microscopy. Studies have shown that borneol has a significant effect on the tight junctions between rat brain capillary endothelial cells. Compared with the control group, the tight junctions between the cells become wider, intermittent, and the structure is reduced. It begins to change 4 hours after the borneol is applied, and peaks at 8 hours. At 48h, it returned to normal, indicating that borneol can reversibly change the ultrastructure of blood-brain barrier in rats.
  • Su Hexiang Rats were divided into sulpiride combined with Su Hexiang group and sulpiride group for one week. After continuous oral administration for 1 week, probes were installed in the brain and neck. After 1 hour of equilibration, blood microdialysis and brain microdialysis were used respectively. The dialysate in the blood and brain hippocampus of the right atrium of the rat was collected at various time points (30, 60, 90, 120, 150, 180 min), and the sulpiride content in the sample was detected by a reversed-phase high performance liquid chromatography system. The sulpiride content in the brain and blood of the two groups after administration was compared by statistical methods.
  • the concentration of sulpiride in the brain and blood of the sulpiride group was significantly higher than that of the sulpiride group in the sulpiride group.
  • the concentration ratio of sulpiride in the blood and brain of the sulpiride group was 1:0.2, and sulpiride and sulpho were combined.
  • the blood-to-brain ratio of the group increased by 1:0.3.
  • the sulpiride concentration in the combination group of sulpiride and Su Hexiang increased by 39% and the blood concentration increased by 69%. It indicated that Su Hexiang can significantly increase the concentration of sulpiride in the brain and blood of rats. Su Hexiang can promote sulpiride through the gastrointestinal barrier and blood-brain barrier.
  • Shichangpu is one of the traditional aromatic medicinal herbs. It is widely used by folks or in combination with other traditional Chinese medicines. It is mainly used for the treatment of encephalopathy. It has a definite curative effect, suggesting that Shichangpu has a certain effect on BBB.
  • the volatile oil was extracted, the extraction rate was about 1.58%, and the oil decoction was concentrated to 1g/ml.
  • Gas chromatography-mass spectrometry (GC/MS) was used to detect the volatile oil samples containing ⁇ -asarone, ⁇ -asarone, guaiacolene and flavonol.
  • the cerebrospinal fluid of rats after taking volatile oil was tested under the same conditions. The results showed that ⁇ -asarone and ⁇ -asarone could enter the cerebrospinal fluid through the BBB.
  • Liquid chromatography-mass spectrometry HPLC/MS was used to determine the deoiled decoction samples and the cerebrospinal fluid samples of the deoiled decoction. Three compounds were detected with molecular weights of 396, 452, 339, respectively. It is not included in the Shichangpu compound.
  • the ultrastructure of BBB in the cerebral cortex of rats before and after administration was observed under transmission electron microscope.
  • the EB content in the brain was significantly higher than that in the blank group (P ⁇ 0.05), and there was a dose-dependent trend.
  • the total dose was 11.7g/kg, the EB content in the brain was significantly higher after 5h.
  • the blank group P ⁇ 0.01.
  • the concentration of phenytoin in the brain was determined by HPLC. The results showed that the content of phenytoin in the brain of Shichangpu group was higher than that of ungiven stone.
  • the content of phenytoin in the brain of the calamus group remained high after 4 hours (P ⁇ 0.01).
  • the concentration of serotonin (5-HT) in brain tissue was detected by HPLC-ECD.
  • Statistical analysis showed that the content of 5-HT in the brain of volatile oil group increased compared with the blank group (P ⁇ 0.05).
  • the content of 5-HT in the liquid group and the total drug group increased significantly (P ⁇ 0.01).
  • the Hela cell line was used as a carrier for evaluating the activity of P-glycoprotein on BBB.
  • Shichangpu volatile oil, decoction, and total drug
  • VCR vincristine
  • Shichangpu can affect the ultrastructure of rat BBB, enhance the permeability of BBB to EB, and promote the increase of the content of CNS drug phenytoin into the brain. It has the pharmacological effect of opening BBB and introducing drugs into the brain.
  • the mechanism of action study showed that the regulation of 5-HT by Shichangpu is one of the mechanisms of its increase in BBB permeability.
  • Shichangpu has a strong inhibitory effect on the efflux of P-glycoprotein on Hela cell membrane. It is also the mechanism by which it increases the permeability of BBB.
  • Curcumin contained in turmeric has the effect of inhibiting dementia in the elderly.
  • the main cause of dementia is the entanglement of amyloid beta.
  • Curcumin can loosen the entangled amyloid.
  • Curcumin is a powerful anti-oxidant and anti-inflammatory chemical molecule that crosses the gated blood-brain barrier, neutralizes oxides and oxidatively toxic copper ions, and is more resistant to a cellular transcription factor that causes inflammation. (NF kappa B). Therefore, curcumin is a key component that protects brain tissue and reduces dementia. In India, dementia patients are only 10% of other countries, and curcumin is indispensable.
  • the primary object of the present invention is to provide 20(R)-ginsenoside Rg3 for preventing or/and treating the performance and efficacy of dementia, and to provide a new medicinal use of 20(R)-ginsenoside Rg3, namely in treatment, conditioning and improvement New applications in dementia drugs or health foods.
  • Another object of the present invention is to provide a ginsenoside Rg3 external preparation for the poor oral absorption of ginsenoside Rg3 and the lack of clinical application such as poor blood-brain barrier.
  • the ginsenoside Rg3 external pharmaceutical preparation of the invention is directly applied to the sides of the nose, the neck and the skin behind the ear, and is used for treating dementia diseases, especially for treating and preventing Alzheimer's disease.
  • one aspect of the present invention provides a use of 20(R)-ginsenoside Rg3 for the preparation of a medicament or a health care product for preventing or/and treating a dementia disorder.
  • the dementia condition is Alzheimer's disease and vascular dementia, preferably Alzheimer's disease.
  • the drug consists of 20(R)-ginsenoside Rg3 and a pharmaceutically acceptable carrier.
  • it also includes one or more of ginseng oil, chuanxiong oil, calamus oil, musk oil or turmeric oil.
  • the 20(R)-ginsenoside Rg3 described in the present invention may be used singly or in the form of a pharmaceutical composition containing 20(R)-ginsenoside Rg3 when used for preventing or treating dementia.
  • the medicament of the present invention is administered by oral, sublingual, transdermal, intramuscular, subcutaneous, skin mucosa, intravenous and the like.
  • the present invention provides a pharmaceutical preparation for preventing or treating dementia and a corresponding pharmaceutical dosage form using 20(R)-ginsenoside Rg3 as an active ingredient.
  • the pharmaceutical preparation is such that the 20(R)-ginsenoside Rg3 is an effective active ingredient and includes other pharmaceutically acceptable carrier components.
  • the 20(R)-ginsenoside Rg3 has a purity of ⁇ 80%, preferably more than 90%, further preferably more than 95%.
  • the medicament of the present invention exists in the form of an oral preparation, an injection, and a topical preparation.
  • the oral preparation comprises a tablet, a capsule, a pill, a powder, a granule, a syrup, a solution;
  • the injection comprises an injection dosage form or a lyophilized powder injection form;
  • the topical preparation comprises a cream, an ointment, Spray, aerosol, patch, gel, nasal drops or cataplasm.
  • the medicament in the present invention is in the form of a tablet, a capsule, a pill, a powder, a granule, a syrup, a solution, an injection, a spray, an aerosol, a patch, a gel, a nasal drop, and a cataplasm.
  • the pharmaceutical preparation includes a tablet, a capsule, a pill, a powder, a granule, a syrup, a solution, an injection, a spray, an aerosol, a patch, a gel, a nasal drop, a cloth, and the like. But not limited to the above form.
  • a medicament or a health care product comprising 20(R)-ginsenoside Rg3 for preventing or/and treating dementia is provided.
  • the medicine or health care product further comprises one of ginseng volatile oil, musk volatile oil, calamus volatile oil, turmeric volatile oil or Chuanxiong volatile oil.
  • the content of the 20(R)-ginsenoside Rg3 in the medicament or health care product is ⁇ 80%, preferably higher than 90%, further preferably higher than 95%.
  • the inventors have extracted the active ingredient 20(R)-ginsenoside Rg3, which is the active ingredient for preventing and treating dementia, from a ginseng medicinal material by a large amount of modern scientific research, and using advanced separation and purification technology, and 20(R)-ginsenoside Rg3 and The corresponding pharmaceutical preparations were tested for pharmacodynamics and pharmacology of anti-dementia. The results showed that the 20(R)-ginsenoside Rg3 monomer has clear pharmacological effects, strong anti-dementia effect, low toxicity and high safety, and anti-aging. Provide a highly effective and low toxicity drug.
  • the present invention Compared with the existing drugs for preventing and treating dementia, the present invention has the following advantages:
  • the present invention excavates a new medicinal value for the known compound 20(R)-ginsenoside Rg3, which is used for preventing and treating dementia, and can be prepared as a medicine for preventing or treating dementia or a health food, thereby It has opened up a new field for the application of ginseng herbs.
  • Ginsenoside Rg3 significantly increased the density of AChE-positive nerve fibers in the cerebral cortex, indicating that ginsenoside Rg3 has protective effects on the brain cholinergic nervous system in Alzheimer's disease; ginsenoside Rg3 in rat model of Alzheimer's disease In the experiment of learning and memory, the rats in the water maze swimming time is short and the number of errors is small, indicating that ginsenoside Rg3 can significantly improve the learning and memory ability of Alzheimer's disease model rats; ginsenoside Rg3 can reduce the cerebrospinal fluid in AD patients A ⁇ content, used in clinical treatment of AD, can effectively improve the intelligence and memory of patients, improve cognitive function and self-care ability in daily life.
  • the 20(R)-ginsenoside Rg3 of the invention has strong pharmacological action, has remarkable efficacy for anti-aging, has quick effect, small toxic and side effects, good safety, and can be taken for a long time, and has good medicinal prospects.
  • the raw materials of the invention have rich sources, low cost, safe clinical use, simple preparation process, can be made into various dosage forms, and have small dosage and convenient use, so it is easy to promote.
  • Figure 1 is a two-stage full-scan mass spectrum of ginsenoside Rg3 (A) and diosgenin (B) excimer ion [M-H]-;
  • Figure 2 is a typical chromatogram of ginsenoside Rg3 (channel I, m/z 783.8 ⁇ 160.8) and internal standard diosgenin (channel II, m/z 867.5 ⁇ 721.5) in liver homogenate by LC/MS/MS; Among them: (A) rat blank liver homogenate; (B) rat blank liver homogenate added 2ng / ml ginsenoside Rg3 and 100ng / ml internal standard diosgenin; (C) rats orally administered 10mg / kg ginsenoside Rg3 oral post-milk brain fluid sample (10.8 ng/g).
  • Test Example 1 Protective effect of ginsenoside Rg3 on the brain cholinergic nervous system in a rat model of Alzheimer's disease
  • the ginsenoside Rg3 was prepared into a topical lotion according to the formulation and method of Example 7, and was prepared by Dalian Fusheng Pharmaceutical Co., Ltd., batch number: 20120425;
  • Huperzine A produced by Shanghai Hongqi Pharmaceutical Factory, batch number: 20111215;
  • D-galactose produced by Shanghai Reagent II, batch number: 20110607.
  • IBO Ibotenic acid
  • Wistar rats aged 5 months old, 60 females, weighing 300-450 g, were purchased from Experimental Animal Center of Dalian Medical University, and the quality certificate number was SCXK (13) 2012-0003.
  • Sixty Wistar rats were randomly divided into 6 groups, 10 in each group. They were named as the normal control group in the first-old age, the AD model group in the old-aged group, and the positive drug huperzine A group according to different treatment methods. High-, medium-, and low-dose groups of ginsenoside Rg3 external preparations. Rats were housed in cages, 4 to 5 per cage, with natural lighting, free access to water and food. Rats in the normal control group were injected with normal saline in the peritoneal cavity and brain.
  • the rats in the old AD model group, the positive drug huperzine A group and the ginsenoside Rg3 topical preparations were intraperitoneally injected with galactose and the rats in the high, medium and low dose groups.
  • Intracerebral injection of IBO; huperzine A and ginsenoside Rg3 high, medium and low dose rats were given D-galactose injection molding, and then huperzine A was given separately.
  • 0.3mg/kg/d and ginsenoside Rg310, 5, 2.5mg/kg/d, ginsenoside Rg3 was applied to both sides of the rat's nose, and they were administered for 6 weeks. Then, bilateral Meynert nuclear injection was performed in the brain.
  • the Meynert nuclear localization referenced the stereotaxic map of the rat brain with the positioning coordinates of AP-0.8mm, Late 2.6mm, and DV 8.2mm.
  • Intracerebral injection the rats were anesthetized with 3% sodium pentobarbital (30mg/kg) and fixed on the brain stereotaxic apparatus. IBO dissolved in physiological saline (1ul per side containing IBO 5ug) or 1ul The saline was slowly injected into the Meynert nucleus, and the injection time was continued for 5 min on each side, leaving the needle for 10 min. All the rats were perfused with 4% paraformaldehyde at the end of modeling, and then fixed for 24 hours.
  • AChE-positive nerve fibers were changed into sucrose phosphate buffer until the brain tissue was sunk.
  • the incubator was frozen and serially sliced, and the thickness was 40 mm.
  • Sections were stained with acetylcholinesterase (AChE) histochemical method according to the method recommended by Hedreen et al.
  • AChE-positive nerve fibers were Observe the AChE-positive nerve fibers in the frontal, parietal, occipital and entorhinal cortex (Layer II) and the hippocampal CA1 region of the polymorphic layer, radiation layer, lacunar molecular layer and dentate gyrus Density, using a microscope micrometer, the number of AChE-positive nerve fibers was counted by the number of intersections of positive fibers and standard grids.
  • AChE-positive nerve fibers was counted by the number of intersections of positive fibers and standard grids.
  • test results of acetylcholinesterase-positive nerve fiber density in rat cortex and hippocampus are shown in Tables 1 and 2.
  • the results are expressed as (X ⁇ S), and SAS 6.11 statistical software package was used for one-way analysis of variance.
  • ginsenoside Rg3 has a protective effect on the brain cholinergic nervous system in the rat model of Alzheimer's disease.
  • Test Example 2 Effect of ginsenoside Rg3 on learning and memory in rat model of Alzheimer's disease
  • the ginsenoside Rg3 was prepared into an oral emulsion (10 mg/ml) according to the formulation and method of Example 17, and was prepared by Dalian Fusheng Pharmaceutical Co., Ltd., batch number: 20120320;
  • D-galactose produced by Shanghai Reagent II, batch number: 20110607.
  • IBO Ibotenic acid
  • Test method After 1w feeding in stable laboratory conditions, the water maze pre-training was carried out, and 60 rats with similar body weight and good flexibility were randomly divided into 6 groups, normal control group and sham control group. Model control group and ginsenoside Rg3 high, medium and low (10, 5, 2.5 mg/kg/d) dose groups, 10 animals per group.
  • the modeling method was performed by intraperitoneal injection of 0.96% D-galactose prepared by physiological saline, 5 mL/kg once a day for 6 weeks.
  • the sham-operated control group was injected with the same amount of normal saline.
  • the model control group and the ginsenoside Rg3 high, medium and low dose group of rats were injected intracranially with IBO, 1 ⁇ L of IBO was slowly injected on each side, and the sham operation group was injected in the same brain area.
  • the normal control group did not do any treatment, eating and drinking normally.
  • the high, medium and low dose groups of ginsenoside Rg3g were administered orally with ginsenoside Rg3 orally, and administered continuously for 4 weeks (weeks).
  • Test Example 3 Effect of ginsenoside Rg3 on ⁇ amyloid content in cerebrospinal fluid of patients with Alzheimer's disease
  • the ginsenoside Rg3 was prepared according to the formulation and method of Example 27 to prepare adult ginsenoside Rg3 oral solution (10 mg/ml), and was prepared by Dalian Fusheng Pharmaceutical Co., Ltd., batch number: 20120321;
  • the clinical efficacy of the two groups was analyzed by ginsenoside Rg3 oral liquid group with increased clinical symptom scores in 14 cases, scores unchanged in 3 cases, and score reduction in 1 case, the effective rate was 77.8%. There were 13 patients with increased symptom scores in the donepezil group, 3 patients with no scores, and 2 patients with decreased scores. The effective rate was 72.2%. There was no difference between the two groups (P>0.05).
  • the ginsenoside Rg3 was prepared according to the formulation and method of Example 29 to prepare adult ginsenoside Rg3 nasal drops (10 mg/ml), which was prepared by Dalian Fusheng Pharmaceutical Co., Ltd., batch number: 20120324.
  • 89 patients with mild to moderate Alzheimer's disease were randomly divided into 2 groups, one group took ginsenoside rg3, the other group used placebo, of which 46 patients were in rg3 group, 21 in male and 25 in female, age 58 ⁇ 87 (72 6 ⁇ 6.8) years old, simple intelligent state detection and removal scale (MMSE) score of l0 ⁇ 24 (17 8 ⁇ 2.3) points; 43 cases of placebo, male 19 cases, female 24 cases, age 57 ⁇ 88 (71.8 ⁇ 8.2) years old, MMSE score of 10 ⁇ 24 (18.2 ⁇ 2.7) points. There were no significant differences in gender, age, and MMSE scores between the ginsenoside Rg3 group and the placebo group. Patients taking ginsenoside rg3 were given ginsenoside Rg3 nasal drops once a day for 10 mg/ml/time for 12 weeks.
  • MMSE simple intelligent state detection and removal scale
  • ginsenoside rg3 Patients who received placebo were treated with the same method as ginsenoside rg3 except for placebo, which used the same color, traits, taste and dosage as ginsenoside Rg3 nasal drops.
  • the placebo was prepared in accordance with the method of preparing a ginsenoside rg3 nasal drop in Example 29, except that the ginsenoside rg3 was not added, and the same as in Example 29.
  • MMSE screening for AD Detecting the patient's cognitive function (orientation, memory, computational power, language ability, visual spatial segment ability, etc.); (2) clinical dementia degree scale (CDR) to detect the degree of dementia in patients; (3) daily living self-care
  • CDR clinical dementia degree scale
  • ADL measures the patient's ability to take care of daily life. Check the results using SPSS statistical software. Before and after treatment and ginsenoside Rg3 nasal drop group and placebo control were tested by t test. The results of MMSE, CDR and ADL scores of AD patients before treatment are shown in Table 5.
  • the MMSE, CDR and ADL scores of AD patients after 12 weeks of taking rg3 and placebo are shown in Table 6.
  • Cognitive function using MMSE score: The efficacy of the placebo-controlled study group showed that the ginsenoside Rg3 group had a significantly higher MMSE score than the placebo group (P ⁇ 0.01); the degree of dementia (using CDR score): treatment The ginsenoside Rg3 nasal drops group had significantly lower CDR scores than the placebo control group (P ⁇ 0.05); daily living self-care ability (using ADL score): ADL score of ginsenoside Rg3 nasal drop group after treatment compared with placebo control group There was a significant decrease in roots (P ⁇ 0.01), and there was a significant decrease in treatment after treatment compared with 7.1 points (P ⁇ 0.01).
  • ginsenoside Rg3 nasal drops are used to treat AD patients, improve the mild and moderate cognitive dysfunction in AD patients, A drug that is safe and effective in reducing the degree of self-care and dementia.
  • the ginsenoside Rg3 was prepared according to the formulation and method of Example 29 to prepare adult ginsenoside Rg3 nasal drops (10 mg/ml), which was prepared by Dalian Fusheng Pharmaceutical Co., Ltd., batch number: 20120324.
  • ginsenoside Rg3 nasal drops Patients with AD were treated with ginsenoside Rg3 nasal drops, and ginsenoside Rg3 nasal drops (10 mg/ml) were used once daily for 12 weeks.
  • MMSE screening for AD Detecting the patient's cognitive function (orientation, memory, computational power, language ability, visual spatial segment ability, etc.); (2) clinical dementia degree scale (CDR) to detect the degree of dementia in patients; (3) daily living self-care
  • CDR clinical dementia degree scale
  • ADL measures the patient's ability to take care of daily life. The results of the examination were analyzed by SPSS statistical software before and after treatment with ginsenoside Rg3 nasal drops and placebo.
  • the MMSE, CDR and ADL scores of AD patients are shown in Table 7.
  • Cognitive function (using MMSE score): After 12 weeks of treatment, there was a significant improvement compared with before treatment, and the MMSE score increased by 3.5 points (P ⁇ 0.01), and the MMSE score was significantly improved at 4 weeks of treatment (P ⁇ 0.05).
  • the degree of dementia (with CDR score): After 12 weeks of treatment, there was a significant decrease from the pre-treatment period, and the CDR score decreased by 0.6 points (P ⁇ 0.05).
  • Self-care ability in daily life (using ADL score): After treatment, there was a significant decrease in the ADL score, and the ADL score decreased by 7.1 (P ⁇ 0.01).
  • ginsenoside Rg3 nasal drops are used to treat AD patients, improve the mild and moderate cognitive dysfunction in AD patients, A drug that is safe and effective in reducing the degree of self-care and dementia.
  • Ginsenoside Rg3 (98%) 2g, azone 2g, polyethylene glycol 400 10ml, ethanol (70%)
  • Ginsenoside Rg3 (98%) 2g, azone 2g, polyethylene glycol 600 10ml, ethanol (70%)
  • Ginsenoside Rg3 (63%) 2g, azone 2g, polyethylene glycol 200 10ml, ethanol (65%)
  • Ginsenoside Rg3 (30%) 1g, glycerol 2g, polyethylene glycol 200 10ml, ethanol (70%)
  • Ginsenoside Rg3 (98%) 1 g, medium chain triglyceride 20 g, propylene glycol 10 ml, phospholipid 20 g, and water for injection.
  • ginsenoside Rg3 medium chain triglyceride, propylene glycol, phospholipid
  • ginsenoside Rg3 medium chain triglyceride, propylene glycol, phospholipid
  • the medium chain triglyceride used in the embodiment of the present invention namely, citric acid glyceride
  • MCT semi-synthetic natural functional fat
  • This product can be widely used in medicine, food, health care products and cosmetics.
  • Ginsenoside Rg3 (98%) 2g, azone 2g, ginseng oil 0.5g, propylene glycol 10ml, ethanol (65%)
  • Ginsenoside Rg3 (98%) 2g, azone 2g, turmeric oil 1g, polyethylene glycol 400 10ml, ethanol (70%)
  • Ginsenoside Rg3 (98%) 2g, azone 2g, musk oil 0.05g, polyethylene glycol 600 10ml, ethanol (70%)
  • Ginsenoside Rg3 (63%) 2g, azone 2g, Chuanxiong oil 0.2g, polyethylene glycol 200 10ml, ethanol (65%)
  • Ginsenoside Rg3 (30%) 1g, glycerol 2g, calamus oil 0.2g, polyethylene glycol 20010ml, ethanol (70%)
  • Ginsenoside Rg3 (98%) 1 g, medium chain triglyceride 20 g, borneol 0.5 g, propylene glycol 10 ml, phospholipid 20 g, water for injection.
  • ginsenoside Rg3 medium chain triglyceride, borneol, propylene glycol, phospholipid
  • ginsenoside Rg3 medium chain triglyceride, borneol, propylene glycol, phospholipid
  • Ginsenoside Rg3 (98%) 2g, azone 2g, artificial bezoar 0.5g, propylene glycol 10ml, ethanol (65%)
  • Ginsenoside Rg3 (98%) 2 g, azone 2 g, Su Hexiang 0.5 g, polyethylene glycol 400 10 ml, 70% ethanol was added to 100 ml.
  • ginsenoside Rg3, 2 g of azone, 0.5 g of sulphate, 10 ml of polyethylene glycol 400, and 70 ml of ethanol were added to 85 ml, and the mixture was placed in a 500 ml flask, stirred by heating for 30 minutes to dissolve, and 70% ethanol was added thereto. 100 ml, a ginsenoside Rg3 external preparation was obtained.
  • Ginsenoside Rg3 (98%) 1 g, soybean oil 20 g, propylene glycol 10 ml, powdered phospholipid 20 g, distilled water in an appropriate amount.
  • ginsenoside Rg3 vegetable oil, propylene glycol, powdered phospholipids, added to a small amount of distilled water, heated to 65-70 °C, stirring to dissolve ginsenoside Rg3, the homogenized mixture is added to the homogenizer for homogenization treatment, cooled to room temperature (15-25 ° C), and then added with distilled water to 100ml to obtain ginsenoside Rg3 oral milk .
  • Ginsenoside Rg3 (98%) 1 g, soybean oil (for injection) 50 g, medium chain triglyceride 100 g.
  • Ginsenoside Rg3 (98%), vegetable oil, medium chain triglyceride is heated to 80-85 ° C, and ginsenoside Rg3 is dissolved by stirring to obtain ginsenoside Rg3 injection.
  • Ginsenoside Rg3 (98%) 1 g, ginseng volatile oil 2 g, propylene glycol 10 ml, Chuanxiong oil 0.5 g, phospholipid 20 g, distilled water amount.
  • ginsenoside Rg3 ginseng volatile oil, propylene glycol, chuanxiong oil, and phospholipid
  • distilled water heat to 65-70 ° C
  • additional distilled water was added to 100 ml to obtain ginsenoside Rg3 oral milk.
  • Ginsenoside Rg3 (85%) 1 g, calamus oil 20 g, propylene glycol 10 ml, powdered phospholipid 20 g, distilled water amount.
  • ginsenoside Rg3 calamus oil, propylene glycol, powdered phospholipid to a small amount of distilled water, heat to 65-70 ° C, stir to dissolve ginsenoside Rg3, add the uniformly mixed mixture to the colloid mill for treatment, and cool After adding to room temperature (15-25 ° C), additional distilled water was added to 100 ml to obtain ginsenoside Rg3 oral milk.
  • Ginsenoside Rg3 (63%) 1g, tulip oil 10g, medium chain triglyceride 100g
  • the ginsenoside Rg3, the tulip oil, the medium chain triglyceride is heated to 50-55 ° C, and the ginsenoside Rg3 is dissolved by stirring to obtain a ginsenoside Rg3 oral solution.
  • Ginsenoside Rg3 (30%) 1g, Chuanxiong oil 10g, medium chain triglyceride 100g
  • Ginsenoside Rg3, Chuanxiong oil, medium chain triglyceride are heated to 50-55 ° C, and ginsenoside Rg3 is dissolved by stirring to obtain ginsenoside Rg3 oral solution.
  • Ginsenoside Rg3 (98%) 1 g, musk oil 2 g, propylene glycol 10 ml, phospholipid 20 g, distilled water in an appropriate amount.
  • Ginsenoside Rg3 (98%) 1 g, medium chain triglyceride 20 g, musk oil 3 g, propylene glycol 10 ml, phospholipid 20 g, water for injection.
  • ginsenoside Rg3 medium chain triglyceride, musk oil, propylene glycol, phospholipid
  • a small amount of water for injection heat to 65-70 ° C, stir to dissolve ginsenoside Rg3, and mix the mixture with uniform stirring. Homogenization was carried out in a homogenizer, and after cooling to room temperature (15-25 ° C), water for injection was added to 100 ml to obtain ginsenoside Rg3 injectable milk.
  • Ginsenoside Rg3 (98%) 1 g, medium chain triglyceride 20 g, calamus oil 2 g, polyethylene glycol 100 10 ml, phospholipid 20 g, water for injection.
  • ginsenoside Rg3 medium chain triglyceride, calamus oil, propylene glycol, phospholipid
  • a small amount of water for injection heat to 65-70 ° C, stir to dissolve ginsenoside Rg3, and mix the mixture with uniform stirring. Homogenization was carried out in a homogenizer, and after cooling to room temperature (15-25 ° C), water for injection was added to 100 ml to obtain ginsenoside Rg3 injectable milk.
  • Ginsenoside Rg3 (98%) 1 g, sucrose 50 g, calamus oil 2 g, dextrin 100 g, silica 5 g.
  • calamus oil Take the prescribed amount of calamus oil, silica, and mix.
  • the silica is adsorbed to the calamus oil completely.
  • a prescribed amount of ginsenoside Rg3, sucrose, dextrin is added.
  • the ginsenoside Rg3 granules were obtained by granulating with 70% ethanol, sieving, and drying at 65-70 °C.
  • Ginsenoside Rg3 (98%) 1 g, sucrose 50 g, Chuanxiong oil 2 g, dextrin 100 g, silica 3 g.
  • Ginsenoside Rg3 (98%) 1 g, starch 100 g, ginseng oil 3 g, microcrystalline cellulose 100 g, betacyclodextrin 10 g, talc powder 1 g.
  • ginsenoside Rg3 (98%) 1 g, 100 g of starch, and 100 g of microcrystalline cellulose were added, and an appropriate amount of ethanol was added to granulate. After sieving the whole granules, 1 g of talc powder was added and tableted to obtain ginsenoside Rg3 tablets.
  • Ginsenoside Rg3 (98%) 1 g, sucrose 50 g, Chuanxiong oil 3 g, betacyclodextrin 10 g, sodium benzoate 2 g, distilled water amount.
  • ginsenoside Rg3 (98%) 1 g, sucrose 50 g, and sodium benzoate 2 g were added, and distilled water was added to 500 ml to obtain a ginsenoside Rg3 oral solution.
  • Ginsenoside Rg3 (98%) 1 g, musk oil 1 g, 90% ethanol 10 ml, glycerin 5 g, F12/F114 (a propellant of a (Freon) compound) was added to 100 g.
  • Ginsenoside Rg3 (98%) 1 g, Chuanxiong oil 1 g, 60% ethanol 10 ml, polyethylene glycol 200 5 ml, glycerin 20 g, water for injection.
  • the ginseng oil, the chuanxiong oil, the musk oil, the turmeric oil, the scallop oil, the borneol, the artificial bezoar, the Su Hexiang and the like are used in the embodiments of the present invention, and the main functions thereof are aromatic sputum, refreshing and returning to the Soviet Union, and clinical treatment. Stroke, mental coma and other evidence. In recent years, studies have found that sputum drugs can exert their effects on the treatment of brain diseases by affecting the blood-brain barrier (BBB). Some sputum drugs can promote the penetration of other drugs, which is the main reason for their development. The mechanism of action is presumed to be the theoretical basis for the drug's return to the heart.
  • BBB blood-brain barrier
  • borneol has the effect of improving the permeability of the blood-brain barrier and promoting the entry of drugs into brain tissue.
  • the medicinal properties of musk, benzoin and Su Hexiang have a certain opening effect on the blood-brain barrier in the physiological state of mice, which can promote the entry of drugs into the brain. organization.
  • rg3 preparation In the preparation of rg3 preparation, azone, polyethylene glycol, ethanol, Chinese medicine volatile oil and other ingredients are added to the prescription. The addition of these ingredients can make the blood-brain barrier open, and let rg3 enter the brain tissue to exert its anti-dementia.
  • the role of the patent also in the "Example 30 ginsenoside Rg3 different dosage forms administered by the blood brain barrier absorption test" again verified that rg3 has entered the physiological state of the rat brain. Therefore, the formulations and processes designed in this patent have prepared anti-dementia effects.
  • the ginseng oil used in the examples of the present invention was purchased from Hualong Spice Oil Co., Ltd., Qingyuan District, Ji'an City, Jiangxi province; the Chuanxiong Oil was purchased from Senhai Spice Co., Ltd., Ji'an City, Jiangxi Province; the eucalyptus oil was purchased from Jiangxi Baicao Herbal Industry Co., Ltd.; The iris was purchased from Xi'an Ruiying Biotechnology Co., Ltd.; the tulip oil was purchased from Jiangxi Herbal Natural Flavor Oil Co., Ltd.; the turmeric oil was purchased from Jiangxi Hengcheng Natural Flavor Oil Co., Ltd.; the ginseng volatile oil was purchased from Jiangxi Global Natural Flavor Co., Ltd.
  • the main component of ginseng oil is essential oil content% ⁇ 11, acid value KOHmg/g ⁇ 30, ginsenoside, ginseng polysaccharide, volatile oil, amino acid and polypeptide, ⁇ -elemene, ⁇ -gutriene, ⁇ -ginsene, ⁇ - ginsengene, syringene, ⁇ -farnesene, palmitic acid;
  • the main component of ginseng volatile oil is sesquiterpene, accounting for about 40%, followed by oxygenates and long-chain alkanes.
  • Rat brain fluid extraction instrument size surgical scissors. Scorpion, hemostat, 1mL syringe. Scale, tray, sterile Cotton ball, iodophor, Angle Two brain stereotaxic instrument (MyNeurolab, USA), low speed dental drill (Grobet, USA), microinjection pump (KdScientific, USA), 10 ⁇ L and 100 ⁇ L microinjector (Hamilton, USA).
  • Rg3 drug content determination instrument in rat brain pulp fluid API 4000 triple quadrupole mass spectrometer with ion spray ion source and Analysis 1.3 data processing system, Applied Biosystem, USA; Agilent 1100 high performance liquid chromatography infusion pump, automatic The sampler was Agilent, USA; the column was a Nucleosil C18 column (50 x 4.6 mm ID, 5 ⁇ m particle size), Dalian Elite Analytical Instruments Co., Ltd.
  • the ginsenoside Rg3 preparations prepared in Examples 1-2, Examples 6-14, Examples 16-21, and Examples 25-29.
  • Oral preparation of ginsenoside Rg3 was intragastrically administered to SD rats, and the high, medium and low dose groups were respectively 20, 10, 5 mg/kg (the above dosage is pure dose of rg3) / bid 18 rats per dose group;
  • ginsenoside Rg3 was administered to SD rats, and the external dosage form drugs were applied to the acupoints of Qinglitouyu or the alar, neck and ear, respectively.
  • the high, medium and low dose groups were respectively: 10 5, 2.5 mg / kg (ie, the amount of rg3 is 10, 5, 2.5 mg / kg) / bid, 18 rats per dose group;
  • ginsenoside Rg3 injection into the SD rats was injected into the tail vein, the high, medium and low dose groups were: 6, 3, 1.5 mg / kg / bid, rats in each dose group 18 only.
  • ginsenoside Rg3 Different preparations of ginsenoside Rg3 were extracted from the rat brain and marrow fluid 4 hours after the above oral, topical and injection administration.
  • the rat cerebrospinal fluid method was performed by cutting the dura mater under direct vision, and the reference literature (improvement of rat cerebrospinal fluid extraction method. Journal of Hebei Medical University, 2010 (31) 2: 125-127) was used. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate.
  • the head of the rat was fixed with the ear straight body and the incisor rod.
  • the head pillow was cut and disinfected, and a longitudinal incision (about 2 cm) was cut along the posterior midline. The blunt dissection separates the dorsal muscles of the item.
  • Rats not administered ginsenoside Rg3 were used as blank controls.
  • the concentration of the drug in the rat brain spinal fluid was determined by LC/MS/MS.
  • Ginsenoside Rg3 reference substance purchased from China National Institute for the Control of Pharmaceutical and Biological Products, batch number: 110804-200603.
  • Ginsenoside Rg3 raw material produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 2012303, HPLC calibrated by ginsenoside Rg3 reference substance, the content is 98.2%;
  • ginsenoside Rg3 high, medium and low doses in other preparations of other ginsenoside Rg3 are the same as those of ginsenoside Rg3 raw materials.
  • the contents of different preparations of ginsenoside Rg3 are shown in the test results.
  • acetonitrile, methanol, and ethyl acetate were chromatographically pure, purchased from Fisher Company of the United States and Tianjin Concord Technology Co., Ltd.; physiological saline was purchased from Shenyang Zhiying Pharmaceutical Factory.
  • Tissue sample treatment Take 200 ⁇ l of the extracted brain pulp solution, add 100 ⁇ l of methanol solution, 100 ⁇ l of internal standard solution (100 ng/ml diosgenin methanol solution), 0.5 ml of water, mix, add 3 ml of ethyl acetate, vortex for 1 min, shake for 10 min, After centrifugation (4000 rpm) for 8 min, the supernatant was separated and dried under a stream of air at 40 ° C, and the residue was dissolved in 200 ⁇ l of the mobile phase, and 20 ⁇ l of the analysis was carried out.
  • the mobile phase was methanol-acetonitrile-10 mmol/l acetic acid amine solution (47.5:47.5:5, v/v), the flow rate was 0.7 ml/min, and the column temperature was room temperature.
  • Mass spectrometry conditions ion ejection voltage: -3500 V; source gas 1 (GS1, N 2 ) pressure: 30 p.si; gas 2 (GS2, N 2 ) pressure: 30 p.si; gas curtain gas (N 2 ) pressure: 15 p. Collision gas (N 2 ) pressure: 3 p.si; source temperature: 500 ° C.
  • Detection method negative ion detection; scanning method: select reaction monitoring (MRM) method, the ion reaction for quantitative analysis is m/z 783.8 ⁇ m/z 160.8 (ginsenoside Rg3), m/z 867.5 ⁇ m/z 721.5 (internal standard, diosgenin).
  • MRM select reaction monitoring
  • Method specificity Take the untreated blank rat brain and marrow fluid, except that no internal standard solution is added and 100 ⁇ l of methanol is additionally added, and the other method is performed according to the method of “tissue sample processing” to obtain a chromatogram of the blank sample; ginsenoside Rg3 control product series solution (2ng/ml) and internal standard solution (100ng/ml) were added to the white rat brain and marrow fluid, and the corresponding chromatogram was obtained according to the same method. The same method was used to obtain the brain medullary fluid after rat administration. Sample chromatogram.
  • Fig. 2 is a chromatogram of a rat brain medullary blank sample, a blank sample added with rg3 control and an internal standard (diamond saponin) solution and a sample after administration.
  • Working curve preparation Take 200 ⁇ l of the tissue homogenate supernatant of the blank control group, add 100 ⁇ l of the ginsenoside Rg3 control solution series solution (add 100 ⁇ l of Rg3 control solution to 200 ⁇ l of the supernatant), and prepare the tissue concentration to be 2, 4, Tissue samples of 10, 20, 40, 100, 200 and 400 ng/g.
  • the operation curve was established by operating under "Organization Sample Processing".
  • the peak area ratio of the chromatogram of the analyte and the internal standard is the ordinate
  • the regression operation is performed by the weighted least squares method, and the obtained linear regression equation is the working curve.
  • the linear range of ginsenoside Rg3 concentration in the brain was determined to be 2-400 ng/g, and the lowest limit of quantification was 2 ng/g.
  • QC samples high-, medium-, and low-quality control samples (QC samples) were prepared, and each sample was subjected to two-sample analysis. According to the working curve of each analysis batch, the concentration of QC sample is calculated. In the 6 QC (18 samples in total) samples, up to 2 samples of different concentrations are allowed to exceed 15% of the theoretical value (the lowest point is 20%), otherwise the batch The data is not accepted.
  • Table 8 Different preparations of ginsenoside Rg3 in rat brain pulp by blood-brain barrier test

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Abstract

一种人参皂苷Rg3在制备用于防治脑痴呆药物或保健品中的用途。所述药物或保健品包括人参皂苷Rg3的外用制剂、口服制剂和注射剂。

Description

人参皂苷Rg3在制备用于预防或/和治疗痴呆病症药物中的应用及药物 技术领域
本发明涉及人参皂苷Rg3外用制剂及其制备方法和用途，属于医药领域。
背景技术
进入二十一世纪，随着人口的老龄化，全世界患老年性痴呆的发病率在逐年增多，据报道全世界约有2000万人患老年性痴呆，在中国约有500-700万人，因患老年性痴呆而死亡的人数在心脏病，癌症，中风之后，居第四位。中国65岁以上，痴呆患病率位6-7％；70岁以上为13-15％，80岁以上为22-25％；然而令人遗憾的是，有效治疗和预防老年性痴呆迄今为止还没有更好的方法。
老年性痴呆有阿尔茨海默病(Alzheimer disease，简称AD)和血管性痴呆(Vascular dementia，简称VD)，现代医学关于AD病因至今不明，目前普遍认为是一种渐进性，神经退化性疾病。而血管性痴呆(VD)是有脑颅内出血，多发性梗塞，动脉硬化性脑病急颅脑外伤，颅内占位性病变，酒精中毒，大脑营养缺乏，代谢内分泌疾病等所致的各种痴呆症。
无论何种原因引发的老年性痴呆，早期症状都是健忘，计算能力和语言能力减退，第二阶段是，严重的记忆力丧失，迷失方向和寡言少语，第三阶段是，严重的思维能力混乱方位模糊。因此研究预防和治疗老年性痴呆，首先是预防记忆力和智力的衰退，人参皂苷Rg3具有明显的改善记忆力，延缓衰老作用，这也是本发明的目的之一。
现在医学大量研究资料证明，老年性痴呆人的大脑皮层和海马胆碱神经元大量丢失，胆碱能传递介质乙酰胆碱(Ach)突触前特异性胆碱能标志胆碱乙酰化转移酶(ChAT)显著减少，这是引发老年痴呆的病因之一，那么如何能增加脑干网状结构上行激活系统的机能，促进乙酰胆碱的合成，是预防和治疗老年性痴呆的一种有效方法，人参皂苷Rg3能够有效激活脑干网状结构上行激活系统的机能，促进促进乙酰胆碱的合成，增加脑供血量，善微循环，因而可以用于治疗和预防老年性痴呆，这也是本发明的目的之二。
目前在预防和治疗痴呆中所用的药物主要有，多奈哌齐(donepezil)，利斯的明(rivastigmine)，加兰他敏(galantamine)，吡拉西坦(piracetam)，阿尼西坦(aniracetam)，长春西汀(vinpocetine)，尼麦角林(nicergoline)，二氢麦角碱(dihydroergotoxine)，石杉碱A(huperaine)，银杏叶制剂(ginkgo leaves agent)，脑蛋白水解物(brain protein hydrolysate)，胞磷胆碱(citicoline)，胆碱酯酶抑制剂，黄嘌呤衍生物(xanthine)，非甾体抗炎药物，雌激素 等等，这些药物都有一定的缓解作用，但是相应的副反应很大，限制了它们的普遍使用。
中医对老年性痴呆用药有1，从肝肾论治，多采用添精益髓方药，如熟地黄，山茱萸，黄精，龟板，阿胶，枸杞子，鹿茸等；2，从痰论治，多采用健脾化湿，祛痰开窍醒神的方药，如郁金，石菖蒲，远志，益智仁，醒神胶囊等；3，从气滞血淤论治，多采用理气活血通窍醒脑之方药。如当归芍药散，补阳还五汤，通脉益智丹等，这类药物使用需要中医因人而异，辨正施治，患者不能单独使用，不易自行掌握用药。本发明的天然植物提取的有效成分人参皂苷Rg3具有纯度高(80％以上)，疗效显著独特，所制成的制剂使用方便，患者可以自行根据自身反应调整使用剂量，其透皮吸收后作用温和持久。
人参为五加科多年生草本植物，中国古老的关于中药的药学典籍《神农本草经》把人参列为上品，言其“主补五脏，安精神，定魂魄，止惊悸，明目，开心益智，久服有轻身延年之功效”。经现代医学研究及化验分析表明，人参内含有一种叫人参皂甙的化学物质，它对调节人的中枢神经系统、强心、抗疲劳、调节物质代谢等有明显功效，所以对治疗神经系统.心血管系统、内分泌系统及生殖系统的多种疾病有很好的治疗作用。
人参能调节中枢神经系统兴奋过程和抑制过程的平衡。通过人参对动物脑电活动影响的研究，结果表明：其对兴奋和抑制两种神经过程均有影响，但主要加强大脑皮层的兴奋过程。由于同时作用于抑制过程，故使抑制趋于集中，使分化加速且更完全。人参可调节神经功能，使紧张造成紊乱的神经过程得以恢复。
有报告认为，人参提取物对樟柳碱和戊巴比妥钠造成的记忆获得不良有拮抗作用，亦能改善环己酰亚胺和亚硝酸钠造成的记忆巩固障碍及40％乙醇造成的记忆再现缺陷。用人参茎叶皂甙200mg/kg、100mg/kg、50mg/kg给小鼠腹腔注射，可明显对抗樟柳碱的作用和改善小鼠的记忆，增加脑内的RNA，但对DNA和蛋白质含量无明显影响。
人参对脑血流量和脑能量代谢亦有明显的影响。人参制剂可增加兔脑葡萄糖的摄取，同时减少乳酸、丙酮酸和乳酸/丙酮酸的比值，并可使葡萄糖的利用从无氧代谢途径转变为有氧代谢。人参亦可使大脑皮层中自由的无机磷增加25％。人参果皂甙能提高脑摄氧能力。人参总皂甙、人参根总皂甙对脑缺血/再灌注损伤均有保护作用。总之，人参能使动物大脑更合理地利用能量物质葡萄糖，氧化产能，合成更多的ATP供学习记忆等活动之用。
人参皂苷Rg3是人参稀有皂甙，在5年以上红参中仅含10万分之3，是人参中最精华的成分。人参皂苷Rg3对于很多疾病有改善和预防作用，对于中老年的常见疾病，心脑血管疾病、冠心病、四肢乏力、腿脚不便、记忆力减退都有很好的疗效。
人参皂苷Rg3是人参二醇类四环三萜皂苷，在实际应用中发现，由于人参皂苷Rg3分子 量较大(785.02)，其脂溶性和水溶性都较差，使其口服吸收百分数约为10.28％，导致该口服制剂吸收较差，动物药代动力学试验结果表明约有80％的原形药物未被吸收经粪便排泄，药物在体内不能得到充分利用，影响其临床疗效的充分发挥。因此，将人参皂苷Rg3制成外用制剂，通过颈部皮肤，耳后粘膜吸收，直接进入淋巴系统，吸收通过血脑屏障，以达到对脑神经系统的治疗作用，获得更好的临床疗效，并进一步通过药理实验及临床应用得到了证实。
冰片：赵保胜等运用免疫组化染色技术研究显示冰片不能诱导正常大鼠脑微血管EC上ICAM-1表达，却能显著减少脑外伤时白细胞和EC的黏附，对EC起到保护作用，证明冰片促血脑屏障开放与ICAM-1无关，冰片对脑外伤大鼠脑组织具有一定的保护作用。葛朝莉等把大鼠分为灌服冰片石蜡油溶液10，13mL·kg－12组，根据取样时间点的不同每组又分为1，2，4，8，24，48h，对照组灌服等量的石蜡油溶液。2组大鼠分别在以上时间点处死取脑，透射电镜观察。研究表明冰片对大鼠脑毛细血管内皮细胞之间的紧密连接影响显著，与对照组比较细胞间紧密连接缝隙变宽、断续、结构减少，灌服冰片后4h开始出现变化，8h时达到高峰，48h时恢复正常，说明冰片可使大鼠血脑屏障的超微结构发生可逆性改变。陈艳明制备含中药材冰片的家兔血清，以维拉帕米作为阳性对照药，用MTT法观察发现在MDCK(犬肾)和Hela(人子宫癌)细胞系上，冰片均能明显地增强长春新碱所致的细胞毒性，作用与维拉帕米相似，说明了冰片对P-糖蛋白的表达有明显的抑制作用。也有研究表明冰片促进血脑屏障生理性开放是因为冰片进入血脑屏障以后与P-糖蛋白亲和力大，结果冰片被泵出细胞外，这样就使与P-糖蛋白亲和力小的血管活性物质5-HT在细胞内蓄积，从而促进血脑屏障开放。
苏合香：将大鼠分为舒必利合用苏合香组和单用舒必利组，连续口服用药1周后，进行脑部与颈部安装探针手术，平衡1h后，利用血微透析法与脑微透析法分别在(30，60，90，120，150，180min)各时间段采集大鼠右心房内血液中与脑海马组织中的透析液，利用反相高效液相荧光色谱系统检测样品中舒必利的含量，利用统计学方法比较2组给药后脑中及血液中的舒必利含量。结果，舒必利合用苏合香组的大鼠脑中和血中舒必利浓度明显高于单用舒必利组，单用舒必利组大鼠血中和脑中舒必利的浓度比值为1∶0.2，舒必利与苏合香合用组血脑比增加为1∶0.3，与单用舒必利组相比，舒必利与苏合香合用组的舒必利脑浓度上升39％，血浓度上升69％。说明苏合香能够明显增加舒必利在大鼠脑中和血中的浓度，苏合香能够促进舒必利透过胃肠屏障与血脑屏障。
麝香：专家实验表明；麝香水剂、混悬剂静脉注射50mg/kg或侧脑室注射2.5mg/kg，可 使安静清醒兔皮质脑电图(EEG)短时间去同步，部分动物拌有行为躁动，处于清醒警戒状态，表明能兴奋大脑皮质增强皮质电活动；麝香水剂对麻醉兔有明显唤醒作用，侧脑室注射比静脉注射更有效，以上说明麝香可能通过血脑屏障直接作用于中枢神经系统。
石菖蒲：石菖蒲是传统的芳香开窍药之一，民间单用或与其他中药配伍使用广泛，主要用于脑病的治疗，有确切的疗效，提示石菖蒲对BBB有一定作用。按照《中国药典》2005版一部附录挥发油提取法甲法，提取挥发油，提取率约为1.58％，去油水煎液浓缩至1g/ml。采用气相色谱-质谱(GC/MS)联用仪检测挥发油样品中含有α—细辛醚、β—细辛醚、愈创木烯、菖蒲烯酮，相同条件下检测服用挥发油后大鼠的脑脊液，结果显示α—细辛醚，β—细辛醚可透过BBB进入脑脊液。采用液相色谱-质谱(HPLC/MS)联用仪测定去油水煎液样品及灌服去油水煎液的大鼠脑脊液样品，检测到三个化合物，分子量分别为396，452，339，已知石菖蒲化合物中尚未包括。透射电镜下观察给药前后大鼠大脑皮层BBB的超微结构，可发现石菖蒲各提取部分均可使BBB内皮细胞之间的紧密连接(tight junction，TJ)疏松，但BBB基本完整，尚未破坏。以冰片为阳性药，初步探讨石菖蒲各提取部分对伊文思兰(EB)透过BBB的促进作用，即以EB在脑内的含量反映BBB的通透性，统计分析表明冰片在灌胃2.5h后脑内EB含量显著高于空白组(P＜0.01)，石菖蒲在11.7g/kg剂量下自给药0.5h至2.5h未见明显作用；延长石菖蒲给药时间，石菖蒲挥发油剂量为23.4g/kg和11.7g/kg时，5h后脑内EB含量显著高于空白组(P＜0.05)，且有剂量依赖性趋势；总药剂量为11.7g/kg时，5h后脑内EB含量显著高于空白组(P＜0.01)。石菖蒲各提取部分给予小鼠灌胃一周后，腹腔注射苯妥英钠，采用HPLC方法检测苯妥英钠在脑内的浓度，结果显示：石菖蒲总组动物脑内苯妥英钠的含量较高于未给石菖蒲组动物脑内的苯妥英钠含量，4h后仍保持较高浓度(P＜0.01)。利用HPLC-ECD检测脑组织内5-羟色胺(5-HT)的浓度，统计分析得：与空白组比较，挥发油组小鼠大脑中5-HT的含量增加(P＜0.05)，石菖蒲水煎液组及总药组中5-HT的含量显著增加(P＜0.01)。应用Hela细胞系作为评价石菖蒲对BBB上P-糖蛋白活性的载体，0.5h时石菖蒲各部分(挥发油、水煎液、总药)可明显增强长春新碱(VCR)的细胞毒性(P＜0.01)；8h后挥发油部分仍可明显增强VCR的细胞毒性(P＜0.01)，挥发油的药效维持时间明显长于阳性药维拉帕米(VER)(P＜0.01)；24h后，阳性药及石菖蒲各部分对VCR的细胞毒性无明显影响(P＞0.05)。综上，石菖蒲能够影响大鼠BBB的超微结构，增强BBB对EB的通透性，促进CNS药苯妥英钠进入脑内的含量增加，具有开启BBB、引药入脑的药理作用。作用机制研究结果显示，石菖蒲调节5-HT的含量是其增加BBB通透性的作用机制之一，此外，石菖蒲有很强的抑制Hela细胞膜上P-糖蛋白的药物外排作用，这也是其增加BBB通透性的作用机制。
郁金：郁金所含的姜黄素有抑制老人痴呆症之效。促成痴呆症的主要原因，使淀粉样蛋白(amyloid beta)纠缠在一起。姜黄素能令已纠缠在一起的淀粉样蛋白松绑。姜黄素是一个强力的抗氧化及消炎的化学分子，能穿越门禁森严的血脑屏障，中和氧化物和有氧化毒性的铜离子，除此之外.更能抑制引起发炎的一个细胞转录因子(NF kappa B)。因此，姜黄素是一个保护脑组织，减少痴呆症的关键成分。在印度，痴呆症患者只有其他国家的百分之十，姜黄素功不可没。
发明内容
本发明的首要目的是提供20(R)-人参皂苷Rg3预防或/和治疗痴呆症的性能和功效，并提供20(R)-人参皂苷Rg3新的药用用途，即在治疗、调理和改善痴呆症的药物或保健食品中的新应用。
本发明的另一目的是针对人参皂苷Rg3口服吸收较差，通过血脑屏障差等临床应用的不足，提供一种人参皂苷Rg3外用制剂。本发明的人参皂苷Rg3外用药物制剂直接涂布于鼻翼两侧，颈部，耳后皮肤，用于治疗痴呆病症，尤其是治疗、预防阿尔茨海默病。
为实现本发明的目的，本发明一方面提供20(R)-人参皂苷Rg3在制备预防或/和治疗痴呆病症药物或保健品中的应用。
其中，所述的痴呆病症为阿尔茨海默病及血管性痴呆，优选为阿尔茨海默病。
其中，所述药物由20(R)-人参皂苷Rg3和药学上可接受的载体组成。
特别是，还包括人参油、川芎油、菖蒲油、麝香油或郁金油中的一种或多种。
本发明中所述的20(R)-人参皂苷Rg3在用于预防或治疗痴呆症时，可以单独使用，也可以通过含有20(R)-人参皂苷Rg3的药物组合物的形式使用。
本发明所述药物是通过口服、舌下、经皮、肌肉、皮下、皮肤黏膜、静脉等途径给药。
本发明提供以20(R)-人参皂苷Rg3为活性成分，用于预防或治疗痴呆症的药物制剂和相应的药物剂型。所述药物制剂是以所述20(R)-人参皂苷Rg3为有效活性成分，并包括了药剂学上可接受的其它载体组分。
其中，所述20(R)-人参皂苷Rg3纯度≥80％，优选为高于90％，进一步优选为高于95％。
本发明所述药物以口服制剂、注射剂、局部给药制剂形式存在。其中所述口服制剂包括片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂；注射剂包括注射液剂型或注射用冻干粉针剂型；局部给药制剂包括霜剂、软膏剂、喷雾剂、气雾剂、贴剂、凝胶剂、滴鼻剂或巴布剂。
本发明中所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、注射剂、喷雾剂、气雾剂、贴剂、凝胶剂、滴鼻剂、巴布剂形式存在，即药物制剂包括片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、注射剂、喷雾剂、气雾剂、贴剂、凝胶剂、滴鼻剂、布剂等形式，但不局限于以上形式。
本发明再一方面提供一种含有20(R)-人参皂苷Rg3的预防或/和治疗痴呆症的药物或保健品。
其中，所述药物或保健品中还包括人参挥发油、麝香挥发油、菖蒲挥发油、郁金挥发油或川芎挥发油中的一种。
特别是，所述药物或保健品中所述20(R)-人参皂苷Rg3的含量≥80％，优选为高于90％，进一步优选为高于95％。
本发明人通过大量的现代科学研究，采用先进的分离纯化技术从人参药材中提取其预防、治疗痴呆症的有效成分20(R)-人参皂苷Rg3，并对20(R)-人参皂苷Rg3及其相应的药物制剂进行了抗痴呆的药效学、药理学研究，结果表明20(R)-人参皂苷Rg3单体药理作用清楚，抗痴呆功效强，毒副作用低，安全性高，为抗衰老提供一种高效低毒的药物。
与现有预防、治疗痴呆症的药物相比，本发明具有如下优点：
1、本发明对已知化合物20(R)-人参皂苷Rg3发掘了新的药用价值，将其用于预防、治疗痴呆症，并可制备成预防、治疗痴呆症的药物或保健食品，从而为人参药材的应用开拓了一个新的领域。
2、本发明的系列试验研究证明20(R)-人参皂苷Rg3对预防、治疗痴呆症的功效显著。人参皂苷Rg3对大脑皮质的AChE阳性神经纤维密度均有明显增高，表明人参皂苷Rg3对阿尔茨海默病脑内胆碱能神经系统具有保护作用；人参皂苷Rg3对阿尔茨海默病大鼠模型学习记忆影响实验中，大鼠在水迷宫游出时间短、错误次数少，说明人参皂苷Rg3能够明显地提高阿尔茨海默病模型大鼠学习记忆能力；人参皂苷Rg3能降低AD患者脑脊液中的Aβ含量，用于临床治疗AD，能有效提高患者的智力和记忆力，改善认知功能和日常生活自理能力。
3、本发明的20(R)-人参皂苷Rg3药理作用强，用于抗衰老的功效显著，见效快、毒副作用小、安全性好，能够长期服用，具有良好的药用前景。
4、本发明的产品原料来源丰富、价廉、临床使用安全，制备工艺简单，可制成各种剂型，且服量小，使用方便，因此易于推广。
说明书附图
图1是人参皂苷Rg3(A)和薯蓣皂苷(B)准分子离子[M-H]-的二级全扫描质谱图；
图2是利用LC/MS/MS法测定肝匀浆中人参皂苷Rg3(通道I，m/z 783.8→160.8)及内标薯蓣皂苷(通道II，m/z 867.5→721.5)的典型色谱图；其中：(A)大鼠空白肝匀浆；(B)大鼠空白肝匀浆中加入2ng/ml人参皂苷Rg3和100ng/ml内标薯蓣皂苷；(C)大鼠口服给予10mg/kg人参皂苷Rg3口服乳后脑髓液样品(10.8ng/g)。
具体实施例：
以下通过试验例来进一步阐述本发明所述药物的有益效果，这些试验例包括了本发明药物的药效学试验。
试验例1人参皂苷Rg3对阿尔茨海默病大鼠模型脑内胆碱能神经系统的保护作用
1.试验材料：
20(R)-人参皂苷Rg3(纯度＞98％)，大连富生天然药物开发有限公司生产，批号：20120316；以中国药品生物制品检定所提供的标准品对照并进行HPLC测定，含量符合标定值，测定值为98.2％；
将人参皂苷Rg3按照实施例7的配方及方法制备成外用洗剂，备用，大连富生制药有限公司试制，批号：20120425；
阳性药物：石杉碱甲，上海红旗制药厂生产，批号：20111215；
D-半乳糖：上海试剂二厂生产，批号：20110607。
鹅膏覃氨酸(Ibotenic acid，IBO)，Sigma公司生产，批号：20101224。
Wistar大鼠，l5月龄初老年雌性，60只，体重300～450g，购自大连医科大学实验动物中心，质量合格证号：SCXK(13)2012-0003。
2.实验方法与结果：
2.1方法
将Wistar大鼠60只，体重300～450g，随机分成6组，每组10只，按不同处理方法分别命名为初老年正常对照组、初老年AD病模型组、阳性药物石杉碱甲组和人参皂苷Rg3外用制剂高、中、低剂量组。大鼠分笼饲养，每笼4～5只，自然照明，随意饮水和进食。初老年正常对照组大鼠腹腔及脑内注射生理盐水；初老年AD病模型组、阳性药物石杉碱甲组和人参皂苷Rg3外用制剂高、中、低剂量组大鼠均腹腔注射半乳糖和脑内注射IBO；石杉碱甲组和人参皂苷Rg3高、中、低剂量组大鼠在D-半乳糖注射造模同时，再分别给予石杉碱甲 0.3mg/kg/d和人参皂苷Rg310、5、2.5mg/kg/d，人参皂苷Rg3涂于大鼠鼻翼两侧，均连用药6周，然后进行脑内双侧Meynert核注射。Meynert核定位参照大鼠脑立体定位图谱，定位坐标为AP-0.8mm，Late 2.6mm，DV 8.2mm。脑内注射时，大鼠先经3％的戊巴比妥钠(30mg/kg)麻醉后固定在脑立体定位仪上，将生理盐水溶解的IBO(每侧注射1ul含IBO 5ug)或1ul的生理盐水缓慢注人Meynert核，注射时间每侧持续5min，留针10min。其中所有大鼠在造模结束后经4％多聚甲醛灌流取脑，后固定24h，改入蔗糖磷酸缓冲液中至脑组织块下沉，恒温箱冰冻连续切片，片厚40mm。切片按Hedreen等推荐的方法行乙酰胆碱酯酶(Acetycholinesterase，AChE)组织化学方法染色。观察额叶、顶叶、枕叶和内嗅皮质(第Ⅱ层)及海马结构中的海马CA1区多形层、辐射层、腔隙分子层和齿状回分子层等部分的AChE阳性神经纤维密度，采用显微镜用测微尺，以阳性纤维与标准网格交点数计数AChE阳性神经纤维密度。
2.2结果
大鼠皮质和海马结构乙酰胆碱酯酶阳性神经纤维密度的测试结果见表1和表2，结果均以(X±S)表示，采用SAS 6.11统计软件包，进行单因素方差分析。显著性水平取a＝0.05。
表1大鼠皮质AchE阳性神经纤维密度(X±S)
Figure PCTCN2014090475-appb-000001
与正常组比较，○P>0.05，#p<0.05，##p<0.01；与模型组比较，△p<0.05，△△p<0.01；与石杉碱组比较，*p<0.05，**p<0.01；
由表1的测试结果可见，人参皂苷Rg3高、中、低剂量组各部位的AChE阳性神经纤维密度均有明显增高，在额叶、枕叶与模型组、石杉碱甲组比较，均有显著性差异(P<0.05和P<0.01)，且基本恢复到正常水平(与正常组比较，P>0.05)。
表2大鼠海马结构AchE阳性神经纤维密度(X±S)
Figure PCTCN2014090475-appb-000002
与正常组比较，○P>0.05，#p<0.05，##p<0.01；与模型组比较，△p<0.05，△△p<0.01；与石杉碱组比较，*p<0.05，**p<0.01；
由表2的测试结果可见，人参皂苷Rg3高、中、低剂量组各部位AChE阳性神经纤维密度则均较模型组增高，在CA1区多形层、辐射层和齿状回分子层与正常组比较没有显著差别(均P>0.05)。
上述实验结果表明，人参皂苷Rg3对阿尔茨海默病大鼠模型脑内胆碱能神经系统具有保护作用。
试验例2人参皂苷Rg3对阿尔茨海默病大鼠模型学习记忆的影响
1.试验材料：
20(R)-人参皂苷Rg3(纯度＞98％)，大连富生天然药物开发有限公司生产，批号：20120316；以中国药品生物制品检定所提供的标准品对照并进行HPLC测定，含量符合标定值，测定值为98.2％；
将人参皂苷Rg3按照实施例17的配方及方法制备成口服乳剂(10mg/ml)，备用，大连富生制药有限公司试制，批号：20120320；
阳性药物。D-半乳糖(上海试剂二厂生产)，批号：20110607。
鹅膏覃氨酸(Ibotenic acid，IBO)，Sigma公司生产，批号：20101224。
Wistar大鼠，6月龄初老年雌性，60只，体重180～250g，购自大连医科大学实验动物中心，质量合格证号：SCXK(13)2012-0003。
2.试验方法：在实验室稳定条件下饲养1w后，进行水迷宫预训练，选取体重相近、灵活性较好的大鼠60只，随机分为6组，正常对照组、假手术对照组、模型对照组和人参皂苷Rg3高、中、低(10、5、2.5mg/kg/d)剂量组，每组10只动物。
造模方法采用大鼠腹腔注射由生理盐水配制的0.96％D-半乳糖，5mL/kg，每日1次，连续6w。假手术对照组注射等量生理盐水。从第43d开始，模型对照组和人参皂苷Rg3高、中、低剂量组大鼠群颅内注射IBO，每侧缓缓注射1μL的IBO，假手术组在相同脑区内注射 等量的生理盐水，(连续4w)。正常对照组不做任何处理，正常进食和饮水。人参皂苷Rg3g高、中、低剂量组于造模的同时，每日灌胃人参皂苷Rg3口服乳，造模后，连续给药4w(周)。
用通道式水迷宫试验测试各组动物行为学的改变，以进入盲端次数、到达终点所需时间来衡量大鼠的学习记忆能力。所有实验结果以均数±标准差表示，组间差异采用SPSS10.0统计软件包进行方差分析。
3.实验结果：试验结果见表3。
表3 Rg3口服乳对阿尔茨海默病大鼠模型学习记忆的影响(X±S)
Figure PCTCN2014090475-appb-000003
与正常组、假手术组比较，*p<0.01；与模型组比较，#p<0.05
动物在水迷宫游出时间越短，错误次数越少反映动物学习记忆能力越强。从表3可以看出，模型组动物游出时间和错误次数明显高于正常组与假手术组，而人参皂苷Rg3高、中、低剂量组明显低于模型组，说明人参皂苷Rg3能够明显地提高模型大鼠学习记忆能力。
试验例3人参皂苷Rg3对阿尔茨海默病患者脑脊液β淀粉样蛋白含量的影响
3.1试验材料：
20(R)-人参皂苷Rg3(纯度＞98％)，大连富生天然药物开发有限公司生产，批号：20120316；以中国药品生物制品检定所提供的标准品对照并进行HPLC测定，含量符合标定值，测定值为98.2％；
将人参皂苷Rg3按照实施例27的配方及方法制备成人参皂苷Rg3口服液(10mg/ml)，备用，大连富生制药有限公司试制，批号：20120321；
阳性对照药物，多奈哌齐(5mg*7，卫材(中国)药业有限公司)。
3.2试验方法：
36例患者均为住院患者，随机分为人参皂苷Rg3口服液组、多奈哌齐组各18例。人参皂苷Rg3口服液组予人参皂苷Rg3口服液(10mg/d)；多奈哌齐组予多奈哌齐5mg/d，睡前 口服。两组均连续服药12周后。腰椎穿刺术采集脑脊液2ml，-80℃保存，采用酶联免疫吸附法(ELISA)测定β-淀粉样蛋白(Aβ)含量。采用改良长谷川痴呆量表记分，治疗前后对比，记分增加为有效，记分不变或下降为无效。计数资料用χ2检验，计量资料比较用t检验，所有数据采用SPSS12.0统计软件处理。测试结果如表4所示。
两组临床疗效分析人参皂苷Rg3口服液组临床症状记分增加者l4例，记分不变者3例，记分下降者1例，有效率为77.8％。多奈哌齐组临床症状记分增加者13例，记分不变者3例，记分下降者2例，有效率为72.2％，两组无差异(P>0.05)。
表4两组治疗前后记分及Aβ含量比较(n＝18，X±s)
Figure PCTCN2014090475-appb-000004
与同组内治疗前比较：#P<0.05，与多奈哌齐组比较：△P<0.01
从表4的实验结果可知，两组治疗后记分较治疗前均明显增加(P<0.05)，人参皂苷Rg3组记分增加显著(P<0.05)。人参皂苷Rg3组治疗后Aβ含量明显降低(P<0.05)，阳性药物多奈哌齐对照组治疗后Aβ含量无明显变化(P>0.05)。临床观察表明，人参皂苷Rg3用于临床治疗AD，能有效提高患者的智力和记忆力，改善认知功能和日常生活自理能力。本研究表明，人参皂苷Rg3能降低AD患者脑脊液中的Aβ含量，这可能是其治疗AD的作用机制之一。
试验例4人参皂苷Rg3对轻中度阿尔茨海默病有效性的临床研究
1.试验材料：
20(R)-人参皂苷Rg3(纯度＞98％)，大连富生天然药物开发有限公司生产，批号：20120316；以中国药品生物制品检定所提供的标准品对照并进行HPLC测定，含量符合标定值，测定值为98.2％；
将人参皂苷Rg3按照实施例29的配方及方法制备成人参皂苷Rg3滴鼻液(10mg/ml)，备用，大连富生制药有限公司试制，批号：20120324。
2.试验方法：
轻中度阿尔茨海默症患者89例，随机分成2组，一组服用人参皂苷rg3，另一组使用安慰剂，其中服用rg3组患者46例，男性2l例，女性25例，年龄58～87(72 6±6.8)岁，简易智能状态检删量表(MMSE)评分为l0～24(17 8±2.3)分；使用安慰剂者43例，男性19例，女 性24例，年龄57～88(71.8±8.2)岁，MMSE评分为10～24(18.2±2.7)分。人参皂苷Rg3组和安慰剂组性别、年龄及MMSE评分无明显差异。服用人参皂苷rg3组患者每日使用人参皂苷Rg3滴鼻液1次，每次10mg/ml/次，连续应用12周。
使用安慰剂的患者除了滴鼻使用的是安慰剂外，其余均按照与人参皂苷rg3的同样的用药方式方法进行，使用的安慰剂的颜色、性状、味道及剂量与人参皂苷Rg3滴鼻液相同，安慰剂为按照实施例29中制备人参皂苷rg3滴鼻剂的方法制备，其中除了不添加人参皂苷rg3之外，其余与实施例29相同。
人参皂苷Rg3滴鼻液10mg/ml，安慰剂的颜色、性状、味道及剂量与人参皂苷Rg3滴鼻液相同。每日使用一次，连续应用12周。用药前和用药后每4周检查一次，检查方法如下：
(1)MMSE筛查AD。检测患者的认知功能(定向力、记忆力、计算力、语言能力、视空间段运用能力等)；(2)临床痴呆程度量表(CDR)检测患者的痴呆程度；(3)日常生活自理量表(ADL)检测患者的日常生活自理能力。检查结果应用SPSS统计软件。治疗前后及人参皂苷Rg3滴鼻液组与安慰剂对照均采用t检验。治疗前AD患者的MMSE、CDR及ADL评分结果见表5。
表5治疗前AD患者的MMSE、CDR、ADL评分结果(x±s，分)。
Figure PCTCN2014090475-appb-000005
注：人参皂苷Rg3外用制剂与安慰剂组比较，MMSE、CDR及ADL差异均无明显意义(P>0.05)。
服用rg3和安慰剂12周后AD患者的MMSE、CDR及ADL评分结果见表6
表6治疗12周后AD患者的MMSE、CDR、ADL评分结果(x±s，分)
Figure PCTCN2014090475-appb-000006
注：人参皂苷Rg3滴鼻液组与安慰剂组比较，*P<0.05，**P<0.01。
认知功能(用MMSE评分)：安慰剂对照研究组疗效观察显示，治疗后人参皂苷Rg3组较安慰剂对照组MMSE评分有很明显提高(P<0.01)；痴呆程度(用CDR评分)：治疗后人参皂苷Rg3滴鼻液组较安慰剂对照组CDR评分有显著减低(P<0.05)；日常生活自理能力(用ADL评分)：治疗后人参皂苷Rg3滴鼻液组较安慰剂对照组ADL评分有根明显减低(P<0.01)，治疗后较治疗前有很明显减低，ADL分数减少7.1分(P<0.01)。
综上，人参皂苷Rg3滴鼻液是治疗AD患者，改善AD患者轻、中度认知功能障碍、生 活自理能力下降及痴呆程度安全而有效的药物。
试验例5人参皂苷Rg3对轻中度阿尔茨海默病有效性的临床研究
1.试验材料：
20(R)-人参皂苷Rg3(纯度＞98％)，大连富生天然药物开发有限公司生产，批号：20120316；以中国药品生物制品检定所提供的标准品对照并进行HPLC测定，含量符合标定值，测定值为98.2％；
将人参皂苷Rg3按照实施例29的配方及方法制备成人参皂苷Rg3滴鼻液(10mg/ml)，备用，大连富生制药有限公司试制，批号：20120324。
2.试验方法：
轻中度阿尔茨海默症患者99例，男性47例，女性52例，年龄55～85(73.5±7.6)岁，MMSE评分为10～24(16.3±2.6)分。
AD患者均使用人参皂苷Rg3滴鼻液，每日使用人参皂苷Rg3滴鼻液(10mg/ml)一次，连续应用12周。
用药前和用药后每4周检查一次，检查方法如下：
(1)MMSE筛查AD。检测患者的认知功能(定向力、记忆力、计算力、语言能力、视空间段运用能力等)；(2)临床痴呆程度量表(CDR)检测患者的痴呆程度；(3)日常生活自理量表(ADL)检测患者的日常生活自理能力。检查结果应用SPSS统计软件治疗前后及人参皂苷Rg3滴鼻液组与安慰剂对照均采用t检验。
AD患者的MMSE、CDR及ADL评分结果见表7。
表7AD患者使用rg3治疗前后MMSE、CDR、ADL评分结果(x±s，分)
Figure PCTCN2014090475-appb-000007
注：自身对照组在治疗前与治疗后比较，*P<0.05，**P<0.01。
认知功能(用MMSE评分)：治疗12周后较治疗前有很明显提高，MMSE分数提高3.5分(P<0.01)，且在用药4周时MMSE评分已有明显改善(P<0.05).痴呆程度(用CDR评分)：治疗12周后较治疗前有显著减低，CDR分数减少0.6分(P<0.05)。日常生活自理能力(用ADL评分)：治疗后较治疗前有很明显减低，ADL分数减少7.1分(P<0.01)。
综上，人参皂苷Rg3滴鼻液是治疗AD患者，改善AD患者轻、中度认知功能障碍、生 活自理能力下降及痴呆程度安全而有效的药物。
下面结合实施例对本发明做进一步的描述。
实施例1
1、按照如下配比准备原料
20(R)-人参皂苷Rg3(含量98％)2g，氮酮2g，丙二醇10ml，乙醇(65％)适量
2、将人参皂苷Rg3、氮酮、丙二醇，加入到乙醇(65％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(65％)加至100ml，得到人参皂苷Rg3擦剂。
实施例2
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，聚乙二醇400 10ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、聚乙二醇400，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例3
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，聚乙二醇600 10ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、聚乙二醇600，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例4
1、按照如下配比准备原料
人参皂苷Rg3(63％)2g，氮酮2g，聚乙二醇200 10ml，乙醇(65％)适量
2、将人参皂苷Rg3、氮酮、聚乙二醇200，加入到乙醇(60％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(65％)加至100ml，得到人参皂苷Rg3洗剂。
实施例5
1、按照如下配比准备原料
人参皂苷Rg3(30％)1g，甘油2g，聚乙二醇200 10ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、聚乙二醇200，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例6
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，中链甘油三酸酯20g，丙二醇10ml，磷脂20g，注射用水适量。
2、将人参皂苷Rg3、中链甘油三酸酯、丙二醇、磷脂，加入到少量的注射用水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加注射用水加至100ml，得到人参皂苷Rg3注射乳。
本发明实施例中使用的中链甘油三酸酯即辛癸酸甘油脂，是半合成的天然功能性油脂，具国内外多年来应用安全可靠，具有很广阔的应用领域和使用价值，欧美国家简称为MCT。该产品可广泛应用于药品、食品、保健品及化妆品等方面。
实施例7
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，人参油0.5g，丙二醇10ml，乙醇(65％)适量
2、将人参皂苷Rg3、氮酮、人参油，丙二醇，加入到乙醇(65％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(65％)加至100ml，得到人参皂苷Rg3洗剂。
实施例8
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，郁金油1g，聚乙二醇400 10ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、郁金油，聚乙二醇400，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例9
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，麝香油0.05g，聚乙二醇600 10ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、麝香油，聚乙二醇600，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例10
1、按照如下配比准备原料
人参皂苷Rg3(63％)2g，氮酮2g，川芎油0.2g，聚乙二醇200 10ml，乙醇(65％)适量
2、将人参皂苷Rg3、氮酮、川芎油，聚乙二醇200，加入到乙醇(65％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(65％)加至100ml，得到人参皂苷Rg3洗剂。
实施例11
1、按照如下配比准备原料
人参皂苷Rg3(30％)1g，甘油2g，菖蒲油0.2g，聚乙二醇20010ml，乙醇(70％)适量
2、将人参皂苷Rg3、氮酮、菖蒲油，聚乙二醇200，加入到乙醇(70％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(70％)加至100ml，得到人参皂苷Rg3洗剂。
实施例12
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，中链甘油三酸酯20g，冰片0.5g，丙二醇10ml，磷脂20g，注射用水适量。
2、将人参皂苷Rg3、中链甘油三酸酯、冰片、丙二醇、磷脂，加入到少量的注射用水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加注射用水加至100ml，得到人参皂苷Rg3注射乳。
实施例13
1、按照如下配比准备原料
人参皂苷Rg3(98％)2g，氮酮2g，人工牛黄0.5g，丙二醇10ml，乙醇(65％)适量
2、将人参皂苷Rg3、氮酮、人工牛黄，丙二醇，加入到乙醇(65％)中，混合后的总体积为85ml；
3、将总体积为85ml的混合物料一并加入到500ml烧瓶中，加热至50-55℃，在保持温度为50-55℃下搅拌，直至20(R)-人参皂苷Rg3完全溶解，搅拌30分钟后，停止加热，冷却至室温(15-25℃)后，再补加乙醇(65％)加至100ml，得到人参皂苷Rg3洗剂。
实施例14
人参皂苷Rg3(98％)2g，氮酮2g，苏合香0.5g，聚乙二醇400 10ml，70％乙醇加至100ml。
将处方量的人参皂苷Rg3、氮酮2g、苏合香0.5g、聚乙二醇400 10ml，70％乙醇加至85ml，加入500ml烧瓶中，加热搅拌30分钟使溶解，放冷补加70％乙醇至100ml，得到人参皂苷Rg3外用制剂。
实施例15
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，大豆油20g，丙二醇10ml，粉末磷脂20g，蒸馏水适量。
2、将人参皂苷Rg3、植物油、丙二醇、粉末磷脂，加入到少量的蒸馏水中，加热至65-70 ℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加蒸馏水加至100ml，得到人参皂苷Rg3口服乳。
实施例16
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，大豆油(注射用)50g，中链甘油三酸酯100g。
2、将人参皂苷Rg3(98％)，植物油，中链甘油三酸酯加热至80-85℃，搅拌使人参皂苷Rg3溶解，得到人参皂苷Rg3注射液。
实施例17
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，人参挥发油2g，丙二醇10ml，川芎油0.5g，磷脂20g，蒸馏水适量。
2、将人参皂苷Rg3、人参挥发油、丙二醇、川芎油、磷脂，加入到少量的蒸馏水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加蒸馏水加至100ml，得到人参皂苷Rg3口服乳。
实施例18
1、按照如下配比准备原料
人参皂苷Rg3(85％)1g，菖蒲油20g，丙二醇10ml，粉末磷脂20g，蒸馏水适量。
2、将人参皂苷Rg3、菖蒲油、丙二醇、粉末磷脂，加入到少量的蒸馏水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入胶体磨中进行处理，冷却至室温(15-25℃)后，再补加蒸馏水加至100ml，得到人参皂苷Rg3口服乳。
实施例19
1、按照如下配比准备原料
人参皂苷Rg3(63％)1g，郁金香油10g，中链甘油三酸酯100g
2、将人参皂苷Rg3，郁金香油，中链甘油三酸酯加热至50-55℃，搅拌使人参皂苷Rg3溶解，得到人参皂苷Rg3口服液。
实施例20
1、按照如下配比准备原料
人参皂苷Rg3(30％)1g，川芎油10g，中链甘油三酸酯100g
2、将人参皂苷Rg3，川芎油，中链甘油三酸酯加热至50-55℃，搅拌使人参皂苷Rg3溶解，得到人参皂苷Rg3口服液。
实施例21
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，麝香油2g，丙二醇10ml，磷脂20g，蒸馏水适量。
2、将人参皂苷Rg3、麝香油、丙二醇、磷脂，加入到少量的蒸馏水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加蒸馏水加至100ml，得到人参皂苷Rg3口服乳。
实施例22
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，中链甘油三酸酯20g，麝香油3g，丙二醇10ml，磷脂20g，注射用水适量。
2、将人参皂苷Rg3、中链甘油三酸酯、麝香油、丙二醇、磷脂，加入到少量的注射用水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加注射用水加至100ml，得到人参皂苷Rg3注射乳。
实施例23
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，中链甘油三酸酯20g，菖蒲油2g，聚乙二醇100 10ml，磷脂20g，注射用水适量。
2、将人参皂苷Rg3、中链甘油三酸酯、菖蒲油、丙二醇、磷脂，加入到少量的注射用水中，加热至65-70℃，搅拌使人参皂苷Rg3溶解，将搅拌均匀的混合物料加入均质机中进行均质处理，冷却至室温(15-25℃)后，再补加注射用水加至100ml，得到人参皂苷Rg3注射乳。
实施例24
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，蔗糖50g，菖蒲油2g，糊精100g，二氧化硅5g。
2、取处方量的菖蒲油，二氧化硅，混匀。使二氧化硅吸附菖蒲油完全。另加入处方量的人参皂苷Rg3，蔗糖，糊精。用70％乙醇制粒，过筛，65-70℃烘干之后得到人参皂苷Rg3颗粒剂。
实施例25
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，蔗糖50g，川芎油2g，糊精100g，二氧化硅3g。
2、取处方量的川芎油，二氧化硅，混匀。使二氧化硅吸附菖蒲油完全。另加入处方量的人参皂苷Rg3，蔗糖，糊精。用70％乙醇制粒，过筛，65-70℃烘干之后得到人参皂苷Rg3颗粒剂。
实施例26
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，淀粉100g，人参油3g，微晶纤维素100g，倍他环糊精10g，滑石粉1g。
2、取处方量的倍他环糊精10g，加水100ml使溶解，在70℃水浴保温，并加入人参油3g，搅拌30min，取出溶液，放冷，过滤沉淀，得到人参油的包合物。加入处方量的人参皂苷Rg3(98％)1g，淀粉100g，微晶纤维素100g，加入适量的乙醇制粒，过筛整粒之后加入滑石粉1g，压片，得到人参皂苷Rg3片剂。
实施例27
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，蔗糖50g，川芎油3g，倍他环糊精10g，苯甲酸钠2g，蒸馏水适量。
2、取处方量的倍他环糊精10g，加水100ml使溶解，在70℃水浴保温，并加入川芎油3g，搅拌30min，取出溶液，放冷，过滤沉淀，得到川芎油的包合物。加入处方量的人参皂苷Rg3(98％)1g，蔗糖50g，苯甲酸钠2g，加蒸馏水至500ml，得到人参皂苷Rg3口服液。
实施例28
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，麝香油1g，90％乙醇10ml，甘油5g，F12/F114(为(Freon)类化合物的抛射剂)加至100g。
2、准确称取处方量人参皂苷Rg3(98％)1g，麝香油1g，加入90％乙醇使溶解，再加入甘油混匀制成溶液，分装于气雾剂容器，安装阀门，轧紧封帽后，充装混合抛射剂F12/F114。
实施例29
1、按照如下配比准备原料
人参皂苷Rg3(98％)1g，川芎油1g，60％乙醇10ml，聚乙二醇200 5ml，甘油20g，注射用水适量。
2、准确称取处方量人参皂苷Rg3(98％)1g，川芎油1g，加入60％乙醇10ml，聚乙二 醇200 5ml，甘油20g，低温加热并搅拌使溶解，加注射用水至100ml，制成人参皂苷Rg3滴鼻剂。
本发明实施例中使用的人参油、川芎油、麝香油、郁金油、菖蒲油、冰片、人工牛黄、苏合香等开窍类药物，其主要功效为芳香开窍,醒神回苏,临床用以治疗脑卒中、神志昏迷等证。近年来研究发现开窍药能通过影响血脑屏障(blood-brain barrier,BBB)发挥其治疗脑部疾患的作用,有些开窍药则可以促进其它药物通透,是其发挥开窍醒神回苏的主要作用机制,推测其也许是该类药归心经的理论基础。如冰片具有提高血脑屏障的通透性，促进药物进入脑组织的作用，麝香，安息香和苏合香等开窍药对小鼠的生理状态下对血脑屏障有一定的开放作用，能促进药物进入脑组织。
在制备rg3制剂时，在处方中均加入了氮酮，聚乙二醇，乙醇，中药挥发油等成分，这些成分的加入，能使血脑屏障处于开放状态，使rg3进入脑组织发挥其抗痴呆的作用，专利中也用“实施例30人参皂苷Rg3不同剂型给药通过血脑屏障吸收药物的检测试验”再次验证了rg3已经进入生理状态的大鼠脑内。因此，本专利中设计的处方及工艺，制备的样品均有抗痴呆的作用。
本发明实施例中使用的人参油购自江西省吉安市青原区华隆香料油有限公司；川芎油购自江西省吉安市森海香料有限公司；麝香油购自江西百草药业有限公司；菖蒲购自西安瑞盈生物科技有限公司；郁金香油购自江西本草天然香料油有限公司；郁金油购自江西恒诚天然香料油有限公司；人参挥发油购自江西环球天然香料有限公司。
人参油的主要成为精油含量％≥11、酸价KOHmg/g≤30、人参皂苷、人参多糖、挥发油、氨基酸和多肽、为β—榄香烯、β—古芸烯、β—人参烯、α—人参烯、丁香烯、β—金合欢烯、棕榈酸；人参挥发油的主要成分为倍半萜烯，约占40％，其次为含氧化合物及长链烷烃类。
实施例30人参皂苷Rg3不同剂型给药通过血脑屏障吸收药物的检测试验
1.试验材料
1.1动物
50日龄SD大鼠，雄性，体质量260g左右，清洁级动物，购自大连医科大学实验动物中心，实验动物许可证号：SCXK(辽)2004-0017。
1.2仪器试剂
大鼠脑髓液抽取仪器：大小手术剪。镊子，止血钳，1mL注射器。刻度尺，托盘，无菌 棉球，碘伏，Angle Two脑立体定位仪(美国MyNeurolab公司)，低速牙科钻(美国Grobet公司)，微量注射泵(美国KdScientific公司)，10μL和100μL微量注射器(美国Hamilton公司)。
大鼠脑髓液中Rg3药物含量测定仪器：API 4000型三重四极杆串联质谱仪，配有离子喷雾离子源以及Analysis 1.3数据处理系统，美国Applied Biosystem公司；Agilent 1100高效液相色谱输液泵，自动进样器，美国Agilent公司；色谱柱为Nucleosil C18柱(50×4.6mm I.D.，5μm粒径)，大连伊利特分析仪器有限公司。
1.3仪器药品
实施例1-2、实施例6-14、实施例16-21、实施例25-29制备的人参皂苷Rg3制剂。
2.试验方法
(1)人参皂苷Rg3不同剂型给药
①口服给药：将人参皂苷Rg3的口服制剂灌胃给予SD大鼠，其高、中、低剂量组均分别为：20、10、5mg/kg(上述剂量是纯的rg3的剂量)/bid，每剂量组大鼠18只；
②外用给药：将人参皂苷Rg3给予SD大鼠，外用剂型药物分别涂布于清利头窍的穴位处或鼻翼、颈部、耳后，其高、中、低剂量组均分别为：10、5、2.5mg/kg(即rg3的量分别为10、5、2.5mg/kg)/bid，每剂量组大鼠18只；
③注射给药：将人参皂苷Rg3的注射乳鼠尾静脉注射给予SD大鼠，其高、中、低剂量组均分别为：6、3、1.5mg/kg/bid，每剂量组大鼠18只。
(2)大鼠脑髓液抽取
人参皂苷Rg3不同制剂经上述口服、外用及注射给药后4小时，进行大鼠脑髓液抽取。采用直视下划破硬脊膜抽取大鼠脑脊液法，参照文献(大鼠脑脊液抽取方法的改良。河北医科大学学报，2010(31)2:125-127)方法。腹腔注射10％水合氯醛麻醉大鼠，利用脑直体定位耳杆、门齿杆固定大鼠头部，头枕部剪毛、消毒，沿后正中线切一纵行切口(约2cm)，用剪刀钝性分离项部背侧肌肉。最深层附着在枕骨L的肌肉用手术刀背刮开，以避免出血，暴露寰枕筋膜后，用蒸馏水冲洗创面，无菌棉球擦净后，用手术刀纵向划破硬脊膜0.3cm，可见清亮的脑脊液流出，将事先准备好100μl微量注射器(圆头)置于破口处抽取脑脊液，每只大鼠抽取2次，每次抽取100μl；每剂量组3只大鼠脑髓液混匀制备为1个样品，每剂量组共制备6个生物样品。
以未给药人参皂苷Rg3的大鼠为空白对照。
(3)不同剂型给药大鼠脑脊液中人参皂苷Rg3含量测定
采用LC/MS/MS法测定大鼠脑髓液中的药物浓度。
药品：
人参皂苷Rg3对照品，购自于中国药品生物制品检定所，批号：110804-200603。
人参皂苷Rg3原料，大连富生天然药物开发有限公司生产，批号：2012303，经人参皂苷Rg3对照品进行HPLC标定，含量为98.2％；
其他人参皂苷Rg3不同制剂中人参皂苷Rg3高、中、低剂量组的含量测定方法与人参皂苷Rg3原料的标定方法相同，不同制剂人参皂苷Rg3的含量详见试验结果。
试剂：乙腈、甲醇、乙酸乙酯均为色谱纯，分别购自美国Fisher公司和天津康科德科技有限公司；生理盐水购自沈阳志鹰制药厂。
组织样品处理  取抽取后的脑髓液200μl，分别加入甲醇溶液100μl，内标溶液100μl(100ng/ml薯蓣皂苷甲醇溶液)，水0.5ml，混匀，加入乙酸乙酯3ml，涡流1min，振荡10min，离心(4000rpm)8min，分离上清液于40℃空气流下吹干，残留物于200μl流动相中溶解，取20μl分析。
色谱条件：流动相为甲醇-乙腈-10mmol/l乙酸胺溶液(47.5：47.5：5，v/v)，流速为0.7ml/min，柱温为室温。
质谱条件：离子喷射电压：-3500V；源内气体1(GS1，N2)压力：30p.s.i.；气体2(GS2，N2)压力：30p.s.i.；气帘气体(N2)压力：15p.s.i.；碰撞气(N2)压力：3p.s.i.；源内温度：500℃。检测方式：负离子检测；扫描方式：选择反应监测(MRM)方式，用于定量分析的离子反应分别为m/z 783.8→m/z 160.8(人参皂苷Rg3)，m/z 867.5→m/z 721.5(内标，薯蓣皂苷)。相应的二级全扫描质谱图如图1所示。
方法专属性  取未给药的空白大鼠脑髓液，除不加内标溶液并另外补加100μl甲醇外，其余按“组织样品处理”项下方法操作，获得空白样品的色谱图；将人参皂苷Rg3对照品系列溶液(2ng/ml)和内标溶液(100ng/ml)加入空白大鼠脑髓液中，依同法操作，获得相应的色谱图；同法处理，得到大鼠给药后脑髓液样品色谱图。
图2为大鼠脑髓液空白样品、空白样品添加rg3对照品及内标(薯蓣皂苷)溶液和给药后样品色谱图。
结果表明组织中内源性物质不干扰人参皂苷Rg3和内标薯蓣皂苷的测定。
工作曲线制备  取空白对照组大鼠组织匀浆上清液200μl，加入人参皂苷Rg3对照品系列溶液100μl(向200μl上清液中加入100μlRg3对照品溶液)，配制相当于组织浓度为2，4， 10，20，40，100，200和400ng/g的组织样品。除不加入100μl甲醇外，按“组织样品处理”项下操作，建立工作曲线。以待测物浓度为横坐标，待测物与内标物色谱图的峰面积比值为纵坐标，用加权最小二乘法进行回归运算，求得的直线回归方程即为工作曲线。
根据工作曲线，脑中人参皂苷Rg3浓度测定的线性范围为：2-400ng/g，最低定量限为2ng/g。工作曲线：y＝0.0014+0.0091x r＝0.9992
同时做高、中、低3个浓度的质控样品(QC样品)，每个浓度进行双样本分析。根据每1分析批的工作曲线计算QC样品的浓度，6个QC(共有18个样品)样品中最多允许2个不同浓度的样品超出理论值的15％(最低点为20％)，否则此批数据不被接受。
不同制剂给药样品测定按“组织样品处理”项下操作，每个分析批制备1条工作曲线，药大鼠脑髓液样品测定结果见表8。
数据处理分别给出每1只大鼠给药后脑髓液中药物浓度数据，计算平均值及标准差。
表8不同制剂透过血脑屏障试验大鼠脑髓液中人参皂苷Rg3的含量
Figure PCTCN2014090475-appb-000008
Figure PCTCN2014090475-appb-000009
表8测定结果表明，本发明研制的人参皂苷Rg3不同制剂(代表型)给药后，在大鼠脑髓液中测定出人参皂苷Rg3的药物浓度，说明本发明的多种剂型皆可通过血脑屏障，进入脑髓内，发挥药物疗效。

Claims (10)

  1. 20(R)-人参皂苷Rg3在制备预防或/和治疗痴呆病症药物或保健品中的应用。
  2. 如权利要求1所述的应用，其特征是所述的痴呆病症为阿尔茨海默病或血管性痴呆。
  3. 如权利要求1或2所述的应用，其特征是所述药物由20(R)-人参皂苷Rg3和药学上可接受的载体组成。
  4. 如权利要求1或2所述的应用，其特征是所述药物是通过口服、舌下、经皮、肌肉、皮下、皮肤黏膜、静脉途径给药。
  5. 如权利要求1或2所述的应用，其特征是所述药物以口服制剂、注射剂或局部给药制剂形式存在。
  6. 如权利要求5所述的应用，其特征是所述口服制剂包括片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂或溶液剂；注射剂包括注射液剂型或注射用冻干粉针剂型；局部给药制剂包括霜剂、软膏剂、喷雾剂、气雾剂、滴鼻剂、凝胶剂或贴剂。
  7. 如权利要求1或2所述的应用，其特征是所述20(R)-人参皂苷Rg3的含量≥80％。
  8. 一种预防或/和治疗痴呆症的药物或保健品，其特征是含有20(R)-人参皂苷Rg3。
  9. 如权利要求8所述的药物或保健品，其特征是还包括人参挥发油、麝香挥发油、 菖蒲挥发油、郁金挥发油或川芎挥发油中的一种。
  10. 如权利要求8所述的药物或保健品，其特征是所述20(R)-人参皂苷Rg3的含量超过80％。
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CN104644658A (zh) 2015-05-27
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