TWI587861B - A pharmaceutical composition for increasing the content and availability of cyclic cyclic adenosine monophosphate and a method for producing the same - Google Patents

Description

Pharmaceutical composition for increasing circulating adenosine monophosphate content and utilization in vivo and preparation method thereof

The present invention relates to a pharmaceutical composition, especially an oral drug and a health food.

Earl Wilbur Sutherland Jr., a scientist who won the 1971 Nobel Prize in Physiology and Medicine, discovered the mechanism of action of intracellular cAMP. Edmond H. Fischer and Edwin G. Krebs, scientists who won the 1992 Nobel Prize in Physiology and Medicine, further discovered The mechanism of intracellular cAMP→PKA (cAMPdependent protein kinase); Eric Kandel, a scientist who won the 2000 Nobel Prize in Physiology and Medicine, further discovered the mechanism of intracellular cAMP→PKA→CREB. It shows that the related role of intracellular cAMP has an absolute and very significant correlation with human physiology and medicine; for example, when Eric Kandel explores the mechanism of short-term memory and long-term memory formation, it relies on intracellular cAMP→PKA→CREB signal. After the transduction pathway (second messenger transduction pathway) and won the Nobel Prize, scientists believe that these results are the key to the invention of memory-enhancing drugs. Although scientists are constantly striving to develop drugs that can rapidly activate the cAMP→PKA→CREB signal transduction pathway in brain cells, enabling humans to strengthen their memory to improve their learning ability, as well as prevent and treat forgetfulness, Alzheimer's disease, Azhai. Cerebral neurodegenerative diseases such as murmur and Parkinson can prevent and treat other diseases related to low cAMP content and utilization in the body, such as depression; however, in 1971, Earl Wilbur Sutherland Jr. won the Nobel Prize in Physiology and Medicine, which has exceeded For 30 years, there are still no related drugs that are suitable for long-term use, low toxicity and high efficiency.

In the prior art, anti-depressant drugs such as SSRI, SNRI, and NDRI have been asked to suppress 5-HT, which has a lower concentration in a body of a depressed patient than a normal person. NE, DA and other first messenger neurotransmitter re-uptake, after the first messenger neurotransmitter concentration and binding to the receptor tend to be normal, then the cAMP second messenger transduction of the transmitter in the patient's cells The tendency to produce is more normal; however, the side effects are high and the efficiency is low, but the anti-depressant drugs are daunting. Therefore, it has never been heard that healthy people take antidepressants for a long time in order to increase their memory.

The National Institutes of Health (NIH) Institute of Mental Health (NIMH) spent $35 million on five representative SSRI antidepressants for more than 2,800 melancholic patients over a six-year period. The clinical remission rat study (STAR ^* D study) of the drugs (Celexa, Zoloft, Wellbutrin, Effexor, Buspar), the results of the study indicated that each drug was only about 30% effective and averaged about 6 to 7 weeks to alleviate the symptoms of depression. What's more, the anti-depressant drugs that have been asked have different degrees of side effects, such as: increased suicide rate, headache, dizziness, dizziness, insomnia, lethargy, tinnitus, dry mouth, anorexia, increased appetite, weight gain, blood pressure rise, Gastrointestinal discomfort, nausea, nausea, vomiting, indigestion, diarrhea, constipation, lower limb pain, skin rash, trembling, cramps, excessive sweating, edema, decreased libido, sexual incompetence, etc. In recent years, anti-depressant drugs such as Prozac have become a serious concern in the society. The US Food and Drug Administration (FDA) in 2004 requested pharmaceutical companies to relabel the main 32 anti-depressants on the market. Part of its side effects and warnings, and stressing to medical staff that these drugs may increase the chances of suicide in children and adolescents.

For many years, scientists in the international medical community have been working hard to develop low-potential, safer, and more effective side effects, which can directly act on the receptors of the first messenger neurotransmitter and more rapidly increase the intracellular cAMP second messenger transduction transmitter. The production and utilization of drugs to prevent and treat diseases associated with low intracellular cAMP. Although, an inhibitor of phosphodiesterase 4 (PDE4), Roleta (Rolipram), which belongs to the regulation mechanism of the post-receptor mechanism, and the test shows that it has obvious anti-depression effect, because the cAMP produced in the cell is degraded by the tetra-type phosphodiesterase, and the tetra-type diphosphate is inhibited. Esterase increases the availability of cAMP; however, it is not widely used because of the side effects such as strong vomiting.

In order to solve the shortcomings of the above-mentioned technologies, we have cooperated with Mr. Zhang Zuoguang to develop a pharmaceutical composition made from three kinds of natural plants, such as human cockroach, licorice and jujube, which can not only increase the intracellular cAMP content, but also inhibit it. Phosphodiesterase can increase the utilization of cAMP by reducing the degradation of cAMP, and can increase the concentration of neurotransmitters such as DA and NE in the brain, and it is safe for long-term use, and is suitable for treating depression that must be taken for a long time. It is suitable for the prevention and treatment of intracellular cAMP hypoplasia, as well as related disorders of neurotransmitters such as DA and NE in the brain. However, natural plants can increase cAMP in each batch of natural plant materials because of different growth seasons, different habitats, different harvest seasons, different preservation methods, and climate change temperatures, rain, and sunlight. The content of the active ingredients of the production and utilization may not be the same; therefore, the clearer the effective ingredients, the more effective and safe the pharmaceutical composition produced each time by controlling and proportioning the active ingredients. More consistent to improve the quality controllability (CMC) of the pharmaceutical composition; in addition, if the type of saponin in the active ingredient can be more clearly defined, the range of raw materials can be expanded, so that the raw materials are not scarce because In addition to the roots, stems, and leaves of human cockroaches, roots, stems, and leaves of plants such as Panax notoginseng and yam are also available in a variety of human saponins. Therefore, in order to further enhance the quality controllability (CMC) of the pharmaceutical composition and to expand the base of raw materials, Mr. Zhang Zuoguang continued to work hard to study the more clarifying main functional ingredients based on the original research and development results. The mechanism of action, while the main efficacy of the research and development is more clear, the human saponins Rg1, Rb1, glycyrrhizic acid (Gan The pharmaceutical composition of oxalic acid and jujube cAMP, and the multi-target receptor mechanism of action regulating drugs, and the selection of human saponins Rg1, Rb1 as the main functional ingredients, can enhance the expression of BDNF.

However, in addition to human saponins Rg1 and Rb1, if you can further use other types of human saponins contained in the roots, stems, and leaves of plants such as human cockroaches, notoginseng, and yam, help human saponins Rg1 and Rb1. By achieving the effectiveness of the aforementioned pharmaceutical composition, the utilization of the active ingredients in the natural plant material can be further increased and the cost can be reduced. In addition, the aforementioned pharmaceutical composition contains glycyrrhizic acid components, and since ancient times, Chinese medicine has long established that people who have vomiting problems may cause vomiting problems if they take licorice.

The post is the reason, we continue to work hard to study and explore on the basis of the original research and development results, to further improve the conventional technology and its lack, and a perseverance spirit, finally conceived the case "can quickly increase the body's cAMP content And the pharmaceutical composition of the utilization degree, the following is a brief description of the case.

In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a group comprising ginsenosides (Rg1+Rb1+Re), glycyrrhizic acid and jujube cAMP as main components, thereby rapidly increasing the content of cyclic adenosine monophosphate in the body and utilizing Pharmaceutical compositions, in particular, can further increase the availability and cost of active ingredients in natural plant materials, and provide stable quality to rapidly enhance intracellular cAMP/PKA/CREB signal transduction pathways and enhance BDNF expression. A new technical solution for oral or health foods.

The solution of the pharmaceutical composition of the present invention is the result of the research and exploration by our people and the full experiment using the examples, and the results are proved by prior art experiments. Saponins Rg1 and Rb1 can increase the expression of BDNF in vivo, so it can be used as the main functional component of the development of related drugs; however, the current technology has not been able to artificially synthesize human saponins Rg1 and Rb1, so it must be from human cockroaches, notoginseng, western glutinous rice, etc. Obtained from the roots, stems and leaves of natural plants. Since there are more than 30 kinds of human saponins, in order to further increase the availability of effective ingredients in natural plant materials and reduce costs, the inventors may use saponins Re to cooperate with human saponins Rg1. Rb1, combined with glycyrrhizic acid (glycyrrhetinic acid) and jujube cAMP as the main functional ingredients to make a pharmaceutical composition, after thorough experimentation, the present invention can be improved in hippocampus after 8 hours in normal rats. cAMP concentration, and enhance PKA activity, and increase CREB phosphorylation level; can still inhibit the activity of PDE in rat hippocampus; and chronic repeated stress test, cAMP concentration, PKA activity, CREB in hippocampus of mice The expression of phosphorylation and the expression of BDNF were significantly higher than those of the untreated model group. In the chronic repeated stress test, cAMP, PKA, and serum BDNF and hypothalamic 5-HT were measured in the hippocampus of rats. The expression of monoamine transmitters such as NE, DA, etc. was also significantly higher than that of the untreated model group; thus, it was confirmed that the pharmaceutical composition of the present invention can rapidly increase in vivo. Monophosphate content and line utilization. It is to be noted that the present invention has good effectiveness, and further increases the utilization of the active ingredients in natural plant materials and reduces the cost, and can effectively perform quality controllability than the prior art. In addition, the present invention has high safety for long-term administration, and is suitable for treating depression which requires long-term medication, as well as prevention and treatment of low cAMP in the body and cells, low expression of BDNF, insufficient neurotransmitters such as DA and NE in the brain, and the like. Related diseases (such as: memory deficiency, dementia, Alzheimer's, Parkinson's and other neurodegenerative diseases, as well as psoriasis, cancer and other diseases in the body and cells with low cAMP, etc.), applicable to a wide range of people; however, because of drugs The composition contains glycyrrhizic acid components, and since ancient times Chinese medicine has long been determined that people who have vomiting problems may induce vomiting if they take licorice; however, since ancient times, Chinese medicine has long established that long-term use of safe ginger is an antiemetic; therefore, the pharmaceutical composition of the present invention Ginger powder or its extract can also be added to improve the deficiencies of the prior art.

The invention discloses a pharmaceutical composition for rapidly increasing the content and utilization of cyclic adenosine monophosphate in the body, which comprises a raw material comprising a main functional ingredient including ginsenoside (Rg1+Rb1+Re), glycyrrhizic acid and jujube cAMP. production.

The pharmaceutical composition for rapidly increasing the content and availability of cyclodextrin monophosphate in the body described in the specification and the scope of the present application is the core of the object of the present invention, and after the disclosure of the present invention, those skilled in the art It is within the scope of the present invention to perform conventional addition and subtraction, which are generally the technical activities of those skilled in the art and the researcher.

The invention will be better understood by reference to the drawings and the detailed description.

The invention will be further illustrated by the following figures and examples. The present invention is primarily directed to the preparation of the medicaments of the present invention using methods well known to those skilled in the art in conjunction with the features of the present invention. The following examples are for illustrative purposes only and are not intended to limit the invention.

In order to accomplish the object of the present invention, the present invention particularly proposes the following technical solutions.

The invention discloses a pharmaceutical composition for rapidly increasing the content and utilization of cyclodextrin monophosphate in the body, which is prepared from a raw material comprising a main functional ingredient including ginsenoside (Rg1+Rb1+Re), glycyrrhizic acid and jujube cAMP. .

Option One:

The invention comprises the raw materials containing ginsenosides (Rg1+Rb1+Re), glycyrrhizic acid or glycyrrhetinic acid and jujube cAMP, and the invention can rapidly increase the content of cyclodextrin monophosphate in the body. And a pharmaceutical composition of availability.

Option II:

The pharmaceutical composition of the present invention is processed by using a raw material containing 2 to 26 parts by weight of ginsenosides (Rg1 + Rb1 + Re), 3 to 48 parts by weight of glycyrrhizic acid or glycyrrhetinic acid, and 0.002 to 0.5 parts by weight of jujube cAMP.

third solution:

The pharmaceutical composition of the present invention is processed by using 4 to 13 parts by weight of total ginsenosides (Rg1 + Rb1 + Re), 5 to 16 parts by weight of glycyrrhizic acid or glycyrrhetinic acid, and 0.01 to 0.1 parts by weight of jujube cAMP.

Option 4:

The pharmaceutical composition of the present invention comprises the addition of a ginger water extract.

Option 5:

The pharmaceutical composition of the present invention comprises any pharmaceutically acceptable oral pharmaceutical dosage form which may contain a pharmaceutically acceptable carrier or additive, and may be formulated into tablets, capsules, powders and the like.

Option six:

The pharmaceutical composition of the present invention can be used to prepare medicines, health foods and nutrients which rapidly increase the content and availability of cyclic adenosine monophosphate in the body.

In order to accomplish the object of the present invention, the following methods for producing the following drugs are proposed.

method one:

Extracts containing ginsenosides (Rg1+Rb1+Re), glycyrrhizic acid and jujube cAMP are extracted from ginseng, licorice and jujube, respectively, or directly prepared using ginsenosides (Rg1+Rb1+Re) ), glycyrrhizic acid or glycyrrhetinic acid and jujube cAMP raw materials, processed to form a rapid increase in the body of cyclodextrin A pharmaceutical composition of phosphoric acid content and availability.

Method Two:

A raw material containing 2 to 26 parts by weight of ginsenoside (Rg1+Rb1+Re), 3 to 48 parts by weight of glycyrrhizic acid or glycyrrhetinic acid, and 0.002 to 0.5 parts by weight of jujube cAMP is processed to prepare a pharmaceutical composition of the present invention.

Method three:

A raw material containing 4 to 13 parts by weight of ginsenoside (Rg1 + Rb1 + Re), 5 to 16 parts by weight of glycyrrhizic acid or glycyrrhetinic acid, and 0.01 to 0.1 part by weight of jujube cAMP is processed to prepare a pharmaceutical composition of the present invention.

Method four:

The pharmaceutical composition of the present invention comprises the addition of a ginger water extract.

Method five:

The pharmaceutical composition of the present invention comprises any pharmaceutically acceptable oral pharmaceutical dosage form which may contain a pharmaceutically acceptable carrier or additive and may be formulated into tablets, capsules, powders and the like.

Method six:

The raw material of the present invention is processed into a health food or a nutrient for rapidly increasing the content and utilization of cyclodextrin monophosphate in the body according to the food management standard or the method according to the health food production and manufacturing standard.

Specific embodiment

The invention will be further described below in conjunction with the drawings and specific embodiments.

Embodiment 1

Please refer to FIG. 1 , which is a schematic flow chart of a method for preparing a medicament according to an embodiment of the present invention. In Fig. 1, 40 kg of ginseng is crushed, extracted with 70% ethanol solution, and separated and purified by column chromatography to obtain 270 g of ginsenoside Rg1+Rb1+Re (Rg1). About 48.4g, Rb1 about 176.9g, Re about 44.7g) of ginseng extract 1.42kg; and 15kg of licorice was crushed and immersed at room temperature for 12 hours, extracted by water extraction and alcohol precipitation, concentrated and dried to obtain 307g containing glycyrrhizic acid. The licorice extract is 3.1kg; and 10kg of jujube is crushed, soaked with water at room temperature, and then extracted with water and alcohol precipitation to obtain jujube extract. Then, the macroporous resin OU-2 and ME-2 are successively applied to the column. Adsorption separation and drying, 40 g of jujube extract containing 0.752 g of jujube cAMP was prepared as a raw material for preparing the medicament of the present invention; after that, 144 g of ginseng extract obtained by the above method, 300 g of licorice extract and 3.6 g of jujube extract were pulverized. After uniformly mixing, 447.6 g (containing 27.4 g of ginsenoside Rg1 + Rb1 + Re, and 29, 7 g of glycyrrhizic acid, and 0.067 g of jujube cAMP) of the pharmaceutical composition of the first aspect of the invention were obtained.

Embodiment 2

Please refer to FIG. 2 , which is a schematic flow chart of a method for preparing the second embodiment of the present invention. In Fig. 2, 120 g of the ginseng extract obtained in Example 1, 200 g of the licorice extract and 0.5 g of the jujube extract were pulverized and mixed to obtain 320.5 g (containing 22.8 g of ginsenoside Rg1 + Rb1 + Re, 19.8 g). Glycyrrhizic acid and 0.009 g jujube cAMP) The pharmaceutical composition of the second embodiment of the present invention.

Embodiment 3

Please refer to FIG. 3, which is a schematic flow chart of a method for preparing the third embodiment of the present invention. In Fig. 3, 3.6 g of ginsenoside Rg1 having a purity of 90%, 3.2 g of ginsenoside Re having a purity of 90%, 15.6 g of ginsenoside Rb1 having a purity of 90%, and 26 g of a purity of 96% were prepared. After the glycyrrhetinic acid and the jujube extract obtained in the first example were pulverized and mixed uniformly, 58.4 g (containing 22.4 g of ginsenoside Rg1 + Rb1, 26 g of glycyrrhetinic acid and 0.188 g of jujube cAMP) of the drug combination of the third embodiment of the present invention was obtained. Things.

Embodiment 4

Please refer to FIG. 4, which is a schematic flow chart of a method for preparing the fourth embodiment of the present invention. Figure. In Fig. 4, 100 g of the pharmaceutical composition of the first aspect of the present invention obtained in Example 1 was mixed with 35 g of a commercially available ginger extract to obtain 135 g of the pharmaceutical composition of the fourth aspect of the present invention.

Experimental Example 1: Animal experiment in which normal rats were administered for 8 hours, the effect of cAMP/PKA/CREB signal transduction pathway.

Normal rats were intragastrically administered for 8 hours after the administration of hippocampus, and Cyclic AMP and Phoclic AMP response element binding protein in hippocampus were determined by enzyme-linked immunosorbent assay (ELISA). Change in the content of CREB), bioluminescent method to measure the change in activity of phosphoprotein kinase A (cAMP-Dependent PKA), and change in activity of phosphodiesterase (PDE) by fluorescence method, revealing the administration of Example 1. The Cyclic AMP concentration was increased in a short time after 8 hours, thereby enhancing cAMP-Dependent PKA activity, increasing CREB phosphorylation level, and inhibiting the molecular pharmacological mechanism of PDE activity in hippocampus. The test data were plotted using Oringin Pro 7.5 software statistical analysis.

Test material

1.1 test animals

Healthy male Sprague-Dawley rats, weighing 180-200 g, 70, were purchased from Beijing Weitong Lihua Animal Testing Center.

1.2 reagent

Positive control drug, anti-depressant, paroxetine hydrochloride (batch number: 08030078, Sino-US Tianjin Shike Pharmaceutical Co., Ltd.); Parameter Cyclic AMP Assay Kit, KGE002 (American R&D Systems, Inc.); DuoSet IC Human/Mouse/Rat Phospho- CREB (S133) ELISA Kit, DYC2510-2 (American R&D Systems, Inc.); PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase Kit, V5340 (Promega Corporation, USA); PDE-Glo Phosphodiesterase Assay Kit, V1361 (Promega Corporation, USA); Pierce BCA Protein Assay Kit, 23227, (Thermo), Cyclic Adenosine, Reference materials such as adenosine were purchased from China National Institute for the Control of Pharmaceutical and Biological Products; reagents such as NaH2PO4, Na2HPO4, KH2PO4, KCl, NaCl, MgCl2, Tris Base, and Tris-HCl were all cell culture grade biochemical reagents purchased from Sigma, USA; E- 64, APROTININ, LEUPEPTIN, Pepstatin A, PMSF, NaF, EDTA, EGTA, DTT, NaVO4, Sodium pyrophosphate, agarose, glycerol and other reagents are high purity grade, purchased from BioBasic, Canada; acetonitrile, methanol (chromatography, Germany) MERCK company); ultrapure water (MilliQ pure water); Example 1.

1.3 test instrument FlexStation 3 multi-function microplate analyzer (Molecular Devices Corporation, USA); Waters600E high performance liquid chromatograph (quaternary pump, online degasser, autosampler, column oven, UV detector, USA Waters); refrigerated centrifuge (BECKMAN, USA); electronic ultrasonic homogenizer (UNTRASOUND TECHNOLOGY, USA); electrophoresis (Beijing Liuyi Instrument Factory); gel imager (SYN GENE); ULTRA LOW ultra-low temperature refrigerator ( Japan SANYO company); mLine single-channel pipette, 8-channel pipette (Biohit, Finland).

2. Administration

Three days after adaptive feeding, the rats were randomly divided into three groups, which were labeled as: A saline group, B paroxetine group, and C group. The paroxetine hydrochloride tablets were crushed and mixed with ultra-pure water to form a certain concentration of suspension. The rats were intragastrically administered at a dose of 5 mg/kg. In the first example, the contents were mixed with water to form a certain concentration of the solution. The gastric administration dose was 50 mg/kg; the saline group was given an equal volume of 0.9% physiological saline; all rats were weighed and labeled with picric acid before administration, and all the drugs were administered after preheating at 37 ° C for 30 minutes.

3. Drawing materials

After 8 hours of administration of the test animals of group A, B and C, the rats were anesthetized with ether, the femoral artery was exsanguinated, the brain was decapitated on ice, and the hippocampus tissue was taken and finely cut into three parts, which were placed in pre-numbered 1.5 mL color caps. In the frozen tube of the screw, it is quickly weighed and quickly frozen in liquid nitrogen for 15 minutes, and then stored in a refrigerator at -80 °C for later use.

4. Sample testing

4.1 Determination of Cyclic AMP content in hippocampus of rats: Thaw the hippocampal tissue samples, rinse with a small amount of physiological saline, and add the cell lysate provided by the kit at a ratio of 1:20 (g:mL) (diluted 5 times concentrated solution) After use, the electronic ultrasonic homogenizer was homogenized for 30 s, and centrifuged at 10,000 rpm for 5 min at 4 ° C. The supernatant was placed in a pre-numbered 1.5 mL color Eppendorf centrifuge tube and placed in an ice box for testing. Samples were taken using the American R&D Systems Parameter Cyclic AMP Assay ELISA kit. The sample was returned to room temperature and the Cyclic AMP content in the sample was determined by competitive ELISA according to the kit instructions. The OD value was measured at 450 nm using a multiwell plate analyzer, and the Cyclic AMP content in the sample was calculated from the standard curve.

4.2 Determination of cAMP-Dependent PKA activity in rat hippocampus: Thaw the hippocampal tissue samples, rinse with a small amount of physiological saline, and then add PKA extraction buffer (according to the formula in the kit) at a ratio of 1:10 (g:mL). The ultrasonic homogenizer was homogenized for 30 s, centrifuged at 10,000 rpm for 5 min at 4 ° C, and the supernatant was placed in a pre-numbered 1.5 mL color Eppendorf centrifuge tube and placed in an ice box for testing. Apply PepTag Assay for Promega, USA Non-Radioactive Detection of cAMP-Dependent Protein Kinase Kit for assay analysis. Add pre-mixed reagents to the pre-coded 200 μL PCR occlusion tube according to the kit instructions. Add 9 μL of each sample to each tube, vortex and mix, centrifuge, and react at room temperature for 30 min, then place in the PCR machine. The enzyme was inactivated at 98 ° C for 5 min. In the test, positive control and negative control tubes were set according to the requirements of the manual, and the test was carried out. A 0.8% agarose gel was prepared, and 10 μL of each sample after the enzyme reaction was added to the gel well, 100 V, 130 mA for 30 min, and the electrophoresis solution was 50 mM Tris-HCl (pH 8.0) buffer. After electrophoresis, the agarose gel was taken out, photographed by a gel imager, and then placed on an ultraviolet analyzer. The phosphorylated PipTag A1 Peptide spot was cut and placed in a pre-numbered 1.5 mL cap screw. In the tube. Heat to melt the agarose gel, dilute to 250 μL with ultrapure water, quickly remove 125 μL into a pre-numbered 1.5 mL color Eppendorf centrifuge tube, add 75 μL of the sol solution provided by the kit and 50 μL of glacial acetic acid, vortex and mix. 200 μL was added to a 96-well microtiter plate, and a negative control tube agarose was used as a blank control, and fluorescence analysis was performed on a multiwell plate analyzer. Set Excitation Wavelength 568nm, Emission Wavelength 592nm. PKA activity is expressed in terms of sample fluorescence intensity.

4.3 Determination of p-CREB content in rat hippocampus: Samples were taken using the US R&D Systems DuoSet IC Human/Mouse/Rat Phospho-CREB (S133) ELISA kit. The hippocampus tissue samples were thawed, rinsed with a small amount of physiological saline, and then added to the tissue homogenate at a ratio of 1:20 (g:mL) (according to IC DELUENT 6# in the kit), and homogenized by an ultrasonic homogenizer for 30 s. After centrifugation at 10,000 rpm for 5 min at 4 ° C, the supernatant was placed in a pre-numbered 1.5 mL color Eppendorf centrifuge tube and placed in an ice box for testing. The sample was returned to room temperature during the assay and tested by sandwich ELISA according to the kit instructions. Determine the p-CREB content in the sample. The OD value was measured at 450 nm using a multiwell plate analyzer, and the p-CREB content in the sample was calculated from the standard curve.

4.4 Determination of total protein in rat hippocampus: In order to more accurately calibrate the amount of p-CREB protein per mg of protein in the sample, the total protein content of the sample needs to be determined. The supernatant after centrifugation of the tissue homogenate in the p-CREB assay was diluted 25 times with PBS, and then used as a test sample, and the OD value was measured at 562 nm using a perforated plate analyzer according to the Pierce BCA Protein Assay Kit reagent specification. Using bovine serum albumin (BSA) as a standard, the total protein content in the sample was calculated from the standard curve.

4.5 Phosphodiesterase (PDE) Activity Assay: The effect of PDE activity in a pair of rat brain hippocampus was determined using the Bioluminescent PDE-Glo Phosphodiesterase Assay kit from Promega, USA.

4.5.1 Preparation of the drug solution: The contents of the first embodiment were prepared into three concentrations of 0.02 mg/mL, 0.05 mg/mL and 1.0 mg/mL.

4.5.2 Sample preparation: Take a certain amount of hippocampus tissue, rinse with a small amount of physiological saline, add PDE-Glo Reaction Buffer (Tris-HCl 40 mM, MgCl 2 10 mM, BSA 0.1 mg/ml in a ratio of 1:10 (g:mL). Further, PMSF 1 mM, leupetin 2 μM/mL, aprotinin 2 μM/mL, E-64 2 μM/mL) were added, and the mixture was homogenized by an ultrasonic machine, and centrifuged at 14,000 rpm for 30 minutes at 4 ° C, and the supernatant was taken as an enzyme solution for use.

4.5.3 Bioluminescence assay: Operate according to the method provided in the kit instructions. Add 1 μL of the solution to the enzyme reaction solution, 1.5 μL of the enzyme solution for a total of 2.5 μL; add 2 μmol of Cyclic AMP. 2.5 μL of substrate solution, mix, react at 37 ° C for 30 min, then add 2.5 μL of reaction stop solution containing IBMX, a strong inhibitor of PDE, and mix; add 2.5 μL of test solution, mix, react at room temperature for 20 min; 10 μL of the reagent was reacted at room temperature for 10 min and then tested on a multi-function microplate analyzer.

5. Test results

5.1 Cyclic AMP content in rat hippocampus: The concentration of Cyclic AMP in the rat hippocampal tissue homogenate was determined by ELISA, and the amount of Cyclic AMP contained in the hippocampus was obtained by dividing the weight of the weighed tissue sample, expressed as pmol/g Tissue (Fig. 5).

(Comparative group compared with saline group and paroxetine group, the content of Cyclic AMP in rat hippocampus was significantly increased, *P<0.05, n=10)

5.2 cAMP-Dependent PKA activity in rat hippocampus: After the enzymatic reaction, the samples were subjected to agarose gel electrophoresis, photographed by a gel imager, and subjected to rough analysis (Fig. 6).

(Swimming lanes from left to right 1-4 saline group; 5-8 paroxetine group; 9-11 example group; 12 positive control sample; 13 negative control sample) Phosphorylated A1 peptide with negative charge to positive electrode The direction moves; the unphosphorylated A1 peptide has a positive charge and moves in the direction of the negative electrode, separating the two. The higher the brightness of the phosphorylated A1 peptide spot moving toward the positive electrode, indicating that the higher the phosphorylation level, the higher the cAMP-Dependent PKA activity in the sample. It can be seen that the brightness of the positive direction spot of a set of samples in the embodiment is higher than that of the physiological saline group and the paroxetine group.

The agarose gel spots were cut, and the volume was adjusted after melting, and the activity of cAMP-Dependent PKA in rat hippocampus was measured by fluorescence method, which was expressed by fluorescence intensity (Fig. 7).

(8h after administration, the activity of cAMP-Dependent PKA was significantly increased in the hippocampus of the rats in the group compared with the blank control group and the paroxetine group, *P<0.05. n=10)

5.3 p-CREB content in rat hippocampus: The concentration of total protein in the rat hippocampal tissue homogenate was determined by BCA method to calibrate the amount of p-CREB per μg of total protein. The concentration of p-CREB in rat hippocampal tissue homogenate was determined by sandwich ELISA. The p-CREB content in rat hippocampus was expressed by p-CREB(pg)/total protein (μg). The results are shown in Figure 8. .

(After administration for 8 hours, the concentration of p-CREB in hippocampus of rats in the group was higher than that in the saline group and the paroxetine group, n=10)

5.4 Effects of drugs on phosphodiesterase (PDE) activity in rat hippocampus: The activity of PDE in hippocampus and the inhibition of PDE activity after drug administration in vitro were determined by bioluminescence. The PDE activity was expressed by the luminescence intensity. Compared with the control group, the luminescence intensity was higher, indicating that the PDE activity was higher; the luminescence intensity was lower, indicating that the PDE activity was inhibited; the results of the assay showed that compared with the blank control group, Example 1 ( 0.5 mg/ml) significantly inhibited the activity of PDE in rat hippocampus, *P<0.05, n=4.

6 Conclusion

(1) After 8 hours of intragastric administration, compared with the saline group and the paroxetine group, the Cyclic AMP content in the hippocampus of the rats in the group was significantly increased; the cAMP-Dependent PKA activity was significantly enhanced; p - CREB content has also increased.

(2) This experiment demonstrates that Example 1 can exert pharmacological effects through the second messenger Cyclic AMP cell signal transduction pathway, and can rapidly initiate the cAMP-PKA-CREB (p-CREB) pathway after 8 hours of administration (with There was a significant difference between the saline group and the paroxetine group, *P<0.05).

(3) The same positive drug administration, paroxetine did not start. cAMP-PKA-CREB (p-CREB) pathway.

(4) The results of this experiment also show that the medium dose group of Example 1 can significantly inhibit the activity of phosphodiesterase in rat hippocampus. Since the phosphodiesterase is an inactivation enzyme of Cyclic AMP, it can be inhibited after being inhibited. The content of Cyclic AMP in the hippocampus of rats increased.

Experimental Example 2: Animal experiment of chronic stress mice administered with Example 10 for 10 days on the expression of cAMP/PKA/CREB signal transduction pathway and BDNF expression.

1. The effect of Example 1 on hippocampal cAMP concentration in mice with repeated stress (Fig. 9): Results: The concentration of cAMP in hippocampus of mice administered in Example 1 was significantly higher than that in the unadministered model group, *P<0.05 .

2. The effect of Example 1 on hippocampal PKA activity in chronically stressed mice (Fig. 10): Results: The PKA activity of the hippocampus of the first embodiment was significantly higher than that of the unadministered model group, *P<0.05 .

3. Effect of Example 1 on CREB phosphorylation in hippocampus of chronically stressed mice (Fig. 11): Results: The expression of CREB phosphorylation in the hippocampus of the administration Example 1 was significantly higher than that of the unadministered model group.

4. Example 1 Effect on hippocampal BDNF in chronically stressed mice (Fig. 12): Results: The expression of BDNF in the hippocampus of the mice of Example 1 was significantly higher than that of the unadministered model group.

Experimental Example 3: Chronic stress rat administration Example 1 for 21 days, hippocampus cAMP, PKA And animal experiments on the effects of serum BDNF and hypothalamic NE, DA, 5HT and other expression levels.

1. The effect of Example 1 on the concentration of cAMP in hippocampus and cortex of chronically stressed rats (Table 1):

2. The effect of Example 1 on the PKA content in hippocampus of chronically stressed rats (Table 2):

3. The effect of Example 1 on serum BDNF content in chronic stress rats (Table 3):

4. Example 1 Effect on the expression of monoamine neurotransmitters in the hypothalamus of chronically stressed rats (Table 4):

5. Conclusion: The expression of monoamine neurotransmitters in rat hippocampus cAMP, PKA, and serum BDNF and hypothalamic 5-HT, NE, DA were significantly higher than those in the unadministered model group (*P). <0.05).

Experimental Example 4: Example 1 Antidepressive pharmacodynamics animal experiment.

1. Behavioral animal experiment: the pathogenesis and clinical symptoms of depression, we selected mouse tail suspension, forced swimming in rats, forced swimming in mice, unpredictable long-term stress in rats, rat olfactory bulb destruction model and other resistance Depressive behavioral animal experiments.

1.1 Example 1 The mice were intragastrically administered with 20 mg/kg/d, 40 mg/kg/d, and 80 mg/kg (rat 15 mg/kg/d, 30 mg/kg/d, 60 mg/kg/d) for 1 week. It can show the effect of anti-experimental depression, in which the middle dose group (mouse 40mg/kg/d, rat 30mg/kg/d) and the positive drug paroxetine group (3mg/kg/d) can significantly shorten the suspension. The immobile mice did not move for a long time, which significantly shortened the immobility time of forced swimming mice and rats, and had significant statistical difference compared with the model group ( P <0.05).

1.2 Example 1 Rats in chronic stress depression model (CU.MS) were intragastrically administered with 15 mg/kg/d, 30 mg/kg/d, 60 mg/kg/d for 21 consecutive days. Results: Compared with the normal group, the model The weight gain of CUMS rats was slow, the water consumption of sucrose was significantly decreased (P<0.01), the level of activity and vertical activity were significantly decreased (P<0.01), and the number of jumping errors in rats was significantly increased (P<0.01). Compared with the model group, the weight gain of the rats in the low-dose group and the positive drug paroxetine group were significantly increased, and the water consumption of sucrose was significantly increased (P<0.01). The small-dose group and the positive drug paroxetine group in the first example were larger. The level and vertical activity of the rats increased significantly (P<0.01). The number of errors in the low-dose group and the positive drug paroxetine group was significantly reduced (P<0.05, P<0.01).

1.3 Example 1 Rats with olfactory bulb destruction model were intragastrically administered with 15 mg/kg/d, 30 mg/kg/d, 60 mg/kg/d for 24 consecutive days. In the open-box test, the large-dose group and the model group were given. In comparison, the level and vertical movement of rats caused by olfactory bulb damage can be significantly improved. The positive reference drug paroxetine (3mg/kg/d) can also significantly reduce the decrease of rat level motion caused by olfactory bulb damage; in passive avoidance test In the large, medium-dose group and the positive drug paroxetine group, the learning and memory dysfunction caused by olfactory bulb damage was significantly improved in rats.

2. Interaction model animal experiment: According to the pathogenesis and clinical symptoms of depression, we selected the mouse reserpine model (decreased body temperature, inability to exercise, drooping eyelids) and serotonin-induced mouse taro.

2.1 Example 1 The mice were intragastrically administered with 20 mg/kg/d, 40 mg/kg/d, 80 mg/kg/d (rat 15 mg/kg/d, 30 mg/kg/d, 60 mg/kg/d), continuous 1 Week, can significantly antagonize the decrease of body temperature, lack of exercise and drooping of eyelid induced by reserpine, indicating that the experimental depression of the first antibody may be related to the influence of monoamine transmitter; it can significantly increase the injection of pentahydroxytryptophan in mice. The number of subsequent taro indicates that the antidepressant effect of Example 1 may be related to inhibition of MAO. In addition, since the test results showed that the autonomous activity of the pair of mice had no significant effect, the first embodiment had no central excitatory effect.

3. Example 1 The results of the main antidepressant pharmacodynamics experiments are summarized in the following table (Table 5):

4. Conclusion: Example 1 has a significant anti-experimental depression effect.

The results of the various experimental examples show:

(1) Rats were administered orally 8 hours later, with saline group and Paro Compared with the statin group, cAMP was significantly increased in hippocampus, PKA activity was significantly increased (*P<0.05), p-CREB content was also increased, and PDE4 activity was significantly inhibited (*P<0.05).

(2) After 10 days of intragastric administration in chronic stress mice, the decrease of cAMP and PKA in hippocampus induced by repeated stress can be significantly increased, and the expression of phosphorylated CREB and BDNF in hippocampus of mice is promoted (*P). <0.05).

(3) After 21 days of intragastric administration of chronically stressed rats, the decrease of cAMP and PKA in hippocampus caused by repeated stress can be significantly increased, and serum BDNF and hypothalamic NE, 5-HT and other monoamines can be promoted. Qualitative expression (*P<0.05), downregulation of serum GSC.

(4) Example 1 has a significant anti-experimental depression effect.

The invention discloses a pharmaceutical composition for rapidly increasing the content and utilization of cyclodextrin monophosphate in the body: 1. The pharmaceutical composition for rapidly increasing the content and utilization of cyclodextrin monophosphate in the invention may comprise a pharmacologically An acceptable additive; 2. The pharmaceutical composition of the present invention for rapidly increasing the content and availability of cyclic adenosine monophosphate in the body can be processed into various conventional dosage forms such as powders, capsules, tablets, and the like; 3. The pharmaceutical composition for rapidly increasing the content and availability of cyclodextrin monophosphate in the present invention can be used for preventing and treating diseases caused by low cAMP content and utilization in vivo and cells, enhancing BDNF expression, and strengthening cells. Internal cAMP/PKA/CREB signal transduction pathways, as well as drugs, health foods and nutrients that increase the levels of DA, NE and 5HT neurotransmitters in the brain and enhance memory.

In summary, the implementation of the present invention is as follows:

A pharmaceutical composition for increasing the content and availability of cyclodextrin monophosphate in the body, comprising: a first main component comprising: ginsenoside Rg1, Rb1 and Re; and a second main component comprising: a glycyrrhizic acid, selected from the group consisting of One of glycyrrhizic acid, monoglycyrrhetic acid, and a combination thereof; and a third main component: comprising a large amount of cyclic adenosine monophosphate.

2. The pharmaceutical composition according to the embodiment 1, wherein the pharmaceutical composition comprises 2 to 26 parts by weight of the ginsenoside Rg1 and Rb1, 3 to 48 parts by weight of the glycyrrhizic acid, and 0.002 to 0.5 part by weight of the large Jujube cyclosporine monophosphate.

3. The pharmaceutical composition according to embodiment 1 or 2, wherein the pharmaceutical composition comprises 4 to 13 parts by weight of the ginsenoside Rg1 and Rb1, 5 to 16 parts by weight of the glycyrrhizic acid, and 0.01 to 0.1 part by weight. The jujube ring adenosine monophosphate.

4. The pharmaceutical composition according to any one of embodiments 1 to 3, wherein the pharmaceutical composition comprises one selected from the group consisting of a pharmaceutically acceptable carrier, an additive, and a combination thereof.

5. The pharmaceutical composition according to any one of embodiments 1 to 4, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of a tablet, a capsule, a powder, a tablet, a powder, One of a solution, a microcapsule, a suspension, an emulsion, a granule, a pill, a pill, and a pharmaceutically acceptable oral pharmaceutical dosage form.

6. The pharmaceutical composition according to any one of embodiments 1 to 5, wherein the pharmaceutical composition is formulated to prevent and treat cAMP content and utilization in vivo and in cells, and to enhance intracellular cAMP/PKA/CREB signal transduction. Guide pathway, enhanced BDNF Amounts of drugs, health foods, and nutrients that increase the amount of DA, NE, and 5HT neurotransmitters in the brain and enhance memory.

7. The pharmaceutical composition according to any one of embodiments 1 to 6, wherein the pharmaceutical composition further comprises a fourth main component selected from the group consisting of a ginger powder and a ginger extract and a combination thereof.

A pharmaceutical composition for increasing the availability of cyclodextrin monophosphate in the body, comprising: a first main component comprising: ginsenoside Rg1, Rb1 and Re; and a second main component comprising: a glycyrrhizic acid selected from the group consisting of glycyrrhizic acid One of a glycyrrhetinic acid and a combination thereof; and a third main component: comprising a large amount of cyclodextrin monophosphate.

9. A method of making a pharmaceutical composition for increasing a cyclic adenosine monophosphate content in a body comprising: providing a first major component, wherein the first major component comprises ginsenoside Rg1, Rb1 and Re; providing a second major component Wherein the second major component comprises a glycyrrhizic acid selected from the group consisting of glycyrrhizic acid, glycyrrhetinic acid, and a combination thereof; and a third major component, wherein the third major component comprises a large jujube cyclic adenosine Monophosphate is used to prepare the pharmaceutical composition.

This case has been modified by people who are familiar with this skill, but it is not to be protected as intended.

Fig. 1 is a schematic flow chart showing a method for preparing a medicament according to a first embodiment of the present invention.

Fig. 2 is a schematic view showing the process of preparing the medicament of the second embodiment of the present invention.

Fig. 3 is a schematic view showing the process of preparing the third drug of the present invention.

Fig. 4 is a schematic flow chart showing the process of preparing the fourth drug of the present invention.

Figure 5 shows the changes in the content of Cyclic AMP in the hippocampus of rats after 8 hours of administration.

Figure 6 is a photograph of a typical gel electrophoresis in the assay for cAMP-Dependent PKA activity.

Figure 7 shows the difference in cAMP-Dependent PKA activity in rat hippocampus after 8 h of administration.

Figure 8 shows the changes in the content of p-CREB in rat hippocampus after 8 hours of administration.

Figure 9 is a graph showing the effect of Example 1 on cAMP concentration in hippocampus of mice after repeated stress.

Figure 10 is a graph showing the effect of Example 1 on PKA activity in hippocampus of chronically stressed mice.

Figure 11 is a graph showing the effect of Example 1 on CREB phosphorylation in hippocampus of chronically stressed mice.

Figure 12 is a graph showing the effect of Example 1 on BDNF in hippocampus of chronically stressed mice.

Claims (7)

The invention relates to a pharmaceutical composition for rapidly increasing the content and utilization of cyclic adenosine monophosphate in the body, comprising: a first main component: comprising ginsenoside Rg1, Rb1 and Re; and a second main component comprising: a glycyrrhizic acid, selected from the group consisting of One of glycyrrhizic acid, monoglycyrrhetic acid, and a combination thereof; and a third main component: comprising a large amount of cyclic adenosine monophosphate, wherein the pharmaceutical composition comprises 2 to 26 parts by weight of the ginsenoside Rg1, Rb1 and Re 3 to 48 parts by weight of the glycyrrhizic acid and 0.002 to 0.5 parts by weight of the jujube cyclic adenosine monophosphate. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises 4 to 13 parts by weight of the ginsenoside Rg1, Rb1 and Re, 5 to 16 parts by weight of the glycyrrhizic acid, and 0.01 to 0.1 parts by weight. The jujube ring adenosine monophosphate. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises one selected from the group consisting of a pharmaceutically acceptable carrier, an additive, and a combination thereof. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of a tablet, a capsule, a powder, a tablet, a powder, a solution, One of a microcapsule, a suspension, an emulsion, a granule, a drop of a pill, a pill, and a pharmaceutically acceptable oral pharmaceutical dosage form. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition can be used for preventing and treating cAMP content and utilization in vivo and cells, enhancing intracellular cAMP/PKA/CREB signal transduction pathway, and strengthening BDNF expression, drugs that increase brain DA, NE, and 5HT neurotransmitters and enhance memory Things, health foods and nutrients. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a fourth main component selected from the group consisting of a ginger powder and a ginger extract and a combination thereof. A method of producing a pharmaceutical composition for rapidly increasing the content of a cyclic adenosine monophosphate in a body, comprising: providing a first major component, wherein the first major component comprises ginsenosides Rg1, Rb1 and Re; providing a second major component, Wherein the second main component comprises a glycyrrhizic acid, one selected from the group consisting of glycyrrhizic acid, glycyrrhetinic acid and a combination thereof; and a third main component, wherein the third main component comprises a large amount of cyclic adenosine monophosphate And mixing the first main component, the second main component and the third main component to produce the pharmaceutical composition, wherein the pharmaceutical composition comprises 2 to 26 parts by weight of the ginsenoside Rg1, Rb1 and Re 3 to 48 parts by weight of the glycyrrhizic acid and 0.002 to 0.5 parts by weight of the jujube cyclic adenosine monophosphate.