CN117860859A - Traditional Chinese medicine composition for preventing and treating stroke and preparation method and application thereof - Google Patents
Traditional Chinese medicine composition for preventing and treating stroke and preparation method and application thereof Download PDFInfo
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- CN117860859A CN117860859A CN202410059579.2A CN202410059579A CN117860859A CN 117860859 A CN117860859 A CN 117860859A CN 202410059579 A CN202410059579 A CN 202410059579A CN 117860859 A CN117860859 A CN 117860859A
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Abstract
The invention provides a traditional Chinese medicine composition for preventing and treating stroke, a preparation method and application thereof, belonging to the technical field of traditional Chinese medicine preparation, and the traditional Chinese medicine composition is prepared from the following raw materials: rhizoma Gastrodiae, radix Panacis Quinquefolii, notoginseng radix, saviae Miltiorrhizae radix, arillus longan, cortex Cinnamomi, curcumae rhizoma, radix Codonopsis, ramulus Uncariae cum Uncis, and Concha Haliotidis. The preparation method comprises the following steps: (1) mixing the raw materials to obtain a mixture; (2) Pulverizing the mixture into coarse powder, sterilizing, and further pulverizing into fine powder; and (3) preparing the pill, integrating the pill, drying and sub-packaging to obtain a finished product. The traditional Chinese medicine composition has the effects of invigorating qi, nourishing blood, activating yang, resolving hard mass, activating blood and removing obstruction in collaterals, and can be used for preventing and treating dementia, dizziness, insomnia, palpitation, chest stuffiness, tinnitus, apoplexy and the like caused by deficiency of qi and blood and accumulation of phlegm and blood stasis.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine preparation, in particular to a traditional Chinese medicine composition for preventing and treating stroke, a preparation method and application thereof.
Background
Stroke is also known as stroke. Stroke is a common name of traditional Chinese medicine for acute cerebrovascular diseases. It is a kind of brain blood circulation disorder disease with sudden syncope, unconsciousness, facial distortion, language handicap and hemiplegia as main symptoms. Because of the characteristics of high incidence rate, high mortality rate, high disability rate, high recurrence rate and many complications of stroke, the medical community has parallel coronary heart disease and cancer as one of three diseases threatening human health. The importance of preventing stroke has attracted attention from the medical community at home and abroad, and medical practitioners are exploring preventive measures for stroke from various aspects. Cerebral congestion refers to non-traumatic internal cerebral hemorrhage, which often forms intracerebral hematoma of different sizes, and sometimes breaks through cerebral parenchyma to form secondary cerebral indoor and/or subarachnoid hematocele. The traditional Chinese medicine composition mainly occurs in patients with hypertension and cerebral arteriosclerosis, and clinically is mainly characterized by the occurrence of stroke, such as headache, vomit, conscious disturbance, migraine, sensory disturbance and the like, also called hemorrhagic cerebral apoplexy, and is a common disease with higher mortality and disability rate.
Because of the rapid onset, the symptoms are multiple in ends, the disease changes rapidly, and the characteristics are similar to those of the wind, so the symptoms are named stroke and apoplexy. The disease often has sequelae and the onset age tends to be younger, so the disease is a serious disease threatening human life and quality of life.
Cerebral apoplexy is a common refractory disease seriously endangering human health and life safety, and is classified as the first four difficult diseases of wind, tuberculosis, tympanites and diaphragm by traditional Chinese medicine, and has the obvious phenomena of three high (high morbidity, high disability rate and high mortality rate). According to statistics, 200 ten thousand patients with cerebral apoplexy occur each year in China. The incidence rate is as high as 120/10 ten thousand. 700 thousands of stroke patients now survived, with 450 thousands of patients losing labor and life to varying degrees being unable to self-care. The disability rate is as high as 75%. Chinese stroke patients die by 120 ten thousand per year. Patients who have suffered from cerebral apoplexy can easily relapse, and once for each relapse, the patients are aggravated. Therefore, effective measures to prevent recurrence are more needed.
Cerebral apoplexy causes great threat to human health and life, brings great pain to patients and has heavy household and social burden. Therefore, how to develop a traditional Chinese medicine composition for preventing and treating stroke is a problem which needs to be solved by the person skilled in the art.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition for preventing and treating stroke, a preparation method and application thereof, and the traditional Chinese medicine composition has good effects of preventing and treating cerebral apoplexy and protecting nerve functions.
The technical scheme of the invention is realized as follows:
the invention provides a traditional Chinese medicine composition for preventing and treating stroke, which is prepared from the following raw materials: rhizoma Gastrodiae, radix Panacis Quinquefolii, notoginseng radix, saviae Miltiorrhizae radix, arillus longan, cortex Cinnamomi, curcumae rhizoma, radix Codonopsis, ramulus Uncariae cum Uncis, and Concha Haliotidis.
As a further improvement of the invention, the invention is prepared from the following raw materials in parts by weight: 5-10 parts of prepared gastrodia tuber, 5-10 parts of American ginseng, 2-5 parts of pseudo-ginseng, 2-5 parts of red sage root, 5-10 parts of longan pulp, 2-5 parts of cinnamon, 2-5 parts of curcuma zedoary, 5-10 parts of radix codonopsis pilosulae, 5-10 parts of uncaria, and 2-5 parts of sea-ear shell.
As a further improvement of the invention, the invention is prepared from the following raw materials in parts by weight: 6-8 parts of prepared gastrodia tuber, 6-8 parts of American ginseng, 3-4 parts of pseudo-ginseng, 3-4 parts of red sage root, 6-8 parts of longan pulp, 3-4 parts of cinnamon, 3-4 parts of curcuma zedoary, 6-8 parts of radix codonopsis pilosulae, 6-8 parts of uncaria, and 3-4 parts of sea-ear shell.
As a further improvement of the invention, the invention is prepared from the following raw materials in parts by weight: 6.666 parts of prepared gastrodia tuber, 6.666 parts of American ginseng, 3.334 parts of pseudo-ginseng, 3.334 parts of red sage root, 6.666 parts of longan pulp, 3.334 parts of cinnamon, 3.334 parts of zedoary, 6.666 parts of pilose asiabell root, 6.666 parts of uncaria and 3.334 parts of sea-ear shell.
The invention further provides a preparation method of a traditional Chinese medicine composition for preventing and treating apoplexy, which comprises the following steps:
(1) Mixing rhizoma Gastrodiae, radix Panacis Quinquefolii, notoginseng radix, saviae Miltiorrhizae radix, arillus longan, cortex Cinnamomi, curcumae rhizoma, radix Codonopsis, ramulus Uncariae cum Uncis, and Concha Haliotidis to obtain mixture;
(2) Pulverizing the mixture into coarse powder, sterilizing, and further pulverizing into fine powder;
(3) Making into pill in clean area, shaping, drying, and packaging to obtain the final product.
As a further development of the invention, the weight is 0.4-0.5g per 10g of the finished product.
As a further improvement of the invention, each gram of the finished product is equivalent to 0.8-1.2g of decoction pieces.
The invention further protects the application of the traditional Chinese medicine composition for preventing and treating stroke in preparing medicines for preventing and treating cerebral stroke.
The invention further protects the application of the traditional Chinese medicine composition for preventing and treating stroke in preparing medicines for protecting nerve functions.
The invention has the following beneficial effects:
monarch drug: in the recipe, the key drugs for treating dizziness and headache, which are the drugs for treating dizziness and headache, are prepared by tall gastrodia tuber, which calms liver yang, dispels external wind and unblocks channels and collaterals, are described in Ben Cao gang mu: the prepared gastrodia elata is a herb for treating wind, so the prepared gastrodia elata is a magic herb for treating wind; ramulus Uncariae cum Uncis has effects of calming endogenous wind and arresting convulsion, clearing heat and suppressing hyperactive liver, and can be used for treating dizziness, both of which are used as monarch drug.
Ministerial drugs: american ginseng and pilose asiabell root, radix codonopsis, have the effects of supplementing qi, nourishing yin and promoting the production of body fluid and are mainly used for treating deficiency of both qi and yin; the longan pulp has the effects of tonifying heart and spleen, nourishing blood and tranquillizing, and the three are ministerial drugs.
Adjuvant drug: notoginseng radix has effects of promoting blood circulation, removing blood stasis, stopping bleeding, removing blood stasis, and eliminating stagnation, and is recorded in the book of Yingzhong Shenxi Ji (Chinese and western medicine book): notoginseng radix is good at resolving blood stasis and stopping bleeding; rou Gui is good at warming channel and promoting blood circulation, improving microcirculation, both of which are adjuvant drugs.
The preparation method comprises the following steps: the salvia miltiorrhiza has the effects of promoting blood circulation, removing blood stasis, promoting tissue regeneration, clearing heart fire, cooling blood, relieving restlessness, and the same four things, is mainly used for treating the symptoms of blood stasis, orifice obstruction, dysphoria, palpitation and insomnia, the sea-ear shell has the effects of calming liver, suppressing yang, and the rhizoma curcumae has the effects of promoting qi circulation and breaking blood, and is compatible with the salvia miltiorrhiza to achieve the effects of calming and relieving arthralgia.
Efficacy: invigorating qi, nourishing blood, activating yang, resolving hard mass, promoting blood circulation, and dredging collaterals.
The main indications are: can be used for treating dementia, vertigo, insomnia, palpitation, thoracic obstruction, tinnitus, apoplexy, vascular dementia, atherosclerosis, heart failure, hypertension, hyperlipidemia, apoplexy sequelae, dementia, and apoplexy. Symptoms are chest distress, discomfort or chest distress, asthma, aggravated movement, short breath, debilitation or slow speech, dizziness, blurred vision, tinnitus, amnesia, insomnia, etc., dark purple face and lips, dark red tongue, or ecchymosis, and thready and unsmooth pulse.
The traditional Chinese medicine composition is mainly used for dementia, dizziness, insomnia, palpitation, chest stuffiness, tinnitus, apoplexy and the like caused by qi and blood deficiency and phlegm and blood stasis, and is used for vascular dementia, atherosclerosis, heart failure, hypertension, hyperlipidemia, apoplexy sequelae and the like caused by qi and blood deficiency and phlegm and blood stasis. Symptoms are chest distress, discomfort or chest distress, asthma, aggravated movement, short breath, debilitation or slow speech, dizziness, blurred vision, tinnitus, amnesia, insomnia, etc., dark purple face and lips, dark red tongue, or ecchymosis, and thready and unsmooth pulse. The prescription can obviously improve clinical symptoms, has no toxic or side effect, and has reasonable composition, simple preparation process, safe use, convenient clinical administration, convenient carrying and storage and better patient compliance. The medicines are combined to achieve the effects of invigorating the vital energy, internal memory and qi and blood circulation of a human body, and the effects of tonifying deficiency, promoting blood circulation, strengthening the body resistance without retaining blood stasis, removing blood stasis without hurting the vital energy, and achieving the effects of promoting blood circulation, removing blood stasis, dispelling wind, dredging collaterals, tonifying qi, nourishing blood, clearing heart and soothing nerves like the theory of qi and blood circulation of the body are disclosed in the general theory of Su Wen, sheng Qi, tong Tian Lun, that qi and blood flow, striae are in close …, and a long life is achieved.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a flow chart of a preparation method of a traditional Chinese medicine composition;
FIG. 2 is a thin layer chromatography identification chart of test example 1;
FIG. 3 is an optical photomicrograph of HE staining of brain and colon tissue from a rat of test example 8;
FIG. 4 is a photograph of a rat brain slice of test example 8.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The red sage root reference medicine (batch number: 120923-201615), the American ginseng reference medicine (batch number: 120997-201810) and the pseudo-ginseng total saponin reference substance (batch number: 110870-201904) are all purchased from Chinese food and drug verification institute.
Example 1 preparation method of a Chinese medicinal composition for preventing and treating Stroke
333.3g of prepared gastrodia tuber, 333.3g of American ginseng, 166.7g of pseudo-ginseng, 166.7g of red sage root, 166.7g of cinnamon, 333.3g of longan pulp, 333.3g of pilose asiabell root, 333.3g of uncaria, 166.7g of sea-ear shell, 166.7g of zedoary,
pulverizing the above materials into coarse powder, sterilizing, pulverizing into fine powder, sieving, mixing, adding appropriate amount of water, making into pill, and drying to obtain the final product. The product is a brown to deep brown watered pill, and has light smell and slightly bitter taste. The preparation flow is shown in figure 1.
Example 2 preparation method of Chinese medicinal composition for preventing and treating Stroke
666.6g of prepared gastrodia tuber, 666.6g of American ginseng, 333.4g of pseudo-ginseng, 333.4g of red sage root, 666.6g of longan pulp, 333.4g of cinnamon, 333.4g of zedoary, 666.6g of pilose asiabell root, 666.6g of uncaria and 333.4g of sea-ear shell.
Pulverizing the above materials into coarse powder, sterilizing, pulverizing into fine powder, sieving, mixing, adding appropriate amount of water, making into pill, and drying to obtain the final product. The preparation flow is shown in figure 1.
Example 3 preparation method of Chinese medicinal composition for preventing and treating Stroke
500g of prepared gastrodia tuber, 500g of American ginseng, 200g of pseudo-ginseng, 200g of red sage root, 500g of longan pulp, 200g of cinnamon, 200g of curcuma zedoary, 500g of pilose asiabell root, 500g of uncaria, and 200g of sea-ear shell.
Pulverizing the above materials into coarse powder, sterilizing, pulverizing into fine powder, sieving, mixing, adding appropriate amount of water, making into pill, and drying to obtain the final product. The preparation flow is shown in figure 1.
Example 4 preparation method of Chinese medicinal composition for preventing and treating Stroke
1000g of prepared gastrodia tuber, 1000g of American ginseng, 500g of pseudo-ginseng, 500g of red sage root, 1000g of longan pulp, 500g of cinnamon, 500g of curcuma zedoary, 1000g of pilose asiabell root, 1000g of uncaria and 500g of sea-ear shell.
Pulverizing the above materials into coarse powder, sterilizing, pulverizing into fine powder, sieving, mixing, adding appropriate amount of water, making into pill, and drying to obtain the final product. The preparation flow is shown in figure 1.
Test example 1:
10g of the Chinese medicinal composition prepared in example 1 was placed in a conical flask with a plug, 40ml of methanol was added, the mixture was sonicated for 30 minutes, the mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 20ml of water. Extracting with diethyl ether for 2 times (25 ml each time), removing diethyl ether solution, adding dilute hydrochloric acid into water solution to adjust pH to 2.5, extracting with diethyl ether for 1 time (25 ml each time), removing diethyl ether solution, extracting water solution with ethyl acetate for 2 times (25 ml each time), mixing ethyl acetate solutions, washing with 30ml water once, evaporating ethyl acetate solution to dryness, and dissolving residue with 1ml methanol to obtain test solution.
The red sage root lack negative sample solution is prepared by weighing the rest medicinal materials except red sage root according to the prescription proportion, preparing a negative preparation according to the preparation method, and adopting the preparation method of the sample solution.
Taking 1g of red sage root reference medicine, adding 40ml of methanol, and preparing a reference medicine solution by the same method.
According to thin layer chromatography (0502 of four general rules of Chinese pharmacopoeia 2020 edition), 5 μl of sample solution and 2 μl of control medicinal material solution are sucked and respectively spotted on the same silica gel G thin layer plate, and are spread with toluene-ethyl acetate-formic acid (volume ratio of 5:6:1) as developing agent, taken out, air dried, fumigated in an ammonia steam cylinder for 15 min, taken out, sprayed with 10% sulfuric acid ethanol test solution, heated at 105deg.C for 5 min, and detected under ultraviolet lamp 365 nm. The results are shown in fig. 2, wherein 1 is the test result of the ischemic son negative sample, 2-4 is the test result of the traditional Chinese medicine composition in different batches, and 5 is the test result of the red sage root reference medicinal material. From the figure, spots of the same color appear on the chromatogram of the sample at positions corresponding to those on the chromatogram of the control.
Test example 2: moisture determination
Three batches of the Chinese medicinal composition prepared in example 1 (batch numbers: 20220614, 20220616, 20220618) were examined according to the general rule 0832 "moisture assay" (pages 103 to 104) of the fourth edition of Chinese pharmacopoeia 2020, and the moisture content was not more than 9.0%. The results are all in accordance with the regulations, see Table 1.
Table 1 moisture test results table of chinese medicinal composition
Test example 3: weight differential determination
Three batches of the samples of the Chinese medicinal composition prepared in example 1 were examined according to the method of weight difference under the term "pills" by the rule 0108, four edition of Chinese pharmacopoeia 2020 (batch numbers: 20220614, 20220616, 20220618). Each batch of 10 pills is taken as 1 part, 10 parts are respectively taken, the weight is respectively weighed, and compared with each part of the marked weight, the weight difference limit is not more than 2 parts, and 1 part is not more than 1 time of the limit. (0.3 g or more to 1.5g, weight difference limit of.+ -. 9%) and the results are shown in Table 2, and the results are all in accordance with the regulations.
TABLE 2 weight differential results of Chinese medicinal compositions
Test example 4: measurement of the amount of load difference
Three samples of the Chinese medicinal composition prepared in example 1 were examined according to the method under the method of the four-part minimum filling inspection method (general rule 0942) of the Chinese pharmacopoeia 2020 edition (lot numbers: 20220614, 20220616, 20220618). The test specimen 10 bags were taken, the weight of the contents of each bag was weighed, and the amount of each bag was not more than 2 bags exceeding the limit of the difference in the amount of the contents compared with the indicated amount of the contents, and 1 bag exceeding the limit by 1 time (2 g or more to 3g, the limit of the difference in the contents being.+ -. 8%). The results were all in compliance. Details are shown in Table 3.
TABLE 3 results of the difference in the amount of the Chinese medicinal composition (labeled 3 g/bag)
Test example 5: determination of the dissolution time limit
Three samples of the Chinese medicinal composition prepared in example 1 were examined according to the method of dissolution time under the term "pill" of the four general rules 0108 in the edition of Chinese pharmacopoeia 2020 (lot numbers: 20220614, 20220616, 20220618). Taking 6 pills of each batch of test article, placing into a hanging basket, and operating according to the method of four 11 pages dissolution time limit inspection method of the Chinese pharmacopoeia 2020 edition, wherein the pills are required to be completely dissolved within 1 hour. The results are all in accordance with the specifications, see Table 4.
Table 4 Chinese medicinal composition dissolution time limit check table
Test example 6: alcohol soluble extract assay
According to the operation under the cold leaching method of the general rule 2201 in the fourth edition of Chinese pharmacopoeia 2020, the prescription Chinese herbal medicines mostly contain saponins, so that the contents of the alcohol-soluble extracts of 50% ethanol, 70% ethanol and 80% ethanol are respectively examined, and the results are shown in the following table 5.
TABLE 5 alcohol soluble extract content of ethanol at various concentrations
From the above results, it is found that the ethanol concentration is 50% and the ethanol-soluble extract content is the highest, so that 50% ethanol is preferable as the optimum concentration of the ethanol-soluble extract.
Three samples of the Chinese medicinal composition prepared in example 1 (lots: 20220614, 20220616, 20220618) were assayed for alcohol-soluble extract using 50% ethanol as a solvent. Taking about 4g of each batch of samples, precisely weighing, placing into a 250ml conical flask, precisely adding 100ml of water, sealing, cold soaking, shaking for 6 hours at any time, standing for 18 hours, rapidly filtering by a drying filter, precisely weighing 20ml of the continuous filtrate, placing into a evaporating dish dried to constant weight, evaporating to dryness on a water bath kettle, drying at 105 ℃ for 3 hours, taking out, cooling in a dryer for 30 minutes, and rapidly precisely weighing. The results are shown in Table 6.
Table 6 content of alcohol-soluble extract of Chinese medicinal composition
Test example 7: microbial limit determination
The traditional Chinese medicine composition is an oral administration solid preparation, and contains raw medicinal material powder of prepared gastrodia tuber, american ginseng, pseudo-ginseng, red sage root and the like. According to the route and prescription of the medicine, the total number of aerobic bacteria, the total number of mould and yeast and the control bacteria, namely Escherichia coli, salmonella and cholate-resistant gram-negative bacteria, should be detected. And (3) performing a counting method applicability test according to the related regulations of the microbiological limit inspection method of non-sterile products of the four-part general rules 1105 and 1106 of the edition of Chinese pharmacopoeia 2020, and establishing a microbiological limit inspection method. The test results are as follows:
1.1 Experimental strains
Staphylococcus aureus [ CMCC (B) 26003], pseudomonas aeruginosa [ CMCC (B) 10104], bacillus subtilis [ CMCC (B) 63501], candida albicans [ CMCC (F) 98001], aspergillus niger [ CMCC (F) 98003], escherichia coli [ CMCC (B) 44102], salmonella paratyphi B (B) 50094. The strain passaging times are not more than 5 generations.
1.2 sample
The Chinese medicinal composition prepared in example 1 (lot numbers: 20220614, 20220616, 20220618).
1.3 Medium and Diluent
Trypticase soy agar medium, trypticase soy liquid medium, saccharum sinensis Roxb dextrose agar medium, makka liquid medium, xylolysine deoxycholate agar medium, pH7.0 sterile sodium chloride-peptone buffer containing 0.05% (ml/ml) polysorbate 80, etc. were all purchased from Guangdong Cycloche microorganism technologies Co.
1.4 instruments
SPX-250B-III computer biochemical incubator, shanghai, medical instruments Inc.; MJ-250-II mould incubator, shanghai-Heng science instruments Co., ltd; YM75 vertical pressure steam sterilizer, medical equipment factory Shen An Shanghai city; an LDZM-80L-I vertical pressure steam sterilizer, a medical instrument factory in Shanghai city Shen An; BSC-1604 IIA 2 second level biological clean safe, air technologies Co., ltd; JJ200Y electronic balance, a well-established market double jetty test instrumentation factory; refrigerators, microwave ovens, and the like.
2. Test of applicability of the method for counting the total number of aerobic bacteria, mold and Yeast
2.1 preparation of bacterial liquid
Taking fresh cultures of staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis and escherichia coli, and preparing bacterial suspension with proper concentration by using a pH7.0 sterile sodium chloride-peptone buffer solution for later use; preparing candida albicans liquid culture into bacterial suspension with proper concentration by using sterile sodium chloride-peptone buffer solution with pH of 7.0 for later use; fresh cultures of Aspergillus niger are taken, 3-5 ml of sterile sodium chloride-peptone buffer pH7.0 containing 0.05% (ml/ml) polysorbate 80 are added, spores are eluted, then the spore suspension is sucked out into a sterile test tube by adopting a proper method, and the sterile sodium chloride-peptone buffer pH7.0 containing 0.05% (ml/ml) polysorbate 80 is used for preparing the Aspergillus niger spore suspension with proper concentration.
2.2 preparation of test solutions
Taking 10g of a sample, adding a pH7.0 sterile sodium chloride-peptone buffer solution to dilute to 100ml, and uniformly dispersing to prepare a 1:10 test solution; and respectively taking a proper amount of 1:10 test solution, and preparing the test solution of 1:50 by using a sterile sodium chloride-peptone buffer solution with pH of 7.0 for later use.
2.3 applicability test method
Test group: taking 9.9ml of test solutions with different dilution levels prepared in 2.2, placing the test solutions into a sterilizing test tube, respectively adding 0.1ml of bacterial solution of each test bacterium, and uniformly mixing to ensure that the bacterial content of each 1ml of test solution is not more than 100cfu. After mixing, 1ml of pouring dishes are taken per tube, 2 dishes are prepared in parallel, tryptone agar (total number of aerobic bacteria) or Saccharum sinensis Roxb glucose agar medium (total number of mould and saccharomycetes) is immediately poured, and after solidification, the bacteria are respectively placed at a specified temperature for 3 days and fungi are cultivated for 5 days.
Bacterial liquid control group: and (3) taking corresponding diluent to replace test liquid, adding test bacterial liquid according to the operation of a test group, and performing legal test.
Test article control group: and taking the prepared test solution, and replacing the bacterial solution with the diluent to operate with the test group.
The calculation formula is as follows: recovery ratio = (average colony count of test group-average colony count of test control group)/average colony count of bacterial liquid control group
2.4 Pre-test
Test solutions of different dilution levels prepared according to 2.2 were used, with staphylococcus aureus and candida albicans as sensitive strains, and the pre-test was performed according to 2.3, and the results are shown in tables 7-8.
Table 7 lot No.: 20220614 sample counting method applicability Pre-test results (1:10 test solution)
Table 8 lot number: 20220614 sample counting method applicability Pre-test results (1:50 test solution)
As a result, the total number of the aerobic bacteria is 1:10, 1:50 test solution is 1 ml/dish for a pre-test of bacteria adding recovery, and the recovery ratio of the staphylococcus aureus of the 1:10 and 1:50 test solutions meets the requirement that the four general rules 1105 of the Chinese pharmacopoeia 2020 edition should be in the range of 0.5-2. Taking 1 ml/dish of 1:10 and 1:50 test solution for adding bacteria and recovering pre-test, wherein the recovery ratio of candida albicans accords with the requirement that the four general rules 1105 of the year 2020 of Chinese pharmacopoeia in the range of 0.5-2.
Based on the pre-test results, the total number of aerobic bacteria, mold and yeast were determined using 1 ml/dish of 1:10 test solution.
2.5 test of applicability of the method for counting the total aerobic bacteria, the total mold and Yeast
3 samples of lot numbers (20220614, 20220616, 20220618) were taken and tested for suitability of the counting method by plate pouring as determined by pre-experiments, the results are shown in tables 9-14.
Table 9 20220614 batch test results-1) total aerobic count
Table 10 20220616 batch test results-1) total aerobic count
Table 11 20220618 batch test results-1) total aerobic count
Table 12 20220614 batch test results-2 Total mold and Yeast
Table 13 20220616 batch test results-2 Total mold and Yeast
Table 14 20220618 batch test results-2 Total mold and Yeast
As can be seen from the results of tables 9, 10 and 11, the ratio of the colony numbers of the three groups of Chinese medicinal compositions is within the range of 0.5-2 of the acceptable standard, which shows that the Chinese medicinal compositions have no inhibition effect on bacteria, and the total number of the aerobic bacteria can be determined by adopting a plate pouring method.
As can be seen from the results of tables 12, 13 and 14, the ratio of the colony numbers of the test groups of three Chinese medicinal compositions is within the range of 0.5-2 of the acceptable standard, which shows that the Chinese medicinal compositions have no inhibition effect on fungi, and the total number of the fungi and the yeasts can be measured by adopting a plate pouring method.
The method for determining the total number of aerobic bacteria, mould and yeast in the traditional Chinese medicine composition comprises the following steps: 10g of the Chinese medicinal composition is taken, and sterile sodium chloride-peptone buffer solution with pH of 7.0 is added to 100ml, so that the Chinese medicinal composition is uniformly dispersed to obtain a 1:10 test solution. 1 ml/dish of 1:10 sample solution (dish method-pouring method) was taken and examined according to law.
3 test for applicability of control fungus inspection method
3.1 establishment of method for examining Escherichia coli and suitability test
3.1.1 preparation of bacterial liquid
Fresh cultures of Escherichia coli were prepared with sterile sodium chloride-peptone buffer pH7.0 to prepare a bacterial suspension containing a bacterial count of not more than 100cfu per 1 ml.
3.1.2 preparation of test solutions
10g of a sample was diluted to 100ml with a sterile sodium chloride-peptone buffer pH7.0 to prepare a 1:10 sample solution having a uniform dissolution.
3.1.3 test methods for applicability test
Test group: 10ml of a 1:10 test solution is inoculated into 100ml of trypticase soy peptone liquid medium, and simultaneously, escherichia coli with a concentration of not more than 100cfu is added, and the mixture is uniformly mixed and cultured for 18 hours at 33 ℃.1ml of the culture was inoculated into 100ml of a Maiconkai liquid medium and cultured at 43℃for 24 hours. Streaking the culture of the MAIKAI on a MAIKAI agar medium plate, and culturing at 33 ℃ for 18 hours.
Test article group: 10ml of the 1:10 test solution was inoculated into 100ml of trypticase soytone liquid medium (TSB) and cultured at 33℃for 18 hours. 1ml of the culture is inoculated into 100ml of a Maiconk liquid culture medium, the culture is cultured for 24 hours at the temperature of 43 ℃, and the culture is streaked and inoculated on a Maiconk agar plate, and the culture is cultured for 18 hours at the temperature of 33 ℃ and is subjected to a legal test.
Positive control: coli not more than 100cfu was inoculated into Trypticase Soy Broth (TSB) and tested according to Law.
Negative control: the diluted solution is used for replacing the test liquid, and inoculated into trypticase soy peptone liquid culture medium (TSB) for legal test.
Three batches 20220614, 20220616 and 20220618 were subjected to the method suitability test, and the test results are shown in table 15.
TABLE 15 test results of E.coli method applicability
Note that: + represents colony growth; -representing sterile colony growth.
The test results of 3 batches of samples show that 10ml of the 1:10 test solution prepared by the method in 2.2 batches is inoculated into 100ml of trypticase soy peptone liquid medium (TSB) to carry out the applicability test of the escherichia coli inspection method, and meets the requirements.
3.2 establishment of the method for checking the bile salt-tolerant gram-negative bacteria and test for applicability
The escherichia coli and the pseudomonas aeruginosa are adopted as test bacteria, and the bacterial liquid preparation is the same as 2.1.
3.2.1 preparation of the test solution and Pre-culture
Taking a test sample, and taking a sterile sodium chloride-peptone buffer with pH7.0 as a diluent to check the microbial limit of a non-sterile product: microorganism count method (general rule 1105) "to prepare 1:10 test solution, mixing, culturing at 20-25deg.C for a period of time sufficient to recover bacteria but not proliferate (about 2 h).
3.2.2 quantitative test
Selection and isolation culture
1ml, 0.1ml and 0.01ml (corresponding to 0.1g, 0.01g and 0.001g of test sample) of 1:10 test solution are respectively inoculated into 100ml of intestinal bacteria enrichment liquid culture medium, and are cultured for 24-48 hours at the temperature of 30-35 ℃. Each culture is respectively streaked and inoculated on a red bile salt glucose agar culture medium plate, and is cultured for 18-24 hours at the temperature of 30-35 ℃.
And (3) judging results: if colony growth exists on the purple red bile salt glucose agar medium plate, the corresponding culture tube is positive, otherwise, the culture tube is negative. Based on the examination results of each culture tube, the number of possible bacteria containing cholate-resistant gram-negative bacteria in 1g of the sample was examined from Table 14.
TABLE 16 possible bacterial count of bile salt-tolerant gram-negative bacteria (N)
Note that: + represents colony growth on purple red bile salt glucose agar plates; representative of sterile colony growth on purple bile salt glucose agar plates.
TABLE 17 examination test results of bile salt-tolerant gram-negative bacteria
Note that: + represents colony growth on purple red bile salt glucose agar plates; representative of sterile colony growth on red bile salt glucose agar plates.
Results: three batches of test results of the test sample: 0.1g, 0.01g and 0.001g of the test sample were grown aseptically on purplish red bile salt dextrose agar plates, according to Table 14, the number of possible bacteria per g of test sample was < 10cfu.
3.3 test for applicability of the salmonella test method
(1) Test group: 10g of a sample is inoculated into 100ml of trypticase soyase liquid culture medium (TSB), salmonella paratyphi with the concentration not more than 100cfu is added, the mixture is cultured for 18 hours at the temperature of 33 ℃, 1ml of the culture is inoculated into 100ml of RV salmonella enrichment medium, the culture is cultured for 24 hours at the temperature of 33 ℃, the culture is streaked and inoculated on a xylose lysine deoxycholate agar plate, the culture is cultured for 18-48 hours at the temperature of 33 ℃, and the colony morphology of the strain is observed. The suspected colony is selected by an inoculating needle and inoculated on the inclined plane of the trisaccharide iron agar culture medium in an inclined plane. Culturing for 18-24h.
(2) Positive control group: salmonella paratyphi B with a concentration of not more than 100cfu was inoculated into 100ml of Trypticase Soy Broth (TSB) and tested according to Law.
(3) Negative control group: the diluted solution is used for replacing the test liquid, and inoculated into 100ml of trypticase soy peptone liquid culture medium (TSB) for legal test.
The detected bacteria were identified by biochemical methods and the experimental results are shown in table 18.
Table 18 examination test results of Salmonella paratyphi (amount of culture medium: 100 ml)
Note that: + represents colony growth; -representing sterile colony growth.
As a result, 10g of the sample was inoculated into 100ml of trypticase soyase liquid medium (TSB), and the added Salmonella typhimurium was examined by law.
According to the experimental results and the related rules of microbiological limit check method for non-sterile products of the four-part rule 1105, 1106 of the edition 2020 of Chinese pharmacopoeia, the standard text of microbiological limit check method for the traditional Chinese medicine composition is formulated as follows:
10g of the sample was diluted to 100ml with a pH7.0 sterile sodium chloride-peptone buffer solution, and mixed uniformly to prepare a 1:10 test solution.
The total number of aerobic bacteria is 1:10 of the sample solution, 1ml of the sample solution is poured into a dish, 2 dishes are prepared in parallel, and the measurement is carried out according to a dish pouring method.
The total amount of mould and saccharomycetes is 1ml of test solution 1:10 of the product, 2 plates are prepared in parallel, and the test solution is measured by a plate pouring method.
The escherichia coli is inoculated with 10ml of a test solution of 1:10 of the escherichia coli product into 100ml of trypticase soytone liquid culture medium, and then the escherichia coli is checked according to a law.
10ml of the sample solution of 1:10 of the cholate-resistant gram-negative bacteria is inoculated into 100ml of trypticase soytone liquid culture medium, and the sample solution is checked according to law.
10g of salmonella samples were inoculated into 100ml of trypticase soy broth and examined as usual.
The total number of aerobic bacteria in the product is less than 3×10 per 1g 4 cfu, total amount of mold and yeast is less than 10 per 1g 2 cfu, escherichia coli should not be detected per 1 g. Every 10g of salmonella should not be detected, and every 1g of cholate-resistant gram-negative bacteria should be less than 10 2 cfu。
Microbial limit inspection results of three batches of Chinese medicinal compositions
The three pilot-test batches were inspected according to the established microbiological limit inspection method for the traditional Chinese medicine composition, and the results all meet the regulations. The results are shown in Table 19.
Table 19 microbial limit check table for Chinese medicinal composition
Test example 8: pharmacodynamics test of Chinese medicinal composition
Animals: 90 clean-class male SD rats without specific pathogens, and the body mass is 160-200 g;
medicament: test example 1A Chinese medicinal composition
Grouping: dividing SD rats into a false operation group, a model group and a high, medium and low dosage group of traditional Chinese medicine composition according to a random digital table method, wherein 30 SD rats are in each group;
model group: the rats are fed with high-fat feed on day 1, the rats are subjected to load swimming (water temperature is 15-20 ℃) for 8 hours on day 2, fasted for 24 hours on day 3, repeated on day 4, repeated on day 1, repeated on day 5, repeated on day 2, repeated on day 6, and repeated on day 3, the rats are anesthetized by adopting a nylon bolt with one end being round after the rats are fasted for 12 hours on day 15, the rats enter the internal carotid artery cavity from the external carotid artery for 15-20 mm until resistance is felt to stop immediately, at the moment, the slipknot at the right carotid artery is tied, the nylon bolt is fixed in a blood vessel, and redundant thread heads and nylon bolts are cut off. The neck skin of the rat was sutured, and the rat was placed on a blanket after iodophor sterilization until consciousness was restored. The 1 st neural function scoring is carried out after the operation of the rat is naturally revived: score 0, no neurological symptoms; 1 minute, the injured contralateral forelimb cannot straighten; 2 minutes, rotating to the opposite side during crawling; 3 minutes, dumping or turning round to the opposite side of the operation when walking; 4 minutes, no voluntary action was taken. A score of 1-3 indicates successful modeling of Middle Cerebral Artery Occlusion (MCAO) in rats.
Group of sham operations: only the right common carotid artery, the external carotid artery, and the internal carotid artery were exposed. And (5) after disinfection and stitching.
The traditional Chinese medicine composition comprises the following components: rats successfully selected for composition were given to continuous lavage rats for 15 days, once to twice daily, with doses of 3.0g/Kg (equivalent to 8 times the clinically equivalent dose of the rats for adults, i.e., conversion of the effective dose), 6.0g/Kg,9.0g/Kg, and 16 days for specimen collection and treatment.
Effects of the Chinese medicinal composition on MCAO rat stroke (cerebral apoplexy): as shown in fig. 3, the results of the sham operation group were clearly and completely observed under an optical microscope, and the neuronal cells had no obvious lesions;
MCAO/R group neuronal nuclei shrink, large necrosis, visible as dark blue morphologically abnormal nikohlrabi; the necrosis of the neuron cells of the high-dose group is obviously improved, and the Neisseria with deep blue morphological abnormality is reduced.
The colon tissue of the false operation group is normal in morphology; MCAO/R group myolayer thinning, goblet cell increase, inflammatory cell infiltration; the myometrium is thinned, inflammatory cell infiltration is obviously improved, and goblet cells are reduced.
As shown in fig. 4, the rat brain slice is stained, the white color indicates the infarcted area, and the calculation results in that the infarcted volume of the model group is about 50%, and the infarcted volume of the high-dose group is significantly reduced by about 30%.
In conclusion, the high-dose group of the traditional Chinese medicine composition can improve the stroke (cerebral apoplexy) condition of the MCAO model rat, and plays a role in protecting the nerve function.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. A traditional Chinese medicine composition for preventing and treating stroke is characterized by being prepared from the following raw materials: rhizoma Gastrodiae, radix Panacis Quinquefolii, notoginseng radix, saviae Miltiorrhizae radix, arillus longan, cortex Cinnamomi, curcumae rhizoma, radix Codonopsis, ramulus Uncariae cum Uncis, and Concha Haliotidis.
2. The traditional Chinese medicine composition for preventing and treating stroke according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 5-10 parts of prepared gastrodia tuber, 5-10 parts of American ginseng, 2-5 parts of pseudo-ginseng, 2-5 parts of red sage root, 5-10 parts of longan pulp, 2-5 parts of cinnamon, 2-5 parts of curcuma zedoary, 5-10 parts of radix codonopsis pilosulae, 5-10 parts of uncaria, and 2-5 parts of sea-ear shell.
3. The traditional Chinese medicine composition for preventing and treating stroke according to claim 2, which is characterized by being prepared from the following raw materials in parts by weight: 6-8 parts of prepared gastrodia tuber, 6-8 parts of American ginseng, 3-4 parts of pseudo-ginseng, 3-4 parts of red sage root, 6-8 parts of longan pulp, 3-4 parts of cinnamon, 3-4 parts of curcuma zedoary, 6-8 parts of radix codonopsis pilosulae, 6-8 parts of uncaria, and 3-4 parts of sea-ear shell.
4. The traditional Chinese medicine composition for preventing and treating stroke according to claim 3, which is characterized by being prepared from the following raw materials in parts by weight: 6.666 parts of prepared gastrodia tuber, 6.666 parts of American ginseng, 3.334 parts of pseudo-ginseng, 3.334 parts of red sage root, 6.666 parts of longan pulp, 3.334 parts of cinnamon, 3.334 parts of zedoary, 6.666 parts of pilose asiabell root, 6.666 parts of uncaria and 3.334 parts of sea-ear shell.
5. A method for preparing a traditional Chinese medicine composition for preventing and treating stroke according to any one of claims 1 to 4, comprising the following steps:
(1) Mixing rhizoma Gastrodiae, radix Panacis Quinquefolii, notoginseng radix, saviae Miltiorrhizae radix, arillus longan, cortex Cinnamomi, curcumae rhizoma, radix Codonopsis, ramulus Uncariae cum Uncis, and Concha Haliotidis to obtain mixture;
(2) Pulverizing the mixture into coarse powder, sterilizing, and further pulverizing into fine powder;
(3) Making into pill in clean area, shaping, drying, and packaging to obtain the final product.
6. The method of claim 5, wherein each 10g of the final product weighs 0.4-0.5g.
7. The method according to claim 5, wherein each gram of the finished product corresponds to 0.8-1.2g of decoction pieces.
8. Use of a Chinese medicinal composition for preventing and treating stroke according to any one of claims 1 to 4 in the preparation of a medicament for preventing and treating stroke.
9. Use of a Chinese medicinal composition according to any one of claims 1-4 for the prevention and treatment of stroke in the preparation of a medicament for protecting neurological function.
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