CN112773852A - Preparation process of Chinese herbal medicine composition with cell protection effect - Google Patents
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Abstract
A preparation process of a Chinese herbal medicine composition with a cell protection effect comprises the following steps: collecting the whole herb of the Chinese violet in a harvesting period, removing impurities, cleaning, airing, completely drying in an oven at 50 ℃, crushing into coarse powder, adding water with the weight being 12 times of the weight of the Chinese violet, stirring for 0.5h, soaking for 1h, decocting for 0.5h with strong fire, decocting for 1.5h with slow fire, passing through sterile gauze, collecting decoction, adding water with the weight being 12 times of the weight of the Chinese violet, decocting for the second time by the same method, combining the decoction of the two times, adding white vinegar with half amount of the decoction, stirring uniformly, standing for precipitation, and standing overnight to obtain a supernatant. The invention is prepared into extract through direct squeezing, filtering, adding white vinegar, decompressing and concentrating, vacuum drying, which is easy to store, can reserve the effective components in the medicinal materials to the maximum extent, and the prepared Chinese medicinal white vinegar extract has the proliferation effect on vascular endothelial cells and immune macrophages, and has wide medicinal application and good safety.
Description
The technical field is as follows:
the invention belongs to the field of traditional Chinese medicinal material processing, and particularly relates to a preparation process of a traditional Chinese medicinal material Chinese violet and an edible white vinegar new composition with a cell protection effect.
Background art:
the herba Violae is dried whole plant of Viola yedoensis Makino of Violaceae (Violae) or Viola, and is also called Viola yedoensis Makino, Viola glabrata, Viola liaotu, etc. Viola yedoensis Makino is usually grown in humid places such as mountain wild grass slopes and fields, and is mainly produced in Jiangsu, Fujian, Yunnan and southern provinces of Yangtze river (city and autonomous region) in China. Viola yedoensis Makino can be harvested in spring, summer and autumn, dried in the sun or cut into pieces for use. The Chinese violet is taken as a medicine by dry whole herbs, has bitter, pungent and cold taste, enters heart and liver channels, is collected in the first part of 2015 edition of Chinese pharmacopoeia, has strong heat-clearing and detoxifying effects, is mainly used for sore yellow furuncle, conjunctival congestion, swelling and pain and the like, and can be used for treating venomous snake bite. Modern medical research finds that the main components of the Chinese violet comprise flavone and glycosides thereof, coumarin, organic acid, phenols, tannin, alkaloid, amino acid, protein or polypeptide, saccharides, steroids, volatile oil and the like. It has effects in inhibiting mycobacteria, Staphylococcus aureus, Streptococcus A, Streptococcus pneumoniae and dermatophytes. At present, herba violae is often used with dandelion, honeysuckle and wild chrysanthemum in clinic, such as Wuwei Xiaodu Yin; or the Chinese violet is processed and extracted to obtain a single component for treating a single disease, the operation technology is complex, the preparation time is long, the traditional Chinese medicine composition has many components, the cost is high, the Chinese violet cannot be widely applied, and a large amount of Chinese violet medicinal materials are needed in the preparation process, so that the waste is serious. Therefore, a preparation process which is simple and convenient to operate and can exert more effective components is urgently needed in clinic.
The white vinegar is an indispensable seasoning in daily life, has wide application in the aspect of medicine, but is mostly used as an auxiliary material to participate in the development of medicinal preparations, and is rarely used as a main medicinal component to play a role, so that the Chinese herbal medicine herba violae and the white vinegar are mainly mixed to obtain an extract, the method is simple and convenient, the storage is easy, and more medicinal values can be stimulated.
Endothelial cells or vascular endothelium are a thin layer of specialized epithelial cells, consisting of a layer of flattened cells, polygonal in shape, with jagged edges of the cells, which are interfitted with one another. It forms the inner wall of a blood vessel and is the interface of blood and other vessel walls (single-layered squamous epithelium) within the lumen of the vessel. Endothelial cells are the capillaries that run along the entire circulatory system, from the heart to the smallest. It is located between blood plasma and blood vessel tissue, and can not only complete the metabolic exchange of blood plasma and tissue fluid, but also synthesize and secrete various bioactive substances to ensure the normal contraction and relaxation of blood vessel, and has the special functions of maintaining blood vessel tension, regulating blood pressure, blood coagulation and anticoagulation balance, etc. so as to keep the normal flow of blood and the long-term unobstructed blood vessel. Therefore, the structural and functional integrity of endothelial cells plays an important role in vascular disease.
Macrophages belong to immune cells that are involved in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. Their primary functions are phagocytosis (i.e., phagocytosis and digestion) of cellular debris and pathogens in the form of fixed or free cells, and activation of lymphocytes or other immune cells in response to pathogens.
The invention content is as follows:
the invention provides a preparation process of a Chinese herbal medicine composition with a cell protection effect, namely a mixture of Chinese herbal medicines including Chinese violet and white vinegar. The process can furthest retain the active ingredients in the Chinese violet medicinal material, avoid the waste of the medicinal material in the processing process and the loss of the active ingredients and the reduction of the quality of the medicinal material caused in the storage and transportation processes, and can reduce the pollution of the medicinal material by mould and the like in the preparation and use processes.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a Chinese medicinal composition with cell protecting effect is prepared from herba Violae and white vinegar. The specific technical scheme is as follows:
(1) picking herba Violae whole plant, removing impurities, and air drying in dry and cool place for 2 days.
(2) Baking fresh herba Violae in an oven at 50 deg.C for 6 hr, turning, baking for 6 hr, taking out after 6 hr, breaking to obtain completely baked herba Violae, and baking for another time until completely baked herba Violae.
(3) Coarsely pulverizing the baked herbs, weighing 200g purified water 1000ml, soaking for 1h, mixing with purified water 1400ml, placing into a 3000ml flask, decocting with strong fire for 0.5h, and decocting with slow fire for 1.5 h.
(4) Filtering the obtained medicinal liquid with sterile gauze to obtain decoction, weighing 2400ml purified water, decocting for 2 hr with the same method, filtering decoction, mixing decoctions,
(5) adding white vinegar of half amount of the decoction, stirring, standing for precipitation, and standing overnight.
(6) And measuring the supernatant, putting the supernatant into a rotary evaporator, performing water extraction, and obtaining an average 80g of extract according to the standard that 1g of extract is equivalent to 2-5g of herbal medicines. Storing at-4 deg.C for use.
(7) The medicine acts on vascular endothelial cells and macrophages according to different concentrations, and the medicinal universality of the traditional Chinese medicine composition is verified.
Further, the reagent including Cell Counting Kit8(CCK-8) Cell proliferation assay Kit was supplied by APExBIO.
Further, the composition extract is prepared into different drug concentrations and is applied to human umbilical vein endothelial cells; and (5) verifying the safety of the medicament in mouse-derived abdominal cavity macrophages.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention cleans and washes the fresh Chinese violet, directly squeezes, filters, adds white vinegar, reduces pressure and concentrates, and dries in vacuum to obtain the extract, which is easy to store and can reserve the effective components in the Chinese violet to the utmost extent.
(2) The traditional Chinese medicine white vinegar extract obtained by the invention has proliferation effect on vascular endothelial cells and immune macrophages, wide medicinal application and good safety.
(3) The traditional Chinese medicine white vinegar extract obtained by the invention has wide protection effect on human-derived and mouse-derived cells, provides theoretical basis for traditional Chinese medicine preparation and clinical medication, and has potential practical value.
(4) The method is simple and easy to implement, low in cost and easy to accept and popularize by the masses.
Drawings
FIG. 1 shows the preparation process of the Chinese medicine herba Violae and white vinegar extract;
FIG. 2 is a comparison table of CCK-8 cell proliferation rates of different drug concentrations acting on human umbilical vein endothelial cells;
FIG. 3 is a table showing the comparison of the cell proliferation rates of CCK-8 cells when different concentrations of drugs are applied to mouse peritoneal macrophages.
The specific implementation mode is as follows:
the technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.
A Chinese medicinal composition with cell protecting effect is prepared from herba Violae and white vinegar. The specific technical scheme is as follows:
(1) picking herba Violae whole plant, removing impurities, and air drying in dry and cool place for 2 days.
(2) Baking fresh herba Violae in an oven at 50 deg.C for 6 hr, turning, baking for 6 hr, taking out after 6 hr, breaking to obtain completely baked herba Violae, and baking for another time until completely baked herba Violae.
(3) Coarsely pulverizing the baked herbs, weighing 200g purified water 1000ml, soaking for 1h, mixing with purified water 1400ml, placing into a 3000ml flask, decocting with strong fire for 0.5h, and decocting with slow fire for 1.5 h.
(4) Filtering the obtained medicinal liquid with sterile gauze to obtain decoction, weighing 2400ml purified water, decocting for 2 hr with the same method, filtering decoction, mixing decoctions,
(5) adding white vinegar of half amount of the decoction, stirring, standing for precipitation, and standing overnight.
(6) And measuring the supernatant, putting the supernatant into a rotary evaporator, performing water extraction, and obtaining an average 80g of extract according to the standard that 1g of extract is equivalent to 2-5g of herbal medicines. Storing at-4 deg.C for use.
(7) The medicine acts on vascular endothelial cells and macrophages according to different concentrations, and the medicinal universality of the traditional Chinese medicine composition is verified.
And putting the obtained supernatant into a rotary evaporator at 55 ℃, and concentrating to obtain an extract.
The traditional Chinese medicine white vinegar extract prepared by the invention verifies the proliferation effect on cells and further verifies the safety of the medicine.
1. Test object
Cell line
Human Umbilical Vein Endothelial Cells (HUVECs), mouse peritoneal macrophages (RAW264.7)
2. Instrument and experimental reagent
2.1 Main Instrument
Clean bench (suzhou, anta); CO22Incubators (Heraeus, germany); model 5415D micro bench centrifuge (Eppendorf, Germany); microplate reader Epoch (Bio Tek, USA).
2.2 Primary reagents
Fetal bovine serum, DMEM medium, RPMI 1640 medium (Gibco, Australia), streptomycin, trypsin digest, and CCK-8 cytotoxicity/proliferation assay kit (Ape × Bio, USA).
3 method
3.1 cell culture
Culturing HUVECs in DMEM medium containing 10% fetal calf serum at 37 deg.C under 5% CO2And (3) in the incubator, when the cell density reaches about 70%, carrying out cell passage, cryopreservation or cell collection for subsequent experiments.
Culturing RAW264.7 in RPMI 1640 culture medium containing 10% fetal calf serum, placing at 37 deg.C and 5% CO2And (3) in the incubator, when the cell density reaches about 70%, carrying out cell passage, cryopreservation or cell collection for subsequent experiments.
3.2 dilution of the liquid medicine
Diluting the extract to obtain 250mg/ml initial concentration medicinal solution, weighing 5g extract in 50ml centrifuge tube, adding hot water from high pressure steam kettle, oscillating, mixing the medicinal liquid, passing through 100 mesh nylon net, packaging the filtered liquid in 15ml centrifuge tube, rotating at 4000 rpm, and centrifuging for 10 min. Taking the supernatant in another 15ml centrifuge tube, placing the medicinal liquid into the cells, filtering with 0.22mm sterile filter to obtain sterile pure medicinal liquid, and storing at-4 deg.C.
The liquid medicine is diluted into different concentrations to intervene in vascular endothelial cells and immune macrophages, which show obvious proliferation capacity to cells.
3.3 CCK-8 detection of cell proliferation
Inoculating 2 kinds of cells into 2 96-well cell culture plates, culturing for 24 hr, discarding supernatant, adding cell culture solution containing different concentrations of medicinal liquid (0 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL) into the culture plates at a concentration of 100 μ l/well, setting 3 multiple wells at 37 deg.C, and 5% CO2After 24h of culture, 10 mul of CCK-8 reagent is added into each hole, the cells are continuously cultured for 2h at 37 ℃ and 5 percent CO2, and the absorbance value of each cell in each concentration is detected at 450nm of an enzyme-labeling instrument. Cell viability was calculated according to the formula: [ (drug addition well OD value) - (blank well OD value)]Div [ (control well OD value) - (blank well OD value)]×100％。
4 statistical methods
The experimental results of the research are all measured data, and are statistically analyzed by prism7.0 statistical software, and the results are shown in the specification
The expression that the comparison between two groups adopts t test, the comparison between multiple sample averages adopts One-way ANOVA test, the comparison between two groups adopts Student-Newman-Keuls test, and P less than 0.05 shows that the difference has statistical significance.
5 results
5.1 Process for preparing herba Violae and white vinegar extract
5.2 extract under preparation process
5.3 CCK-8 detection of human umbilical vein endothelial cell proliferation rate
The drug concentration is 0 mug/ml, 10 mug/ml, 50 mug/ml, 100 mug/ml, 250 mug/ml and 500 mug/ml, the cell survival rate is continuously increased along with the increase of the drug concentration, the cell survival rate reaches one hundred percent at the concentration of 250 mug/ml, and the drug composition is proved to have stronger endothelial cell proliferation capacity.
5.4 CCK-8 detection of mouse peritoneal macrophage proliferation rate
The medicine concentration is 0 mug/ml, 10 mug/ml, 50 mug/ml, 100 mug/ml, 250 mug/ml and 500 mug/ml, the cell survival rate is continuously increased along with the increase of the medicine concentration, the cell survival rate reaches one hundred percent at the concentration of 100 mug/ml, and the medicine composition is proved to have stronger proliferation capacity on endothelial cells.
Conclusion 6
The Chinese herbal medicine herba violae and the white vinegar extract obtained by the specific operation method have obvious proliferation effects in endothelial cells and macrophages, have good medicine safety and wide application, provide a new medicine direction for endothelial cell related vascular diseases and macrophage related immune diseases, and provide a new direction for preventing and treating vascular diseases and immune related diseases.
The above description is only a preferred and specific implementation of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art can easily change the technical solution or substitute the same within the technical scope of the present invention.
Claims (4)
1. A preparation process of a Chinese herbal medicine composition with a cell protection effect is characterized in that a medicinal extract of a Chinese herbal medicine herba violae and white vinegar composition is prepared, and the specific preparation method comprises the following steps:
(1) picking whole herb of Chinese violet, removing impurities, and airing in a dry and cool place for 2 days;
(2) baking fresh herba Violae in an oven at 50 deg.C for 6 hr, turning, baking for 6 hr, taking out after 6 hr, breaking when it is broken, baking completely, and baking if it still has shape, and observing once per hour until baking completely;
(3) coarsely pulverizing the baked herbs, weighing 200g of purified water with the amount of 1000ml, soaking for 1h, then uniformly mixing the purified water with the amount of 1400ml and the soaked herbs, putting into a flask with the amount of 3000ml, decocting for 0.5h with strong fire, and decocting for 1.5h with slow fire;
(4) filtering the obtained medicinal liquid with sterile gauze to obtain water decoction, weighing 2400ml purified water, decocting for 2 hr with the same method, filtering decoction, and mixing decoctions;
(5) adding white vinegar of half amount of the decoction, stirring, standing for precipitation, and standing overnight;
(6) and measuring the supernatant, putting the supernatant into a rotary evaporator, performing water extraction, and obtaining an average 80g of extract according to the standard that 1g of extract is equivalent to 2-5g of herbal medicines. Storing at-4 deg.C for use;
(7) the medicine acts on vascular endothelial cells and macrophages according to different concentrations, and the medicinal universality of the traditional Chinese medicine composition is verified.
2. The process for preparing a Chinese herbal medicine composition with cell protection effect as claimed in claim 1, wherein the supernatant is concentrated to obtain an extract by putting the supernatant into a rotary evaporator at 55 ℃.
3. The process for preparing a Chinese herbal medicine composition with cell protection effect as claimed in claim 1, wherein the extract is diluted into a drug solution with an initial concentration of 250mg/ml, i.e. 5g of the extract is weighed into a 50ml centrifuge tube, hot water taken out from a high pressure steam cooker is added, the mixture is shaken and fully mixed, the mixture is passed through a nylon net of 100 meshes, the filtered liquid is subpackaged into 15ml centrifuge tubes, and the centrifugation is carried out at 4000 rpm for 10 minutes. Taking the supernatant in another 15ml centrifuge tube, placing the medicinal liquid into the cells, filtering with 0.22mm sterile filter to obtain sterile pure medicinal liquid, and storing at-4 deg.C.
4. The preparation process of Chinese herbal medicine composition with cell protecting effect as set forth in claim 1, wherein the medicine liquid is diluted into different concentrations for intervening vascular endothelial cells and immune macrophages, and has obvious cell proliferating capacity.
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2021
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Application publication date: 20210511 |