CN108324749B - Traditional Chinese medicine composition for treating acne and preparation method and application thereof - Google Patents
Traditional Chinese medicine composition for treating acne and preparation method and application thereof Download PDFInfo
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- CN108324749B CN108324749B CN201810247026.4A CN201810247026A CN108324749B CN 108324749 B CN108324749 B CN 108324749B CN 201810247026 A CN201810247026 A CN 201810247026A CN 108324749 B CN108324749 B CN 108324749B
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Abstract
The invention discloses a traditional Chinese medicine composition for treating acne and a preparation method and application thereof. The traditional Chinese medicine composition provided by the invention comprises the following raw materials in parts by weight: 200-600 parts of honeysuckle, 100-300 parts of chrysanthemum, 100-300 parts of dandelion and 50-150 parts of liquorice. Furthermore, the invention also provides a preparation method of the traditional Chinese medicine composition. The invention firstly adopts the treatment mode of combining the microbial degradation method with the rapid leaching method to treat the medicinal materials, shortens the treatment time, reduces the heavy metal content and solves the problem of pesticide residue. In addition, the medicinal materials are treated and processed by a microwave enzyme deactivation method, so that the activity of enzyme and the content of chlorogenic acid are improved. Experiments prove that the traditional Chinese medicine composition can fundamentally treat acne, has few raw material varieties, simple preparation process and controllable quality, and has the effects of clearing away heat and toxic materials, dispelling wind heat, and relieving spasm and pain under the synergistic effect of the raw material medicines. The invention provides a convenient and effective treatment method for treating acne.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, a preparation method and application thereof, in particular to a traditional Chinese medicine composition for treating acne, a preparation method and application thereof, and belongs to the field of natural medicines or health-care foods.
Background
Acne is a chronic inflammatory skin disease of a hair follicle sebaceous gland unit, and is commonly called as acne, lung wind comedo, acne and the like. It usually occurs in seborrheic areas of the face, chest and back, manifested as acne, blackhead, inflammatory papules, nodules, cysts, scars, and the like, and is also seen in adolescence, both in children and adults. Some teenagers suffer from the acne in different degrees, and serious acne patients can leave depressed scars and keloids in the later stage of adolescence, and can also cause secondary infection except the influence on the appearance.
Foreign research shows that the incidence rate of acne in 12-24 years old teenagers is as high as 85%. Therefore, the research on a new medicine for treating acne is an urgent need of the majority of acne patients. Acne is treated by a plurality of methods, and physical therapy: electrotherapy, phototherapy, microwave, laser, etc., but the physical therapy treatment costs expensive medicine and has complex process, and most importantly, the treatment is temporary and permanent. A plurality of medicines for treating acne are marketed at home and abroad, and mainly comprise two dosage forms of oral administration and external application. The acne oral treatment medicine has the advantages of good curative effect and almost no stimulation to skin, the medicines for treating acne by oral administration which are clinically applied at present mainly comprise antibacterial medicines, medicines for inhibiting follicular sebaceous gland duct parakeratosis, antiandrogenic medicines, anti-inflammatory medicines, antiseborrheic medicines, Chinese herbal medicines and the like, wherein western medicines are not thoroughly cured, a plurality of adverse reactions exist, and the medicines are dependent and easy to relapse and cannot be fundamentally cured.
Chinese patent application with the application number of CN00114872.9 discloses a compound acne tea and a preparation method thereof, wherein the compound acne tea comprises the following raw material components: 10-30 parts of coix seeds, 10-30 parts of phaseolus calcaratus, 10-30 parts of fried black beans, 5-15 parts of angelica dahurica, 10-30 parts of dandelion, 5-15 parts of ginkgo seeds, 10-30 parts of mulberry leaves, 5-15 parts of oldenlandia diffusa, 5-15 parts of calendula officinalis, 5-15 parts of beauveria bassiana, 5-15 parts of kudzu roots and 5-15 parts of gardenia. The treatment method comprises the following steps: cleaning and sterilizing the raw materials respectively or mixing the raw materials, then sterilizing, and finally decocting the sterilized raw materials in water and filtering to obtain the acne-removing tea. The disadvantages are that: 1) the content and activity of the active ingredients of the medicinal materials are reduced due to cleaning and sterilization. 2) The method is convenient and quick to take, but the curative effect is still discounted compared with the drinking mode in the prior art, and the effect is worse when the medicine is stored for a longer time, especially the medicine for relieving exterior syndrome and regulating the body condition. 3. The raw materials of the prescription are various, the final liquid product is obtained through a series of procedures of cleaning, sterilizing, disinfecting, water decocting and filtering, the process is complex, the operation is complicated, and the production and transportation cost is high.
Patent application No. CN201610703243.0 discloses a medicine for treating acne, which is composed of the following raw material components: 6-10 parts of coptis chinensis, 8-10 parts of scutellaria baicalensis, 12-18 parts of phellodendron amurense, 8-12 parts of loquat leaves, 8-12 parts of mulberry leaves, 8-12 parts of herba violae, 12-18 parts of wild chrysanthemum flowers, 12-18 parts of honeysuckle flowers, 6-10 parts of folium isatidis, 8-12 parts of dandelion, 12-18 parts of andrographis paniculata, 8-12 parts of cortex moutan, 6-10 parts of red paeony roots, 8-10 parts of fructus forsythiae and 5-7 parts of liquorice. The preparation method comprises the following steps: weighing the raw materials according to the weight ratio, crushing all the raw materials into coarse powder, mixing the coarse powder and the coarse powder together, placing the mixture in a container, adding ethanol with the concentration of 45 percent which is 5 times of that of the raw materials, soaking the mixture for 6 days, filtering and removing slag, and decompressing and recovering the ethanol from the filtrate until no ethanol smell exists, thus obtaining concentrated solution. Adding water and potassium sorbate into the concentrated solution, stirring, standing, and filtering to obtain filtrate; boiling sucrose in water for 35-45min, and filtering; mixing the above medicinal liquids, placing in a container, adding water, cooling, bottling, sealing, and sterilizing to obtain the final product. The disadvantages are that: 1) various raw materials, complex process, difficult operation and high production cost. 2) The medicine is added with antiseptic potassium sorbate.
The natural pure plant Chinese herbal medicines such as honeysuckle, chrysanthemum and the like have rich experience in treating the disease, have definite curative effect and high safety, and are widely applied to clinic. The traditional Chinese medicine considers that the acne is caused by yin deficiency and internal heat, flaring up of deficient fire or fumigation of skin by damp-heat and internal toxin. Due to improper diet, overeating the food with a fat, sweet and thick flavor, generating damp-heat accumulation, and steaming damp-heat on the face along with yangming meridians to cause acne; excessive yang heat, gradually-hot nutritive blood, and blood heat accumulating externally, and blood flowing with heat accumulating in the chest and face, which are the cause of the disease. The invention researches the pathogenesis of the acne on the basis of treatment based on syndrome differentiation, belongs to the origin, fundamentally treats the acne, purges fire for detoxification, clears heat and dries dampness, and selects a Chinese medicinal composition for treating the acne according to theoretical research of modern pharmacological effects and actual effect research of each raw material, wherein the Chinese medicinal composition consists of honeysuckle, chrysanthemum, dandelion and liquorice. The four pure natural medicinal materials are selected, pretreated by combining a microbial degradation method and a rapid leaching method, subjected to microwave enzyme deactivation, dried, crushed, mixed and packaged to prepare the traditional Chinese medicine composition, so that the heavy metal content and the pesticide residue are reduced, the activity of enzyme and the content of chlorogenic acid are improved, and the traditional Chinese medicine composition is a novel safe pure natural medicine for treating acne. Has the functions of purging fire, detoxifying, clearing heat and drying dampness, and can achieve good effect of treating acne.
Disclosure of Invention
The invention aims to solve the technical problem of providing a traditional Chinese medicine composition for treating and preventing acne and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a traditional Chinese medicine composition for treating acne, which comprises the following raw materials in parts by weight: 200-600 parts of honeysuckle, 100-300 parts of chrysanthemum, 100-300 parts of dandelion and 50-150 parts of liquorice.
The traditional Chinese medicine composition is characterized by comprising the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice.
Wherein, the pharmacology and the efficacy of the raw material medicines are respectively as follows:
honeysuckle (also known as honeysuckle, honeysuckle flower, etc.) is a dried bud or a flower that is just bloomed of Lonicera japonica Thunb. Collected before blooming in summer and dried. Researches show that the types of chemical components separated and identified from honeysuckle are more, the chemical components mainly comprise volatile oil, organic acids, flavonoids, triterpenoid saponins, iridoids and the like, and the honeysuckle extract and the chemical components contained in the honeysuckle extract have various pharmacological activities, including anti-inflammatory activity, antibacterial activity, antiviral activity, antioxidant activity, liver protection activity, anti-tumor activity and the like. The honeysuckle has strong broad-spectrum antibacterial action, and modern pharmacological and clinical researches show that the honeysuckle has strong antibacterial action and good treatment effect on various germs. Qing dynasty xylol, Zhong zhu she li yuan (source of meridian), jin Yin Hua dominates diarrhea and pus blood, and is the essential herb for swelling inside and outside. Herbs that are Sheng Yao after abscess, deep-rooted carbuncle and ulceration, for removing toxicity and pus, tonics in purgation.
The dandelion is an important common clinical Chinese herbal medicine, and the dandelion is considered by the traditional Chinese medicine to have the effects of clearing heat and removing toxicity, relieving swelling and resolving masses, and inducing diuresis for treating stranguria. The 2010 version of the Chinese pharmacopoeia records that dandelion is a dried whole herb of dandelion (Taraxacum mongolicum hand. -Mazz.) which is a Compositae (Compositae), Taraxacum sinicum Kitag @, and various plants of the same genus. It is mainly used for treating acute mastitis, scrofula, furuncle, sore, pharyngalgia, pulmonary abscess, intestinal abscess, conjunctival congestion, jaundice due to damp-heat pathogen, stranguria with damp-heat pathogen, etc. Pharmacological tests prove that the traditional Chinese medicine dandelion has the functions of broad-spectrum antibiosis, liver protection, cholagogue, endotoxin resistance, stomach strengthening and immunity promotion.
The flos Chrysanthemi is dried flower of Chrysanthemum morifolium Ramat of Compositae, and is a rare ornamental flower. The chrysanthemum is mainly used for treating wind-heat type common cold, headache, dizziness, conjunctival congestion, swelling and pain, dim eyesight, sore and carbuncle and pyogenic infections clinically. Flos Chrysanthemi is used as main ingredient in various Chinese medicinal preparations, such as SANGJUYIN granule, QIJUDIHUANG pill, and JUHUACHATIAO powder; the chrysanthemum can also be made into a large number of health care products for people to use, such as chrysanthemum medical pillow, chrysanthemum mattress and the like. Chrysanthemum is various in variety, and 8 main stream commodities of chrysanthemum morifolium, chrysanthemum tribute, chrysanthemum morifolium, chrysanthemum keemun, chrysanthemum morifolium and chrysanthemum morifolium exist in the medicinal material market. Is also a medicinal and edible plant. The existing research shows that the chrysanthemum mainly contains chemical components such as flavone, terpenoid, organic acid and the like. Besides the main components, the chrysanthemum also contains a small amount of anthraquinone, fatty acid, fatty alcohol, trace elements and other substances. The traditional Chinese medicine theory considers that the chrysanthemum has the effects of clearing away heat and toxic materials, dispelling wind and heat, calming the liver and improving eyesight. Modern pharmacological research mainly focuses on the aspects of cardiovascular protection, tumor resistance, blood fat regulation and the like, and the protection effect and the pain relieving effect on the liver are newly discovered in recent years.
Licorice (Radix Glycyrrhizae) is a tonifying Chinese herbal medicine. Is derived from dry tough root and rhizome of Glycyrrhiza uralensis G.uralensis Fisch, Glycyrrhiza inflata G.in float Bat, or Glycyrrhiza glabra G.glabra l. Licorice root, radix Glycyrrhizae is neutral in nature and sweet in flavor, entering twelve meridians. Has the pharmacological actions of detoxification, phlegm elimination, pain relief, spasmolysis and cancer resistance. The liquorice can tonify spleen and qi, relieve cough and moisten lung, relieve urgency and detoxify and harmonize a plurality of medicines. Clinical applications are divided into unprocessed and honey-processed. Unprocessed is used for treating swollen and sore throat, carbuncle, deep-rooted carbuncle, pyocutaneous disease, gastrointestinal ulcer, drug toxicity, food poisoning and the like. For abscess, deep-rooted carbuncle, sores and ulcers, it is combined with jin Yin Hua and Lian Qiao to achieve the actions of clearing heat and removing toxicity, such as Xian Fang Huo Ming Yin. Mainly produced in inner Mongolia, Gansu and Xinjiang provinces of China. Chinese licorice is one of the commodities which are long-standing reputed in the international licorice market, is widely applied to flavoring agents and excipients in the pharmaceutical and food industries, and has the effects of tonifying spleen and qi, clearing heat and removing toxicity, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the drugs. According to traditional Chinese medicine, liquorice can enter qi along with qi and blood, and cannot enter blood along with blood, so that the liquorice is called as 'the old age'. By virtue of its sweet and slow property, it can make the drug potency slow and durable, pass through twelve meridians, rise and fall, and cooperate with tonics, purgatives, cold, heat, warm and cool drugs to regulate the various drugs well, so that all the drugs can be harmonious without the disadvantage of conflict, so it is said to have the effect of 'regulating all the drugs'.
The invention researches the pathogenesis of the acne on the basis of treatment based on syndrome differentiation, belongs to the origin, fundamentally treats the acne, purges fire for detoxification, clears heat and dries dampness, and selects four pure natural medicinal materials of honeysuckle, chrysanthemum, dandelion and liquorice to prepare a novel safe traditional Chinese medicine composition according to theoretical research of modern pharmacological efficacy and actual efficacy research of each raw material.
Further, the invention also provides a method for preparing the traditional Chinese medicine composition, which comprises the following steps:
(1) sorting: manually selecting impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts from honeysuckle, chrysanthemum, dandelion and liquorice;
(2) pretreating the raw material by microbial degradation
1) Preparing a seed culture solution:
respectively inoculating pseudomonas aeruginosa (P.Aeruginosa), Bacillus subtilis (Bacillus subtilis), Clostridium butyricum (Clostridium butyricum) and Escherichia coli (Escherichia coli) into a liquid culture medium, and culturing to respectively obtain seed culture solutions of the pseudomonas aeruginosa, the Bacillus subtilis, the Clostridium butyricum and the Escherichia coli;
2) preparing a compound microbial preparation:
mixing the prepared seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli according to the following volume percentages: 30% of a pseudomonas aeruginosa seed culture solution, 30% of a bacillus subtilis seed culture solution, 20% of a clostridium butyricum seed culture solution and 20% of an escherichia coli seed culture solution, mixing, inoculating the mixed bacteria solution into a liquid culture medium under an aseptic condition according to an inoculation amount of 2-10% (v/v), and culturing to obtain a composite microbial preparation;
3) pretreatment:
respectively adding the composite microbial preparation prepared in the step 2) into the screened medicinal material raw materials, soaking for 30 minutes, and quickly washing for later use;
(3) microwave enzyme deactivation and drying:
placing the honeysuckle, the chrysanthemum, the dandelion and the liquorice processed in the step 3) into a microwave oven respectively, carrying out microwave enzyme deactivation for 10-20 minutes, then taking out, heating at 65-100 ℃ for 5-10 minutes, suspending, rapidly turning over for 1 time, continuing to heat for 3-5 minutes, and taking out for later use;
(4) crushing and sieving:
respectively crushing the medicinal materials treated in the step (3) and sieving the medicinal materials with a 20-mesh sieve;
(5) mixing and subpackaging:
the crushed medicinal materials are weighed according to the weight parts of the invention, mixed evenly and subpackaged to obtain the traditional Chinese medicine.
Among them, preferably, the composition of the liquid culture medium for culturing pseudomonas aeruginosa in the step 2) is as follows: 4.0g of beef extract, 10.5g of peptone, 5.0g of NaCl, 20.0g of agar, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h;
the composition of the liquid medium for the cultivation of Bacillus subtilis was: 4.0g of beef extract, 10.1g of peptone, 5.0g of glucose, 5g of ammonium sulfate, 2.0g of calcium carbonate, 1.0L of distilled water, pH 7.0, fermentation temperature of 26-30 ℃ and culture time of 36-48 h;
the composition of the liquid medium for the culture of clostridium butyricum is as follows: 5.0g of beef extract, 10.0g of glucose, 6.0g of peptone, 3g of yeast powder, 5.0g of NaCl, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h;
the composition of the liquid culture medium for culturing the Escherichia coli is as follows: 4.0g of beef extract, 10.5g of peptone, 5.0g of glucose, 5.0g of yeast powder, 5.0g of NaCl, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h.
Preferably, in step 2), the liquid medium for culturing the mixed bacterial liquid comprises the following components: 5.0g of beef extract, 10g of peptone, 5.0g of glucose, 5g of ammonium sulfate, 2.0g of calcium carbonate, 1.0L of distilled water, pH 7.0, fermentation temperature of 26-32 ℃, and culture time of 5-8 days, wherein the pH of the culture medium is reduced to 3-5, and the compound microbial preparation is obtained.
Among them, preferred is the case of step 3). The rapid leaching is to put the medicinal materials into a high-pressure spray cleaning machine and rapidly leach the raw materials of the medicinal materials once by using clear water.
Furthermore, the invention also provides application of the traditional Chinese medicine composition 2 in preparing medicines or health-care foods for treating or preventing acne.
Wherein, preferably, the traditional Chinese medicine composition is a health food.
Compared with the prior art, the invention has the beneficial effects that:
1. the traditional Chinese medicine composition disclosed by the invention can be used for fundamentally treating acne, the raw materials are few in variety, the preparation process is simple, the quality is controllable, and the raw materials have the effects of clearing away heat and toxic materials, dispelling wind heat and relieving spasm and pain under the synergistic effect.
2. The invention adopts a treatment mode combining a rapid leaching method after treating the medicinal materials by a microbial degradation method for the first time, shortens the treatment time, reduces the content of heavy metal and solves the problem of pesticide residue.
3. The microwave enzyme deactivation method is adopted to treat and process the medicinal materials, so that the activity of enzyme and the content of chlorogenic acid are improved.
The enzyme deactivation means that the oxidase activity in the fresh leaves is destroyed and passivated by high temperature or other methods, the enzymatic oxidation of tea polyphenol and the like in the fresh leaves is inhibited, the color change in the drying process is prevented, and the formation of good aroma is promoted. Aims to destroy the activity of polyphenol oxidase by using high temperature, inhibit the oxidative red change of polyphenol oxidase and fix the quality of fresh leaves. When the water is removed, the low boiling point aromatic substances are volatilized along with the evaporation of the water, and the faint scent is revealed. The microwave enzyme deactivation has the characteristics of high temperature rising speed, uniform temperature, high thermal efficiency, short enzyme deactivation time, high enzyme deactivation quality and the like. Has no pollution to the environment. Deactivating enzymes contained in fresh flowers by enzyme deactivation, and drying to obtain high-quality flos Lonicerae and flos Chrysanthemi. Honeysuckle contains enzymes such as polyphenol oxidase, and the purpose of enzyme deactivation is to rapidly raise the internal temperature of honeysuckle by rapid temperature rise, and to pass through a stage with strong enzyme activity in a short time to reach the critical temperature of enzyme deactivation, so that polyphenol oxidase basically loses activity at 85 ℃. Therefore, the chlorogenic acid content is high. The 4 raw materials with the same mesh number are accurately weighed according to a certain proportion to be 2.0g, so that the defects of decoction and extraction of the traditional Chinese medicine in the prior art are overcome, and meanwhile, the raw materials are treated by the quick leaching method and the microwave enzyme deactivation method, so that the unique and pure natural taste is kept, and the praise of vast testers is obtained.
4. The raw materials of the traditional Chinese medicine composition disclosed by the invention all belong to a list of traditional Chinese medicines published by the national ministry of health, which are both food and medicinal materials, the traditional Chinese medicine composition is safe and non-toxic, the product is not added with any pigment, spice and synthetic preservative, in order to overcome the defect that the traditional Chinese medicine is decocted and has poor taste, the content of chlorogenic acid is increased on the basis of reducing the heavy metal content of honeysuckle, and meanwhile, the unique fragrance of honeysuckle and dandelion, the faint scent of chrysanthemum and the sweet taste of liquorice are also kept.
5. The method of the invention is easy to operate, saves time and reduces production cost; the medicine is convenient to take, and is a treatment mode which is easy to accept by people.
Detailed Description
The invention is further described below by experiments with specific examples. The advantages and features of the present invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) And (3) sterilization and drying: placing the medicinal materials in a drying oven, drying at 60 deg.C for 24 hr, and taking out for use.
(3) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(4) Mixing: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, and the crushed medicinal materials are weighed and mixed uniformly.
(4) And (6) subpackaging.
EXAMPLE 2 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) And (3) sterilization and drying: respectively putting the honeysuckle, the chrysanthemum, the dandelion and the liquorice into a high-pressure spray cleaning machine, quickly rinsing the medicinal raw materials with clear water for one time, respectively putting the 4 medicinal raw materials into a microwave oven, carrying out microwave enzyme deactivation for 10 minutes, heating at 85 ℃ for 5 minutes, pausing, rapidly turning for 1 time, continuing to heat for 5 minutes, and taking out for later use.
(3) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(4) Mixing: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, and the crushed medicinal materials are weighed and mixed uniformly.
(5) And (6) subpackaging.
EXAMPLE 3 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreating the raw material by microbial degradation
1) Preparing a seed culture solution:
preparation of a culture solution of pseudomonas aeruginosa (p.aeroginosa) cmcc (b)10104 seeds: inoculating pseudomonas aeruginosa in a liquid culture medium under the aseptic condition, wherein the culture medium comprises the following components: beef extract 4.0g, peptone 10.5g, NaCl 5.0g, agar 20.0g, distilled water 1.0L, pH 7.0, fermentation temperature: culturing at 28-32 deg.C for 48h to obtain culture solution of Pseudomonas aeruginosa seed.
Preparation of seed culture solution of Bacillus subtilis CMCC (B) 6350: inoculating bacillus subtilis into a liquid culture medium under an aseptic condition, wherein the culture medium comprises the following components: 4.0g of beef extract, 10.1g of peptone, 5.0g of glucose, 5g of ammonium sulfate, 2.0g of calcium carbonate, 1.0L of distilled water, pH 7.0, fermentation temperature: culturing at 26-30 deg.C for 48h to obtain seed culture solution of Bacillus subtilis.
Clostridium butyricum (Clostridium butyricum) BNCC 213262, which is inoculated into a liquid culture medium under aseptic conditions, wherein the culture medium comprises: beef extract 5.0g, glucose 10.0g, peptone 6.0g, yeast powder 3g, NaCl 5.0g, distilled water 1.0L, pH 7.0, fermentation temperature: culturing at 28-32 deg.C for 48h to obtain seed culture solution of Clostridium butyricum, and keeping.
Escherichia coli (Escherichia coli) CMCC (B)44102, which is inoculated into a liquid medium under aseptic conditions, the medium composition being: 4.0g of beef extract, 10.5g of peptone, 5.0g of glucose, 5.0g of yeast powder, 5.0g of NaCl, 1.0L of distilled water, pH 7.0, and fermentation temperature: culturing at 28-32 deg.C for 48h to obtain seed culture solution of Escherichia coli.
2) Preparing a compound microbial preparation:
mixing the prepared seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli according to the following volume percentages: 30% of pseudomonas aeruginosa seed culture solution, 30% of bacillus subtilis seed culture solution, 20% of clostridium butyricum seed culture solution and 20% of escherichia coli seed culture solution, and after mixing, inoculating the mixed bacterial solution into a liquid culture medium under the aseptic condition according to the inoculum size of 2-10% (v/v), wherein the liquid culture medium is as follows: beef extract 5.0g, peptone 10g, glucose 5.0g, ammonium sulfate 5g, calcium carbonate 2.0g, distilled water 1.0L, pH 7.0, fermentation temperature: culturing at 26-32 deg.C for 7 days until pH reaches 3.5 to obtain compound microbial preparation.
3) Pretreatment:
respectively adding the composite microbial preparation prepared in the step 2) into the screened medicinal material raw materials, and soaking for 30 minutes for later use;
(3) microwave enzyme deactivation and drying: putting the 4 medicinal materials of honeysuckle, chrysanthemum, dandelion and liquorice in a microwave oven, deactivating enzyme for 10 minutes by microwave, heating for 5 minutes at 85 ℃, suspending, rapidly turning for 1 time, continuing to heat for 5 minutes, and taking out for later use.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 4 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 10min, heating at 85 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, and the raw materials are uniformly mixed and subpackaged.
EXAMPLE 5 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 10min, heating at 85 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 600 parts of honeysuckle, 400 parts of chrysanthemum, 400 parts of dandelion and 200 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 6 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 10min, heating at 85 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 200 parts of honeysuckle, 100 parts of chrysanthemum, 100 parts of dandelion and 50 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 7 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 15min, heating at 85 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 8 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in microwave oven, deactivating enzyme with microwave for 20 min, heating at 85 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 9 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 10min, heating at 100 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, uniformly mixing and subpackaging.
EXAMPLE 10 preparation of the Chinese medicinal composition
(1) Sorting: the honeysuckle, the chrysanthemum, the dandelion and the liquorice are respectively selected from impurities, foreign matters, non-medicinal parts and worm-eaten and mildewed parts by manual work.
(2) Pretreatment: respectively adding the composite microbial preparation prepared in the step 2) of the embodiment 3 into the screened medicinal materials, soaking for 30 minutes, putting the medicinal materials into a high-pressure spray cleaning machine, and quickly rinsing the medicinal materials with clear water for one time for later use.
(3) Microwave enzyme deactivation and drying: processing flos Lonicerae, flos Chrysanthemi, herba Taraxaci, and Glycyrrhrizae radix by rapid leaching, placing 4 medicinal materials in a microwave oven, deactivating enzyme with microwave for 10min, heating at 65 deg.C for 5min, suspending, rapidly turning over for 1 time, heating for 5min, and taking out.
(4) Crushing and sieving: the 4 medicinal materials are respectively crushed and sieved by a 20-mesh sieve for later use.
(5) Mixing and subpackaging: weighing the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice, uniformly mixing and subpackaging.
Experimental example 1 screening of preparation method of Chinese medicinal composition and determination of Process parameters
The preparation method and the process parameters of the traditional Chinese medicine composition are screened and determined by investigating indexes such as chlorogenic acid content, medicinal material color, heavy metal content, pesticide residue and the like.
The detection method comprises the following steps:
1. determination of chlorogenic acid content (2015 edition Chinese pharmacopoeia)
Preparation of a test solution: respectively taking about 0.5g of the sample powder prepared in the above examples 1-10, precisely weighing, placing in a conical flask, adding 50ml of 50% methanol, precisely weighing, treating with ultrasound (power 250W, frequency 35kHz) for 30min, taking out, cooling, weighing, supplementing the lost weight with 50% methanol, filtering, discarding the primary filtrate, precisely weighing 5ml of the subsequent filtrate, diluting to 25ml with 50% methanol, and filtering with 0.45 μm filter membrane to obtain each sample solution.
Preparation of control solutions: taking appropriate amount of chlorogenic acid reference substance, precisely weighing, placing in brown measuring flask, and making into 40% solution per lml with 50% methanol (storing below 10 deg.C).
Content determination: chlorogenic acid content was determined by high performance liquid chromatography (general rule 0512).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.4% phosphoric acid solution (13:87) is used as a mobile phase; the detection wavelength was 327 nm. The number of theoretical plates is not less than 1000 calculated according to chlorogenic acid peak.
Precisely sucking 5ul of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring. The product contains chlorogenic acid (C) calculated on dried product16H1809) Not less than 1.5%.
2. Method for measuring heavy metal
2.1 heavy Metal Limit test of samples from different examples
Preparation of standard lead solution: 0.1598g of lead nitrate dried to constant weight at 105 ℃ is precisely weighed, placed in a 1000ml measuring flask, added with 5ml of nitric acid and 50ml of water for dissolution, diluted to scale with water and shaken up to be used as stock solution.
Before use, precisely measuring 10ml of stock solution, placing into a 100ml measuring flask, adding water to dilute to scale, and shaking up to obtain the final product (each ml is equivalent to 10 μ g of Pb).
Preparing a thioacetamide test solution: 4g of thioacetamide is taken, water is added to dissolve the thioacetamide into 100ml, and the thioacetamide is stored in a refrigerator. The mixed solution (composed of 1mol/L sodium hydroxide solution 15ml, water 5.0ml and glycerin 20 ml) 5.0ml is taken before use, added with the thioacetamide solution 1.0ml, placed on a water bath for heating for 20s, cooled and used immediately.
Preparing acetate buffer solution (pH3.5) by dissolving 25g ammonium acetate in 25ml water, adding 38ml 7mol/L hydrochloric acid solution, adjusting pH to 3.5 with 2mol/L hydrochloric acid solution or 5mol/L ammonia solution (potentiometric indication), and diluting with water to 100 ml.
The checking method comprises the following steps: about 1.0g of the Chinese medicinal composition powder prepared in examples 1-10 was weighed out precisely. Slowly burning until the mixture is completely carbonized (or slowly heating on an electric furnace until white smoke is emitted but open fire cannot be generated), cooling to room temperature, adding 0.5ml of sulfuric acid to moisten the mixture, heating at low temperature until sulfuric acid vapor is completely removed, burning in a muffle furnace at 500-600 ℃ until the weight is constant, cooling, adding 0.5ml of nitric acid to evaporate to dryness until nitrogen oxide vapor is completely removed, cooling, adding 2ml of hydrochloric acid, placing the mixture on a water bath to evaporate to dryness, adding 15ml of water, dropwise adding an ammonia test solution until phenolphthalein indicator solution is neutral, adding 2ml of acetate buffer solution (pH3.5), slightly dissolving by heat, transferring the mixture to a complete nano colorimetric tube, and adding water to dilute the mixture to 25 ml. Taking another reagent for preparing a sample solution, placing the reagent in a porcelain dish, evaporating to dryness, adding 2ml of acetate buffer solution (pH3.5) and 15ml of water, slightly heating to dissolve, transferring to a Nami colorimetric tube, adding 2.5ml of standard lead solution, diluting with water to 25ml, adding 2ml of thioacetamide test solution into the Nami colorimetric tube of the sample and the reference product respectively, shaking uniformly, and standing for 2 min. The results are shown in Table 2.
2.2 arsenic salt Limit test of samples from different examples
Preparation of standard arsenic solution: 0.1324g of arsenic trioxide is weighed and placed in a 1000ml measuring flask, 5ml of 20 percent sodium hydroxide solution is added for dissolution, then an appropriate amount of dilute sulfuric acid is used for neutralization, 10ml of dilute sulfuric acid is added for dilution with water to the scale, and the mixture is shaken up to be used as stock solution. Before use, precisely measuring 10ml of stock solution, placing into a 1000ml measuring flask, adding 10ml of dilute sulfuric acid, diluting with water to scale, and shaking up to obtain the final product (each ml is equivalent to 1 μ g of arsenic).
Preparing a potassium iodide test solution: taking 16.5g of potassium iodide, adding water to dissolve the potassium iodide into 100ml, and obtaining the potassium iodide. The liquid should be newly prepared. Preparing an acidic stannous chloride test solution: taking 20g of stannous chloride, adding hydrochloric acid to dissolve the stannous chloride into 50ml, and standing overnight to obtain the stannous chloride.
Preparation of standard arsenic plaques: precisely measuring 2ml of standard arsenic solution, placing the solution in a bottle A, adding 5ml of hydrochloric acid and 21ml of water, adding 5ml of potassium iodide test solution and 5 drops of acidic stannous chloride test solution, placing the solution at room temperature for 10min, adding 2g of zinc particles, immediately sealing a gas guide tube C arranged in the bottle A with a plug, placing the bottle A in a water bath at 25-40 ℃ for reacting for 45min, and taking out mercury bromide test paper to obtain the mercury bromide test paper.
Preparing a test article arsenic spot: respectively taking about 2.0g of the Chinese medicinal composition powder prepared in the examples 1-10, precisely weighing, adding an equal amount of calcium hydroxide, adding a small amount of water, uniformly mixing to form paste, drying, carbonizing by using a small fire, burning at 550-600 ℃ until the paste is completely ashed, taking out, and cooling. And (3) placing the ashed sample into a bottle A, adding hydrochloric acid for neutralization, and adding 5ml of hydrochloric acid and a proper amount of water to obtain 28ml, which is the same as the preparation method of the standard arsenic spot.
3. The pesticide residue determination method comprises the following steps:
using a rapid test card method (paper method): paper sheets of the intrinsic cholinesterase and indophenol acetate reagents. Eluent (PH7.5 buffer): 15.0g of disodium hydrogen phosphate and 1.59g of anhydrous potassium dihydrogen phosphate were taken in 500ml of distilled water.
(1) Weighing 5g of sample, placing the sample into a bottle with a cover, adding 10ml of buffer solution, shaking for 50 times, and standing for more than 2 min.
(2) Taking a piece of rapid test card, dipping the white tablet in the extractive solution, placing in a pesticide residue rapid test instrument for 10min, and keeping the surface of the tablet after pre-reaction wet.
(3) The tache was folded in half and operated with a pesticide residue velocimeter for 3min to allow the red and white tablets to overlap and react.
(4) A blank control card of purified water or buffer solution is arranged for each batch of measurement.
(II) detection results:
1. the content determination result is as follows: the product contains chlorogenic acid (C) calculated on dried product16H1809) Not less than 1.5%.
2. And (3) heavy metal detection results:
2.1 lead detection: the sample tube and the reference tube are placed on white paper and are seen through from top to bottom, the color of the sample tube is lighter than that of the reference tube when the sample tube is compared with the reference tube, and the color of the sample tube is basically consistent, so the limit of heavy metal is less than 10 mu g/g.
2.2 arsenic detection: the arsenic spots generated by the test article were compared with the standard arsenic spots, and the colors were lighter than the standard arsenic spots, and the color shades were, in order, example 1 sample > example 2 sample > example 3 sample > example 5 sample > example 10 sample > example 9 sample > example 8 sample > example 7 sample > example 6 sample > example 4 sample.
3. And (3) detecting a pesticide residue: compared with a blank control card, the white tablets do not change color or have slight bluish color, both positive results are obtained, the unchanged bluish color is a strong positive result, which indicates that the pesticide residue is higher, the bluish color is a weak positive result, which indicates that the pesticide residue is relatively lower, and the white tablets become sky blue or are the same as the blank control card, which indicates that the white tablets have negative results.
The results of the above tests are shown in table 1 below:
TABLE 1 comparison of results of different treatment methods
By comparing the treatment methods of the composition, the experimental result of the composition treated by the enzyme deactivating method is better than that of the drying method in time, smell, heavy metal, moisture and chlorogenic acid content, and the enzyme deactivating method saves the treatment time and saves the time for enlarging the production by analyzing the treatment time. From the analysis of heavy metal angle, the heavy metal index of the fixation method combined with the rapid leaching method is obviously reduced. From the perspective of pesticide residue analysis, the heavy metal indexes and pesticide residue of the composition are obviously reduced after the composition is treated by combining a microbial degradation method and a rapid leaching method and then is subjected to microwave enzyme deactivation, sterilization and drying. Thus, example 4 is superior to the other example groups in the treatment method.
4. Influence of different fixation time and temperature on composition
The results are shown in Table 2.
TABLE 2 comparison of the results of the different treatment methods
Through analysis of detection results of odor, heavy metals, moisture, chlorogenic acid content and pesticide residue, the experimental result of example 4 is due to other example groups.
Experimental example 2 screening of the amount of the composition of the present invention prescribed
The dosage of the prescription components is determined by comparing the detection results of the chlorogenic acid content of the composition. The content determination method comprises the following steps: chlorogenic acid is determined by high performance liquid chromatography (general rule 0512).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.4% phosphoric acid solution (13:87) is used as a mobile phase; the detection wavelength was 327 nm. The number of theoretical plates is not less than 1000 calculated according to chlorogenic acid peak.
Preparation of control solutions: taking appropriate amount of chlorogenic acid reference substance, precisely weighing, placing in brown measuring flask, and making into 40% solution per lml with 50% methanol (storing below 10 deg.C).
Preparation of a test solution: taking about 0.5g of the Chinese medicinal composition powder (sieved by a sieve with the number four) of the embodiment 4-6, precisely weighing, placing in a conical flask with a plug, precisely adding 50% methanol to 50ml, weighing, carrying out ultrasonic treatment (power 250W and frequency 35kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 50% methanol, shaking up, filtering, precisely weighing or continuing to filtrate 5ml, placing in a brown measuring flask with the volume of 25ml, adding 50% methanol to the scale, and shaking up to obtain the Chinese medicinal composition.
The determination method comprises the following steps: precisely sucking 5ul of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.
The traditional Chinese medicine composition contains chlorogenic acid (C16H1809) not less than 1.5% calculated on a dry product. The results are shown in Table 3.
TABLE 3 determination of chlorogenic acid content in the compositions
As a result, example 4 was formulated better than examples 5 and 6, and example 4 was selected accordingly.
Experimental example 3 chlorogenic acid content of the composition of the invention was compared to that of the prior art one and two compositions.
Samples were prepared according to the prior art one, two experimental methods:
prior art I (patent application No. 00114872.9) prescription process: 10-30 parts of coix seeds, 10-30 parts of phaseolus calcaratus, 10-30 parts of fried black beans, 5-15 parts of angelica dahurica, 10-30 parts of dandelion, 5-15 parts of ginkgo seeds, 10-30 parts of mulberry leaves, 5-15 parts of oldenlandia diffusa, 5-15 parts of calendula officinalis, 5-15 parts of beauveria bassiana, 5-15 parts of kudzu roots and 5-15 parts of gardenia, and the raw materials are respectively cleaned and sterilized or mixed, then cleaned and sterilized, then sterilized, and finally decocted in water and filtered to obtain a sample 1.
Prescription process of prior art two (application No. 201610703243.0): 6-10 parts of coptis chinensis, 8-10 parts of scutellaria baicalensis, 12-18 parts of phellodendron amurense, 8-12 parts of loquat leaves, 8-12 parts of mulberry leaves, 8-12 parts of herba violae, 12-18 parts of wild chrysanthemum flowers, 12-18 parts of honeysuckle flowers, 6-10 parts of folium isatidis, 8-12 parts of dandelion, 12-18 parts of andrographis paniculata, 8-12 parts of cortex moutan, 6-10 parts of red paeony roots, 8-10 parts of fructus forsythiae and 5-7 parts of liquorice. Pulverizing all the raw materials into coarse powder, mixing, placing in a container, adding 45% ethanol 5 times the concentration of the raw materials, soaking for 6 days, filtering to remove residue, and recovering ethanol from the filtrate under reduced pressure until no ethanol smell exists to obtain concentrated solution. Adding water and potassium sorbate into the concentrated solution, stirring, standing, and filtering to obtain filtrate; boiling sucrose in water for 35-45min, and filtering; mixing the above medicinal liquids, placing in a container, adding water, cooling, bottling, sealing, and sterilizing to obtain sample 2. A sample was prepared according to the invention of example 4, giving sample 3.
The chlorogenic acid content of samples 1, 2, 3 was compared by a chlorogenic acid content detection method. The results are shown in Table 4.
TABLE 4 comparison of the compositions of the invention with the prior art I and II
The results in table 4 show that the chlorogenic acid content of the composition is higher than that of the samples 1 and 2, the number of the medicinal materials contained in the composition is simple and is far lower than that of the medicinal materials in the first and second prior art, and the pesticide residue problem and the heavy metal problem are effectively solved by adopting 2 pretreatment modes combined with the microwave enzyme deactivation method for treatment.
Experimental example 4 pharmacodynamic test of the Chinese medicinal composition of the present invention
The research provides the therapeutic effect of the composition for treating the acne through the experimental observation of the influence of the pharmaceutical composition on the propionibacterium induced rat auricle acne inflammation model.
1. Material
1.1 animals
Wistar rats with the weight of 160-200g were selected and provided by Heilongjiang university of traditional Chinese medicine.
1.2 drugs
The test drugs are: a Chinese medicinal composition for the treatment of acne prepared according to example 4;
positive control drug: acne removing pills (Jiangxi Hui ren pharmaceutical Co., Ltd.);
the experimental administration doses are all expressed by crude drugs (g.kg)-1) When the medicine is administrated by gastric lavage, the normal saline is used for preparing 0.5 g.mL-1To (3) is prepared.
1.3 strains
Propionibacterium (provided by the institute of microorganisms, Heilongjiang); the Propionibacterium test bacteria is extracted and separated from the focus of a patient suffering from acne.
2. Method of producing a composite material
Taking Wistar rats, each half of male and female, randomly dividing the Wistar rats into 6 groups, each group comprises 10 rats, and preparing the traditional Chinese medicine composition prepared in the embodiment 4 into three different dosage specifications of high, medium and low according to dosage, wherein the high dosage group comprises 4g/kg, the medium dosage group comprises 2g/kg, the low dosage group comprises 1g/kg, and the positive control group is given with detoxifying acne pills with dosage of 2g/kg, the blank control group and the auricular acne model group with equal volume of physiological saline every day.
The administration is performed by gavage 1 time daily for 10 days. On the 5 th day after each group of rats started gavage, 50uL of sterile normal saline was injected into the left auricle of the blank control group, and 50uL of propionibacterium solution was injected into the left auricle of the other groups (Propionibacterium stock solution: lyophilized Propionibacterium, after being enriched with sodium thioglycollate liquid medium under anaerobic conditions, was washed with normal saline for 3 times, and the concentration of Propionibacterium was adjusted to 6000 trill mL-1And inactivating in water bath at 95 ℃ for 15min for later use). After injection of the prepared model, the thickness of the auricle of the rat was measured with a micrometer every 24 hours for 5 times continuously. The swelling rate of auricles of each mouse was calculated by the following formula:
auricle swelling rate ═ thickness of auricle after injection-thickness of auricle before injection)/thickness of auricle before injection × 100%
After further dosing to 12 days, rat ears were excised, fixed with 10% formaldehyde, sections embedded in paraffin, HE purple, and observed under light.
3. Results
The traditional Chinese medicine composition and the detoxifying acne pill can reduce the auricle swelling rate of rats, but have more obvious effect when the traditional Chinese medicine composition is used in high and medium doses. The results are shown in Table 5.
TABLE 5 Effect of a composition for treating acne on the rat auricle acne model
Note: in comparison with the set of models,**P<0.01。
pathological diagnosis results under a light microscope show that no obvious lesions are found on auricle epidermis, muscle, sebaceous gland and cartilage of rats in the blank control group; a plurality of abscesses are formed under the skin of the model group, inflammatory cell diffuse infiltration is mainly performed by a large number of neutrophils, and part of inflammatory cells are mononuclear and lymphocytes, peripheral tissue capillaries are dilated, hyperemia and exudation are performed, tissue edema is obvious, muscle degeneration and fracture are performed, inflammatory cell infiltration in muscle gaps and muscle layers is performed, and the like, so that the establishment of an acne model is prompted; the detoxified acne pill has obviously reduced organized purulent inflammation; the traditional Chinese medicine composition has no suppurative lesion in each dosage group, the inflammation is reduced along with the increase of the administration dosage, and the tissues are basically normal in the high-dosage groups.
Experimental results show that the traditional Chinese medicine composition can obviously inhibit the swelling rate and pathological changes of a rat auricle acne model caused by propionibacterium. The experimental result provides the pharmacodynamic experimental evidence for the traditional Chinese medicine composition to treat the acne.
Experimental example 5 toxicological study of the Chinese medicinal composition of the present invention
Mouse gavage LD by using traditional Chinese medicine composition prepared in example 450The value is (1.98 +/-0.233) g/kg, which is equivalent to 165 times of the accepted common dosage, 3 dosages (0.05, 0.5 and 1.0g/kg) are taken to carry out a 4-week administration experiment on rats, and obvious irreversible toxic reaction is not seen in observation of weight gain, hemogram, liver and kidney functions, ten blood biochemical index detections and animal organ detection of the rats.
Experimental example 6 clinical study of the Chinese medicinal composition of the present invention on patients with acne
120 acne patients are selected to be subjected to clinical test, 120 acne patients are randomly divided into two groups, and 62 patients in the treatment group are infused with the traditional Chinese medicine composition prepared in the example 4 and are taken 1-2 bags at a time 2 times a day. The control group 58 used no drug. After one month of treatment, the treatment group comprises 62 cases, 24 cases are basically cured, 19 cases are obviously effective, 9 cases are improved, 10 cases are not effective, and the effective (basic cure + obvious effect) rate is 69.35%; 58 cases of the control group have 17 cases of basic cure, 11 cases of obvious effect, 14 cases of improvement and 16 cases of no effect, and the effective rate is 48.28%. The curative effect of the treatment group is better than that of the control group (P < 0.01). The results are shown in Table 6.
TABLE 6 statistics of clinical efficacy of Chinese medicinal compositions for treating acne patients
| Grouping | Number of people | Basic cure | Show effect | Improvement of life | Invalidation | Effective rate% |
| Control group | 58 | 17 | 11 | 14 | 16 | 48.28 |
| Treatment group | 62 | 24 | 19 | 9 | 10 | 69.35 |
Claims (8)
1. The traditional Chinese medicine composition for treating acne is characterized by being prepared by the following steps:
(1) sorting: manually selecting impurities, worm-eaten parts and mildewed parts of the honeysuckle, the chrysanthemum, the dandelion and the liquorice;
(2) pretreating the raw material by microbial degradation
1) Preparing a seed culture solution:
respectively treating Pseudomonas aeruginosaP.Aeruginosa) Bacillus subtilis preparation (B)Bacillus subtilis) Clostridium butyricum: (Clostridium butyricum) And Escherichia coli (E.coli) ((R))Escherichia coli) Inoculating the strain in a liquid culture medium, and culturing to obtain seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli respectively;
2) preparing a compound microbial preparation:
mixing the prepared seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli according to the following volume percentages: 30% of a pseudomonas aeruginosa seed culture solution, 30% of a bacillus subtilis seed culture solution, 20% of a clostridium butyricum seed culture solution and 20% of an escherichia coli seed culture solution, mixing, inoculating the mixed bacteria solution into a liquid culture medium under an aseptic condition according to an inoculation amount of 2-10% (v/v), and culturing to obtain a composite microbial preparation;
3) pretreatment:
respectively adding the composite microbial preparation prepared in the step 2) into the screened medicinal material raw materials, soaking for 30 minutes, and quickly washing for later use;
(3) microwave enzyme deactivation and drying:
placing the honeysuckle, the chrysanthemum, the dandelion and the liquorice processed in the step 3) into a microwave oven respectively, carrying out microwave enzyme deactivation for 10-20 minutes, then taking out, heating at 65-100 ℃ for 5-10 minutes, suspending, rapidly turning over for 1 time, continuing to heat for 3-5 minutes, and taking out for later use;
(4) crushing and sieving:
respectively crushing the medicinal materials treated in the step (3) and sieving the medicinal materials with a 20-mesh sieve;
(5) mixing and subpackaging:
weighing the crushed medicinal materials according to the following weight parts, uniformly mixing and subpackaging to obtain the traditional Chinese medicine composition;
200-600 parts of honeysuckle, 100-300 parts of chrysanthemum, 100-300 parts of dandelion and 50-150 parts of liquorice.
2. The traditional Chinese medicine composition for treating acne as claimed in claim 1, which is prepared from the following raw materials in parts by weight: 400 parts of honeysuckle, 200 parts of chrysanthemum, 200 parts of dandelion and 100 parts of liquorice.
3. A method of preparing the traditional Chinese medicine composition for treating acne as claimed in claim 1 or 2, comprising the steps of:
(1) sorting: manually selecting impurities, worm-eaten parts and mildewed parts of the honeysuckle, the chrysanthemum, the dandelion and the liquorice;
(2) pretreating the raw material by microbial degradation
1) Preparing a seed culture solution:
respectively treating Pseudomonas aeruginosaP.Aeruginosa) Bacillus subtilis preparation (B)Bacillus subtilis) Clostridium butyricum: (Clostridium butyricum) And Escherichia coli (E.coli) ((R))Escherichia coli) Inoculating the strain in a liquid culture medium, and culturing to obtain seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli respectively;
2) preparing a compound microbial preparation:
mixing the prepared seed culture solutions of pseudomonas aeruginosa, bacillus subtilis, clostridium butyricum and escherichia coli according to the following volume percentages: 30% of a pseudomonas aeruginosa seed culture solution, 30% of a bacillus subtilis seed culture solution, 20% of a clostridium butyricum seed culture solution and 20% of an escherichia coli seed culture solution, mixing, inoculating the mixed bacteria solution into a liquid culture medium under an aseptic condition according to an inoculation amount of 2-10% (v/v), and culturing to obtain a composite microbial preparation;
3) pretreatment:
respectively adding the composite microbial preparation prepared in the step 2) into the screened medicinal material raw materials, soaking for 30 minutes, and quickly washing for later use;
(3) microwave enzyme deactivation and drying:
placing the honeysuckle, the chrysanthemum, the dandelion and the liquorice processed in the step 3) into a microwave oven respectively, carrying out microwave enzyme deactivation for 10-20 minutes, then taking out, heating at 65-100 ℃ for 5-10 minutes, suspending, rapidly turning over for 1 time, continuing to heat for 3-5 minutes, and taking out for later use;
(4) crushing and sieving:
respectively crushing the medicinal materials treated in the step (3) and sieving the medicinal materials with a 20-mesh sieve;
(5) mixing and subpackaging:
weighing the crushed medicinal materials according to the parts by weight of the raw materials in the claim 1 or 2, uniformly mixing, and subpackaging to obtain the traditional Chinese medicine composition.
4. The process for preparing the traditional Chinese medicine composition for treating acne according to claim 1 or 2, characterized in that, the composition of the liquid culture medium for the culture of pseudomonas aeruginosa in step 2) is: 4.0g of beef extract, 10.5g of peptone, 5.0g of NaCl, 20.0g of agar, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h;
the composition of the liquid medium for the cultivation of Bacillus subtilis was: 4.0g of beef extract, 10.1g of peptone, 5.0g of glucose, 5g of ammonium sulfate, 2.0g of calcium carbonate, 1.0L of distilled water, pH 7.0, fermentation temperature of 26-30 ℃ and culture time of 36-48 h;
the composition of the liquid medium for the culture of clostridium butyricum is as follows: 5.0g of beef extract, 10.0g of glucose, 6.0g of peptone, 3g of yeast powder, 5.0g of NaCl, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h;
the composition of the liquid culture medium for culturing the Escherichia coli is as follows: 4.0g of beef extract, 10.5g of peptone, 5.0g of glucose, 5.0g of yeast powder, 5.0g of NaCl, 1.0L of distilled water, pH 7.0, fermentation temperature of 28-32 ℃ and culture time of 36-48 h.
5. The method for preparing the traditional Chinese medicine composition for treating acne as claimed in claim 1 or 2, wherein, in step 2), the composition of the liquid culture medium for mixed bacterial liquid culture is as follows: 5.0g of beef extract, 10g of peptone, 5.0g of glucose, 5g of ammonium sulfate, 2.0g of calcium carbonate, 1.0L of distilled water, pH 7.0, fermentation temperature of 26-32 ℃, and culture time of 5-8 days, wherein the pH of the culture medium is reduced to 3-5, and the compound microbial preparation is obtained.
6. The method for preparing the Chinese medicinal composition for treating acne as claimed in claim 1 or 2, wherein in step 3), the quick washing means that the medicinal materials are put into a high pressure spray washer and washed with clean water quickly.
7. Use of the traditional Chinese medicine composition for treating acne according to claim 1 or 2 in the preparation of a medicament for treating or preventing acne.
8. Use of the traditional Chinese medicine composition for treating acne according to claim 1 or 2 in the preparation of health food for preventing acne.
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Address after: 150025 Zhuhai Road, Limin District, Hulan District, Heilongjiang, Harbin Applicant after: Letai Pharmaceutical Co.,Ltd. Address before: 150025 Zhuhai Road, Limin District, Hulan District, Heilongjiang, Harbin Applicant before: HARBIN KUAIHAO PHARMACEUTICAL Co.,Ltd. |
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