CN116726077B - Gynecological external lotion and preparation method thereof - Google Patents
Gynecological external lotion and preparation method thereof Download PDFInfo
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- CN116726077B CN116726077B CN202310910694.1A CN202310910694A CN116726077B CN 116726077 B CN116726077 B CN 116726077B CN 202310910694 A CN202310910694 A CN 202310910694A CN 116726077 B CN116726077 B CN 116726077B
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Abstract
The invention provides a gynecological external lotion and a preparation method thereof, and belongs to the technical field of lotions. Extracting flos Caryophylli and herba Rosmarini officinalis with supercritical fluid to obtain mixed essential oil, collecting the rest solid, extracting Galla chinensis, radix Angelicae Dahuricae, coptidis rhizoma, herba Pteridis Multifidae, polygoni chinensis, and herba Blumeae Balsamiferae with water, precipitating with ethanol, filtering, concentrating the filtrate to obtain concentrated solution, extracting the solid with ethanol-propanol mixed water solution, filtering, drying to obtain ethanol extract, mixing the solid with the rest solid, inoculating activated lactobacillus plantarum and lactobacillus paracasei seed solution, fermenting, lyophilizing, embedding to obtain fermentation product microcapsule, dissolving propolis and Borneolum Syntheticum in ethanol, adding PEG400 or propylene glycol, dissolving, adding mixed essential oil, ethanol extract, surfactant and fermentation product microcapsule, and making into external lotion with antiinflammatory, toxic substance removing, astringing, and hemostasis effects.
Description
Technical Field
The invention relates to the technical field of lotion, in particular to a gynecological external lotion and a preparation method thereof.
Background
With the development of economy and the improvement of living standard, the health care consciousness of people is enhanced, and women pay more attention to the health of the women. Gynecopathy is a natural enemy of female health, 90% of adult females have different degrees of gynecopathy, vaginitis is a common disease and frequently-occurring disease in gynecopathy, and according to the research and statistics of the Ministry of health, the incidence rate of symptomatic vaginitis in the life of females is 75%, wherein about half of females experience multiple recrudescence, mainly manifested by symptoms such as vulva and vaginal itching, red swelling, leucorrhea increase and the like, and the vulva itching is hard after illness, vaginal secretion increases, has peculiar smell and repeatedly attacks, influences the work and sleep of females, brings great inconvenience to life, can mutually infect, and brings serious consequences to the physical and mental health of females.
At present, most of similar medicines for treating colpitis mainly adopt western medicines, some of the medicines have a certain stimulation effect on skin, are not suitable for partial people, and few of the medicines for treating the diseases are prepared by adopting a hot reflux method to extract medicinal components, the extraction time is long, the energy is consumed, the effective components unstable when the medicines are subjected to heat are also easily damaged by decoction, and the extraction rate is low.
The traditional Chinese medicine also reports on treating gynecological diseases, for example, chinese patent No. CN2216416Y discloses a Mingmen navel therapy body-building bag which comprises hundreds of crude drugs such as radix aconiti and is used for treating chronic diseases such as dizziness, gynecological diseases, climacteric syndrome and the like; in addition, chinese patent application CN104547919a discloses a traditional Chinese medicine composition for treating condyloma acuminatum, which comprises tens of crude drugs such as astragalus, codonopsis pilosula, angelica, szechwan chinaberry fruit, nutgrass galingale rhizome, zedoary, rhizoma sparganii, medicinal cyathula root, red sage root, dittany bark and the like, and is orally taken for treating the viral disease of condyloma acuminatum. The traditional Chinese medicines have various crude drugs, complex components and difficult production quality control, and limit the prospect of practical application.
Disclosure of Invention
The utility model aims to provide a gynecological external lotion and a preparation method thereof, which have the effects of diminishing inflammation, detoxifying, astringing, stopping bleeding and the like, do not generate side effects on human bodies, have good sterilization, diminishing inflammation and detoxifying effects, reduce vaginal lipstick swelling after use, eliminate purulent secretion, reduce inflammatory cells infiltrated in vaginal epithelial tissues, have no damage to the vaginal epithelial tissues, can relieve vaginal tissue inflammation caused by bacteria, trichomonas, mould and other infections, promote tissue repair, and have wide application prospects.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of gynecological external lotion, which comprises the steps of extracting flos caryophylli and rosemary through supercritical fluid to obtain mixed essential oil, reserving the rest solid, extracting gallnut, radix angelicae, coptis chinensis, white phoenix vegetable, charcoal mother and blumea balsamifera with water, precipitating with alcohol, filtering, concentrating filtrate to obtain concentrated solution, extracting the solid through ethanol-propanol mixed aqueous solution, filtering, drying to obtain alcohol extract, mixing the solid with the rest solid, adding the solid into water, inoculating activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, performing anaerobic fermentation culture, freeze-drying to obtain fermentation products, embedding to obtain fermentation product microcapsules, dissolving propolis and borneol in ethanol to obtain additive solution, adding PEG400 or propylene glycol into the concentrated solution to dissolve, adding the mixed essential oil, the alcohol extract, the surfactant and the fermentation product microcapsules, and uniformly mixing to obtain the gynecological external lotion.
As a further improvement of the invention, the method comprises the following steps:
s1, supercritical fluid extraction: cleaning flos Caryophylli and herba Rosmarini officinalis, pulverizing, drying, extracting with supercritical fluid, and extracting with ethyl acetate as entrainer to obtain mixed essential oil;
S2, water extraction: cleaning Galla chinensis, radix Angelicae Dahuricae, coptidis rhizoma, herba Pteridis Multifidae, polygoni chinensis, and herba Blumeae Balsamiferae, mixing, pulverizing to obtain Chinese medicinal powder, boiling with water, extracting for 2-3 times, filtering, collecting residue, mixing filtrates, precipitating with ethanol, collecting supernatant, recovering ethanol, and concentrating to obtain concentrated solution;
s3, alcohol extraction: adding the solid residue obtained in the step S2 into an ethanol-propanol mixed aqueous solution, heating and extracting, filtering, reserving the solid residue, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
s4, fermenting: adding the rest solid in the step S1 and the solid residue in the step S3 into water, sterilizing, inoculating activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, performing anaerobic fermentation culture, and freeze-drying to obtain a fermentation product;
s5, embedding: adding the fermentation product obtained in the step S4 into water, adding sodium alginate and carrageenan, stirring and mixing uniformly to obtain a water phase, adding the water phase into fish oil, quickly emulsifying by a membrane, regulating the pH value of the solution to be a first pH value, stirring and reacting, regulating the pH value to be a second pH value, adding glutamine transaminase and calcium chloride, stirring and reacting, centrifuging, washing and freeze-drying to obtain a fermentation product microcapsule;
S6, preparing an additive: dissolving propolis and Borneolum Syntheticum in ethanol to obtain additive solution;
s7, preparing gynecological external lotion: adding the additive solution prepared in the step S6 into the concentrated solution prepared in the step S2, adding PEG400 or propylene glycol for dissolution, adding the mixed essential oil prepared in the step S1, the alcohol extract prepared in the step S3 and the surfactant, stirring and mixing uniformly, adding the fermentation product microcapsule prepared in the step S5, and dispersing uniformly by ultrasonic waves to prepare the gynecological external lotion.
As a further improvement of the present invention, the conditions for supercritical fluid extraction in step S1 are extraction pressure: 20-30MPa, extraction temperature: 35-45 ℃, CO 2 Flow rate: 7-12L/h, extraction time: 1-3h, wherein the adding amount of the entrainer is 2-3wt%, and the mass ratio of the flos caryophylli to the rosemary is 5-7:2; in the step S2, the mass ratio of the Chinese gall, the dahurian angelica root, the golden thread, the white phoenix-tail fern, the Chinese knotweed herb and the blumea balsamifera is 5-7:2-3:3-5:1-2:2-3:3-5, the solid-liquid ratio of the Chinese medicinal powder to the water is 1:5-10g/mL, the boiling extraction time is 2-3h, the content of ethanol added into a system is 50-60wt%, and the relative density of the concentrated solution is 1.05-1.08.
As a further improvement of the invention, in the step S3, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed aqueous solution is 30-40:15-20:50-70, the solid-liquid ratio of the solid slag and the ethanol-propanol mixed aqueous solution is 1:3-5g/mL, and the heating extraction temperature is 50-70 ℃ and the time is 2-4h.
As a further improvement of the present invention, the lactobacillus plantarum strain seed solution in the step S4 has a bacterial content of 10 8 -10 9 cfu/mL, wherein the mass ratio of the solid remained in the step S1 to the solid slag and water in the step S3 is 5-7:12-15:120-150, the inoculation amounts of lactobacillus plantarum and lactobacillus paracasei strain seed liquid are 2-3% and 1-2%, respectively, and the anaerobic fermentation culture condition is that CO is 3-5v/v% 2 And fermenting and culturing for 36-48h at the temperature of 35-38 ℃ in the environment of 50-70r/min with the balance of nitrogen.
As a further improvement of the invention, the mass ratio of the fermentation product, sodium alginate, carrageenan, ammonia amide transaminase and calcium chloride in the step S5 is 12-15:7-10:3-5:7-12:2-3, the pore diameter of the membrane emulsified membrane is 3-5 μm, the first pH value is 4.2-4.5, and the second pH value is 6.3-6.7.
As a further improvement of the invention, the mass ratio of the propolis to the borneol to the ethanol in the step S6 is 10-12:5-7:30-50.
As a further improvement of the invention, the mass ratio of the additive solution, the concentrated solution, the PEG400 or the propylene glycol, the mixed essential oil, the alcohol extract, the surfactant and the fermentation product microcapsule in the step S7 is 10-12:50-70:3-5:3-5:5-6:1-2:4-6, and the surfactant is at least one selected from tween-20, tween-40, tween-60, tween-80, span-20, span-40, span-60 and span-80.
As a further improvement of the present invention, the method specifically comprises the following steps:
s1, supercritical fluid extraction: cleaning 5-7 parts by weight of flos caryophylli and 2 parts by weight of rosemary, crushing, drying, extracting by supercritical fluid, and taking ethyl acetate as an entrainer to prepare mixed essential oil;
the conditions of the supercritical fluid extraction are the extraction pressure: 20-30MPa, extraction temperature: 35-45 ℃, CO 2 Flow rate: 7-12L/h, extraction time: 1-3h, wherein the adding amount of the entrainer is 2-3wt%;
s2, water extraction: cleaning 5-7 parts by weight of gallnut, 2-3 parts by weight of angelica dahurica, 3-5 parts by weight of coptis chinensis, 1-2 parts by weight of white phoenix-headed vegetable, 2-3 parts by weight of Chinese knotweed herb and 3-5 parts by weight of blumea balsamifera, mixing, crushing to obtain Chinese medicinal powder, adding water, boiling and extracting for 2-3 hours, wherein the solid-to-liquid ratio of the Chinese medicinal powder to the water is 1:5-10g/mL, repeatedly extracting for 2-3 times, filtering, retaining solid residues, mixing filtrate, adding ethanol until the content of system ethanol is 50-60wt%, precipitating for 6-10 hours, collecting supernatant, recovering ethanol, and concentrating until the relative density is 1.05-1.08 to obtain concentrated solution;
s3, alcohol extraction: adding the solid slag obtained in the step S2 into an ethanol-propanol mixed water solution, wherein the solid-to-liquid ratio of the solid slag to the ethanol-propanol mixed water solution is 1:3-5g/mL, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed water solution is 30-40:15-20:50-70, heating to 50-70 ℃, extracting for 2-4h, filtering, reserving the solid slag, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
S4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 8 -10 9 cfu/mL of activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, wherein the inoculation amount of the lactobacillus plantarum and lactobacillus paracasei strain seed liquid is 2-3% and 1-2%, and the inoculation amount of CO is 3-5v/v%, respectively 2 Fermenting and culturing for 36-48h at the temperature of 35-38 ℃ in the environment of 50-70r/min with the balance of nitrogen, and freeze-drying to obtain a fermentation product;
s5, embedding: adding 12-15 parts by weight of the fermentation product obtained in the step S4 into 100 parts by weight of water, adding 7-10 parts by weight of sodium alginate and 3-5 parts by weight of carrageenan, stirring and mixing uniformly to obtain a water phase, adding the water phase into fish oil, emulsifying by a rapid membrane, adjusting the pore diameter of the membrane to 3-5 mu m, adjusting the pH value of the solution to 4.2-4.5, stirring and reacting for 1-2 hours, adjusting the pH value to 6.3-6.7, adding 7-12 parts by weight of glutamine transaminase and 2-3 parts by weight of calcium chloride, stirring and reacting for 20-30 minutes, centrifuging, washing, and freeze-drying to obtain the fermentation product microcapsule;
s6, preparing an additive: dissolving 10-12 parts by weight of propolis and 5-7 parts by weight of borneol in 30-50 parts by weight of ethanol to obtain an additive solution;
s7, preparing gynecological external lotion: adding 10-12 parts by weight of the additive solution prepared in the step S6 into 50-70 parts by weight of the concentrated solution prepared in the step S2, adding 3-5 parts by weight of PEG400 or propylene glycol for dissolution, adding 3-5 parts by weight of the mixed essential oil prepared in the step S1, 5-6 parts by weight of the alcohol extract prepared in the step S3 and 1-2 parts by weight of the surfactant, stirring and mixing uniformly, adding 4-6 parts by weight of the fermentation product microcapsule prepared in the step S5, and dispersing uniformly by ultrasonic waves to prepare the gynecological external lotion.
The invention further protects the gynecological external lotion prepared by the preparation method.
The invention has the following beneficial effects: anaerobic pathogenic bacteria such as candida albicans are taken as conditional pathogenic bacteria, the bacteria are easy to form a biological film on various biological materials, the drug resistance of candida albicans is greatly enhanced after the biological film is formed, and the sensitivity of the candida albicans is greatly reduced to clinically common candida albicans resisting drugs such as clotrimazole and the like. Flos Caryophylli and herba Rosmarini officinalis are rich in volatile oil, mainly contain eugenol and rosmarinic acid, and have effects on membrane permeability of Candida albicans, so that Candida albicans content is leaked, microorganism death is caused, and good killing effect on trichomonas and mould is also achieved.
The gallnut is insect gall on Rhus chinensis, populus chinensis or Populus rubra leaf of Rhus chinensis, is mainly parasitic by gallnut aphid, and has obvious broad-spectrum antibacterial activity. The radix angelicae is the dry root of the radix angelicae of the Umbelliferae, has the effects of inhibiting pathogenic microorganisms and the like, and the radix angelicae alcohol extract can have obvious inhibition effect on the growth activity of candida albicans biomembrane. The main medicinal components of blumea balsamifera are volatile components and flavonoid compounds, and also contain sesquiterpene and the like, so that the blumea balsamifera has better pharmacological effects of resisting inflammation, easing pain, resisting bacteria, resisting fungi, reducing fever and the like. Coptidis rhizoma is a traditional antibacterial agent, and can be used for treating eczema by external application, and has effects of relieving fever and pain, and inhibiting infection of many bacteria, fungi, viruses and intestinal bacteria. The white phoenix-tail fern has the special effects of relieving fever, detoxifying, diminishing inflammation, promoting urination, reducing blood pressure and the like, can be used for treating external injuries, traumatic injuries, wounds, toxic insect bite, non-swelling toxin and the like, and has the inhibition effect on gram positive bacteria and gram negative bacteria. The Chinese knotweed herb has the effects of clearing heat and promoting diuresis, cooling blood and detoxicating, calming liver and improving eyesight, and activating blood and relaxing tendons, and the Chinese knotweed herb total flavone can effectively treat bacterial vaginitis, has an inhibiting effect on NO released by stimulated RAW264.7 cells, and shows remarkable in-vitro anti-infective activity; the Sancao decoction total flavone can reduce the release of interleukin-1 and interleukin-6 as inflammatory mediators. Different from the clinical common medicines such as antibiotics, hormone and the like, the medicine resistance is easy to generate when the medicine is used, and various components interfere the growth of pathogenic microorganisms from various links such as ribose, protein, energy conversion and the like.
The invention extracts gallnut, angelica dahurica, coptis chinensis, white phoenix-tail fern, chinese knotweed and blumea balsamifera with water to obtain a large amount of water-soluble antibacterial and anti-inflammatory active components, extracts a large amount of total flavonoids in the traditional Chinese medicine components by ethanol-propanol mixed water solution, has low toxicity and no irritation of propylene glycol, has better solubility for a plurality of organic active components, and has a certain promoting effect on the absorption of the active components on skin and mucous membrane. Therefore, the ethanol-propanol mixed aqueous solution is used for extracting the Chinese medicine solid residues, so that the solubility of various active components is greatly improved, and the extraction efficiency is improved.
The female vagina is a complex micro-ecological system, which can lead to bacterial vaginosis if pathogenic bacteria multiply. Bacterial vaginosis is mainly characterized by a reduced number of lactobacilli vaginalis and by the massive proliferation of other vaginal microorganisms such as gardnerella or mixed anaerobes that cause dysbacteriosis. The probiotics are planted into the vagina, so that the mildew and bacterial vaginosis can be effectively prevented and treated, the microecological balance of the vagina can be maintained after the planting, and pathogenic bacteria are prevented from being adhered by adhering human epithelial cells; the secretion of metabolic secretion such as lactic acid inhibits the growth of pathogenic bacteria, enhances the cleanliness of vagina, generates substances such as bacteriocin, hydrogen peroxide, biological surface active protein and the like, stimulates a host to generate local immune response, thereby eliminating part of pathogenic bacteria, recovering the microecology of the vagina and reducing the risk of vagina diseases.
Pathogenic bacteria, after invading the vagina, adhere to epithelial cells and form a competing relationship with the probiotics, reducing the number of the latter. Overgrowth of pathogenic bacteria results in a disruption of the equilibrium relationship of the microenvironment, ultimately leading to bacterial vaginosis. If the number of probiotics in the vagina is increased through field planting, the vaginal microecological balance is recovered, and the effect of preventing and treating bacterial vaginosis is achieved. Lactobacillus, including Lactobacillus plantarum and Lactobacillus paracasei according to the invention, may also reduce complications of bacterial vaginosis, such as venereal disease, infertility, premature delivery, etc., after colonization. Lactobacillus can decompose glycogen to produce lactic acid, maintain lower pH value of female vagina, and inhibit proliferation of pathogenic bacteria. The lactobacillus can also produce bacteriocin, surface active substances and other antibacterial substances. Bacteriocins have strong killing power on homologous and near-edge bacteria and even on microorganisms of other species, and have stronger killing power under acidic conditions; the surface active substances have broad-spectrum antibacterial effect. Compared with traditional antibiotics, lactobacillus has few side effects.
The ability of lactobacillus to adhere to these tissues is related to the presence of adhesion receptors such as glycoproteins, carbohydrates, etc. on the bacterial wall surface. In vitro experiments show that lactobacillus plantarum and lactobacillus paracasei strains can be well adhered to the cell surface, and especially have good adhesion to the epithelial cells of the urogenital tract. Lactobacillus plantarum is capable of producing bacteriocin-like substances other than lactic acid and hydrogen peroxide, which substances are capable of inhibiting the growth of uropathogenic E.coli. Lactobacillus plantarum can inhibit the activity of the escherichia coli adhesion factor (type 1 and p pili) promoters, up-regulate the activity of the 2 outer membrane porins involved in escherichia coli membrane stress, and all of these mechanisms are involved in inhibiting pathogenic bacterial infection.
Anaerobic pathogens are able to form biofilms, the difficult penetrability of which can lead to poor therapeutic effects of traditional antibiotics. The surface tension-reducing surface-active substances secreted by lactobacillus can destroy the adhesion of urinary tract pathogenic bacteria and the formation of biological films. The lactobacillus paracasei strain can generate surface active substances which can be combined with the epithelial cell collagens III and IV to generate more stable biological films, so that the biological films formed by gardnerella are removed and replaced. Treatment of candida-infected cells with lactobacillus paracasei strain supernatant up-regulates the production of interleukin 8 and chemokine 10 by the infected cells, attracting more immune cells to the infected site, helping to clear the infection quickly. Lactobacillus paracasei also can increase macrophage to produce anti-inflammatory cytokine IL-10, thereby reducing inflammatory reaction of organism, inhibiting lipopolysaccharide-induced tumor necrosis factor alpha generation, and increasing IL-10 generation through transcriptional activator 3 and mitogen activated protein kinase p38 phosphorylation.
The traditional Chinese medicine solid residue, the flos caryophylli and the rosemary solid residue are mixed and inoculated to the lactobacillus plantarum and the lactobacillus paracasei for fermentation, on one hand, the plant cell wall is broken through fermentation, the dissolution of active components is promoted, the extraction rate of the active components is improved, on the other hand, nutrients of the traditional Chinese medicine solid residue can promote the proliferation of the lactobacillus plantarum and the lactobacillus paracasei, meanwhile, the amounts of active substances produced by probiotics are promoted, the active substances are subjected to complex coacervation reaction, high molecular polymers with opposite charges form an embedding shell layer through electrostatic interaction or intermolecular hydrogen bond, hydrophobic bond and polarity induction, and meanwhile, calcium ions form a stable shell layer through promoting the crosslinking of sodium alginate, so that the prepared slow-release fermentation product microcapsule can resist severe environment, improve the survival rate of the probiotics, prolong the service cycle of the external lotion, and ensure good antibacterial and anti-inflammatory effects.
In addition, propolis and borneol are added into the lotion, wherein the propolis contains flavonoid, phenols, lactone, aldehyde, ketone, steroid and other compounds, is nontoxic, non-irritating and non-carcinogenic, has the effects of resisting bacteria, resisting viruses, stimulating immunity, easing pain, promoting tissue regeneration and the like, has the effects of moisturizing skin, promoting granulation, detoxifying, sterilizing, dispelling wind and relieving pain, has broad-spectrum antimicrobial effect, has antioxidant effect, enhances phagocytic function of macrophages, promotes apoptosis of tumor cells and the like. The borneol has the effects of clearing heat, relieving itching and promoting granulation, can also have the effects of clearing heat and easing pain, has cool feeling, and can further have the effects of relieving pain, diminishing inflammation, resisting oxidation and cooling by cooperation of the borneol and the borneol, so that the effect and experience of the lotion are enriched.
The gynecological external lotion prepared by the invention has the effects of diminishing inflammation, detoxifying, astringing, stopping bleeding and the like, does not produce side effects on human bodies, has good sterilization, anti-inflammation and detoxification effects, reduces vaginal lipstick swelling after use, eliminates purulent secretion, reduces inflammatory cells in vaginal epithelial tissues, has no damage to the vaginal epithelial tissues, can relieve vaginal tissue inflammation caused by bacteria, trichomonas, mould and other infections, promotes tissue repair, and has wide application prospect.
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.
Lactobacillus plantarum, JYLP-326, 100 hundred million cfu/g, lactobacillus paracasei, JLPF-176, 100 hundred million cfu/g, purchased from Jia Yi bioengineering Co., ltd.
The preparation method of the activated lactobacillus plantarum and lactobacillus paracasei strain seed solution comprises the following steps: inoculating Lactobacillus plantarum and Lactobacillus paracasei respectively to Gao's medium, and inoculating at 3v/v% CO 2 Fermenting and culturing at 37deg.C for 18-24 hr in nitrogen atmosphere at 50r/min to obtain strain seed solution with bacterial content of 10 8 -10 9 cfu/mL。
Example 1
The embodiment provides a preparation method of gynecological external lotion, which specifically comprises the following steps:
s1, supercritical fluid extraction: cleaning 5 parts by weight of flos caryophylli and 2 parts by weight of rosemary, crushing, drying, extracting by using a supercritical fluid, and preparing mixed essential oil by using ethyl acetate as an entrainer;
The conditions of the supercritical fluid extraction are the extraction pressure: 20MPa, extraction temperature: 35 ℃, CO 2 Flow rate: 7L/h, extraction time: 1h, wherein the adding amount of the entrainer is 2wt%;
s2, water extraction: cleaning, mixing and crushing 5 parts by weight of gallnut, 2 parts by weight of radix angelicae, 3 parts by weight of coptis chinensis, 1 part by weight of white phoenix-tail fern, 2 parts by weight of Chinese knotweed herb and 3 parts by weight of blumea balsamifera to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 2 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:5g/mL, repeatedly extracting for 2 times, filtering, reserving solid residues, merging filtrate, adding ethanol until the content of system ethanol is 50wt%, precipitating for 6 hours, collecting supernatant, recovering ethanol, and concentrating until the relative density is 1.05 to obtain concentrated solution;
s3, alcohol extraction: adding the solid slag obtained in the step S2 into an ethanol-propanol mixed water solution, wherein the solid-to-liquid ratio of the solid slag to the ethanol-propanol mixed water solution is 1:3g/mL, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed water solution is 30:15:50, heating to 50 ℃, extracting for 2 hours, filtering, reserving the solid slag, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
s4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 8 cfu/mL of activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, wherein the inoculation amount of the lactobacillus plantarum and lactobacillus paracasei strain seed liquid is 2 percent and 1 percent respectively, and the inoculation amount of CO is 3v/v percent 2 Fermenting and culturing for 36h at the temperature of 35 ℃ in the environment of 50r/min and the balance of nitrogen, and freeze-drying to obtain a fermentation product;
s5, embedding: adding 12 parts by weight of the fermentation product obtained in the step S4 into 100 parts by weight of water, adding 7 parts by weight of sodium alginate and 3 parts by weight of carrageenan, stirring and mixing for 20min to obtain a water phase, adding the water phase into fish oil, quickly emulsifying by a membrane, adjusting the pore diameter of the membrane to be 3 mu m, adjusting the pH value of the solution to be 4.2, stirring and reacting for 1h, adjusting the pH value to be 6.3, adding 7 parts by weight of glutamine transaminase and 2 parts by weight of calcium chloride, stirring and reacting for 20min, centrifuging, washing and freeze-drying to obtain a fermentation product microcapsule;
s6, preparing an additive: dissolving 10 parts by weight of propolis and 5 parts by weight of borneol in 30 parts by weight of ethanol to obtain an additive solution;
s7, preparing gynecological external lotion: adding 10 parts by weight of the additive solution prepared in the step S6 into 50 parts by weight of the concentrated solution prepared in the step S2, adding 3 parts by weight of PEG400 for dissolution, adding 3 parts by weight of the mixed essential oil prepared in the step S1, 5 parts by weight of the alcohol extract prepared in the step S3 and 1 part by weight of Tween-20, stirring and mixing for 30min, adding 4 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological external lotion.
Example 2
The embodiment provides a preparation method of gynecological external lotion, which specifically comprises the following steps:
s1, supercritical fluid extraction: cleaning 7 parts by weight of flos caryophylli and 2 parts by weight of rosemary, crushing, drying, extracting by using a supercritical fluid, and preparing mixed essential oil by using ethyl acetate as an entrainer;
the conditions of the supercritical fluid extraction are the extraction pressure: 30MPa, extraction temperature: 45 ℃, CO 2 Flow rate: 12L/h, extraction time: 3h, wherein the adding amount of the entrainer is 3wt%;
s2, water extraction: cleaning, mixing and crushing 7 parts by weight of gallnut, 3 parts by weight of radix angelicae, 5 parts by weight of coptis chinensis, 2 parts by weight of white phoenix-tail fern, 3 parts by weight of Chinese knotweed herb and 5 parts by weight of blumea balsamifera to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:10g/mL, repeatedly extracting for 3 times, filtering, reserving solid residues, combining filtrates, adding ethanol until the content of system ethanol is 60wt%, precipitating for 10 hours, collecting supernatant, recovering ethanol, and concentrating until the relative density is 1.08 to obtain concentrated solution;
s3, alcohol extraction: adding the solid slag obtained in the step S2 into an ethanol-propanol mixed water solution, wherein the solid-to-liquid ratio of the solid slag to the ethanol-propanol mixed water solution is 1:5g/mL, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed water solution is 40:20:70, heating to 70 ℃, extracting for 4 hours, filtering, reserving the solid slag, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
S4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 9 cfu/mL of activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, wherein the inoculation amount of the lactobacillus plantarum and lactobacillus paracasei strain seed liquid is 3 percent and 2 percent respectively, and the inoculation amount of CO is 5v/v percent 2 Fermenting and culturing for 48 hours at the temperature of 38 ℃ at 70r/min in the environment of nitrogen with the balance, and freeze-drying to obtain a fermentation product;
s5, embedding: adding 15 parts by weight of the fermentation product obtained in the step S4 into 100 parts by weight of water, adding 10 parts by weight of sodium alginate and 5 parts by weight of carrageenan, stirring and mixing for 20min to obtain a water phase, adding the water phase into fish oil, quickly emulsifying by a membrane, adjusting the pH value of the solution to 4.5 with the pore diameter of the membrane to be 5 mu m, stirring and reacting for 2h, adjusting the pH value to 6.7, adding 12 parts by weight of glutamine transaminase and 3 parts by weight of calcium chloride, stirring and reacting for 30min, centrifuging, washing and freeze-drying to obtain a fermentation product microcapsule;
s6, preparing an additive: dissolving 12 parts by weight of propolis and 7 parts by weight of borneol in 50 parts by weight of ethanol to obtain an additive solution;
s7, preparing gynecological external lotion: adding 12 parts by weight of the additive solution prepared in the step S6 into 70 parts by weight of the concentrated solution prepared in the step S2, adding 5 parts by weight of propylene glycol for dissolution, adding 5 parts by weight of the mixed essential oil prepared in the step S1, 6 parts by weight of the alcohol extract prepared in the step S3 and 2 parts by weight of tween-60, stirring and mixing for 30min, adding 6 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological lotion for external use.
Example 3
The embodiment provides a preparation method of gynecological external lotion, which specifically comprises the following steps:
s1, supercritical fluid extraction: cleaning 6 parts by weight of flos caryophylli and 2 parts by weight of rosemary, crushing, drying, extracting by using a supercritical fluid, and preparing mixed essential oil by using ethyl acetate as an entrainer;
the conditions of the supercritical fluid extraction are the extraction pressure: 25MPa, extraction temperature: 40 ℃, CO 2 Flow rate: 10L/h, extraction time: 2h, wherein the adding amount of the entrainer is 2.5wt%;
s2, water extraction: cleaning, mixing and crushing 6 parts by weight of gallnut, 2.5 parts by weight of angelica dahurica, 4 parts by weight of coptis chinensis, 1.5 parts by weight of white phoenix-tail fern, 2.5 parts by weight of Chinese knotweed herb and 4 parts by weight of blumea balsamifera to obtain traditional Chinese medicine powder, adding water to boil and extract for 2.5 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:7g/mL, repeatedly extracting for 3 times, filtering, retaining solid residues, merging filtrate, adding ethanol until the content of ethanol in the system is 55wt%, precipitating for 8 hours, taking supernatant, recovering ethanol, concentrating until the relative density is 1.07, and obtaining concentrated solution;
s3, alcohol extraction: adding the solid slag obtained in the step S2 into an ethanol-propanol mixed water solution, wherein the solid-to-liquid ratio of the solid slag to the ethanol-propanol mixed water solution is 1:4g/mL, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed water solution is 35:17:60, heating to 60 ℃, extracting for 3 hours, filtering, reserving the solid slag, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
S4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 9 cfu/mL of activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, wherein the inoculation amount of the lactobacillus plantarum and lactobacillus paracasei strain seed liquid is 2.5 percent and 1.5 percent respectively, and the inoculation amount of CO is 4v/v percent 2 Fermenting and culturing for 42h at the temperature of 37 ℃ in the environment of 60r/min with the balance of nitrogen, and freeze-drying to obtain a fermentation product;
s5, embedding: adding 13.5 parts by weight of the fermentation product obtained in the step S4 into 100 parts by weight of water, adding 8.5 parts by weight of sodium alginate and 4 parts by weight of carrageenan, stirring and mixing for 20min to obtain a water phase, adding the water phase into fish oil, quickly emulsifying by a membrane, adjusting the pore diameter of the membrane to 4 mu m, adjusting the pH value of the solution to 4.3, stirring and reacting for 1.5h, adjusting the pH value to 6.5, adding 10 parts by weight of glutamine transaminase and 2.5 parts by weight of calcium chloride, stirring and reacting for 25min, centrifuging, washing and freeze-drying to obtain a fermentation product microcapsule;
s6, preparing an additive: dissolving 11 parts by weight of propolis and 6 parts by weight of borneol in 40 parts by weight of ethanol to obtain an additive solution;
s7, preparing gynecological external lotion: adding 11 parts by weight of the additive solution prepared in the step S6 into 60 parts by weight of the concentrated solution prepared in the step S2, adding 4 parts by weight of propylene glycol for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1, 5.5 parts by weight of the alcohol extract prepared in the step S3 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, adding 5 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological lotion.
Comparative example 1
The difference from example 3 is that no flos Caryophylli was added in step S1.
The method comprises the following steps:
s1, supercritical fluid extraction: 8 parts by weight of rosemary is cleaned, crushed, dried, extracted by supercritical fluid, and ethyl acetate is taken as entrainer to prepare the mixed essential oil.
Comparative example 2
In comparison with example 3, the difference is that no rosemary was added in step S1.
The method comprises the following steps:
s1, supercritical fluid extraction: 8 parts by weight of flos caryophylli is washed, crushed, dried, extracted by supercritical fluid, and ethyl acetate is used as entrainer to prepare the mixed essential oil.
Comparative example 3
The difference from example 3 is that no mixed essential oil was added in step S7.
The method comprises the following steps:
s7, preparing gynecological external lotion: adding 11 parts by weight of the additive solution prepared in the step S6 into 60 parts by weight of the concentrated solution prepared in the step S2, adding 4 parts by weight of propylene glycol for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1, 5.5 parts by weight of the alcohol extract prepared in the step S3 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, adding 5 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological lotion.
Comparative example 4
Compared with example 3, the difference is that the ethanol-propanol mixed aqueous solution in step S3 is replaced by an equal amount of ethanol aqueous solution, and the mass ratio of ethanol to water in the ethanol aqueous solution is 52:60.
Comparative example 5
The difference compared to example 3 is that no alcohol extract was added in step S7.
The method comprises the following steps:
s7, preparing gynecological external lotion: adding 11 parts by weight of the additive solution prepared in the step S6 into 60 parts by weight of the concentrated solution prepared in the step S2, adding 4 parts by weight of propylene glycol for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, adding 5 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological external lotion.
Comparative example 6
The difference compared to example 3 is that lactobacillus plantarum was not inoculated in step S4.
The method comprises the following steps:
s4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 9 cfu/mL of activated Lactobacillus paracasei strain seed liquid, the inoculation amount of the Lactobacillus paracasei strain seed liquid is 4%, and the CO is 4v/v% 2 And (3) fermenting and culturing for 42 hours at the temperature of 37 ℃ in the environment of 60r/min with the balance of nitrogen, and freeze-drying to obtain a fermentation product.
Comparative example 7
The difference compared to example 3 is that lactobacillus paracasei was not inoculated in step S4.
The method comprises the following steps:
s4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 9 cfu/mL of activated lactobacillus plantarum seed solution, the inoculum size of the lactobacillus plantarum seed solution is 4 percent, and CO is 4v/v percent 2 And (3) fermenting and culturing for 42 hours at the temperature of 37 ℃ in the environment of 60r/min with the balance of nitrogen, and freeze-drying to obtain a fermentation product.
Comparative example 8
The difference compared to example 3 is that the fermentation product microcapsules are not added in step S7.
The method comprises the following steps:
s7, preparing gynecological external lotion: adding 11 parts by weight of the additive solution prepared in the step S6 into 60 parts by weight of the concentrated solution prepared in the step S2, adding 4 parts by weight of propylene glycol for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1, 5.5 parts by weight of the alcohol extract prepared in the step S3 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, and adding 1000W for ultrasonic dispersion for 15min to prepare the gynecological external lotion.
Comparative example 9
The difference compared to example 3 is that the fermentation product microcapsules added in step S7 are replaced by the fermentation product produced in step S4.
The method comprises the following steps:
s7, preparing gynecological external lotion: adding 11 parts by weight of the additive solution prepared in the step S6 into 60 parts by weight of the concentrated solution prepared in the step S2, adding 4 parts by weight of propylene glycol for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1, 5.5 parts by weight of the alcohol extract prepared in the step S3 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, adding 5 parts by weight of the fermentation product prepared in the step S4, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological external lotion.
Comparative example 10
The difference from example 3 is that no propolis was added in step S6.
The method comprises the following steps:
s6, preparing an additive: 17 parts by weight of borneol is dissolved in 40 parts by weight of ethanol to obtain an additive solution.
Comparative example 11
In comparison with example 3, the difference is that no borneol is added in step S6.
The method comprises the following steps:
s6, preparing an additive: 17 parts by weight of propolis was dissolved in 40 parts by weight of ethanol to obtain an additive solution.
Comparative example 12
The difference compared to example 3 is that no additive solution is added in step S7.
The method comprises the following steps:
s7, preparing gynecological external lotion: adding 4 parts by weight of propylene glycol into 60 parts by weight of the concentrated solution prepared in the step S2 for dissolution, adding 4 parts by weight of the mixed essential oil prepared in the step S1, 5.5 parts by weight of the alcohol extract prepared in the step S3 and 1.5 parts by weight of tween-80, stirring and mixing for 30min, adding 5 parts by weight of the fermentation product microcapsule prepared in the step S5, and performing 1000W ultrasonic dispersion for 15min to prepare the gynecological external lotion.
Test example 1 Sterilization experiment
Staphylococcus aureus (ATCC 29213), escherichia coli (ATCC 25922) and candida albicans (ATCC 90028) were selected as test bacteria. The gynecological external lotion prepared in examples 1 to 3 and comparative examples 1 to 12 of the present invention was diluted with distilled water at a ratio of 1:20 for a duration of 30s. 0.1ml of the bacterial liquid is added into 5.0ml of diluted washing liquid, evenly mixed and acted for a preset time, then 0.5ml of the bacterial liquid mixture is added into a test tube with 4.5ml of neutralizing agent (PBS solution of 0.5wt% Tween and 0.5wt% lecithin) for neutralization for 10min, and then living bacteria are cultivated and counted. Each group was repeated 3 times.
The results are shown in Table 1.
TABLE 1
As shown in the table above, the gynecological external lotion prepared in the examples 1-3 of the invention has good and rapid disinfection effect on staphylococcus aureus, escherichia coli and candida albicans.
Test example 2 stability test
The plastic bottles filled with the gynecological external washing solutions prepared in examples 1-3 and comparative examples 1-12 of the present invention were stored in a 54 ℃ incubator for 14 days, and the active ingredient content and the number of live probiotics were measured before and after the storage, respectively, and the degradation rate and the survival rate of the probiotics were calculated.
Survival (%) =n t /N 0 ×100％
Wherein N is t To the probiotic concentration (cfu/g) surviving the experiment, N 0 The original concentration (cfu/g) of probiotic surviving before the experiment.
Degradation rate (%) = (1-W) t /W 0 )×100％
In which W is t To the concentration of the effective ingredient (cfu/g) after the experiment, W 0 The original concentration (cfu/g) of the effective components before the experiment.
The results are shown in Table 2.
TABLE 2
As can be seen from the above table, the gynecological external lotion prepared in examples 1-3 of the present invention has good stability.
Test example 3
Preparing candida albicans fungus liquid: the standard strain candida albicans (CMCC 98001) is cultured in a glucose liquid culture medium, and the concentration of the bacterial liquid is adjusted to be 1.7X10 during inoculation 8 cfu/mL。
And (3) molding: after SPF-grade Kunming female mice were adaptively bred for 1 week, 10 female mice were randomly selected as normal groups, and the remaining mice were subcutaneously injected with estradiol benzoate at 0.05mL each and with a double-antibody solution (penicillin and streptomycin mixture, containing 10000U/mL penicillin and 10mg/mL streptomycin) at intervals of 2d 1 for 3 consecutive times. After the last injection, the mice are washed 3 times with 0.9wt% sodium chloride solution at about 1cm in the vagina, then 0.03mL of bacterial liquid is injected, and the animals are placed obliquely according to the head-low-tail high-position for 5min and then placed back into the cages. Molding 1 time every 2d, and continuously 3 times. Taking vaginal secretion at 14 days after the first inoculation, performing microscopic examination, finding candida albicans as positive, culturing the secretion by using a glucose agar culture medium of Saccharomycetes, confirming the existence of candida albicans, combining with naked eyes to observe the occurrence of inflammation such as erythema, edema, erosion, secretion increase and the like of vaginal mucosa, and determining that the modeling is successful.
Grouping: grouping small random groups with successful molding into a model group, a gynecological inflammation cleaning lotion group (10 mL/kg), examples 1-3 groups (10 mL/kg of gynecological cleaning lotion correspondingly prepared), and comparative examples 1-12 groups (10 mL/kg of gynecological cleaning lotion correspondingly prepared); 10 mice per group, all mice from the experimental group were rinsed vaginally, and the wash was diluted 20-fold with water, 2 times daily, and continuously dosed for 14d. Normal and model groups were given equal amounts of physiological saline.
The inflammatory response scoring criteria are shown in table 3.
TABLE 3 Table 3
The results of edema, secretion sand culture medium culture scoring for each group of mice after the experiment are shown in Table 4.
TABLE 4 Table 4
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
As is clear from the above table, the gynecological external lotion prepared in the examples 1-3 can well eliminate symptoms of colpitis such as edema and excessive secretion, and reduce the bacterial content in the secretion.
Determination of NO, IL-6 levels in serum of vaginitis mice: mice after the last administration were fasted for 12 hours, blood was collected from eyeballs, serum was isolated, and the contents of NO and IL-6 in the serum were measured, and the results are shown in Table 5.
TABLE 5
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
As shown in the table above, the gynecological external washing solutions prepared in examples 1-3 of the present invention can well reduce the decrease of serum NO and IL-6.
In comparative examples 1 and 2, compared with example 3, no flos Caryophylli or herba Rosmarini officinalis was added in step S1. Comparative example 3 in comparison with example 3, no mixed essential oil was added in step S7. The sterilization rate is reduced, and the inflammation score is improved. Anaerobic pathogenic bacteria such as candida albicans are taken as conditional pathogenic bacteria, the bacteria are easy to form a biological film on various biological materials, the drug resistance of candida albicans is greatly enhanced after the biological film is formed, and the sensitivity of the candida albicans is greatly reduced to clinically common candida albicans resisting drugs such as clotrimazole and the like. Flos Caryophylli and herba Rosmarini officinalis are rich in volatile oil, mainly contain eugenol and rosmarinic acid, and have effects on membrane permeability of Candida albicans, so that Candida albicans content is leaked, microorganism death is caused, and good killing effect on trichomonas and mould is also achieved.
Comparative example 4 compared with example 3, the ethanol-propanol mixed aqueous solution in step S3 was replaced with an ethanol aqueous solution in which the mass ratio of ethanol to water was 52:60. The sterilization rate is reduced, the stability is reduced, the inflammation score is improved, and the NO and IL-6 contents are improved. Comparative example 5 in contrast to example 3, no alcohol extract was added in step S7. The invention extracts gallnut, angelica dahurica, coptis chinensis, white phoenix-tail fern, chinese knotweed and blumea balsamifera with water to obtain a large amount of water-soluble antibacterial and anti-inflammatory active components, extracts a large amount of total flavonoids in the traditional Chinese medicine components by ethanol-propanol mixed water solution, has low toxicity and no irritation of propylene glycol, has better solubility for a plurality of organic active components, and has a certain promoting effect on the absorption of the active components on skin and mucous membrane. Therefore, the ethanol-propanol mixed aqueous solution is used for extracting the Chinese medicine solid residues, so that the solubility of various active components is greatly improved, and the extraction efficiency is improved.
Comparative examples 6 and 7 in comparison with example 3, lactobacillus plantarum or lactobacillus paracasei was not inoculated in step S4. Comparative example 8 in contrast to example 3, no fermentation product microcapsules were added in step S7. The sterilization rate is reduced, the inflammation score is improved, and the NO and IL-6 contents are improved. Pathogenic bacteria, after invading the vagina, adhere to epithelial cells and form a competing relationship with the probiotics, reducing the number of the latter. Overgrowth of pathogenic bacteria results in a disruption of the equilibrium relationship of the microenvironment, ultimately leading to bacterial vaginosis. If the number of probiotics in the vagina is increased through field planting, the vaginal microecological balance is recovered, and the effect of preventing and treating bacterial vaginosis is achieved. Lactobacillus, including Lactobacillus plantarum and Lactobacillus paracasei according to the invention, may also reduce complications of bacterial vaginosis, such as venereal disease, infertility, premature delivery, etc., after colonization. Lactobacillus can decompose glycogen to produce lactic acid, maintain lower pH value of female vagina, and inhibit proliferation of pathogenic bacteria. The lactobacillus can also produce bacteriocin, surface active substances and other antibacterial substances. Bacteriocins have strong killing power on homologous and near-edge bacteria and even on microorganisms of other species, and have stronger killing power under acidic conditions; the surface active substances have broad-spectrum antibacterial effect. Compared with traditional antibiotics, lactobacillus has few side effects. The ability of lactobacillus to adhere to these tissues is related to the presence of adhesion receptors such as glycoproteins, carbohydrates, etc. on the bacterial wall surface. In vitro experiments show that lactobacillus plantarum and lactobacillus paracasei strains can be well adhered to the cell surface, and especially have good adhesion to the epithelial cells of the urogenital tract. Lactobacillus plantarum is capable of producing bacteriocin-like substances other than lactic acid and hydrogen peroxide, which substances are capable of inhibiting the growth of uropathogenic E.coli. Lactobacillus plantarum can inhibit the activity of the escherichia coli adhesion factor (type 1 and p pili) promoters, up-regulate the activity of the 2 outer membrane porins involved in escherichia coli membrane stress, and all of these mechanisms are involved in inhibiting pathogenic bacterial infection. Anaerobic pathogens are able to form biofilms, the difficult penetrability of which can lead to poor therapeutic effects of traditional antibiotics. The surface tension-reducing surface-active substances secreted by lactobacillus can destroy the adhesion of urinary tract pathogenic bacteria and the formation of biological films. The lactobacillus paracasei strain can generate surface active substances which can be combined with the epithelial cell collagens III and IV to generate more stable biological films, so that the biological films formed by gardnerella are removed and replaced. Treatment of candida-infected cells with lactobacillus paracasei strain supernatant up-regulates the production of interleukin 8 and chemokine 10 by the infected cells, attracting more immune cells to the infected site, helping to clear the infection quickly. Lactobacillus paracasei also can increase macrophage to produce anti-inflammatory cytokine IL-10, thereby reducing inflammatory reaction of organism, inhibiting lipopolysaccharide-induced tumor necrosis factor alpha generation, and increasing IL-10 generation through transcriptional activator 3 and mitogen activated protein kinase p38 phosphorylation. The traditional Chinese medicine solid residue, the flos caryophylli and the rosemary solid residue are mixed and inoculated to the lactobacillus plantarum and the lactobacillus paracasei for fermentation, on one hand, the plant cell wall can be broken through fermentation, the dissolution of active components is promoted, the extraction rate of the active components is improved, and on the other hand, the nutrient substances can promote the proliferation of the lactobacillus plantarum and the lactobacillus paracasei, and meanwhile, the amount of active substances produced by probiotics is promoted.
Comparative example 9 compared with example 3, the fermentation product microcapsules added in step S7 were replaced with the fermentation product produced in step S4. The sterilization rate is reduced, the stability is reduced, the inflammation score is improved, and the NO and IL-6 contents are improved. The fermentation product of the invention forms an embedding shell layer through complex coacervation reaction and electrostatic interaction or intermolecular hydrogen bond, hydrophobic bond and polarity induction between high molecular polymers, and meanwhile, calcium ions form a stable shell layer through enhancing the crosslinking of sodium alginate, so that the prepared slow-release fermentation product microcapsule can resist severe environment, improve the survival rate of probiotics, prolong the service cycle of the external washing liquid and ensure good antibacterial and anti-inflammatory effects.
In comparative examples 10 and 11, no propolis or borneol was added in step S6, as compared with example 3. Comparative example 12 compared with example 3, no additive solution was added in step S7. The sterilization rate is reduced, the inflammation score is improved, and the NO and IL-6 contents are improved. Propolis and borneol are also added in the lotion, wherein the propolis contains flavonoid, phenols, lactone, aldehyde, ketone, steroid and other compounds, is nontoxic, nonirritating and cancerogenic, has the effects of resisting bacteria, resisting viruses, stimulating immunity, easing pain, promoting tissue regeneration and the like, has the effects of moisturizing skin, promoting granulation, detoxifying, sterilizing, dispelling wind and relieving pain, has broad-spectrum antimicrobial effect, has antioxidant effect, enhances phagocytic function of macrophages, promotes apoptosis of tumor cells and the like. The borneol has the effects of clearing heat, relieving itching and promoting granulation, can also have the effects of clearing heat and easing pain, has cool feeling, and can further have the effects of relieving pain, diminishing inflammation, resisting oxidation and cooling by cooperation of the borneol and the borneol, so that the effect and experience of the lotion are enriched.
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 (8)
1. The preparation method of the gynecological external lotion for resisting bacteria and diminishing inflammation is characterized by comprising the following steps of:
s1, supercritical fluid extraction: cleaning flos Caryophylli and herba Rosmarini officinalis, pulverizing, drying, extracting with supercritical fluid, and extracting with ethyl acetate as entrainer to obtain mixed essential oil; the mass ratio of the flos caryophylli to the rosemary is 5-7:2;
s2, water extraction: cleaning Galla chinensis, radix Angelicae Dahuricae, coptidis rhizoma, herba Pteridis Multifidae, polygoni chinensis, and herba Blumeae Balsamiferae, mixing, pulverizing to obtain Chinese medicinal powder, boiling with water, extracting for 2-3 times, filtering, collecting residue, mixing filtrates, precipitating with ethanol, collecting supernatant, recovering ethanol, and concentrating to obtain concentrated solution; the mass ratio of the Chinese gall, the dahurian angelica root, the golden thread, the white phoenix-colored cabbage, the charcoal mother and the blumea balsamifera is 5-7:2-3:3-5:1-2:2-3:3-5, and the solid-liquid ratio of the Chinese medicinal powder and the water is 1:5-10g/mL;
s3, alcohol extraction: adding the solid residue obtained in the step S2 into an ethanol-propanol mixed aqueous solution, heating and extracting, filtering, reserving the solid residue, recovering ethanol from the filtrate, and drying to obtain an alcohol extract; the mass ratio of ethanol to propanol to water in the ethanol-propanol mixed aqueous solution is 30-40:15-20:50-70, the solid-liquid ratio of the solid slag to the ethanol-propanol mixed aqueous solution is 1:3-5g/mL, the heating extraction temperature is 50-70 ℃ and the time is 2-4h;
S4, fermenting: adding the rest solid in the step S1 and the solid residue in the step S3 into water, sterilizing, inoculating activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, performing anaerobic fermentation culture, and freeze-drying to obtain a fermentation product;
s5, embedding: adding the fermentation product obtained in the step S4 into water, adding sodium alginate and carrageenan, stirring and mixing uniformly to obtain a water phase, adding the water phase into fish oil, quickly emulsifying by a membrane, regulating the pH value of the solution to be a first pH value, stirring and reacting, regulating the pH value to be a second pH value, adding glutamine transaminase and calcium chloride, stirring and reacting, centrifuging, washing and freeze-drying to obtain a fermentation product microcapsule;
s6, preparing an additive: dissolving propolis and Borneolum Syntheticum in ethanol to obtain additive solution;
s7, preparing antibacterial and anti-inflammatory gynecological external lotion: adding the additive solution prepared in the step S6 into the concentrated solution prepared in the step S2, adding PEG400 or propylene glycol for dissolution, adding the mixed essential oil prepared in the step S1, the alcohol extract prepared in the step S3 and the surfactant, stirring and mixing uniformly, adding the fermentation product microcapsule prepared in the step S5, and performing ultrasonic dispersion uniformly to prepare the antibacterial and anti-inflammatory gynecological external lotion, wherein the mass ratio of the additive solution to the concentrated solution to the PEG400 or the propylene glycol to the mixed essential oil to the alcohol extract to the surfactant to the fermentation product microcapsule is 10-12:50-70:3-5:3-5:5-6:1-2:4-6.
2. The method according to claim 1, wherein the conditions for supercritical fluid extraction in step S1 are extraction pressure: 20-30MPa, extraction temperature: 35-45 ℃, CO 2 Flow rate: 7-12L/h, extraction time: 1-3h, wherein the adding amount of the entrainer is 2-3wt%; the boiling extraction time in the step S2 is 2-3h, the content of the ethanol added to the system is 50-60wt% and the relative density of the concentrated solution is 1.05-1.08.
3. The preparation method according to claim 1, characterized in thatIn the step S4, the seed solution of Lactobacillus plantarum and Lactobacillus paracasei has a bacterial content of 10 8 -10 9 cfu/mL, wherein the mass ratio of the solid remained in the step S1 to the solid slag and water in the step S3 is 5-7:12-15:120-150, the inoculation amounts of lactobacillus plantarum and lactobacillus paracasei strain seed liquid are 2-3% and 1-2%, respectively, and the anaerobic fermentation culture condition is that CO is 3-5v/v% 2 And fermenting and culturing for 36-48h at the temperature of 35-38 ℃ in the environment of 50-70r/min with the balance of nitrogen.
4. The preparation method according to claim 1, wherein the mass ratio of the fermentation product, sodium alginate, carrageenan, glutamine transaminase and calcium chloride in the step S5 is 12-15:7-10:3-5:7-12:2-3, the pore diameter of the membrane emulsified membrane is 3-5 μm, the first pH value is 4.2-4.5, and the second pH value is 6.3-6.7.
5. The preparation method according to claim 1, wherein the mass ratio of propolis, borneol and ethanol in step S6 is 10-12:5-7:30-50.
6. The method according to claim 1, wherein the surfactant in step S7 is at least one selected from the group consisting of tween-20, tween-40, tween-60, tween-80, span-20, span-40, span-60, span-80.
7. The preparation method according to claim 1, comprising the specific steps of:
s1, supercritical fluid extraction: cleaning 5-7 parts by weight of flos caryophylli and 2 parts by weight of rosemary, crushing, drying, extracting by supercritical fluid, and taking ethyl acetate as an entrainer to prepare mixed essential oil;
the conditions of the supercritical fluid extraction are the extraction pressure: 20-30MPa, extraction temperature: 35-45 ℃, CO 2 Flow rate: 7-12L/h, extraction time: 1-3h, wherein the adding amount of the entrainer is 2-3wt%;
s2, water extraction: cleaning 5-7 parts by weight of gallnut, 2-3 parts by weight of angelica dahurica, 3-5 parts by weight of coptis chinensis, 1-2 parts by weight of white phoenix-headed vegetable, 2-3 parts by weight of Chinese knotweed herb and 3-5 parts by weight of blumea balsamifera, mixing, crushing to obtain Chinese medicinal powder, adding water, boiling and extracting for 2-3 hours, wherein the solid-to-liquid ratio of the Chinese medicinal powder to the water is 1:5-10g/mL, repeatedly extracting for 2-3 times, filtering, retaining solid residues, mixing filtrate, adding ethanol until the content of system ethanol is 50-60wt%, precipitating for 6-10 hours, collecting supernatant, recovering ethanol, and concentrating until the relative density is 1.05-1.08 to obtain concentrated solution;
S3, alcohol extraction: adding the solid slag obtained in the step S2 into an ethanol-propanol mixed water solution, wherein the solid-to-liquid ratio of the solid slag to the ethanol-propanol mixed water solution is 1:3-5g/mL, the mass ratio of ethanol, propanol and water in the ethanol-propanol mixed water solution is 30-40:15-20:50-70, heating to 50-70 ℃, extracting for 2-4h, filtering, reserving the solid slag, recovering ethanol from the filtrate, and drying to obtain an alcohol extract;
s4, fermenting: adding the rest solid in the step S1 and the solid slag in the step S3 into water, sterilizing, and inoculating with a bacterial content of 10 8 -10 9 cfu/mL of activated lactobacillus plantarum and lactobacillus paracasei strain seed liquid, wherein the inoculation amount of the lactobacillus plantarum and lactobacillus paracasei strain seed liquid is 2-3% and 1-2%, and the inoculation amount of CO is 3-5v/v%, respectively 2 Fermenting and culturing for 36-48h at the temperature of 35-38 ℃ in the environment of 50-70r/min with the balance of nitrogen, and freeze-drying to obtain a fermentation product;
s5, embedding: adding 12-15 parts by weight of the fermentation product obtained in the step S4 into 100 parts by weight of water, adding 7-10 parts by weight of sodium alginate and 3-5 parts by weight of carrageenan, stirring and mixing uniformly to obtain a water phase, adding the water phase into fish oil, emulsifying by a rapid membrane, adjusting the pore diameter of the membrane to 3-5 mu m, adjusting the pH value of the solution to 4.2-4.5, stirring and reacting for 1-2 hours, adjusting the pH value to 6.3-6.7, adding 7-12 parts by weight of glutamine transaminase and 2-3 parts by weight of calcium chloride, stirring and reacting for 20-30 minutes, centrifuging, washing, and freeze-drying to obtain the fermentation product microcapsule;
S6, preparing an additive: dissolving 10-12 parts by weight of propolis and 5-7 parts by weight of borneol in 30-50 parts by weight of ethanol to obtain an additive solution;
s7, preparing antibacterial and anti-inflammatory gynecological external lotion: adding 10-12 parts by weight of the additive solution prepared in the step S6 into 50-70 parts by weight of the concentrated solution prepared in the step S2, adding 3-5 parts by weight of PEG400 or propylene glycol for dissolution, adding 3-5 parts by weight of the mixed essential oil prepared in the step S1, 5-6 parts by weight of the alcohol extract prepared in the step S3 and 1-2 parts by weight of the surfactant, stirring and mixing uniformly, adding 4-6 parts by weight of the fermentation product microcapsule prepared in the step S5, and dispersing uniformly by ultrasonic waves to prepare the gynecological external lotion for resisting bacteria and diminishing inflammation.
8. An antibacterial and antiinflammatory gynecological lotion prepared by the preparation method as claimed in any one of claims 1 to 7.
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