CN110743005A

CN110743005A - Traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma and preparation method thereof

Info

Publication number: CN110743005A
Application number: CN201911117090.1A
Authority: CN
Inventors: 杨爱岗
Original assignee: Shanghai Songhao Biotechnology Co Ltd
Current assignee: Shanghai Songhao Biotechnology Co Ltd
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-02-04

Abstract

The invention relates to the related field of treating chloasma, and particularly provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma and a preparation method thereof. The invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which comprises the following raw materials, by weight, 3-15% of a water phase component, 0.5-3% of an oil phase component, 3-12% of recombinant hirudin fermentation liquor, 0.5-2% of a traditional Chinese medicine component, 0.1-1% of an auxiliary agent and the balance water; wherein the aqueous phase component comprises arginine. Compared with the prior art, the external composition for treating chloasma has a good treatment effect on different age groups by combining the recombinant hirudin fermentation broth and the traditional Chinese medicine components, has stable physicochemical properties and good storage stability, and can well prolong the storage time of related products.

Description

Traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma and preparation method thereof

Technical Field

The invention relates to the related field of treating chloasma, and particularly provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma and a preparation method thereof.

Background

Chloasma is a common facial pigmented skin disorder with symmetrical, well-defined, pale brown, brown or light black patches of skin lesions. The disease is frequently seen in middle-aged and young women, is in a chronic process, has a stubborn course of disease and is lingering and difficult to heal, seriously influences the beauty of patients and brings much trouble and pain to the spirit and life of the patients. The incidence of this disease is high, and in recent years, the incidence of the disease tends to increase year by year due to the influence of various factors, and the number of patients is increasing. With the change of living environment and the improvement of living standard of people, chloasma is becoming one of the hot spots of common attention and research in the medical and cosmetic fields at present.

The pathogenesis of chloasma is relatively complex, and the exact pathogenesis of chloasma is not known so far. At present, the increase of estrogen and progesterone is considered to stimulate melanocyte to cause chloasma, and factors such as abnormal indexes of hemorheology, increase of blood viscosity and the like are also considered. Domestic and foreign research shows that the disease is generally related to pregnancy, oral contraceptives, ultraviolet radiation, oxygen free radicals, estrogen, high progestational hormone level, mental factors, ultraviolet radiation, improper use of cosmetics and the like. Endocrine disturbance, heredity and ultraviolet irradiation are the main causes of diseases.

In view of the current situation of treating chloasma, western medicine treatment mostly adopts an external treatment method, has certain effect, but can generate certain toxic and side effects after long-term use, such as 'spot-like' caused by exogenous chloasma, skin atrophy and permanent decoloration, pigment rebound and the like. The traditional Chinese medicine treatment shows certain advantages, has the advantages of prominent overall regulation, stable curative effect, small side effect, easy acceptance by patients and the like, and becomes an effective ideal way for treating chloasma. Some traditional Chinese medicines need to be taken orally, and have the problems of bitter taste, slow response, increase of intestinal tract and liver burden and the like.

The invention discloses a composition for treating facial chloasma by combining traditional Chinese medicine components with hirudin biological fermentation broth, a preparation method and application.

Disclosure of Invention

In order to solve the technical problems, the first aspect of the invention provides a traditional Chinese medicine composition for external use containing recombinant hirudin for treating chloasma, which comprises the following raw materials, by weight, 3-15% of a water phase component, 0.5-3% of an oil phase component, 3-12% of recombinant hirudin fermentation liquor, 0.5-2% of a traditional Chinese medicine component, 0.1-1% of an auxiliary agent and the balance water; wherein the aqueous phase component comprises arginine.

As a preferred technical scheme of the invention, the preparation raw materials of the traditional Chinese medicine component comprise a component A and a component B, wherein the component A comprises at least one of angelica, ligusticum chuanxiong hort, safflower, peach kernel, white paeony root, prepared rehmannia root, red paeony root and twotooth achyranthes root; the component B comprises at least one of radix Platycodi, bupleuri radix, fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, radix Codonopsis, Atractylodis rhizoma, Coicis semen, Poria, rhizoma Cyperi, and fructus Hordei Germinatus.

As a preferable technical solution of the present invention, wherein the aqueous phase component further comprises a small molecule alcohol and a complexing agent.

As a preferable technical scheme of the present invention, the oil phase component includes a humectant, and the humectant is selected from any one or a combination of a plurality of xylitol substances, glycolic acid, xanthan gum, glyceryl caprylate, polyethylene glycol, trehalose, erythritol, sodium pyrrolidone carboxylate, ethoxy glucose derivatives, and sodium hyaluronate.

As a preferable technical scheme of the invention, the oil phase component further comprises flavonoid and/or a sulfhydryl-containing compound.

As a preferred technical scheme of the invention, the preparation process of the recombinant hirudin fermentation liquor comprises the following steps: placing the seed solution in an initial culture medium, and dropwise adding a feed supplement culture medium when the DO value is more than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

In a preferred embodiment of the present invention, the sugar solution comprises an amino-free yeast nitrogen source, a carbon source and an amino acid solution.

As a preferable technical solution of the present invention, the trace metal solution includes at least one of zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, and copper salt.

The second aspect of the invention provides a preparation of the traditional Chinese medicine external composition containing the recombinant hirudin for treating chloasma.

The third aspect of the invention provides a preparation method of the traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which comprises the following steps:

(1) mixing the water phase component and water uniformly, and heating;

(2) mixing the oil phase components uniformly, and heating;

(3) adding the oil phase component into the water phase component, and shearing at high speed;

(4) cooling, and adding recombinant hirudin fermentation liquid and Chinese medicinal components;

(5) cooling, adding adjuvant, and stirring.

Compared with the prior art, the external composition for treating chloasma has a good treatment effect on different age groups by combining the recombinant hirudin fermentation broth and the traditional Chinese medicine components, has stable physicochemical properties and good storage stability, and can well prolong the storage time of related products.

Drawings

FIG. 1: facial condition before and after the first volunteer used the composition of example 1;

FIG. 2: the second volunteer used the facial condition before and after the composition of example 1.

Detailed Description

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto.

The words "preferred", "preferably", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention. The sources of components not mentioned in the present invention are all commercially available.

The invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which comprises the following raw materials, by weight, 3-15% of a water phase component, 0.5-3% of an oil phase component, 3-12% of recombinant hirudin fermentation liquor, 0.5-2% of a traditional Chinese medicine component, 0.1-1% of an auxiliary agent and the balance water; preferably, the preparation raw materials comprise 5-10% of water phase components, 1-2% of oil phase components, 5-10% of recombinant hirudin fermentation liquor, 1-1.5% of traditional Chinese medicine components, 0.3-0.8% of auxiliary agents and the balance of water; more preferably, the preparation raw materials comprise 7.5% of water phase components, 1.5% of oil phase components, 8% of recombinant hirudin fermentation liquor, 1.2% of traditional Chinese medicine components, 0.7% of auxiliary agents and the balance of water.

In one embodiment, the Chinese medicinal component is prepared from a raw material comprising a component A and a component B, wherein the component A comprises at least one of angelica, ligusticum chuanxiong hort, safflower, codonopsis pilosula, bighead atractylodes rhizome, coix seed, platycodon grandiflorum, radix bupleuri, radix paeoniae rubra and radix achyranthis bidentatae; the component B comprises at least one of fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, semen Persicae, radix Paeoniae alba, radix rehmanniae Preparata, Poria, rhizoma Cyperi, and fructus Hordei Germinatus; preferably, the weight ratio of the component A to the component B is 1: (0.8 to 1.2); more preferably, the weight ratio of the component a to the component B is 1: 1.

in one embodiment, the component A comprises angelica, ligusticum chuanxiong hort, safflower, platycodon grandiflorum and radix bupleuri, and the component B comprises fructus aurantii, liquorice, motherwort and peach kernel; the weight ratio of the angelica, the ligusticum chuanxiong hort, the safflower, the platycodon grandiflorum and the radix bupleuri is 1: (0.8-1.2): (0.8-1.2): (0.8 to 1.2); the weight ratio of the bitter orange, the liquorice, the motherwort and the peach kernel is 1: (0.8-1.2): (0.8-1.2): (0.8 to 1.2); more preferably, the weight ratio of the angelica, the ligusticum chuanxiong hort, the safflower, the platycodon grandiflorum and the radix bupleuri is 1: 1: 1: 1; the weight ratio of the bitter orange, the liquorice, the motherwort and the peach kernel is 1: 1: 1: 1.

in one embodiment, the component A comprises safflower, radix codonopsitis, rhizoma atractylodis macrocephalae and semen coicis, and the component B comprises tuckahoe, rhizoma cyperi and malt; preferably, the weight ratio of the safflower to the codonopsis pilosula to the atractylodes macrocephala koidz to the coix seed is 1: (0.8-1.2): (0.8-1.2): (0.8 to 1.2); the weight ratio of the tuckahoe, the rhizoma cyperi and the malt is 1: (0.8-1.2): (0.8 to 1.2); more preferably, the weight ratio of the safflower to the codonopsis pilosula to the atractylodes macrocephala koidz to the coix seed is 1: 1: 1: 1; the weight ratio of the tuckahoe, the rhizoma cyperi and the malt is 1: 1: 1.

in one embodiment, the aqueous phase component comprises arginine; preferably, the water phase component also comprises small molecule alcohol and a complexing agent; further preferably, the weight ratio of the small molecule alcohol to the complexing agent is 1: (0.01 to 0.003); more preferably, the weight ratio of the small molecule alcohol to the complexing agent is 1: 0.005.

in one embodiment, the small molecule alcohol is selected from any one or combination of propylene glycol, 1, 3-butylene glycol, 1, 2-hexanediol, glycerol; preferably, the small molecule alcohol is 1, 3-butanediol and 1, 2-hexanediol; further preferably, the weight ratio of 1, 3-butanediol to 1, 2-hexanediol is 1: (0.05-0.2); more preferably, the weight ratio of 1, 3-butanediol to 1, 2-hexanediol is 1: 0.1.

in one embodiment, the complexing agent is disodium EDTA and/or tetrasodium EDTA; preferably, the complexing agent is disodium EDTA.

In one embodiment, the weight ratio of arginine to complexing agent is 1: (1-2); preferably, the weight ratio of arginine to complexing agent is 1: 1.5.

in one embodiment, the oil phase component comprises a humectant selected from any one or a combination of xylitol, glycolic acid, xanthan gum, glyceryl caprylate, polyethylene glycol, trehalose, erythritol, sodium pyrrolidone carboxylate, ethoxylated glucose derivatives, sodium hyaluronate; preferably, the humectant is xylitol, xanthan gum and sodium hyaluronate; further preferably, the weight ratio of the xylitol substances, the xanthan gum and the sodium hyaluronate is 1: (0.1-0.6): (0.05-0.15); more preferably, the weight ratio of the xylitol substances, the xanthan gum and the sodium hyaluronate is 1: 0.35: 0.1.

in one embodiment, the xylitol-based material includes xylitol, anhydrous xylitol, and xylitol-based glucoside, the present invention is not limited by the manufacturer of the xylitol-based material, and in one embodiment, the xylitol-based material is purchased from SEPPIC, France, AQUAXYL xylitol.

The invention is not particularly limited to the manufacturer of sodium hyaluronate, and in one embodiment, sodium hyaluronate NanoHA is purchased from Huaxi Furida.

In one embodiment, the oil phase component further comprises flavonoids and/or mercapto compounds; preferably, the weight ratio of the flavonoid substance, the sulfhydryl-containing compound and the sodium hyaluronate is (0.005-0.02): (0.005-0.02): 1; more preferably, the weight ratio of the flavonoid substance, the sulfhydryl-containing compound and the sodium hyaluronate is (0.008-0.015): (0.008-0.015): 1; more preferably, the weight ratio of the flavonoid substance, the sulfhydryl-containing compound and the sodium hyaluronate is 0.01: 0.01: 1.

in one embodiment, the flavonoid is selected from any one of glabridin, flavanone, flavanonol, isoflavone, dihydroisoflavone, chalcone, and dihydrochalcone.

In one embodiment, the thiol-containing compound is selected from any one of ergothioneine, tiopronin, penicillamine, cysteine.

In one embodiment, the method of preparing the herbal composition is as follows:

(1) soaking the raw materials for preparing the traditional Chinese medicine components in cold water, and drying for later use;

(2) putting the product obtained in the step (1) into distilled water for cooking, filtering to obtain filtrate and filter residue, and concentrating the filtrate to obtain a component A extracting solution;

(3) and (3) putting the filter residue obtained in the step (2) into a mixed solution of ethanol \ distilled water, performing reflux extraction, and concentrating to obtain a component B extracting solution, wherein the volume ratio of ethanol to distilled water is 1: (3-5).

Radix Angelicae sinensis

The product is dried root of Angelica sinensis (Oliv.) Diels) of Umbelliferae. Digging in late autumn, removing fibrous root and silt, bundling into small bundle after water is slightly evaporated, putting on shed, and slowly smoking with firework. Dried root of Angelica sinensis (Oliv.) Diels of Umbelliferae. Digging in late autumn, removing fibrous root and silt, bundling into small bundle after water is slightly evaporated, putting on shed, and slowly smoking with firework. The root of the whole angelica root is slightly cylindrical, and the upper end of the root is called 'return'; the main root is called "returning to the body" or "cun-sheng". The branch root is called the tail or leg, and the whole is called the whole return. Whole Chinese angelica can nourish blood and promote blood circulation, which is commonly called as blood-harmonizing; dang Gui can nourish blood and Dang Gui can break blood at its tail. Angelica sinensis enriches the blood; promoting blood circulation; regulating menstruation and relieving pain; moistening dryness and smoothing intestine. The main symptoms are blood deficiency; menoxenia; amenorrhea; dysmenorrhea; the accumulation of symptoms; (ii) metrorrhagia and metrostaxis; abdominal pain due to deficiency-cold; flaccidity and paralysis; numbness of the skin; intestinal dryness and difficult defecation; severe dysentery with diarrhea; carbuncle, cellulitis, sore and ulcer; injury from falling.

Ligusticum wallichii

The other names are the ligusticum wallichii tip and the small-leaf ligusticum wallichii; the product is dried rhizome of Ligusticum chuanxiong Hort (or Ligusticum wallichi Franch) belonging to Umbelliferae. In summer, when the node disc on the stem is prominent and is slightly purple, the stem is dug, silt is removed, the stem is dried in the sun, and fibrous roots are removed. For common cold with headache due to wind-cold, it can be used with Jing Jie, Fang Feng and Bai Zhi; for wind-heat syndrome, it is combined with Ju Hua and Bo He. The effect of the ligusticum wallichii on human bodies is very much in life. Rhizoma Ligustici Chuanxiong has tranquilizing effect. The volatile oil of rhizoma Ligustici Chuanxiong has effects of inhibiting animal brain activity, and exciting brain-extending respiratory center, blood vessel motor center and spinal cord reflex center. The eye product is mainly used for promoting qi circulation, activating blood circulation and promoting blood circulation of eyes.

Safflower carthamus

Mainly produced in Henan, Hunan, Sichuan, Xinjiang, Tibet, etc. Promoting blood circulation, dredging channels, removing blood stasis, and relieving pain, and can be used for treating amenorrhea, dysmenorrhea, lochiorrhea, thoracic obstruction, cardialgia, blood stasis, abdominal pain, pricking pain in chest and hypochondrium, traumatic injury, and pyocutaneous disease with swelling and pain. Has effects of promoting blood circulation, removing blood stasis, eliminating dampness, and relieving swelling.

Peach kernel

The product is dried mature seed of Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch of Rosaceae. Harvesting after fruit ripening, removing pulp and nucleocapsid, taking out seed, and drying in the sun. Peach kernel, semen Persicae is bitter and sweet in taste and neutral in nature, enters heart, liver and large intestine channels, and has the effects of promoting blood circulation and removing blood stasis, and is used for treating various kinds of open-ends due to blood stasis. Lipid is rich and moist, and has the effect of moistening intestines and relaxing bowels; can be used for treating lump, pulmonary abscess, acute appendicitis, traumatic injury, amenorrhea, dysmenorrhea, and puerperal pain due to blood stasis. It is commonly used for blood stasis syndrome because it has a wide action of activating blood and resolving stasis. For pulmonary abscess, it is combined with Lu Gen and Yi ren; for intestinal abscess, it is combined with Da Huang and Dan Pi; for abdominal mass and lump, it is combined with Da Huang and Yu Chong; for traumatic injury, it is combined with chai Hu and pangolin scales; for amenorrhea and dysmenorrhea, it is combined with hong Hua and Dang Gui, etc.; for postpartum pain due to blood stasis, it is combined with Dang Gui and Pao Jiang, etc.

White peony root

Radix Paeoniae as alternative name; the product is dried root of Paeonia lactiflora pall. Collected in summer and autumn, cleaned, removed head, tail and fine root, boiled in boiling water, peeled or boiled again, and dried in the sun. White peony root is slightly cold in nature, bitter and sour in taste. Chinese angelica root-barkLiver meridian、Spleen meridianThe medicine contains paeoniflorin, hydroxyl paeoniflorin, benzoylpaeoniflorin, albiflorin, oxypaeoniflorin, paeoniflorin, hederagenin, paeoniflorin ketone, galloyl paeoniflorin, kaempferol-3, 7-di-O- β -D-glucoside, daucosterol, β -sitosterol, paeoniflorin and the like, and volatile oil mainly contains benzoic acid, paeonol and the like.

Prepared rehmannia root

The medicine is prepared from rehmannia glutinosa or rehmannia glutinosa Libosch of Scrophulariaceae by processing, steaming and drying, and has the functions of nourishing yin, enriching blood, treating yin deficiency and blood deficiency, weakness of waist and knees, fatigue cough and steaming bone, spermatorrhea, metrorrhagia, menoxenia, thirst quenching, scanty urine, deafness, dizziness, ① pearl sac (pearl sac) for treating deficiency of large blood, promoting blood circulation, tonifying qi, ② Wang ancient diseases, treating desire for sitting, no vision deficiency and abortion, ③ (compendium of compendium), filling marrow, muscle and muscle growth, promoting essence and blood production, tonifying five internal organs, internal injury deficiency, promoting blood circulation, benefiting ear day, black beard hair, male five-seven-strain injuries, uterus leakage, menoxenia, fetal birth diseases, ④ (herbal) nourishing kidney water, sealing and filling bone marrow, benefiting blood vessels, promoting blood circulation, hearing and hearing loss, tonifying beard, nourishing yin, nourishing kidney, tonifying kidney, pus, treating deficiency, deficiency of spleen, deficiency of stomach yin, deficiency of spleen, deficiency of kidney, deficiency of yin, deficiency of spleen, cough, constipation.

Radix Paeoniae Rubra

The extract is prepared from dried root of Paeonia lactiflora pall or Paeonia veitchii Lynch of Ranunculaceae, collected in spring and autumn, and sun-dried, contains paeoniflorin, neopaeoniflorin, deoxypaeoniflorin, erythrosin, palmitic acid, dihydroapigenin, paeoniflorin, β -sitosterol, camphorhydrocarbon, vanillic acid, etc., has antithrombotic, platelet aggregation inhibiting, blood fat reducing, arteriosclerosis resisting, small-dose mild heart inhibiting, large-dose heart inhibiting, conduction blocking, coronary blood flow increasing, peripheral resistance reducing, tumor resisting, liver protecting, oxygen free radical removing, enterospasm relieving, bacteriostasis, etc., has sodium expelling effect, and has strong inhibition effect on epinephrine, ADP, iron head and arachidonic acid induced platelet aggregation.

Root of bidentate achyranthes

Radix Achyranthis bidentatae, herba Amaranthi Tricoloris, herba Ardisiae Japonicae, radix Achyranthis bidentatae, and radix Achyranthis bidentatae; the product is dried root of Achyranthus bidentis Blume. Digging when stem and leaf wither in winter, removing fibrous root and silt, bundling into small bundle, drying until it is dry and wrinkled, cutting top end, and drying in the sun. The roots are used as the medicine, unprocessed, and activate blood and dredge channels; treating puerperal abdominal pain, menoxenia, amenorrhea, epistaxis, toothache due to deficiency fire, loempe and edema; for use in cooking, liver and kidney are tonified, waist and knee are strengthened; it is indicated for soreness and pain of waist and knees, liver and kidney deficiency, traumatic injury, blood stasis and pain. The veterinary drug is used for treating cattle foot softening disease, bone fracture due to falling injury, and the like.

Root of balloonflower

Alias-burden flower, lily of the valley, dalargin; the source product is dried root of Platycodogondriflorum (Jacq.) A.DC. of Campanulaceae. Collected in spring and autumn, cleaned, and then the fibrous root is removed, and the husk is stripped or not stripped when the fibrous root is fresh, and then dried. Disperse lung qi, relieve sore throat, dispel phlegm, and expel pus. Can be used for treating cough with excessive phlegm, chest distress, pharyngalgia, hoarseness, lung carbuncle, suppuration, and pyocutaneous disease with pus formation; has expectorant, antitussive, blood sugar lowering, antiinflammatory, gastric secretion inhibiting, antiulcer, tranquilizing, analgesic, and antipyretic effects.

Radix bupleuri

The product is dried root of Bupleurum chinense DC or Bupleurum scorzonerifolium Willd. According to the different characteristics, it is called "Bei chai Hu" and "nan chai Hu" respectively. Collected in spring and autumn, removed stems and leaves and silt, and dried. Bupleuri radix contains valeric acid, linolenic acid, palmitic acid, stearic acid, kaempferol, kaempferitrin, quercetin, rutin, and bergamotene, and has slightly cold nature and bitter taste. It enters liver meridian, gallbladder meridian and lung meridian; bupleuri radix has antiinflammatory, antipyretic, tranquilizing, analgesic, antitussive, and anticonvulsive effects; can relieve liver injury and promote bile secretion; has effects in lowering blood pressure, reducing serum cholesterol, and promoting hemolysis; has antiulcer, antibacterial, antiviral, antitumor, blood sugar increasing, blood fat content reducing, and radiation injury resisting effects.

Fructus Aurantii

The product is dried immature fruit of Citrus aurantium L.of Rutaceae and its cultivar. Harvesting when 7 months of pericarp is green, transversely cutting into two halves from the middle part, and drying in the sun or at low temperature. Various fructus Aurantii contain volatile oil and flavonoid glycoside. The outer pericarp of mature fruit of Citrus aurantium contains volatile oil and orange yellow, and the oil mainly contains d-limonene and small amount of decanal, citral, citronellal, methyl anthranilate and linalool. In addition, it contains hesperidin, naringin, neohesperidin, bitter aurantiin, etc. Contains flavonoids such as naringin, hesperidin, neohesperidin, and narirutin; also contains volatile oil and alkaloids. Has effects in regulating gastrointestinal function, cardiovascular system and uterus. Can be used for treating qi stagnation in chest and hypochondrium, fullness and pain, dyspepsia, and phlegm retention. It is used to treat acute and chronic hepatitis, bacillary dysentery, acute and chronic bronchitis, emphysema, gastrectasia, gastroptosis, proctoptosis, and uterine prolapse.

Licorice root, radix Glycyrrhizae

The product is dried root of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L. Collected in spring and autumn, removed fibrous root, and dried in the sun. The Glycyrrhrizae radix contains multiple chemical components, such as glycyrrhizic acid and liquiritin. The chemical composition of licorice is very complex, and the compounds separated from licorice so far include several dozen compounds such as glycyrrhizin, glycyrrhetinic acid, liquiritin, isoliquiritin, neoliquiritin, neoisoliquiritin, liquiritigenin, isoliquiritigenin, glycyl alcohol, isoliquiritigenin, 7-methyl coumarine, umbelliferone, etc.; radix Glycyrrhizae can be used for treating heart-qi deficiency, palpitation, intermittent pulse, deficiency of spleen-stomach qi, listlessness, and asthenia; carbuncle, cellulitis, sore and ulcer, and sore throat; it can also be used for anti-inflammatory and antiallergic effects, and can protect the inflamed throat and trachea mucosa; licorice also inhibits the conversion of cortisol, leading to increased blood pressure and hypokalemia.

Motherwort herb

The other names are artemisia selengensis, safflower artemisia, motherwort fruit, flax, Lespedeza tetrandra and motherwort: the product is fresh or dried aerial parts of Leonurus japonicus Houtt. of Labiatae. Harvesting fresh products from a spring seedling stage to an early summer flower stage; collecting the dried product in summer when stem and leaf flourish, flower do not bloom or bloom, sun-drying, or cutting and sun-drying. Motherwort: pungent, bitter and slightly cold, entering heart, liver and bladder meridians; promoting blood circulation, removing blood stasis, inducing diuresis and relieving edema. Herba Leonuri can be used as medicine, and contains leonurine as effective component, which contains various alkaloids such as leonurine, stachydrine, leonurine, benzoic acid, potassium chloride, etc.

The function of motherwort includes regulating immunity; inhibiting platelet aggregation; the uterine stimulation effect is achieved; inhibiting dermatophytes, promoting blood circulation of ocular capillary vessel, promoting muscle movement, and relieving fatigue.

Codonopsis pilosula

Radix Codonopsis, and radix Codonopsis; the product is dried root of radix Codonopsis pilosula (Franch.) Nannf., Codonopsis Pilosula Hance (West Codonopsis Pilosula) of Campanulaceae, Codonopsis pilosula Nannf., var. modesta (Nannf.), L.T.Shen or Codonopsis tangshen Oliv. Collected in autumn, washed and dried in the sun. Radix Codonopsis is neutral in nature and sweet in taste, has effects of invigorating spleen and replenishing qi, invigorating spleen and benefiting lung, resisting cancer, lowering blood pressure, resisting anoxia, resisting aging, enhancing immunity, improving activity of superoxide dismutase, enhancing ability of eliminating free radicals, regulating gastrointestinal motility, resisting ulcer, inhibiting gastric acid secretion, and reducing pepsin activity. It is suitable for treating spleen and lung deficiency, short breath, palpitation, anorexia, loose stool, ulcer, anemia, asthma, cough, internal heat, and diabetes. The effective components of radix Codonopsis have exciting effect on nervous system, and can enhance organism resistance and increase erythrocyte and hemoglobin; the zinc contained in the product can promote growth and development and improve sexual function.

White atractylodes rhizome

Is also named as rhizoma Atractylodis Macrocephalae, winter technique, Zhejiang technique and seed technique; the product is dried rhizome of Atractylodes macrocephala Koidz of Compositae. In winter, the lower leaves are withered and yellow, and the upper leaves are brittle, picked and dug, sand and sand are removed, and then the leaves are dried or dried in the sun, and fibrous roots are removed. Bai Zhu is bitter and sweet in flavor and warm in nature. It enters spleen and stomach meridians. Invigorate spleen, replenish qi, dry dampness, induce diuresis, stop sweating, prevent abortion. Can be used for treating spleen deficiency, anorexia, abdominal distention, diarrhea, phlegm retention, dizziness, palpitation, edema, spontaneous perspiration, and threatened abortion. Tonify middle energizer and dry dampness, quench thirst and promote fluid production, benefit spleen essence, nourish stomach qi, descend turbid yin to eat and drink, relieve vomiting, ascend clear yang to digest food, and can purge and benefit. Mainly wind-cold-dampness arthralgia, spasm and jaundice, hidroschesis, heat removal and digestion promotion, and is used as a fried bait. The antibacterial effect is as follows: the Atractylodis rhizoma decoction and Sijunzi decoction have different degrees of antibacterial effects on typhoid bacillus, paratyphoid bacillus A, shigella flexneri, Escherichia coli, Pseudomonas aeruginosa, etc., but have no bactericidal effect. Has obvious and lasting diuretic effect, not only increases the excretion of water, but also promotes the excretion of electrolytes, particularly sodium, and the excretion of sodium is better than that of water.

Coix seed

Adlay Coix lacryma-jobi, Coix lacryma-jobi seed, and Tou Zi rice; the product is dried mature kernel of Coicis semen Coix lacryma-jobil.var.ma-yuen (Roman.) Stapf of Gramineae. Harvesting plants when fruits are mature in autumn, drying in the sun, removing fruits, drying in the sun again, removing shells, tawny seed coats and impurities, and collecting kernels. The coix seeds are sweet, light and cool; for retention of damp-heat in the interior, it can be used with Talcum and medulla Tetrapanacis for scanty and brownish urine; for damp-warm disease with pathogenic dampness in qi system and predominant dampness, it is combined with xing ren, Dou kou ren, Zhu Ye and mu Tong, etc. It is also indicated for edema due to spleen deficiency, swelling and pain of beriberi, and combined with Fu Ling, Bai Jing, mu Gua and Wu Zhu Yu, etc. It is used for diarrhea and leukorrhagia due to spleen deficiency with dampness, and can be combined with rhizoma Atractylodis Macrocephalae and Poria. Used for arthralgia pain and spasm caused by dampness stagnation in skin, flesh and tendons, and is often used in combination with ramulus cinnamomi, rhizoma atractylodis and the like. For pulmonary abscess, chest pain, vomiting with purulent phlegm, it is combined with fresh rhizoma Phragmitis, semen Benincasae, semen Persicae, and herba Houttuyniae; for intestinal abscess, it is combined with Bai Jiang Cao and Fu Zi, etc.

Poria cocos (Schw.) wolf

Is named as Poria, Poria peel, Poria block, Poria cocos, and Poria alba; the product is dry sclerotium of Wolf of Polyporaceae fungus Poria poriacos (Schw.). Digging for more than 7-9 months, removing silt after digging, piling up to generate sweat, spreading and drying until the surface is dry, then generating sweat, repeating for a plurality of times until wrinkles appear and most of internal water is lost, and drying in the shade to obtain Poria cocos; or cutting fresh Poria according to different parts, and drying in shade to obtain Poria peel and Poria block. Poria contains abundant pachyman, vitamins, selenium, etc., and has effects of enhancing immunity of human body cell and body fluid, and improving bone marrow hemopoiesis. Modern medical experiments prove that pachyman has obvious inhibiting effect on mouse sarcoma. It can also stimulate appetite of tumor patients, control symptoms, and relieve side effects of radiotherapy and chemotherapy.

The tuckahoe contains rich dietary fiber, and the dietary fiber can prolong the retention time of food in the gastrointestinal tract, so that people feel full, on one hand, people with diabetes can be prevented from eating too much, on the other hand, the pancreas of the patients can have a rest, and the effect of reducing blood sugar is achieved.

Poria has no adverse side effects, and can be used as food, and common edible methods include decocting soup, cooking porridge, making tea, soaking wine, and optionally making Poria cake, Poria paste, and Poria crisp etc. with Poria.

Rhizoma Cyperi

Herba Cyperi, rhizoma Cyperi, herba Lespedezae Cuneatae, rhizoma Sparganii, herba Lespedezae Bicoloris, folium Ilicis Rotundae, and brometwork; the product is dried rhizome of Cyperus rotundus L. Plucked in autumn, singed to remove hair, boiled in boiling water or dried in the sun after steaming, or singed and dried in the sun directly. The rhizoma cyperi contains glucose, fructose, 40-41.1 starch and volatile oil and is used for treating chest and hypochondrium and stomach and abdomen distending pain caused by stagnation of liver qi. It is combined with chai Hu and Qing Pi to treat chest and hypochondriac pain. It is combined with galangal (rhizoma Alpiniae Officinarum pill) to treat stomach cold pain; it can also be used for treating menoxenia and lower abdomen distention and pain due to stagnation of liver-qi. It is combined with ai Ye to treat abdominal pain during menstruation due to cold accumulation and qi stagnation; in addition, it also has antipyretic, analgesic and cooling effects.

Malt

Barley malt, alias; the product is obtained by germinating and drying mature fruit of Hordeum vulgare L. Soaking wheat grains in water, keeping at proper temperature and humidity, and drying in the sun or at low temperature until young buds grow to about 0.5 cm. The malt water is particularly suitable for people with weakness of the spleen and the stomach, because the digestion of food can cause the phenomenon of difficult digestion, and can supplement nutrient components in the body, the malt water can help to relieve various problems of stagnation of liver qi, chest distress, discomfort of the spleen and the stomach and the like, and can help to promote digestion, stimulate the appetite and promote lactation.

Especially for women in lactation period, the phenomenon of milk expansion exists, and the malt can inhibit the release of prolactin, so that the treatment of delactation by malt water can be realized. The malt water can help spleen and stomach and supplement water in a body, and is preferably eaten together with bighead atractylodes rhizome, dried orange peel and the like, so that the curative effect is optimal.

In one embodiment, the adjuvant is selected from the group consisting of oleyl polyether, polyacrylate, stearyl alcohol, hydrogenated castor oil, tween, span, any one or more in combination; preferably, the adjuvant is oleyl polyether.

In one embodiment, the oleyl polyether is selected from any one or a combination of oleyl polyether-12, oleyl polyether-15, oleyl polyether-20; preferably, the oleyl polyether includes oleyl polyether-12, oleyl polyether-15, oleyl polyether-20; further preferably, the weight ratio of oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20 is 1: (0.8-1.2): (0.8 to 1.2); more preferably, the weight ratio of oleyl polyether-12, oleyl polyether-15, oleyl polyether-20 is 1: 1: 1.

the applicant finds that the stability of the combination can be improved when the specific oleyl polyether is selected, and the hydrophilic group and the hydrophobic group of the oleyl polyether-12, the oleyl polyether-15 and the oleyl polyether-20 are balanced with the hydrophilicity and the lipophilicity of the traditional Chinese medicine components in the system, so that the interfacial force among different substances is small, and the acting force among different components is balanced, so that the composition system is uniform and stable.

Recombinant hirudin fermentation liquor

Hirudin is the strongest natural specific thrombin inhibitor found in the world to date. It has excellent effects on lowering blood viscosity, blood fat, blood sugar, blood pressure and uric acid, and has obvious effects on treating hypertension, hyperglycemia, hyperlipidemia, cardiovascular and cerebrovascular diseases, gout, stroke, diabetes and other diseases. However, the natural hirudin only exists in saliva of leeches, the content of the hirudin is extremely low, and the hirudin is obtained by fishing out and artificially culturing the leeches in the field, so that the problems of high cost, more impurities, low activity and the like exist.

The gene engineering technology is adopted to clone hirudin genes into a saccharomyces cerevisiae strain, and recombinant hirudin fermentation liquor is generated through fermentation culture, wherein hirudin in the fermentation liquor can promote blood circulation of the inner layer of skin, accelerate metabolism, improve the capacity of removing free radicals and cell activity of the skin, and the fermentation liquor is rich in sugar, small molecular peptides and vitamins and provides nutritional ingredients for the skin.

In the process of the technical scheme completed by the applicant, the recombinant hirudin fermentation liquid and the traditional Chinese medicine components are utilized to act together, so that the treatment effect on the hyperplasia of mammary glands can be effectively improved, and the recombinant hirudin fermentation liquid is high in activity, so that the absorption of the traditional Chinese medicine composition by skin is facilitated, the blood circulation is promoted, and the treatment effect is further improved.

The recombinant hirudin fermentation liquor mainly plays a role in promoting blood circulation of facial skin and relieving muscles, and peach kernels and safflower in the traditional Chinese medicine composition can promote blood circulation to regulate menstruation, remove blood stasis and dissipate stagnation; the Chinese angelica and the prepared rehmannia root can regulate and supplement liver and kidney, nourish yin and blood, the ligusticum wallichii is warm and passes through the upper and lower parts, and can regulate and smooth qi and blood, the white paeony root can soften liver and astringe yin, and the peach-red decoction has the greatest characteristics of taking blood circulation and stasis removal as a core, promoting blood circulation and promoting qi circulation as well as promoting blood and blood circulation to remove blood stasis and simultaneously promote generation of new blood, and the blood stasis removal and new generation are combined with biologically fermented hirudin, so that the effects of regulating qi and blood, promoting skin metabolism, whitening and removing spots can be achieved.

In one embodiment, the recombinant hirudin fermentation broth is prepared by: placing the seed solution in an initial culture medium, and dropwise adding a feed supplement culture medium when the DO value is more than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

In a preferred embodiment, the recombinant hirudin is prepared by a process comprising: placing the seed liquid in an initial culture medium, introducing sterile air, and keeping the DO value to be more than 40%; when the DO value is more than 55%, the feeding medium is added dropwise in the initial medium in a feeding mode.

In one embodiment, the ventilation amount of the sterile air is 3-7L/min; preferably, the ventilation of sterile air is 5L/min.

In one embodiment, the temperature of the initial medium is 25 to 40 ℃; preferably, the temperature of the initial culture medium is 27-35 ℃; more preferably, the temperature of the initial medium is 30 ℃.

In one embodiment, the recombinant hirudin is prepared in a manner and under conditions conventional or well known in the art.

In one embodiment, the recombinant hirudin is prepared in a shake flask at a rotation speed of 300-1000 r/min; preferably, the rotation speed is 650 r/min.

In one embodiment, the recombinant hirudin is prepared in a 5L fermenter.

In one embodiment, the pH of the initial culture medium is controlled to be 4.5-5.5 by dropwise adding ammonia water; preferably, the pH of the initial medium is controlled to 5.0 by dropwise addition of ammonia water.

In one embodiment, the concentration of the ammonia water is 20-30 wt%; preferably, the concentration of ammonia is 25 wt%.

In one embodiment, the inoculation amount of the seed solution is 10-15%; preferably, the inoculation amount of the seed liquid is 11-13%.

In one embodiment, the seed liquid is prepared by: inoculating engineering bacteria glycerol preservation bacteria into a bottle filled with a seed culture medium, and culturing to obtain a seed solution to be inoculated.

Preferably, the preparation method of the seed solution comprises the steps of inoculating ① engineering bacteria glycerol preservation bacteria into a 100mL triangular flask filled with 5mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ② engineering bacteria glycerol preservation bacteria into a 250mL triangular flask filled with 50mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ③ engineering bacteria glycerol preservation bacteria into a 1000mL triangular flask filled with 250mL seed culture medium (two flasks in total), and carrying out shake culture at 30 ℃ and 220rpm for 12 hours to obtain the seed solution to be inoculated.

In one embodiment, the seed medium comprises glucose, YNB, amino acids, and water.

In one embodiment, the glucose content in the seed culture medium is 10-30 g/L of the total volume of the solution of the initial culture medium; preferably, the sucrose content in the seed medium is 20g/L of the total volume of the solution of the initial medium.

In one embodiment, the weight ratio of glucose, YNB and amino acid is (10-30): (5-9): (1-2); preferably, the weight ratio of glucose, YNB and amino acid is 20: 7: 1.5.

in one embodiment, the water content in the seed culture medium is 400-550 mL; preferably, the water content in the seed medium is 480 mL.

In one embodiment, the amino acid is selected from the group consisting of any one or more of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; more preferably, the weight ratio of the amino acid to the L-arginine, the L-histidine and the L-tryptophan is 1: (0.8-1.2): (0.8 to 1.2); more preferably, the weight ratio of L-arginine, L-histidine and L-tryptophan is 1: 1: 1.

in one embodiment, the inoculated engineered bacteria is a strain of INVSC1 Saccharomyces cerevisiae.

In one embodiment, the sugar solution comprises an amino-free yeast nitrogen source (YNB), a carbon source, and an amino acid solution; preferably, the sugar solution further comprises at least one of ammonium salt, phosphate, magnesium salt, zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt and copper salt; more preferably, the sugar solution further comprises an antifoaming agent.

In one embodiment, the sugar solution comprises an amino-free yeast nitrogen source (YNB), a carbon source, an amino acid solution, an ammonium salt, a phosphate salt, a magnesium salt, and an antifoaming agent.

In one embodiment, the content of the nitrogen source (YNB) without amino yeast is 4 to 10g/L of the total volume of the solution of the initial medium; preferably, the content of the nitrogen source (YNB) without amino yeast is 6-8 g/L of the total volume of the solution of the initial culture medium; more preferably, the content of nitrogen source (YNB) without amino yeast is 7g/L of the total volume of the solution of the initial medium.

In one embodiment, the carbon source content is 20-50 g/L of the total volume of the solution of the initial culture medium; preferably, the content of the carbon source is 30-40 g/L of the total volume of the solution of the initial culture medium; more preferably, the carbon source content is 35g/L of the total volume of the solution of the initial medium.

In one embodiment, the amino acid solution is 10-40 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the amino acid solution is 15-30 mL/L of the total volume of the solution of the initial culture medium; more preferably, the amino acid solution content is 20mL/L of the total volume of the solution of the starting medium.

In one embodiment, the weight ratio of ammonium salt, phosphate salt and magnesium salt in the sugar solution is (1-1.5): (1.5-3): 1; preferably, the weight ratio of ammonium salt, phosphate salt and magnesium salt in the sugar solution is 1.3: 2.5: 1.

in one embodiment, the content of ammonium salt in the sugar solution is 10-30 g/L of the total volume of the solution of the initial culture medium; preferably, the ammonium salt content of the sugar solution is 15g/L of the total volume of the solution of the starting medium.

In one embodiment, the content of the antifoaming agent is 0.1-1 mL/L of the total volume of the solution of the initial medium; preferably, the antifoaming agent content is 0.5mL/L of the total volume of the solution of the starting medium.

In one embodiment, the defoamer is a silicone-based defoamer and/or a polyether-based defoamer; preferably, the antifoaming agent is tween 80.

In one embodiment, the amino acid solution comprises at least one of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine; preferably, the amino acid solution comprises L-histidine, L-leucine, L-lysine and L-methionine; further preferably, the weight ratio of L-histidine, L-leucine, L-lysine and L-methionine is 1: (2-4): (1-3): (0.5 to 1.5); more preferably, the weight ratio of L-histidine, L-leucine, L-lysine and L-methionine is 1: 3: 1.5: 1.

in one embodiment, the L-histidine content is 1-3 g/L of a 100X amino acid solution; preferably, the L-histidine content is 2g/L of the amino acid solution.

In one embodiment, the content of the trace metal solution in the preparation raw material of the initial culture medium is 5-20 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the trace metal solution in the starting material for the preparation of the starting medium is 10mL/L of the total volume of the solution of the starting medium.

In one embodiment, the content of the vitamin solution in the raw material for preparing the initial culture medium is 5-20 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the vitamin solution in the raw materials for preparing the initial culture medium is 10-15 mL/L of the total volume of the solution of the initial culture medium; the content of the vitamin solution in the starting material for the preparation of the starting medium was 12mL/L of the total volume of the solution of the starting medium.

In one embodiment, the feed medium comprises a trace metal solution, a vitamin solution, and a carbon source; preferably, the feed medium further comprises at least one of phosphate, ammonium salt, phosphate, magnesium salt, zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, copper salt and potassium salt.

In one embodiment, the feed medium comprises a trace metal solution, a vitamin solution, a carbon source, phosphate, magnesium salt, and potassium salt.

The applicant has found that, surprisingly, the hirudin obtained by the preparation method of the invention has a high activity under the synergistic effect of a specific initial culture medium and a supplemented culture medium, and the yield of the hirudin which is expressed effectively finally is high, probably because the hirudin is beneficial to the sufficiency of nutrient components in different stages of the thallus fermentation process and the high yield of active substances, and the decomposition of the newly-generated hirudin is avoided.

In one embodiment, the content of the carbon source in the feed medium is 300-500 g/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the carbon source in the feed culture medium is 350-450 g/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the carbon source content in the feed medium is 400g/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of the vitamin solution in the feed medium is 5-20 mL/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the vitamin solution in the feed culture medium is 10-15 mL/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the content of the vitamin solution in the feed medium is 12mL/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of the trace metal solution in the feed medium is 3-15 mL/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the trace metal solution in the feed culture medium is 8-12 mL/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the content of the trace metal solution in the feed medium is 10mL/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of potassium salt in the feed medium is 1.5-6 g/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the potassium salt in the feed culture medium is 3-5 g/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the potassium salt content in the feed medium is 3.5g/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the potassium salt, magnesium salt, and phosphate are present in the feed medium in a weight ratio of 1: (1-3): (2-5); preferably, the weight ratio of potassium salt, magnesium salt and phosphate in the feed medium is 1: 1.5: 3.5.

in one embodiment, the carbon source is selected from any one or a combination of more of glucose, sucrose, galactose, starch, maltose, lactose.

In one embodiment, the trace metal solution comprises at least one of a zinc salt, a manganese salt, a cobalt salt, an iron salt, a calcium salt, a sodium salt, a copper salt, and a potassium salt; preferably, the trace metal solution includes sodium, zinc, calcium and manganese salts; further preferably, the weight ratio of the sodium salt, the zinc salt, the calcium salt and the manganese salt is 1: (50-90): (20-40): (3-10); more preferably, the weight ratio of the sodium salt, zinc salt, calcium salt and manganese salt is 1: 80: 30: 5.

in one embodiment, the content of sodium salt in the trace metal solution is 0.05-0.2 g/L of the trace metal solution; preferably, the content of the sodium salt in the trace metal solution is 0.1g/L of the trace metal solution.

In one embodiment, the trace metal solution further comprises 0.5M EDTA; further preferably, the content of EDTA is 70-90 mL/L of the trace metal solution; more preferably, the EDTA content is 80mL/L of the trace metal solution.

In one embodiment, zinc salts that may be mentioned include, but are not limited to: zinc sulfate, zinc chloride; manganese salts that may be mentioned include, but are not limited to: manganese sulfate, manganese dichloride and manganese glycinate; cobalt salts that may be mentioned include, but are not limited to: cobalt dichloride; iron salts that may be mentioned include, but are not limited to: ferrous sulfate, ferrous chloride; calcium salts that may be mentioned include, but are not limited to: calcium chloride, calcium glycinate; sodium salts that may be mentioned include, but are not limited to: sodium chloride, sodium molybdate; copper salts that may be mentioned include, but are not limited to: anhydrous copper sulfate, copper glycinate; illustrative potassium salts include, but are not limited to: potassium sulfate, potassium chloride, potassium molybdate; ammonium salts that may be mentioned include, but are not limited to: ammonium nitrate, ammonium sulfate, ammonium chloride; exemplary phosphates include, but are not limited to: potassium dihydrogen phosphate, sodium dihydrogen phosphate, and calcium dihydrogen phosphate.

In one embodiment, the vitamin solution comprises at least one of biotin, niacin, thiamine hydrochloride, vitamin B2; preferably, the vitamin solution comprises biotin, niacin, and thiamine hydrochloride; further preferably, the weight ratio of biotin, nicotinic acid and thiamine hydrochloride is 1: (15-30): (10-30); more preferably, the weight ratio of biotin, nicotinic acid and thiamine hydrochloride is 1: 22: 20.

in one embodiment, the content of biotin is 0.03-0.1 g/L of the vitamin solution; preferably, the biotin content is 0.05g/L of the vitamin solution.

In one embodiment, the initial medium is prepared by a process comprising the steps of:

(1) sterilizing the sugar solution, and adding into a fermentation tank;

(2) filtering the trace metal solution for sterilization, and adding the solution into a fermentation tank;

(3) filtering the vitamin solution for sterilization, and adding into a fermentation tank to obtain the initial culture medium.

In a preferred embodiment, the preparation of the initial medium comprises the following steps:

(1) sterilizing the sugar solution, and adding the sugar solution into a fermentation tank in a feeding manner;

(2) filtering and sterilizing the trace metal solution by a sterile filter of 0.22 mu m, and adding the solution into a fermentation tank;

(3) filtering vitamin solution with 0.22 μm sterile filter, sterilizing, and adding into fermentation tank to obtain initial culture medium.

(1) mixing the water phase component and water uniformly, and heating;

(2) mixing the oil phase components uniformly, and heating;

(5) cooling, adding adjuvant, and stirring.

Preferably, the preparation method of the traditional Chinese medicine external composition containing the recombinant hirudin for treating chloasma comprises the following steps:

(1) uniformly mixing the water-phase component with water, and heating to 70-80 ℃;

(2) uniformly mixing the oil phase components, and heating to 70-80 ℃;

(3) adding the oil phase component into the water phase component, and shearing at a high speed for 4-10 min;

(4) cooling to 50 deg.C, and adding recombinant hirudin fermentation liquid and Chinese medicinal components;

(5) cooling to 40 deg.C, adding adjuvant, and stirring.

Example 1

The embodiment 1 of the invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which comprises the following raw materials, by weight, 7.5% of a water phase component, 1.5% of an oil phase component, 8% of recombinant hirudin fermentation liquor, 1.2% of a traditional Chinese medicine component, 0.7% of an auxiliary agent and the balance of water;

the preparation raw materials of the traditional Chinese medicine component comprise a component A and a component B, wherein the weight ratio of the component A to the component B is 1: 1; the A component comprises radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Carthami flos, radix Platycodi, bupleuri radix, and the B component comprises fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, and semen Persicae; the weight ratio of the angelica, the ligusticum chuanxiong hort, the safflower, the platycodon grandiflorum and the radix bupleuri is 1: 1: 1: 1; the weight ratio of the bitter orange, the liquorice, the motherwort and the peach kernel is 1: 1: 1: 1;

the water phase component comprises arginine, small molecular alcohol and a complexing agent; the weight ratio of the micromolecular alcohol to the complexing agent is 1: 0.005; the weight ratio of arginine to complexing agent is 1: 1.5;

the small molecular alcohol is 1, 3-butanediol and 1, 2-hexanediol, and the weight ratio of the 1, 3-butanediol to the 1, 2-hexanediol is 1: 0.1; the complexing agent is EDTA disodium;

the oil phase component comprises humectant, flavonoid and sulfhydryl-containing compound;

the humectant is xylitol substances, xanthan gum and sodium hyaluronate, and the weight ratio of the xylitol substances to the xanthan gum to the sodium hyaluronate is 1: 0.35: 0.1; the weight ratio of the flavonoids, the sulfhydryl-containing compound and the sodium hyaluronate is 0.01: 0.01: 1;

the flavonoid is glabridin, and the sulfhydryl-containing compound is ergothioneine;

the preparation method of the traditional Chinese medicine components comprises the following steps:

(3) and (3) putting the filter residue obtained in the step (2) into a mixed solution of ethanol \ distilled water, performing reflux extraction, and concentrating to obtain a component B extracting solution, wherein the volume ratio of ethanol to distilled water is 1: 4;

the auxiliary agent is oleyl polyether, and the oleyl polyether comprises oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20; the weight ratio of oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20 is 1: 1: 1;

the preparation process of the recombinant hirudin fermentation liquor comprises the following steps: placing the seed liquid in an initial culture medium, introducing sterile air, and keeping the DO value to be more than 40%; when the DO value is more than 55%, dropwise adding a feed culture medium into the initial culture medium in a feeding mode; wherein the ventilation capacity of sterile air is 5L/min, the temperature of the initial culture medium is 30 ℃, the pH of the initial culture medium is controlled to be 5.0 by dropwise adding ammonia water, and the concentration of the ammonia water is 25 wt%;

the preparation process of the recombinant hirudin is carried out in a shake flask, and the rotating speed is 650 r/min;

the inoculation amount of the seed liquid is 11 percent;

the preparation method of the seed solution comprises the steps of inoculating ① engineering bacteria glycerol preservation bacteria into a 100mL triangular flask filled with 5mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ② engineering bacteria glycerol preservation bacteria into a 250mL triangular flask filled with 50mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ③ engineering bacteria glycerol preservation bacteria into a 1000mL triangular flask filled with 250mL seed culture medium (two bottles in total), and carrying out shake culture at 30 ℃ and 220rpm for 12 hours to obtain the seed solution to be inoculated;

the seed culture medium comprises glucose, YNB, amino acid and water, and the sucrose content in the seed culture medium is 20g/L of the total volume of the solution of the initial culture medium; the weight ratio of glucose, YNB and amino acid is 20: 7: 1.5; the water content in the seed culture medium is 480 mL; the amino acids are L-arginine, L-histidine and L-tryptophan; the weight ratio of L-arginine, L-histidine and L-tryptophan is 1: 1: 1; inoculating the engineering bacteria into an INVSC1 saccharomyces cerevisiae strain;

the preparation raw materials of the initial culture medium comprise a sugar solution, a trace metal solution and a vitamin solution;

the sugar solution comprises an amino-free yeast nitrogen source (YNB), a carbon source, an amino acid solution, an ammonium salt, a phosphate, a magnesium salt and an antifoaming agent, wherein the content of the amino-free yeast nitrogen source (YNB) is 7g/L of the total volume of the solution of the initial culture medium, the content of the carbon source is 35g/L of the total volume of the solution of the initial culture medium, the content of the amino acid solution is 20mL/L of the total volume of the solution of the initial culture medium, and the content of the ammonium salt is 15g/L of the total volume of the solution of the initial culture medium;

the weight ratio of ammonium salt, phosphate and magnesium salt in the sugar solution is 1.3: 2.5: 1;

the antifoaming agent is tween 80; the content of the antifoaming agent is 1mL/L of the total volume of the solution of the initial culture medium;

the amino acid solution comprises L-histidine, L-leucine, L-lysine and L-methionine, wherein the weight ratio of the L-histidine to the L-leucine to the L-lysine to the L-methionine is 1: 3: 1.5: 1; wherein the content of L-histidine is 2g/L of the amino acid solution;

the content of the trace metal solution in the preparation raw material of the initial culture medium is 10mL/L of the total volume of the solution of the initial culture medium, and the content of the vitamin solution in the preparation raw material of the initial culture medium is 12mL/L of the total volume of the solution of the initial culture medium;

the supplementary culture medium comprises a trace metal solution, a vitamin solution, a carbon source, phosphate, magnesium salt and potassium salt, wherein the content of the carbon source in the supplementary culture medium is 400g/L of the total volume of the solution of the initial culture medium and the solution of the supplementary culture medium; the content of the vitamin solution in the feed culture medium is 12mL/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; the content of the trace metal solution in the supplemented medium is 10mL/L of the total volume of the solution of the initial medium and the solution of the supplemented medium; the content of potassium salt in the supplemented medium is 3.5g/L of the total volume of the solution of the initial medium and the solution of the supplemented medium; the weight ratio of potassium salt, magnesium salt and phosphate in the feed medium is 1: 1.5: 3.5;

the carbon source is galactose;

the trace metal solution comprises sodium salt, zinc salt, calcium salt and manganese salt, wherein the weight ratio of the sodium salt to the zinc salt to the calcium salt to the manganese salt is 1: 80: 30: 5; wherein the content of sodium salt in the trace metal solution is 0.1g/L of the trace metal solution;

the trace metal solution also comprises 0.5M EDTA, and the content of the EDTA is 80mL/L of the trace metal solution;

zinc salt is zinc sulfate, sodium salt is sodium molybdate, calcium salt is calcium chloride, and manganese salt is manganese dichloride; the potassium salt is potassium sulfate, the magnesium salt is magnesium sulfate, and the phosphate is potassium dihydrogen phosphate; the ammonium salt is ammonium sulfate;

the vitamin solution comprises biotin, nicotinic acid and thiamine hydrochloride, wherein the weight ratio of the biotin to the nicotinic acid to the thiamine hydrochloride is 1: 22: 20; wherein the content of biotin is 0.05g/L of the vitamin solution;

the preparation process of the initial culture medium comprises the following steps:

(3) filtering vitamin solution with sterile filter of 0.22 μm for sterilization, and adding into fermentation tank to obtain initial culture medium;

the preparation method of the traditional Chinese medicine external composition containing the recombinant hirudin for treating chloasma comprises the following steps:

(1) mixing the water phase component and water uniformly, and heating to 80 ℃;

(2) mixing the oil phase components uniformly, and heating to 80 ℃;

(5) cooling to 40 deg.C, adding adjuvant, and stirring.

Example 2

The embodiment 2 of the invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which is prepared from the following raw materials, by weight, 15% of a water phase component, 3% of an oil phase component, 12% of recombinant hirudin fermentation liquor, 2% of a traditional Chinese medicine component, 1% of an auxiliary agent and the balance of water;

the preparation raw materials of the traditional Chinese medicine component comprise a component A and a component B, wherein the weight ratio of the component A to the component B is 1: 1.2; the A component comprises radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Carthami flos, radix Platycodi, bupleuri radix, and the B component comprises fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, and semen Persicae; the weight ratio of the angelica, the ligusticum chuanxiong hort, the safflower, the platycodon grandiflorum and the radix bupleuri is 1: 1.2: 1.2: 1.2; the weight ratio of the bitter orange, the liquorice, the motherwort and the peach kernel is 1: 1.2: 1.2: 1.2; the preparation method of the traditional Chinese medicine components is the same as that of the example 1;

the water phase component comprises arginine, small molecular alcohol and a complexing agent; the weight ratio of the micromolecular alcohol to the complexing agent is 1: 0.01; the weight ratio of arginine to complexing agent is 1: 2;

the small molecular alcohol is 1, 3-butanediol and 1, 2-hexanediol, and the weight ratio of the 1, 3-butanediol to the 1, 2-hexanediol is 1: 0.2; the complexing agent is EDTA disodium;

the humectant is xylitol substances, xanthan gum and sodium hyaluronate, and the weight ratio of the xylitol substances to the xanthan gum to the sodium hyaluronate is 1: 0.6: 0.15; the weight ratio of the flavonoids, the sulfhydryl-containing compound and the sodium hyaluronate is 0.02: 0.02: 1;

the auxiliary agent is oleyl polyether, and the oleyl polyether comprises oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20; the weight ratio of oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20 is 1: 1.2: 1.2;

the preparation method of the recombinant hirudin fermentation broth is the same as that in example 1;

the preparation method of the traditional Chinese medicine external composition containing the recombinant hirudin for treating chloasma is the same as that in example 1.

Example 3

The embodiment 3 of the invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which comprises the following raw materials, by weight, 3% of a water phase component, 0.5% of an oil phase component, 3% of recombinant hirudin fermentation liquor, 0.5% of a traditional Chinese medicine component, 0.1% of an auxiliary agent and the balance of water;

the preparation raw materials of the traditional Chinese medicine component comprise a component A and a component B, wherein the weight ratio of the component A to the component B is 1: 0.8; the A component comprises radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Carthami flos, radix Platycodi, bupleuri radix, and the B component comprises fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, and semen Persicae; the weight ratio of the angelica, the ligusticum chuanxiong hort, the safflower, the platycodon grandiflorum and the radix bupleuri is 1: 0.8: 0.8: 0.8; the weight ratio of the bitter orange, the liquorice, the motherwort and the peach kernel is 1: 0.8: 0.8: 0.8; the preparation method of the traditional Chinese medicine components is the same as that of the example 1;

the water phase component comprises arginine, small molecular alcohol and a complexing agent; the weight ratio of the micromolecular alcohol to the complexing agent is 1: 0.003; the weight ratio of arginine to complexing agent is 1: 1;

the small molecular alcohol is 1, 3-butanediol and 1, 2-hexanediol, and the weight ratio of the 1, 3-butanediol to the 1, 2-hexanediol is 1: 0.05; the complexing agent is EDTA disodium;

the humectant is xylitol substances, xanthan gum and sodium hyaluronate, and the weight ratio of the xylitol substances to the xanthan gum to the sodium hyaluronate is 1: 0.1: 0.05; the weight ratio of the flavonoids, the sulfhydryl-containing compound and the sodium hyaluronate is 0.005: 0.005: 1;

the auxiliary agent is oleyl polyether, and the oleyl polyether comprises oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20; the weight ratio of oleyl polyether-12, oleyl polyether-15 and oleyl polyether-20 is 0.8: 0.8: 0.8;

Example 4

The embodiment 4 of the invention provides a traditional Chinese medicine composition for external use containing recombinant hirudin for treating chloasma, the specific implementation mode of the composition is the same as that of the embodiment 1, and the difference is that the component A comprises safflower, codonopsis pilosula, bighead atractylodes rhizome and coix seed, and the component B comprises poria cocos, rhizoma cyperi and malt; the weight ratio of the safflower to the codonopsis pilosula to the white atractylodes rhizome to the coix seed is 1: 1: 1: 1; the weight ratio of the tuckahoe, the rhizoma cyperi and the malt is 1: 1: 1.

example 5

The embodiment 5 of the invention provides a traditional Chinese medicine composition for external use containing recombinant hirudin for treating chloasma, which has the same specific implementation mode as the embodiment 1, and is characterized in that the content of recombinant hirudin fermentation liquor is 0.

Example 6

Embodiment 6 of the present invention provides a recombinant hirudin-containing external traditional Chinese medicine composition for treating chloasma, which has the same specific implementation manner as embodiment 1, except that the content of the traditional Chinese medicine components is 0.

Example 7

The embodiment 7 of the invention provides a traditional Chinese medicine composition for external use containing recombinant hirudin for treating chloasma, which has the same specific implementation manner as the embodiment 1, and is characterized in that the contents of the traditional Chinese medicine components and the recombinant hirudin fermentation broth are both 0.

Example 8

The embodiment 8 of the invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which has the specific implementation mode as the embodiment 1, and is characterized in that the content of oleyl polyether-12 is 0.

Example 9

Embodiment 9 of the present invention provides a recombinant hirudin-containing external traditional Chinese medicine composition for treating chloasma, which has the same specific implementation manner as embodiment 1, except that the content of oleyl polyether-20 is 0.

Example 10

The embodiment 10 of the invention provides a traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma, which has the same specific implementation manner as the embodiment 1, and is characterized in that the contents of oleyl polyether-20 and oleyl polyether-12 are both 0.

Performance evaluation

1. Evaluation of physicochemical properties:

a-the compositions obtained in examples 1 to 4 are all yellowish homogeneous emulsions by visual observation;

b-determination of the pH of the composition obtained in example 1 at 25 ℃ of 6.52;

c-carrying out a centrifugation experiment on the samples of the examples 1 to 4 and 8 to 10, namely centrifuging the samples at room temperature for 30min at 3000r/min, and observing whether a layering phenomenon exists or not, wherein the experimental results are shown in a table 2;

2. evaluation of the therapeutic effect of the obtained composition on chloasma:

the evaluation method comprises the following steps: selecting 420 volunteers with chloasma, and 130 females with the age of 30-60 years and the age of 30-40 years; women 165 aged 41-50 years, women 125 aged 51-60 years, were randomized into 7 groups of 60 people each.

The medication method comprises the following steps: the judgment standard of chloasma is as follows:

the method is drawn up according to the clinical diagnosis and curative effect standard of chloasma which are set by the pigment pathology group of the skin disease professional committee of the Chinese traditional and western medicine integration.

① face is light brown to dark brown, has clear spots, is basically distributed symmetrically, and has no inflammation and scales.

② has no obvious subjective symptom, and is suitable for women with severe summer and mild winter.

③ has no obvious endocrine diseases, and can eliminate pigmentation caused by other diseases.

Each group of 60 persons is randomly divided into 7 groups, the first group uses the embodiment 1, the second group uses the embodiment 2, the third group uses the embodiment 3, the fourth group uses the embodiment 4, the fifth group uses the embodiment 5, the sixth group uses the embodiment 6, the seventh group uses the embodiment 7, the seven groups are compared in the aspects of sex, age, course of disease and chloasma part, and the like, the difference has no statistical significance, the volunteers apply the affected part once every morning, noon and evening and rub for a few minutes, the continuous uninterrupted use is carried out for 4 weeks, the use of the product can be stopped if the patients are healed within 4 weeks, and the curative effect is judged according to the curative effect and compared.

The determined curative effect standard is as follows:

1. the chloasma skin damage change scoring method and standard are shown in table 1;

the clinical diagnosis and curative effect standard of chloasma is established according to the pigmentary pathology group of the skin disease professional committee of the Chinese traditional and western medicine integration

TABLE 1 chloasma peel change scoring method and standard

Scoring device	0	1	2	3
					Size of area	0	＜3cm²	3-6cm²	＞6cm²
Degree of pigment	Is free of	Light brown	Brown colour	Dark brown color

The total skin loss integral is the skin loss area integral plus the skin loss pigment degree integral

2. Clinical efficacy evaluation criteria

Skin damage: the clinical diagnosis and curative effect standard of chloasma is established according to the pigmentary pathology group of the skin disease professional committee of the Chinese traditional and western medicine integration

Base healing: the area of the visual stain is faded by more than 90 percent by naked eyes, and the color basically disappears; the reduction index after treatment is calculated by a grading method to be more than or equal to 0.8; the effect is shown: the area of the eye-color spots fades to be more than 60 percent, and the color becomes obviously light; the reduction index after treatment is calculated by a grading method to be more than or equal to 0.5; improvement: the area of the eye-color spots fades more than 30 percent, and the color becomes light; the reduction index after treatment is calculated by a grading method to be more than or equal to 0.3; and (4) invalidation: the area of the eye-color spots fades less than 30 percent, and the color change is not obvious; the decline index after treatment is calculated by a grading method to be less than or equal to 0.3. The total effective rate (number of cure cases + number of significant cases + number of improvement cases)/total number of cases × 100%, and the therapeutic effect of the patients with support is shown in table 3.

During the course of treatment of the first group of volunteers, two volunteers were observed randomly for treatment on the tenth day, as shown in FIG. 1, A-1 was the case before the composition was applied to one side of the face, A-2 was the case after the composition was applied to the corresponding position of the face; b-1 is the condition before the composition is applied to the other side of the face, and B-2 is the condition after the composition is applied to the corresponding position on the other side of the face; as shown in FIG. 2, A-1 is the case before the composition was applied to the face side of the second volunteer, and A-2 is the case after the composition was applied to the corresponding position on the face.

TABLE 2

	Phenomenon of delamination
		Example 1	Without delamination
Example 2	Without delamination
		Example 3	Without delamination
Example 4	Without delamination
		Example 8	Layering
Example 9	Layering
		Example 10	Layering

TABLE 3

	Example number (human)	Jiyu (human)	Display effect (human)	Improvement (human)	Invalid (human)	Total effective rate (%)
							Example 1	60	8	40	10	2	96
Example 2	60	6	38	13	3	95
							Example 3	60	6	37	14	3	95
Example 4	60	5	35	15	5	91
							Example 5	60	0	12	23	25	58
Example 6	60	0	10	34	16	73
							Example 7	60	0	6	4	50	16

From the test results, the invention provides a composition for treating chloasma, a preparation method and application thereof, which have obvious treatment and improvement effects on the chloasma diseases of the skin of women of all ages, are green and environment-friendly, do not contain corticosteroid hormone and antibiotics, so that the side effect of the hormone can not appear, and the problems of cross sensitization and cross drug resistance with other antibiotics can not exist.

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.

Claims

1. The traditional Chinese medicine external composition containing recombinant hirudin for treating chloasma is characterized by comprising 3-15% of a water phase component, 0.5-3% of an oil phase component, 3-12% of recombinant hirudin fermentation liquor, 0.5-2% of a traditional Chinese medicine component, 0.1-1% of an auxiliary agent and the balance of water in percentage by weight; wherein the aqueous phase component comprises arginine.

2. The topical Chinese medicinal composition of claim 1, wherein the raw materials for preparing the Chinese medicinal components comprise component A and component B, wherein component A comprises at least one of radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Carthami flos, semen Persicae, radix Paeoniae alba, radix rehmanniae Preparata, radix Paeoniae Rubra, and Achyranthis radix; the component B comprises at least one of radix Platycodi, bupleuri radix, fructus Aurantii, Glycyrrhrizae radix, herba Leonuri, radix Codonopsis, Atractylodis rhizoma, Coicis semen, Poria, rhizoma Cyperi, and fructus Hordei Germinatus.

3. The topical Chinese medicinal composition of claim 2, wherein the aqueous phase further comprises a small molecule alcohol and a complexing agent.

4. The topical Chinese medicinal composition according to claim 2, wherein the oil phase component comprises a humectant selected from xylitol, glycolic acid, xanthan gum, glyceryl caprylate, polyethylene glycol, trehalose, erythritol, sodium pyrrolidone carboxylate, ethoxy glucose derivatives, and sodium hyaluronate.

5. The topical Chinese medicinal composition for treating according to claim 4, wherein the oil phase component further comprises flavonoids and/or compounds containing sulfhydryl group.

6. The topical Chinese medicinal composition according to any one of claims 1 to 5, wherein the recombinant hirudin fermentation broth is prepared by: placing the seed solution in an initial culture medium, and dropwise adding a feed supplement culture medium when the DO value is more than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

7. The topical Chinese medicinal composition of claim 6, wherein the sugar solution comprises nitrogen source, carbon source and amino acid solution of non-amino yeast.

8. The topical Chinese medicinal composition of claim 6, wherein the trace metal solution comprises at least one of zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, and copper salt.

9. A preparation containing the traditional Chinese medicine composition for external use as claimed in any one of claims 1 to 8.

10. A method for preparing the external traditional Chinese medicine composition according to any one of claims 1 to 8, which comprises the following steps:

(1) mixing the water phase component and water uniformly, and heating;

(2) mixing the oil phase components uniformly, and heating;

(5) cooling, adding adjuvant, and stirring.