A kind of pharmaceutical peptide and preparation method thereof

Abstract

The invention discloses a drug peptide and a preparation method thereof, and belongs to the technical field of drug peptides. The drug peptide includes the following raw materials in parts by weight: 15-25 parts of scutellaria, 10-20 parts of andrographis paniculata, 10-15 parts of gallnut, 5-10 parts of honeysuckle, 4-8 parts of licorice, 2-6 parts of Shenqu, 2-4 parts of red ginseng, 5-10 parts of pulsatilla, and 8-12 parts of yam; the present invention uses Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum to ferment the above-mentioned Chinese medicine components, so that the nutrients and active ingredients in each Chinese medicine component can be fully released, which is more conducive to the absorption and utilization of active ingredients in Chinese medicine. The drug peptide prepared by the present invention can effectively stimulate local blood circulation, promote metabolism, relieve discomfort symptoms such as pain and muscle stiffness, promote local and overall immunity, and improve body resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical peptides, and in particular to a pharmaceutical peptide and a preparation method thereof. Background Art

[0002] Cupping uses a cup as a tool, and uses methods such as burning fire and pumping air to generate negative pressure, so that it is adsorbed on the body surface, causing local blood stasis, so as to achieve the effects of dredge meridians, promote qi and blood circulation, reduce swelling and relieve pain, and dispel wind and cold. The most widely used one at present is the vacuum cup. The vacuum cup uses a piston on the top of the cup to control the exhaust of air, and uses the mechanical pumping principle to form a negative pressure in the cup. However, after cupping with existing tools, blood stasis appears in the cupping area, which will not be eliminated for a period of time, and some people have a resistance to this. Therefore, spraying Chinese medicine sprays during and after the cupping process can effectively and quickly discharge local blood stasis, and can also provide drug treatment for the human body. However, many Chinese medicine sprays are rich in polyphenols. During the storage process, a certain amount of black or dark brown precipitate will be produced in a short period of half a month or a long period of 1-2 months, and the amount of precipitation will continue to increase with the extension of storage time. The formation of precipitation will, on the one hand, lead to a decrease in the content of the active ingredients, affecting the effectiveness and stability of the spray, and on the other hand, it will make consumers mistakenly believe that the product is deteriorating, affecting the patient's psychology.

[0003] The birth of probiotic fermentation Chinese medicine technology has pointed out the way forward for better prevention and treatment of swelling, pain relief, and blood stasis. Chinese medicine fermentation pharmaceutical technology is a high-tech Chinese medicine pharmaceutical technology that inherits the fermentation of Chinese medicine, absorbs the results of microecological research, and combines the fermentation technology of bioengineering to form a new Chinese medicine pharmaceutical technology. It is to find new efficacy of drugs from Chinese medicine pharmaceuticals. Probiotics have the characteristics of broad spectrum, high efficiency, and non-toxicity. Fermented Chinese medicine can improve the therapeutic effect and provide a theoretical basis for effective swelling, pain relief, and blood stasis. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a pharmaceutical peptide and a preparation method thereof.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A pharmaceutical peptide comprising the following raw materials in parts by weight:

[0007] Scutellaria: 15-25 parts, Andrographis paniculata 10-20 parts, Galla chinensis 10-15 parts, Honeysuckle 5-10 parts, Licorice root 4-8 parts, Shenqu 2-6 parts, Red Ginseng 2-4 parts, Pulsatilla 5-10 parts, Chinese Yam 8-12 parts;

[0008] A method for preparing a pharmaceutical peptide comprises the following steps:

[0009] S1: Weigh 15-25 parts of Scutellaria baicalensis, 10-20 parts of Andrographis paniculata, 10-15 parts of Galla chinensis, 5-10 parts of Honeysuckle, 4-8 parts of Licorice, 2-6 parts of Shenqu, 2-4 parts of Red Ginseng, 5-10 parts of Pulsatilla chinensis, and 8-12 parts of Dioscorea opposita by weight, and grind each Chinese medicine raw material separately and mix them evenly; then add purified water for decoction to obtain a decoction;

[0010] S2: inoculating the activated strains of Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum into the culture medium respectively, and culturing with shaking at 37° C. overnight to obtain a culture solution;

[0011] S3: Mix the decoction with the culture medium of each bacterial species in step S2, stir evenly, and then culture in a closed manner at 30-37° C. for 24-36 hours; filter, purify, concentrate, add 2-4 parts by weight of a surfactant, and sterilize to obtain the pharmaceutical peptide.

[0012] In step S2, the inoculation amount of Lactobacillus bulgaricus is 4-6 v / v%, the inoculation amount of Lactobacillus rhamnosus is 2-4 v / v%, and the inoculation amount of Bifidobacterium longum is 1-3 v / v%.

[0013] In step S3, the surfactant is prepared by the following method:

[0014] Under nitrogen protection, tetrahydrofuran and 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine were added to the reactor, stirred, and heated to 70-80°C. 2-methacryloyloxyethylphosphocholine and initiator AIBN were slowly added dropwise, and then heated to 100-120°C for reaction for 16-24h. The surfactant was obtained by post-treatment. The reaction equation is shown as follows:

[0015]

[0016] Among them, m and n are natural numbers.

[0017] The feed mass ratio of tetrahydrofuran, 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine, 2-methacryloyloxyethylphosphocholine and AIBN is 50:(5-10):(6-12):(0.01-0.03).

[0018] Scutellaria baicalensis is bitter, neutral, and non-toxic. Functions and indications: purging excess fire, removing dampness and heat, stopping bleeding, and calming the fetus. It is used to treat high fever and thirst, cough due to lung heat, diarrhea due to damp heat, jaundice, stranguria due to heat, vomiting, epistaxis, metrorrhagia, bleeding, red and swollen eyes, restless fetus, carbuncle, furuncle, and sore.

[0019] Andrographis paniculata is bitter and cold in nature. It has the effects of clearing away heat and detoxifying, reducing inflammation and swelling. Functions and indications: clearing away heat and detoxifying, cooling blood, and reducing swelling. It is used for colds and fever, sore throat, mouth and tongue sores, sudden coughs and fatigue coughs, diarrhea and dysentery, hot and painful stranguria, carbuncles, sores, and snake bites.

[0020] Gallnut, sour, astringent, cold in nature. Functions and indications: astringes the lungs and reduces fire, astringes the intestines and stops diarrhea, astringes sweat, stops bleeding, absorbs moisture and heals sores.

[0021] Honeysuckle is sweet and cold in nature. Functions and indications: fever caused by exogenous wind-heat or febrile disease, heatstroke, pyretic bloody diarrhea, carbuncle, furuncle, throat paralysis, and various infectious diseases.

[0022] Licorice root, sweet in taste, neutral in nature. Functions and indications: soothing the stomach, moistening the lungs, detoxifying, and harmonizing various medicines. Roasted, it can treat spleen and stomach weakness, poor appetite, abdominal pain and loose stools, fatigue and fever, pulmonary tuberculosis and cough, palpitations, and epilepsy; raw, it can treat sore throat, peptic ulcer, carbuncle, and ulcer, and detoxify drug poisoning and food poisoning.

[0023] Shenqu, sweet, spicy, warm in nature. Enters the spleen and stomach meridians. Functions and indications: Digestion and stomach. Indications: Food stagnation, abdominal distension, poor appetite.

[0024] Red ginseng, the root is sweet, slightly cold, and non-toxic. Functions and indications: greatly replenishes vital energy, restores the pulse and consolidates the body, replenishes qi and retains blood. It is used for physical weakness and fainting, cold limbs and weak pulse, qi and blood retention, metrorrhagia, heart failure, and cardiogenic shock.

[0025] Pulsatilla: clearing away heat and detoxifying, cooling blood and stopping diarrhea, antibacterial. Functions and indications: bloody diarrhea caused by heat toxins in livestock and poultry, jaundice caused by damp heat, and bacterial enteritis.

[0026] Chinese Yam, sweet and warm. Functions and indications: Strengthen the spleen, nourish the lungs, strengthen the kidneys, and benefit the essence. Treat spleen deficiency diarrhea, chronic dysentery, consumptive cough, thirst, spermatorrhea, leucorrhea, and frequent urination. Tonify the spleen and stomach, promote fluid production and benefit the lungs, and nourish the kidneys and astringe the essence. Used for spleen deficiency, poor appetite, chronic diarrhea, lung deficiency, wheezing and coughing, kidney deficiency, spermatorrhea, leucorrhea, frequent urination, and thirst due to deficiency heat.

[0027] Due to the adoption of the above technical solution, the beneficial effects of the present invention include:

[0028] (1) The present invention selects Chinese medicinal raw materials that have the functions of unblocking blood vessels and dissipating blood stasis, such as scutellaria baicalensis, andrographis paniculata, gallnut, honeysuckle, liquorice, Shenqu, red ginseng, pulsatilla, yam, etc., and scientifically combines them. By utilizing their synergistic effects, the present invention can effectively stimulate local blood circulation, promote metabolism, relieve discomfort symptoms such as pain and muscle stiffness, promote local and overall immunity, and improve body resistance.

[0029] (2) By using Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum to ferment the Chinese medicine components, the nutrients and active ingredients in each Chinese medicine component can be fully released, and a large amount of active peptide components can be generated through the fermentation process. They can be absorbed through the skin and directly act on local muscles and nerves, eliminating blood stasis and relieving pain.

[0030] (3) The pharmaceutical peptide prepared by the present invention is solubilized by adding a surfactant to its active ingredients, thereby solving the problem that the active ingredients of Chinese herbal medicines have low solubility in aqueous solutions. At the same time, Chinese herbal pharmaceutical peptides often precipitate during storage. Adding a surfactant to the system overcomes the turbidity phenomenon caused by long-term storage.

[0031] (4) The surfactant prepared by the present invention contains quaternary ammonium salt molecules, which contain positively charged quaternary ammonium groups, which can attract and bind to negatively charged molecules such as phosphatidylcholine on the surface of bacterial cell membranes. In addition, the surfactant contains phosphorylcholine groups, which increases the compatibility with bacterial cell membranes, thereby increasing the ability of quaternary ammonium salt functional groups to damage and destroy cell membranes. In addition, quaternary ammonium salts can also affect the synthesis of bacterial proteins and nucleic acids, further enhancing their bactericidal effects. DETAILED DESCRIPTION

[0032] The invention will be further described below in conjunction with the embodiments, but the invention is not limited to these embodiments.

[0033] Example 1 Preparation of surfactant:

[0034] Under nitrogen protection, 500g of tetrahydrofuran and 50g of 1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine were added to the reactor, stirred, and heated to 70°C. At the same time, 60g of 2-methacryloyloxyethyl phosphorylcholine and 0.1g of initiator AIBN were slowly added dropwise for 10min, and then the temperature was raised to 100°C for reaction for 24h, cooled to room temperature, 800g of deionized water was added and stirred evenly, centrifuged, washed with 40g of anhydrous ethanol and then with 20g of deionized water, and dried in vacuo at 80°C for 5h to obtain a surfactant.

[0035] Example 2 Preparation of surfactant:

[0036] Under nitrogen protection, 500g of tetrahydrofuran and 80g of 1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine were added to the reactor, stirred, and heated to 75°C. At the same time, 90g of 2-methacryloyloxyethyl phosphorylcholine and 0.2g of initiator AIBN were slowly added dropwise for 15min, and then the temperature was raised to 110°C for reaction for 20h, cooled to room temperature, 800g of deionized water was added, stirred evenly, centrifuged, washed with 40g of anhydrous ethanol and then with 20g of deionized water, and dried under vacuum at 90°C for 4h to obtain a surfactant.

[0037] Example 3 Preparation of surfactant:

[0038] Under nitrogen protection, 500g of tetrahydrofuran and 100g of 1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine were added to the reactor, stirred, and heated to 80°C. At the same time, 120g of 2-methacryloyloxyethyl phosphorylcholine and 0.3g of initiator AIBN were slowly added dropwise for 20min, and then the temperature was raised to 120°C for reaction for 16h, cooled to room temperature, 800g of deionized water was added, stirred evenly, centrifuged, washed with 40g of anhydrous ethanol and then with 20g of deionized water, and dried in vacuo at 100°C for 3h to obtain a surfactant.

[0039] Example 4 Preparation of pharmaceutical peptides:

[0040] S1: Weigh 150g of Scutellaria baicalensis, 100g of Andrographis paniculata, 100g of Galla chinensis, 50g of Honeysuckle, 40g of Licorice, 20g of Shenqu, 20g of Red Ginseng, 50g of Pulsatilla chinensis, and 80g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 5kg of purified water for decoction, and decoct at 95℃ for 7h to obtain a decoction;

[0041] S2: The activated Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum were inoculated into their respective culture media. The inoculation amount of Lactobacillus bulgaricus was 4% (v / v), the inoculation amount of Lactobacillus rhamnosus was 2% (v / v), and the inoculation amount of Bifidobacterium longum was 1% (v / v). The cell concentration of Lactobacillus bulgaricus after inoculation was 2×10 7 cfu / g, the concentration of Lactobacillus rhamnosus was 2×10 7 cfu / g, the cell concentration of Bifidobacterium longum was 2×10 7 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0042] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus bulgaricus, 20 ml of the culture solution of Lactobacillus rhamnosus, and 20 ml of the culture solution of Bifidobacterium longum prepared in step S2, stirred evenly, and then cultured in a closed manner at 30° C. for 36 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 2 g of a surfactant (prepared in Example 1) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0043] Example 5 Preparation of pharmaceutical peptides:

[0044] S1: Weigh 200g of Scutellaria baicalensis, 150g of Andrographis paniculata, 120g of Galla chinensis, 80g of Honeysuckle, 60g of Licorice, 40g of Shenqu, 30g of Red Ginseng, 80g of Pulsatilla chinensis, and 100g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 7kg of purified water for decoction, and decoct at 100℃ for 6h to obtain a decoction;

[0045] S2: The activated Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum were inoculated into the culture medium respectively. The inoculation amount of Lactobacillus bulgaricus was 5% (v / v), the inoculation amount of Lactobacillus rhamnosus was 3% (v / v), and the inoculation amount of Bifidobacterium longum was 2% (v / v). The cell concentration of Lactobacillus bulgaricus after inoculation was 5×10 8 cfu / g, the concentration of Lactobacillus rhamnosus was 3×10 8 cfu / g, the cell concentration of Bifidobacterium longum was 3×10 8 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0046] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus bulgaricus, 20 ml of the culture solution of Lactobacillus rhamnosus, and 20 ml of the culture solution of Bifidobacterium longum prepared in step S2, and stirred evenly, and then cultured in a closed manner at 35° C. for 30 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 3 g of a surfactant (prepared in Example 2) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0047] Example 6 Preparation of pharmaceutical peptides:

[0048] S1: Weigh 250g of Scutellaria baicalensis, 200g of Andrographis paniculata, 150g of Galla chinensis, 100g of Honeysuckle, 80g of Licorice, 60g of Shenqu, 40g of Red Ginseng, 100g of Pulsatilla chinensis, and 120g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 10kg of purified water for decoction, and decoct at 105℃ for 5h to obtain a decoction;

[0049] S2: The activated Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum were inoculated into the culture medium respectively. The inoculation amount of Lactobacillus bulgaricus was 6% (v / v), the inoculation amount of Lactobacillus rhamnosus was 4% (v / v), and the inoculation amount of Bifidobacterium longum was 3% (v / v). The cell concentration of Lactobacillus bulgaricus after inoculation was 6.8×10 8 cfu / g, and the concentration of Lactobacillus rhamnosus was 3.4×10 8 cfu / g, and the cell concentration of Bifidobacterium longum was 3.4×10 8 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0050] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus bulgaricus, 20 ml of the culture solution of Lactobacillus rhamnosus, and 20 ml of the culture solution of Bifidobacterium longum prepared in step S2, and stirred evenly, and then cultured in a closed manner at 37° C. for 24 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 4 g of a surfactant (prepared in Example 3) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0051] In the above-mentioned Examples 4-6, the culture medium used for Lactobacillus bulgaricus is as follows: beef extract peptone medium: 3g beef extract, 100g peptone, 50g sodium chloride, 1000ml distilled water, 10ml 1mol / L sodium hydroxide solution, pH adjusted to 7.4, and filtered at 121°C, 0.1MPa for 20min to obtain.

[0052] The culture medium used for Lactobacillus rhamnosus is as follows: MRS culture medium: peptone 100g, beef extract 100g, yeast extract 50g, glucose 200g, diammonium citrate 20g, sodium acetate 50g, dipotassium hydrogen phosphate 20g, corn steep liquor 30g, magnesium sulfate 2g, manganese sulfate 2g, cysteine ​​hydrochloride 4g, Tween 80: 10ml, add distilled water to 10L. Add 10ml 1mol / L sodium hydroxide solution, adjust pH=6.8, filter and dispense into anaerobic tubes, fill with nitrogen, and sterilize at 121℃ and 0.1MPa for 30min.

[0053] The culture medium used for Bifidobacterium longum is as follows: BBL agar medium: 150g peptone, 200g glucose, 20g yeast extract powder, 5g soluble starch, 50g sodium chloride, 5g L-cysteine, 50g tomato extract powder, 20g liver extract powder, 200g agar, 10ml Tween 80, add distilled water to 10L. Add 10ml 1mol / L sodium hydroxide solution, adjust pH=7, filter and dispense into anaerobic tubes, fill with nitrogen, and sterilize at 115℃ and 0.1MPa for 20min.

[0054] Comparative Example 1

[0055] A pharmaceutical peptide, the raw material composition and process are basically the same as those in Example 5, except that no surfactant is added during the preparation of the pharmaceutical peptide.

[0056] Comparative Example 2

[0057] A pharmaceutical peptide, the raw material composition and process are basically the same as those of Example 5, except that the surfactant is replaced by an equal weight of commercially available polyethylene glycol laurate (PEG200DL).

[0058] Comparative Example 3

[0059] A pharmaceutical peptide, the raw material composition and process are basically the same as those in Example 5, except that the surfactant is replaced by 2g 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine and 2g 2-methacryloyloxyethylphosphocholine.

[0060] Comparative Example 4

[0061] A pharmaceutical peptide, the raw material composition and process are basically the same as those in Example 5, except that the surfactant is replaced with an equal weight of a surfactant prepared by the following method:

[0062] Under nitrogen protection, 500 g of tetrahydrofuran and 80 g of 1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine were added to the reactor, stirred, and heated to 75°C. At the same time, 90 g of acrylamidopropyl dimethylbutylammonium bromide and 0.2 g of initiator AIBN were slowly added dropwise for 15 min, and then the temperature was raised to 110°C for reaction for 20 h, cooled to room temperature, 800 g of deionized water was added, stirred evenly, centrifuged, washed with 40 g of anhydrous ethanol and then with 20 g of deionized water, and dried under vacuum at 90°C for 4 h to obtain a surfactant.

[0063] Comparative Example 5

[0064] A pharmaceutical peptide, the raw material composition and process are basically the same as those of Example 5, except that the surfactant is replaced by an equal weight of 2,3-dipalmitoyl-sn-glycero-1-phosphocholine.

[0065] Comparative Example 6

[0066] A pharmaceutical peptide, the raw material composition and process are basically the same as those in Example 5, except that the surfactant is replaced with an equal weight of a surfactant prepared by the following method:

[0067] Under nitrogen protection, 500g of tetrahydrofuran and 80g of 2-acrylamide-2-methylpropanesulfonic acid were added to the reactor, stirred, and heated to 75°C. At the same time, 90g of 2-methacryloyloxyethyl phosphorylcholine and 0.2g of initiator AIBN were slowly added dropwise for 15min, and then the temperature was raised to 110°C for reaction for 20h, cooled to room temperature, 800g of deionized water was added and stirred evenly, centrifuged, washed with 40g of anhydrous ethanol and then with 20g of deionized water, and dried under vacuum at 90°C for 4h to obtain a surfactant.

[0068] Comparative Example 7

[0069] A pharmaceutical peptide, the raw material composition and process are basically the same as those in Example 5, except that the amount of surfactant added in step S3 is 1 g.

[0070] Inhibition precipitation test:

[0071] Take 3 groups of the pharmaceutical peptides prepared in Examples 4-6 of the present application and Comparative Examples 1-7, each group with 30 mL, and place them at room temperature away from light. Take one group of them for filtration every 30 days, dry the precipitate with a nitrogen blower, and weigh it. The test results are shown in Table 1.

[0072] Table 1

[0073]

[0074] It can be seen from Table 1 that after adding the surfactant, the amount of precipitation of the peptide spray was significantly reduced within 3 months. After 3 months of placement, the amount of precipitation was about 10 mg / 30 ml, indicating that the prepared surfactant can effectively inhibit the formation of precipitation after addition.

[0075] Comparative Example 1 is different from Example 5 in that no surfactant is added before filling. As can be seen from Table 1, after standing for 3 months, the amount of precipitation reaches 27.96 mg / 30 ml, indicating that when no surfactant is added, the amount of precipitation is large after standing for a period of time.

[0076] The surfactant added in Comparative Example 2 is commercially available polyethylene glycol laurate. As can be seen from Table 1, after 3 months of storage, the amount of precipitation reaches 17.67 mg / 30 ml, indicating that the commercially available surfactant has a poor effect in inhibiting precipitation.

[0077] In Comparative Example 3, the added surfactant was replaced with 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine and 2-methacryloyloxyethylphosphocholine. As can be seen from Table 1, after 3 months of storage, the precipitation amount reached 17.98 mg / 30 ml.

[0078] The surfactant added in Comparative Example 4 is a polymer of 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine and acrylamidopropyldimethylbutylammonium bromide. As can be seen from Table 1, after standing for 3 months, the precipitation amount is 13.52 mg / 30 ml.

[0079] The surfactant added in Comparative Example 5 is 2,3-dipalmitoyl-sn-glycero-1-phosphocholine. It can be seen from Table 1 that after 3 months of storage, the precipitation amount reaches 18.39 mg / 30 ml; after a period of storage, the precipitation amount is large and the precipitation inhibition effect is poor.

[0080] The surfactant added in Comparative Example 6 is prepared from 2-acrylamide-2-methylpropanesulfonic acid and 2-methacryloyloxyethyl phosphorylcholine. As can be seen from Table 1, after 3 months of storage, the amount of precipitation reaches 15.78 mg / 30 ml; the precipitation inhibition effect is not as good as that in Example 5.

[0081] The mass of surfactant added in Comparative Example 7 is 1 g. It can be seen from Table 1 that after 3 months of placement, the amount of precipitation reaches 16.45 mg / 30 ml, indicating that when the concentration of surfactant is low, the precipitation inhibition effect is not as ideal as in Example 5.

[0082] Comparative Example 8 Preparation of drug peptide:

[0083] S1: Weigh 200g of Scutellaria baicalensis, 150g of Andrographis paniculata, 120g of Galla chinensis, 80g of Honeysuckle, 60g of Licorice, 40g of Shenqu, 30g of Red Ginseng, 80g of Pulsatilla chinensis, and 100g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 7kg of purified water for decoction, and decoct at 100℃ for 6h to obtain a decoction;

[0084] S2: The activated Lactobacillus rhamnosus and Bifidobacterium longum strains were inoculated into the culture medium, respectively. The inoculation amount of Lactobacillus rhamnosus was 3% (v / v) and the inoculation amount of Bifidobacterium longum was 2% (v / v). The cell concentration of Lactobacillus rhamnosus after inoculation was 3×10 8 cfu / g, the cell concentration of Bifidobacterium longum was 3×10 8 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0085] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus rhamnosus and 20 ml of the culture solution of Bifidobacterium longum prepared in step S2, and stirred evenly, and then cultured in a closed manner at 35° C. for 30 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 3 g of a surfactant (prepared in Example 2) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0086] Comparative Example 9 Preparation of drug peptide:

[0087] S1: Weigh 200g of Scutellaria baicalensis, 150g of Andrographis paniculata, 120g of Galla chinensis, 80g of Honeysuckle, 60g of Licorice, 40g of Shenqu, 30g of Red Ginseng, 80g of Pulsatilla chinensis, and 100g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 7kg of purified water for decoction, and decoct at 100℃ for 6h to obtain a decoction;

[0088] S2: The activated Lactobacillus bulgaricus and Bifidobacterium longum strains were inoculated into the culture medium, respectively. The inoculation amount of Lactobacillus bulgaricus was 5% (v / v) and the inoculation amount of Bifidobacterium longum was 2% (v / v). The cell concentration of Lactobacillus bulgaricus after inoculation was 5×10 8 cfu / g, the cell concentration of Bifidobacterium longum was 3×10 8 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0089] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus bulgaricus and 20 ml of the culture solution of Bifidobacterium longum prepared in step S2, and stirred evenly, and then cultured in a closed manner at 35° C. for 30 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 3 g of a surfactant (prepared in Example 2) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0090] Comparative Example 10 Preparation of drug peptide:

[0091] S1: Weigh 200g of Scutellaria baicalensis, 150g of Andrographis paniculata, 120g of Galla chinensis, 80g of Honeysuckle, 60g of Licorice, 40g of Shenqu, 30g of Red Ginseng, 80g of Pulsatilla chinensis, and 100g of Dioscorea opposita, and grind each of the Chinese medicinal materials and mix them evenly; then add 7kg of purified water for decoction, and decoct at 100℃ for 6h to obtain a decoction;

[0092] S2: The activated Lactobacillus bulgaricus, Lactobacillus casei and Bifidobacterium breve were inoculated into the culture medium respectively. The inoculation amount of Lactobacillus bulgaricus was 5% (v / v), the inoculation amount of Lactobacillus casei was 3% (v / v), and the inoculation amount of Bifidobacterium breve was 2% (v / v). The cell concentration of Lactobacillus bulgaricus after inoculation was 5×10 8 cfu / g, the cell concentration of Lactobacillus casei was 3×10 8 cfu / g, the bacterial concentration of Bifidobacterium breve was 3×10 8 cfu / g, cultured overnight at 37°C with shaking at 200 rpm to obtain the culture fluid of each bacterial species;

[0093] S3: 100 g of the decoction was mixed with 20 ml of the culture solution of Lactobacillus bulgaricus, 20 ml of the culture solution of Lactobacillus casei, and 20 ml of the culture solution of Bifidobacterium breve prepared in step S2, and stirred evenly, and then cultured in a closed manner at 35° C. for 30 h; filtered using a horizontal screw centrifuge with a rotation speed of 3500 rpm, and the obtained filtrate was centrifuged and purified using a three-phase centrifuge with a rotation speed of 15000 rpm, and then concentrated using a double-effect concentrator, 3 g of a surfactant (prepared in Example 2) was added and mixed evenly, and finally sterilized at 121° C. and 0.1 MPa for 20 min to obtain the pharmaceutical peptide.

[0094] The culture medium used for Bifidobacterium breve is the same as that for Bifidobacterium longum, and the culture medium used for Lactobacillus casei is the same as that for Lactobacillus rhamnosus.

[0095] The efficacy of the present invention is described below with specific tests:

[0096] A total of 280 outpatient health care patients were selected, including 140 males and 140 females, aged between 30 and 38 years old. There were 35 people in each of the experimental group and the control group.

[0097] How to use the peptide:

[0098] 1. Clean the skin and spray the peptide on the skin (about 0.4 ml per 5 square centimeters).

[0099] 2. Press the cupping device against the skin so that it adheres to the skin; tap and move the cupping device until the skin area adhered to by the cupping device reaches the desired value. The cupping process should continue for 30 minutes.

[0100] 3. Pat the cupping device to loosen the skin, then remove the cupping device, clean the skin, and spray the medicinal peptide on the cupping position of the skin (about 0.4 ml per 5 square centimeters); spray again after 24 hours (about 0.4 ml per 5 square centimeters).

[0101] The test groups 1-3 of the present application used the drug peptides prepared in Examples 4-6, the control group 1 did not use any drugs, the control group 2 used the black ant active oil produced by Nanjing Tongrentang, and the control groups 3-5 used the drug peptides prepared in Comparative Examples 8-10 respectively. After cupping, the disappearance of the skin redness, swelling and congestion symptoms was observed 3 days later. The test results are shown in Table 2.

[0102] Table 2

[0103]

[0104] In Table 2, "cured" means that the symptoms of redness, swelling and congestion have completely disappeared, "remarkably effective" means that the symptoms of redness, swelling and congestion have basically disappeared, and "effective" means that the symptoms of redness, swelling and congestion have improved.

[0105] It can be seen from the data in Table 2 that the number of people cured by the drug peptides prepared in the embodiments of the present invention for eliminating redness, swelling and congestion is greater than 28, which is more than the number of cured people in the blank group, the drug peptides prepared by fermentation of other strains, and the control group using active oil.

[0106] No adverse reactions occurred in the experimental group or the control group during the clinical study.

[0107] Antibacterial property test of drug peptides: The drug peptides prepared in Examples 4-6 and Comparative Examples 1-7 of the present application were tested for antibacterial properties. The antibacterial property of the drug peptides was detected by the oscillation method according to GB / T 20944.3-2008 standard. A plant fiber membrane with strong adsorption properties was used as a carrier for drug peptide attachment. A 10 mm × 10 mm plant fiber membrane (thickness of about 0.2 mm) was taken and spread horizontally so that it did not touch the table. The drug peptide was sprayed evenly, and the unit spraying amount of the drug peptide on the sample was about 0.008 ml / cm 2 The antibacterial performance test results are shown in Table 3.

[0108] Table 3

[0109]

[0110] It can be seen from Table 3 that the drug peptide prepared in the present application has a highly effective antibacterial property against Escherichia coli 8099 and Staphylococcus aureus ATCC6538. However, when the drug peptide does not contain the surfactant prepared in the present application, contains a surfactant without phosphorylcholine, or other types of phosphorylcholine surfactants, the 24h antibacterial rate is less than 98%, and the antibacterial property is poor.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. However, any equivalent changes, modifications and evolutions made by ordinary technicians in the field without departing from the scope of the technical solution of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a pharmaceutical peptide, characterized in that: The following steps are involved: S1: Weigh 15-25 parts of Scutellaria baicalensis, 10-20 parts of Andrographis paniculata, 10-15 parts of Galla chinensis, 5-10 parts of Honeysuckle, 4-8 parts of Licorice, 2-6 parts of Shenqu, 2-4 parts of Red Ginseng, 5-10 parts of Pulsatilla chinensis, and 8-12 parts of Dioscorea opposita by weight, and grind each Chinese medicine raw material separately and mix them evenly; then add purified water for decoction to obtain a decoction; S2: inoculating the activated strains of Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium longum into the culture medium respectively, and culturing with shaking at 37° C. overnight to obtain a culture solution; S3: mixing the decoction with the culture solution of each bacterial species in step S2, stirring evenly, and then culturing in a closed manner at 30-37° C. for 24-36 hours; filtering, purifying, concentrating, adding a surfactant, and sterilizing to obtain the drug peptide; In step S3, the surfactant is prepared by the following method: Under nitrogen protection, tetrahydrofuran and 1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine are added to the reactor, stirred, and heated to 70-80°C. At the same time, 2-methacryloyloxyethyl phosphorylcholine and initiator AIBN are slowly added dropwise, and then the temperature is raised to 100-120°C for reaction for 16-24h, and a surfactant is obtained by post-treatment.

2. The method for preparing a pharmaceutical peptide according to claim 1, characterized in that: In the step S2, the inoculation amount of Lactobacillus bulgaricus is 4-6 v / v%, the inoculation amount of Lactobacillus rhamnosus is 2-4 v / v%, and the inoculation amount of Bifidobacterium longum is 1-3 v / v%.

3. The method for preparing a pharmaceutical peptide according to claim 2, characterized in that: The feed mass ratio of tetrahydrofuran, 1-O-1'-(Z)-octadecene-2-hydroxy-sn-glycero-3-phosphocholine, 2-methacryloyloxyethylphosphocholine and AIBN is 50:(5-10):(6-12):(0.01-0.03).

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