Chinese and western combined qi and blood nourishing composition and preparation method thereof

Abstract

The invention belongs to the field of biological medicine manufacturing, and particularly relates to a Chinese and western combined qi and blood nourishing composition and a preparation method thereof. The Chinese and western combined qi and blood nourishing composition comprises three functional components, namely a modified sponge microneedle, a nano-liposome active substance and marine-derived oligopeptide. Wherein the modified sponge microneedle plays a role in mechanical support and introduction promotion, and is obtained by modifying mulberry silk protein by adopting polyglutamic acid tyrosine sodium salt. Nano-liposome active substances are main fatigue relieving action substances, specifically, bighead atractylodes rhizome, radix bupleuri and the like are extracted and then encapsulated into a liposome form, and rapid absorption is facilitated. The marine source oligopeptide liquid mainly plays a moisturizing effect, and relieves slight pain of the Chinese and western combined qi and blood nourishing composition in use. The traditional Chinese and western combined qi and blood nourishing composition provided by the invention is verified by a mouse weight-bearing exhaustive swimming experiment to have a relatively good fatigue relieving effect, and the effect is relatively good after the traditional Chinese and western combined qi and blood nourishing composition is applied to corresponding acupuncture points.

Description

Technical Field

[0001] The invention belongs to the field of biological medicine manufacturing, and specifically relates to a Chinese and Western combined qi and blood nourishing composition and a preparation method thereof. Background Art

[0002] Traditional Chinese medicine has the basic theory of qi, blood, essence, and body fluids. Essence, qi, blood, and body fluids are the basic substances that constitute and maintain human life activities, the products of the functional activities of the internal organs, and the material basis of the functional activities of the internal organs. Spirit is the general term for the master of human life activities and its external overall manifestation. Spirit is based on essence, qi, blood, and body fluids, and regulates the generation and operation of these basic substances. "Lingshu·Benzang" records: "Human blood, qi, and spirit are what support life and circulate in nature and life." In addition, the generation of human qi depends on the comprehensive function of all internal organs in the body, and is particularly closely related to the kidney, spleen, stomach, and lungs. The kidney is the root of qi, the spleen and stomach are the source of qi, and the lungs are the master of qi. The normal movement of qi is called "smooth qi movement", including the balanced coordination and unobstructed state of ascending, descending, entering, and exiting. "Lingshu·Jueqi" records that "the middle jiao receives qi and takes juice, which changes and becomes red, which is called blood." In addition, the spleen and stomach are the source of blood biochemistry, and the nutrient qi and body fluids produced by the transformation of water and grain are the main components of blood. The circulation of blood depends on the warming and consolidation of Qi. "Medical Biography·Qi and Blood" says: "Blood cannot circulate without Qi." The integrity and unobstructedness of the meridians are also important factors to ensure the normal circulation of blood. For example, "Suwen·Bazheng Shenming Lun" records that "Blood and Qi are the spirit of man, and must be carefully nurtured." Therefore, TCM methods for nurturing Qi and blood include exercise, diet therapy, acupuncture, Chinese medicine treatment, etc.

[0003] Qi and blood in traditional Chinese medicine correspond to the human body's circulation and immune system. When the blood circulation system is not good, it often leads to slow metabolism of local tissues, reduced heat production, cold hands and feet, dizziness and edema, etc. When the immune system is underfunctioning, it often leads to fatigue, inflammatory reactions, allergic diseases, gastrointestinal infections, etc. Western medicine often treats the above problems with drugs, which can be taken orally, injected or applied externally.

[0004] In modern society, people often suffer from poor circulation of Qi and blood due to unbalanced diet, high stress in life, irregular work and rest, lack of exercise, etc. Symptoms include pale or sallow complexion, fatigue, lack of physical strength, dizziness, cold hands and feet, irregular heartbeat, poor appetite, emotional instability, etc. The above symptoms involve the complications of many diseases or exist as the main symptoms. In traditional Chinese medicine, the pathogenesis of chronic fatigue syndrome is mostly attributed to the imbalance and weakness of the internal organs, Qi and blood. It is defined as long-term (more than 6 consecutive months) unexplained fatigue or physical discomfort, which is difficult to relieve with rest, accompanied by headache, insomnia, low fever, anxiety, memory loss, etc. Clinical examinations often show no obvious organic lesions. Conventional treatment is based on specific and prominent symptoms. Auxiliary treatment methods include nutritional supplements, graded exercise therapy and cognitive behavioral therapy, but the effects are often different. Summary of the invention

[0005] Based on the above problems, the present invention provides a Chinese and Western combination of qi and blood maintenance composition, comprising three functional components, namely modified sponge microneedles, nanoliposome active substances and marine oligopeptides. Among them, the modified sponge microneedles play a role of mechanical support and promotion of introduction, and are obtained by modifying mulberry silk protein with polyglutamic acid tyrosine sodium salt. Nanoliposome active substances are the main fatigue relief agents, specifically, Atractylodes macrocephala, Bupleurum chinense, etc. are extracted, and then encapsulated into liposome form, which helps rapid absorption. Marine source oligopeptide liquid mainly plays a moisturizing effect, alleviating the slight pain of the Chinese and Western combination of qi and blood maintenance composition when it is used. The Chinese and Western combination of qi and blood maintenance composition obtained by the present invention has a good fatigue relief effect after being verified by the mouse weight-bearing exhaustive swimming experiment, and the effect is better after being used on the corresponding acupoints.

[0006] The invention provides a Chinese and Western combined qi and blood nourishing composition, comprising the following raw materials in parts by mass: 3-8 parts of modified sponge microneedles, 2-5 parts of nanoliposome active substances, and 3-6 parts of marine oligopeptide liquid.

[0007] The preparation method of the modified sponge microneedle is as follows: S1. Weigh mulberry silk and sodium carbonate-sodium bicarbonate buffer according to a mass volume ratio of 1g:50-60mL, heat to 80-90℃ and keep for 50-60min after mixing, rinse with deionized water after completion, dry at 30-40℃ to obtain a dried product, weigh the dried product and lithium bromide solution according to a mass volume ratio of 1g:8-10mL, heat to 50-60℃ and keep for 4-5h, then dialyze with deionized water for 1-2d and reverse dialyze with polyethylene glycol solution for 1-2d to obtain a concentrated solution; S2. The concentrated solution obtained in step S1 is freeze-dried in vacuo to obtain substance 1, substance 1, ethanol solution, and calcium chloride are weighed according to a mass volume ratio of 1 g: 20-30 mL: 0.5-0.8 g, and the mixture is stirred at a speed of 150-200 rpm for 10-15 min. After the mixture is washed with deionized water, centrifuged at a centrifugal force of 6000-8000 g for 8-10 min, and a precipitate is obtained after the mixture is dried at 30-40° C. to obtain substance 2; S3. Dissolve polyglutamic acid tyrosine sodium salt in Tris-hydrochloric acid buffer to obtain solution 1 with a mass volume concentration of 4-6 mg / mL, weigh the substance 2 obtained in step S2 and solution 1 according to the mass volume ratio of 1g:10-15mL, ultrasonically treat at a power of 180-240w for 5-8min, keep in a 35-40℃ water bath for 20-30h, centrifuge at a centrifugal force of 6000-8000g for 10-15min, wash the precipitate with deionized water and resuspend it, sterilize it under high pressure, precool it at -20℃ for 8-10h, and freeze-dry it for 2-3d to obtain the modified sponge microneedle.

[0008] Preferably, in step S1, the concentration of the sodium carbonate-sodium bicarbonate buffer is preferably 0.01 M, pH = 9.0, the concentration of the lithium bromide solution is preferably 8-9 M, the deionized water dialysis preferably uses a dialysis bag with a molecular weight cutoff of 5-6 KD, the mass percentage concentration of the polyethylene glycol solution is 30-35%, and the average molecular weight of the polyethylene glycol used is 10000.

[0009] Preferably, the volume fraction of the ethanol solution in step S2 is preferably 50%.

[0010] Preferably, in step S3, the molar ratio of glutamic acid:tyrosine in the polyglutamic acid tyrosine sodium salt is 1:4, the molecular weight of the polyglutamic acid tyrosine sodium salt is preferably 5000-10000, the concentration of the Tris-hydrochloric acid buffer is preferably 1M, and the pH is 8.0.

[0011] The preparation method of the nanoliposome active substance is as follows: L1. Grind dried Atractylodes macrocephala, Bupleurum chinense, Magnolia officinalis, Chuanxiong and Acorus calamus into powder, pass through a 2-mesh sieve and mix in a mass ratio of 5-8:3-5:2-6:1:1-2 to obtain a mixed powder, add 8-10 times the mass of water of the mixed powder, steam distill for 3-5h, and after the end, the obtained extract is dried over anhydrous sodium sulfate and set aside; L2. Cholesterol, sunflower lecithin, the extract obtained in step L1, and dichloromethane were weighed according to a mass volume ratio of 1-1.2 g: 1-1.5 g: 0.8-1 g: 10-15 L, and stirred until uniformly dispersed to obtain a mixed solution, and the mixed solution was heated to 37 ° C and rotary evaporated until the dichloromethane was completely removed to obtain a thin lipid film; L3. Add distilled water to the thin lipid film obtained in step L2, heat to 40-50°C, hydrate at a speed of 150-200rpm for 30-50min, cool naturally to room temperature, and then ultrasonically treat at a power of 100-150w for 30-40min. After the end, the obtained solution is filtered through 0.45μm and 0.22μm filter membranes in turn, and the filtrate is the nanoliposome active substance.

[0012] Preferably, the thin lipid film will adhere to the wall of the distillation flask, so the amount of distilled water added in step L3 is based on the volume of the mixed solution in step L2, and the added amount is preferably 0.2-0.25 times the volume of the mixed solution.

[0013] The preparation method of the marine oligopeptide liquid is as follows: V1. Take bonito meat, freeze-dry it in vacuum and grind it into powder, pass it through a 60-mesh sieve to obtain bonito powder, weigh bonito powder and water according to a mass volume ratio of 1g: 80-100mL, soak it at room temperature for 2-3h, boil it and keep it for 30-40min, and then cool it naturally to room temperature to obtain solution 2; V2. Place the solution 2 obtained in step V1 in a 45-50°C water bath environment until the temperature is balanced, add alkaline protease, and treat at a speed of 100-150rpm for 1-2h. After the treatment, quickly take it out and inactivate the enzyme at high temperature, then centrifuge at a centrifugal force of 5000-6000g for 20-30min, separate with an ultrafiltration membrane, and freeze-dry the isolate to obtain the marine oligopeptide; V3. Weigh the marine oligopeptide and trehalose solution obtained in step V2 according to a mass volume ratio of 1g:3-5mL, and stir until they are evenly dispersed to obtain the marine oligopeptide solution.

[0014] Preferably, the enzymatic activity of the alkaline protease in step V2 is preferably 20000 U / g, the amount of alkaline protease added is 0.3-0.5 g / mL, and the ultrafiltration membrane preferably has a pore size of 3 kD.

[0015] Preferably, the mass percentage concentration of the trehalose solution in step V3 is preferably 8-10%.

[0016] The present invention also provides a method for preparing a Chinese and Western combined qi and blood nourishing composition, the specific steps of which are as follows: The modified sponge microneedles, nanoliposome active substances, and marine oligopeptide liquid are weighed according to the corresponding mass fractions, the nanoliposome active substances and the marine oligopeptide liquid are stirred until they are evenly dispersed, and then the modified sponge microneedles are added, stirred until they are evenly dispersed and stored at 4°C to obtain a Chinese and Western combined qi and blood nourishing composition.

[0017] The beneficial effects of the present invention are as follows: Chronic fatigue syndrome is a disease that is consistent with the traditional Chinese medicine theory of poor circulation of qi and blood, and also involves the situation of poor circulation and immune system in Western medicine, but its complexity lies in the fact that it involves many symptoms and is difficult to treat symptomatically. Based on this, the present application provides a Chinese and Western combination of qi and blood maintenance composition, which is mainly based on daily promotion to slow down the development of chronic fatigue syndrome and improve related symptoms. The Chinese and Western combination of qi and blood maintenance composition obtained by the present invention comprises three functional components, namely modified sponge microneedles, nanoliposome active substances and marine oligopeptides. Among them, the modified sponge microneedles play a role of mechanical support and promotion of introduction, and are obtained by modifying mulberry silk protein with polyglutamic acid tyrosine sodium salt. Nanoliposome active substances are the main fatigue-relieving agents. Specifically, they are extracted from Atractylodes macrocephala and Bupleurum chinense, and then encapsulated in liposome form, which helps to quickly absorb. Among them, Atractylodes macrocephala can stimulate local acupoints to strengthen the spleen and replenish qi, Bupleurum chinense can relieve liver depression, Magnolia officinalis can enhance immunity and promote blood circulation, Chuanxiong can promote blood circulation, and Acorus calamus can relieve itching, inhibit bacteria, and reduce inflammation and dampness. The marine source oligopeptide liquid mainly plays a moisturizing effect, relieving the slight pain when the Chinese and Western combined qi and blood care composition is used.

[0018] The Chinese and Western combined qi and blood nourishing composition obtained by the present invention has been verified by a mouse weight-bearing exhaustive swimming experiment to have a good fatigue relief effect, and has a better effect after being used at corresponding acupoints. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 This is the result of the exhaustive swimming experiment on mice in Housanli; Figure 2 This is the result of the exhaustive swimming experiment of mice in Neiguan; Figure 3 The following is a graph showing the results of the exhaustive swimming experiment on mice in Qiansanli. DETAILED DESCRIPTION

[0020] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0021] Example 1: This example provides a method for preparing a modified sponge microneedle, the specific contents are as follows: S1. Weigh mulberry silk and sodium carbonate-sodium bicarbonate buffer (0.01M, pH=9.0) in a mass-volume ratio of 1g:50mL, heat to 80°C and keep for 50min after mixing, rinse with deionized water after completion, dry at 30°C to obtain a dried product, weigh the dried product and lithium bromide solution in a mass-volume ratio of 1g:8mL, the concentration of the lithium bromide solution is 8M, heat to 50°C and keep for 4h, then dialyze with deionized water for 1d, using a dialysis bag with a molecular weight cutoff of 5KD, and then reverse dialyze with a polyethylene glycol solution for 1d, the mass percentage concentration of the polyethylene glycol solution is 30%, and the average molecular weight of the polyethylene glycol is 10000, to obtain a concentrated solution; S2. The concentrated solution obtained in step S1 is freeze-dried in vacuo to obtain substance 1, substance 1, ethanol solution, and calcium chloride are weighed according to a mass volume ratio of 1 g: 20 mL: 0.5 g, the volume fraction of the ethanol solution is 50%, and the mixture is stirred at a speed of 150 rpm for 10 min. After the mixture is stirred, it is washed with deionized water and centrifuged at a centrifugal force of 6000 g for 8 min. After the mixture is stirred, a precipitate is obtained, and the precipitate is dried at 30° C. to obtain substance 2; S3. Dissolve polyglutamic acid tyrosine sodium salt in Tris-hydrochloric acid buffer (1M, pH=8.0) to obtain solution 1 with a mass volume concentration of 4 mg / mL, the molar ratio of glutamic acid: tyrosine in polyglutamic acid tyrosine sodium salt is 1:4, and the molecular weight of polyglutamic acid tyrosine sodium salt is 5000. Weigh the substance 2 obtained in step S2 and solution 1 at a mass volume ratio of 1g:10mL, and treat with ultrasound at a power of 180w for 5min. After the end, keep it in a water bath at 35℃ for 20h, centrifuge it at a centrifugal force of 6000g for 10min, wash the precipitate with deionized water and resuspend it, sterilize it under high pressure, precool it at -20℃ for 8h, and freeze-dry it for 2d to obtain the modified sponge microneedle.

[0022] Embodiment 2: This embodiment provides a method for preparing a modified sponge microneedle, the specific contents are as follows: S1. Weigh mulberry silk and sodium carbonate-sodium bicarbonate buffer (0.01M, pH=9.0) in a mass-volume ratio of 1g:55mL, heat to 85°C and keep for 54min after mixing, rinse with deionized water after completion, dry at 35°C to obtain a dried product, weigh the dried product and lithium bromide solution in a mass-volume ratio of 1g:9mL, the concentration of the lithium bromide solution is 8.5M, heat to 54°C and keep for 4.5h, then dialyze with deionized water for 1.5d, using a dialysis bag with a molecular weight cutoff of 5.5KD, and then reverse dialyze with a polyethylene glycol solution for 1.5d, the mass percentage concentration of the polyethylene glycol solution is 32%, and the average molecular weight of the polyethylene glycol is 10000, to obtain a concentrated solution; S2. The concentrated solution obtained in step S1 is freeze-dried in vacuo to obtain substance 1, substance 1, ethanol solution, and calcium chloride are weighed according to a mass volume ratio of 1 g: 25 mL: 0.7 g, the volume fraction of the ethanol solution is 50%, and the mixture is stirred at a speed of 180 rpm for 12 min. After the mixture is stirred, it is washed with deionized water and centrifuged at a centrifugal force of 7000 g for 9 min. After the mixture is stirred, a precipitate is obtained, and the precipitate is dried at 35° C. to obtain substance 2; S3. Dissolve polyglutamic acid tyrosine sodium salt in Tris-hydrochloric acid buffer (1M, pH=8.0) to obtain solution 1 with a mass volume concentration of 5 mg / mL, the molar ratio of glutamic acid: tyrosine in polyglutamic acid tyrosine sodium salt is 1:4, and the molecular weight of polyglutamic acid tyrosine sodium salt is 8000. Weigh the substance 2 obtained in step S2 and solution 1 according to the mass volume ratio of 1g:12mL, and treat with ultrasound at a power of 200w for 6min. After the end, keep it in a water bath at 38℃ for 25h, centrifuge it at a centrifugal force of 7000g for 12min, wash the precipitate with deionized water and resuspend it, sterilize it under high pressure, precool it at -20℃ for 9h, and freeze-dry it for 2.5d to obtain the modified sponge microneedle.

[0023] Embodiment 3: This embodiment provides a method for preparing a modified sponge microneedle, the specific contents are as follows: S1. Weigh mulberry silk and sodium carbonate-sodium bicarbonate buffer (0.01M, pH=9.0) in a mass volume ratio of 1g:60mL, heat to 90℃ and keep for 60min after mixing, rinse with deionized water after completion, dry at 40℃ to obtain a dried product, weigh the dried product and lithium bromide solution in a mass volume ratio of 1g:10mL, the concentration of the lithium bromide solution is 9M, heat to 60℃ and keep for 5h, then dialyze with deionized water for 2d, using a dialysis bag with a molecular weight cutoff of 6KD, and then reverse dialyze with polyethylene glycol solution for 2d, the mass percentage concentration of the polyethylene glycol solution is 35%, and the average molecular weight of polyethylene glycol is 10000, to obtain a concentrated solution; S2. The concentrated solution obtained in step S1 is freeze-dried in vacuo to obtain substance 1, substance 1, ethanol solution, and calcium chloride are weighed according to a mass volume ratio of 1 g: 30 mL: 0.8 g, the volume fraction of the ethanol solution is 50%, and the mixture is stirred at a speed of 200 rpm for 15 min. After the mixture is stirred, it is washed with deionized water and centrifuged at a centrifugal force of 8000 g for 10 min. After the mixture is stirred, a precipitate is obtained, and the precipitate is dried at 40° C. to obtain substance 2; S3. Dissolve polyglutamic acid tyrosine sodium salt in Tris-hydrochloric acid buffer (1M, pH=8.0) to obtain solution 1 with a mass volume concentration of 6 mg / mL, the molar ratio of glutamic acid: tyrosine in polyglutamic acid tyrosine sodium salt is 1:4, and the molecular weight of polyglutamic acid tyrosine sodium salt is 10000. Weigh the substance 2 obtained in step S2 and solution 1 according to the mass volume ratio of 1g:15mL, and treat with ultrasound at a power of 240w for 8min. After the end, keep it in a water bath at 40℃ for 30h, centrifuge it at a centrifugal force of 8000g for 15min, wash the precipitate with deionized water and resuspend it, sterilize it under high pressure, precool it at -20℃ for 10h, and freeze-dry it for 3d to obtain the modified sponge microneedle.

[0024] Embodiment 4: This embodiment provides a method for preparing nanoliposome active substances, the specific contents are as follows: L1. Grind dried Atractylodes macrocephala, Bupleurum chinense, Magnolia officinalis, Chuanxiong and Acorus calamus into powder, pass through a 2-mesh sieve and mix in a mass ratio of 5:3:2:1:1 to obtain a mixed powder, add water 8 times the mass of the mixed powder, steam distill for 3 hours, and after the end, the obtained extract is dried over anhydrous sodium sulfate and set aside; L2. Cholesterol, sunflower lecithin, the extract obtained in step L1, and dichloromethane were weighed according to a mass volume ratio of 1g:1g:0.8g:10L, and stirred until uniformly dispersed to obtain a mixed solution. The mixed solution was heated to 37°C and rotary evaporated until all dichloromethane was removed to obtain a thin lipid film; L3. Add distilled water to the thin lipid film obtained in step L2, the amount of distilled water added is 0.2 times the volume of the mixed solution, heat to 40°C, hydrate at a speed of 150rpm for 30min, cool naturally to room temperature, and then ultrasonically treat at a power of 100w for 30min. After the end, the obtained solution is filtered through 0.45μm and 0.22μm filter membranes in turn, and the filtrate is the nanoliposome active substance.

[0025] Example 5: This example provides a method for preparing a nanoliposome active substance, the specific contents are as follows: L1. Grind dried Atractylodes macrocephala, Bupleurum chinense, Magnolia officinalis, Chuanxiong and Acorus calamus into powder, pass through a 2-mesh sieve and mix in a mass ratio of 6:4:3:1:1.5 to obtain a mixed powder, add 9 times the mass of water of the mixed powder, steam distill for 4 hours, and after the end, the obtained extract is dried over anhydrous sodium sulfate and set aside; L2. Cholesterol, sunflower lecithin, the extract obtained in step L1, and dichloromethane were weighed according to a mass volume ratio of 1.1 g: 1.2 g: 0.9 g: 12 L, and stirred until uniformly dispersed to obtain a mixed solution, and the mixed solution was heated to 37 ° C and rotary evaporated until the dichloromethane was completely removed to obtain a thin lipid film; L3. Add distilled water to the thin lipid film obtained in step L2, the amount of distilled water added is 0.22 times the volume of the mixed solution, heat to 45°C, hydrate at a speed of 180rpm for 40min, cool naturally to room temperature, and then ultrasonically treat at a power of 120w for 35min. After the end, the obtained solution is filtered through 0.45μm and 0.22μm filter membranes in turn, and the filtrate is the nanoliposome active substance.

[0026] Example 6: This example provides a method for preparing a nanoliposome active substance, the specific contents are as follows: L1. Grind dried Atractylodes macrocephala, Bupleurum chinense, Magnolia officinalis, Chuanxiong and Acorus calamus into powder, pass through a 2-mesh sieve and mix in a mass ratio of 8:5:6:1:2 to obtain a mixed powder, add 10 times the mass of water of the mixed powder, steam distill for 5 hours, and after the end, the obtained extract is dried over anhydrous sodium sulfate and set aside; L2. Cholesterol, sunflower lecithin, the extract obtained in step L1, and dichloromethane were weighed according to a mass volume ratio of 1.2 g: 1.5 g: 1 g: 15 L, and stirred until uniformly dispersed to obtain a mixed solution. The mixed solution was heated to 37 ° C and rotary evaporated until the dichloromethane was completely removed to obtain a thin lipid film; L3. Add distilled water to the thin lipid film obtained in step L2, the amount of distilled water added is 0.25 times the volume of the mixed solution, heat to 50°C, hydrate at a speed of 200rpm for 50min, cool naturally to room temperature, and then ultrasonically treat at a power of 150w for 40min. After the end, the obtained solution is filtered through 0.45μm and 0.22μm filter membranes in turn, and the filtrate is the nanoliposome active substance.

[0027] Example 7: This example provides a method for preparing a marine oligopeptide solution, the specific contents are as follows: V1. Take bonito meat, freeze-dry it in vacuum, grind it into powder, pass it through a 60-mesh sieve to obtain bonito powder, weigh bonito powder and water according to a mass volume ratio of 1g:80mL, soak it at room temperature for 2h, boil it and keep it for 30min, and then cool it naturally to room temperature to obtain solution 2; V2. Place the solution 2 obtained in step V1 in a 45°C water bath until the temperature is balanced, add alkaline protease in an amount of 0.3 g / mL, and the enzyme activity of alkaline protease is 20,000 U / g, and treat at a speed of 100 rpm for 1 hour. After the treatment, quickly take out and inactivate the enzyme at high temperature, then centrifuge at a centrifugal force of 5,000 g for 20 minutes, separate with an ultrafiltration membrane (3 kD), and freeze-dry the isolate to obtain marine oligopeptides; V3. Weigh the marine oligopeptide obtained in step V2 and the trehalose solution according to a mass volume ratio of 1g:3mL. The mass percentage concentration of the trehalose solution is 8%. Stir until it is evenly dispersed to obtain the marine oligopeptide solution.

[0028] Example 8: This example provides a method for preparing a marine oligopeptide solution, the specific contents are as follows: V1. Take bonito meat, freeze-dry it in vacuum, grind it into powder, pass it through a 60-mesh sieve to obtain bonito powder, weigh bonito powder and water according to a mass volume ratio of 1g:90mL, soak it at room temperature for 2.5h, boil it and keep it for 35min, and then cool it naturally to room temperature to obtain solution 2; V2. Place the solution 2 obtained in step V1 in a 48°C water bath until the temperature is balanced, add alkaline protease in an amount of 0.4 g / mL, and the enzyme activity of alkaline protease is 20,000 U / g, and treat at a speed of 120 rpm for 1.5 hours. After the treatment, quickly take out and inactivate the enzyme at high temperature, then centrifuge at a centrifugal force of 5,500 g for 25 minutes, separate with an ultrafiltration membrane (3 kD), and freeze-dry the isolate to obtain marine oligopeptides; V3. Weigh the marine oligopeptide obtained in step V2 and the trehalose solution according to a mass volume ratio of 1g:4mL. The mass percentage concentration of the trehalose solution is 9%. Stir until it is evenly dispersed to obtain the marine oligopeptide solution.

[0029] Example 9: This example provides a method for preparing a marine oligopeptide solution, the specific contents are as follows: V1. Take bonito meat, freeze-dry it in vacuum, grind it into powder, pass it through a 60-mesh sieve to obtain bonito powder, weigh bonito powder and water according to a mass volume ratio of 1g:100mL, soak it at room temperature for 3h, boil it and keep it for 40min, and then cool it naturally to room temperature to obtain solution 2; V2. Place the solution 2 obtained in step V1 in a 50°C water bath until the temperature is balanced, add alkaline protease in an amount of 0.5 g / mL, and the enzyme activity of alkaline protease is 20,000 U / g, and treat at a speed of 150 rpm for 2 hours. After the treatment, quickly take out and inactivate the enzyme at high temperature, then centrifuge at a centrifugal force of 6,000 g for 30 minutes, separate with an ultrafiltration membrane (3 kD), and freeze-dry the isolate to obtain marine oligopeptides; V3. Weigh the marine oligopeptide obtained in step V2 and the trehalose solution according to a mass volume ratio of 1g:5mL. The mass percentage concentration of the trehalose solution is 10%. Stir until it is evenly dispersed to obtain the marine oligopeptide solution.

[0030] Embodiment 10: This embodiment provides a preparation method of a Chinese and Western combined qi and blood nourishing composition, the specific contents are as follows: The Chinese and Western combined qi and blood nourishing composition of this embodiment comprises the following raw materials in parts by weight: 3 parts of modified sponge microneedles, 2 parts of nanoliposome active substances, and 3 parts of marine oligopeptide liquid, wherein the modified sponge microneedles are prepared by Example 1, the nanoliposome active substances are prepared by Example 4, and the marine oligopeptide liquid is prepared by Example 7; The modified sponge microneedles, nanoliposome active substances, and marine oligopeptide liquid are weighed according to the above-mentioned mass fractions, the nanoliposome active substances and the marine oligopeptide liquid are stirred until they are evenly dispersed, and then the modified sponge microneedles are added, stirred until they are evenly dispersed and stored at 4°C to obtain a Chinese and Western combined qi and blood nourishing composition.

[0031] Embodiment 11: This embodiment provides a preparation method of a Chinese and Western combined qi and blood nourishing composition, the specific contents are as follows: The Chinese and Western combined qi and blood nourishing composition of this embodiment comprises the following raw materials in parts by weight: 5 parts of modified sponge microneedles, 4 parts of nanoliposome active substances, and 4 parts of marine oligopeptide liquid, wherein the modified sponge microneedles are prepared by Example 2, the nanoliposome active substances are prepared by Example 5, and the marine oligopeptide liquid is prepared by Example 8; The modified sponge microneedles, nanoliposome active substances, and marine oligopeptide liquid are weighed according to the above-mentioned mass fractions, the nanoliposome active substances and the marine oligopeptide liquid are stirred until they are evenly dispersed, and then the modified sponge microneedles are added, stirred until they are evenly dispersed and stored at 4°C to obtain a Chinese and Western combined qi and blood nourishing composition.

[0032] Embodiment 12: This embodiment provides a method for preparing a Chinese and Western medicine-based Qi and blood nourishing composition, the specific contents of which are as follows: The Chinese and Western combined Qi and blood nourishing composition of this embodiment comprises the following raw materials in parts by weight: 8 parts of modified sponge microneedles, 5 parts of nanoliposome active substances, and 6 parts of marine oligopeptide liquid, wherein the modified sponge microneedles are prepared by Example 3, the nanoliposome active substances are prepared by Example 6, and the marine oligopeptide liquid is prepared by Example 9; The modified sponge microneedles, nanoliposome active substances, and marine oligopeptide liquid are weighed according to the above-mentioned mass fractions, the nanoliposome active substances and the marine oligopeptide liquid are stirred until they are evenly dispersed, and then the modified sponge microneedles are added, stirred until they are evenly dispersed and stored at 4°C to obtain a Chinese and Western combined qi and blood nourishing composition.

[0033] Comparative Example 1: This comparative example provides a method for preparing a composition, the specific contents are as follows: Compared with Example 10, the composition of this comparative example does not contain modified sponge microneedles, and is obtained by mixing 2 parts by mass of nanoliposome active substances with 3 parts by mass of marine oligopeptide liquid, wherein the sources of the nanoliposome active substances and the marine oligopeptide liquid are consistent with those in Example 10, thereby obtaining the composition of this comparative example.

[0034] Experimental test: 1. Experimental Animals: Male SPF Kunming mice, weighing 18-22 g, were cultured for one week before the experiment, with an ambient temperature of 23±2°C, a humidity of 50±10%, and a 12-hour day-night alternation pattern. The animals were fasted for 12 hours the day before the experiment, but drinking water was not restricted.

[0035] Experimental groups: The mice were randomly divided into 15 groups, which were sequentially recorded as Group 1, Group 2, Group 3 ... Group 15, wherein Group 1 was a blank group, Group 2 was a model group, and the other groups were all experimental groups, corresponding to Examples 1-12 and Comparative Example 1, respectively; Taking Example 1 as an example, the modified sponge microneedle obtained in Example 1 was used for Hou Sanli of mice, once a day, with a dosage of 200 μL each time (dissolved in deionized water, the dosage of the modified sponge microneedle is referred to Example 10), and gently massaged for 10 seconds. In addition, another mouse was changed to treat Neiguan and Qiansanli respectively; The blank group was treated with the same amount of saline solution.

[0036] Animal modeling: A weight-bearing exhaustive swimming test was used. The water depth of the swimming box was set to 30 cm, and the water temperature was maintained at 22.0±2°C. 30 minutes after administration, a 7% lead bar was loaded on the base of the mouse's tail, and then it was gently placed in the small animal swimming box and the timing was started. Finally, the time from the start of the mouse's swimming to the time when the nostrils sank into the water and remained without floating for 3-5 seconds was recorded. This period of time was defined as the mouse's exhaustive swimming time. Once the mouse reached exhaustion, it was immediately fished out and dried.

[0037] 4. Experimental results: The experimental results are as follows Figure 1-3 As shown in the following order, they correspond to Housanli, Neiguan, and Qiansanli. Figures 1 to 3 It can be seen that the Chinese and Western combined qi and blood maintenance compositions obtained in Examples 9-12 have the greatest fatigue improvement effect on mice, followed by the nanoliposome active substances obtained in Examples 4-6, and the modified sponge microneedles are slightly better than the marine oligopeptide liquid. When the modified sponge microneedles are not present (Comparative Example 1), the fatigue improvement effect on mice is close to that of Examples 4-6, or slightly lower than that of Examples 4-6. It can be seen that the Chinese and Western combined qi and blood maintenance composition obtained by the present invention can better improve the symptoms of chronic fatigue, and the modified sponge microneedles have a good introduction effect, and after application at the corresponding acupoints, they can promote the nanoliposome active substances to play a role.

[0038] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A Chinese and Western medicine combined Qi and blood nourishing composition, characterized in that: The invention comprises the following raw materials in parts by weight: 3-8 parts of modified sponge microneedles, 2-5 parts of nanoliposome active substances, and 3-6 parts of marine oligopeptide liquid.

2. The Chinese and Western combined Qi and blood nourishing composition according to claim 1, characterized in that: The preparation method of the modified sponge microneedle is as follows: S1. Weigh mulberry silk and sodium carbonate-sodium bicarbonate buffer according to a mass volume ratio of 1g:50-60mL, heat and maintain after mixing, rinse with deionized water, dry to obtain a dried product, weigh the dried product and lithium bromide solution according to a mass volume ratio of 1g:8-10mL, heat and maintain, dialyze with deionized water, and reversely dialyze with a polyethylene glycol solution to obtain a concentrate; S2. The concentrated solution obtained in step S1 is freeze-dried in vacuo to obtain substance 1, substance 1, ethanol solution, and calcium chloride are weighed according to a mass volume ratio of 1 g: 20-30 mL: 0.5-0.8 g, stirred, washed with deionized water, centrifuged, and precipitated and dried to obtain substance 2; S3. Dissolve polyglutamic acid tyrosine sodium salt in Tris-hydrochloric acid buffer to obtain solution 1 with a mass volume concentration of 4-6 mg / mL, weigh the substance 2 obtained in step S2 and solution 1 according to a mass volume ratio of 1 g: 10-15 mL, ultrasonically treat, centrifuge, wash the obtained precipitate with deionized water and resuspend, sterilize under high pressure, and obtain the modified sponge microneedle after freeze-drying.

3. The Chinese and Western combined Qi and blood nourishing composition according to claim 2, characterized in that: In step S1, the concentration of the lithium bromide solution is 8-9M, and the mass percentage concentration of the polyethylene glycol solution is 30-35%; The molar ratio of glutamic acid to tyrosine in the polyglutamic acid tyrosine sodium salt in step S3 is 1:

4.

4. The Chinese and Western combined Qi and blood nourishing composition according to claim 3, characterized in that: The preparation method of the nanoliposome active substance is as follows: L1. Grind dried Atractylodes macrocephala, Bupleurum chinense, Magnolia officinalis, Chuanxiong and Acorus calamus into powder, sieve and mix in a mass ratio of 5-8:3-5:2-6:1:1-2 to obtain a mixed powder, add 8-10 times the mass of water of the mixed powder, steam distill, and dry the obtained extract for later use; L2. Cholesterol, sunflower lecithin, the extract obtained in step L1, and dichloromethane were weighed according to a mass volume ratio of 1-1.2 g: 1-1.5 g: 0.8-1 g: 10-15 L, and the mixture was stirred until uniformly dispersed to obtain a mixed solution, and the mixed solution was heated and rotary evaporated to obtain a thin lipid film; L3. Add distilled water to the thin lipid film obtained in step L2, heat and stir to hydrate, cool naturally to room temperature, and then ultrasonically treat. Filter the obtained solution through a filter membrane, and the filtrate is the nanoliposome active substance.

5. The Chinese and Western combined Qi and blood nourishing composition according to claim 4, characterized in that: The amount of distilled water added in step L3 is based on the volume of the mixed solution in step L2, and the amount added is 0.2-0.25 times the volume of the mixed solution.

6. The Chinese and Western combined Qi and blood nourishing composition according to claim 5, characterized in that: The preparation method of the marine oligopeptide liquid is as follows: V1. Take bonito meat, freeze-dry it in vacuum and grind it into powder, sieve it to obtain bonito powder, weigh bonito powder and water according to the mass volume ratio of 1g: 80-100mL, soak and boil it, and cool it naturally to room temperature to obtain solution 2; V2. The solution 2 obtained in step V1 is placed in a water bath, alkaline protease is added, stirred, and quickly taken out after completion and the enzyme is inactivated at high temperature, centrifuged, separated by ultrafiltration membrane, and the separated product is freeze-dried to obtain marine oligopeptides; V3. Weigh the marine oligopeptide and trehalose solution obtained in step V2 according to a mass volume ratio of 1g:3-5mL, and stir until they are evenly dispersed to obtain the marine oligopeptide solution.

7. The Chinese and Western combined Qi and blood nourishing composition according to claim 6, characterized in that: The amount of alkaline protease added in step V2 is 0.3-0.5 g / mL.

8. A method for preparing the Chinese and Western combined qi and blood nourishing composition according to any one of claims 1 to 7, characterized in that: The modified sponge microneedles, nanoliposome active substances, and marine oligopeptide liquid are weighed according to the corresponding mass fractions, the nanoliposome active substances and the marine oligopeptide liquid are stirred until they are evenly dispersed, and then the modified sponge microneedles are added, stirred until they are evenly dispersed and stored at 4°C to obtain a Chinese and Western combined qi and blood nourishing composition.

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