CN118593566B - A blood-activating and stasis-removing compound containing lotus stem extract and preparation method thereof - Google Patents

Abstract

The present invention relates to the field of medical technology, and in particular to a blood-activating and stasis-removing complex containing lotus stem extract and a preparation method thereof, wherein the blood-activating and stasis-removing complex comprises the following raw materials in parts by weight: 10-15 parts of lotus stem extract preparation, 8-12 parts of traditional Chinese medicine compound preparation, 60-90 parts of lotus stem fiber carrier, 20-30 parts of myrrh oil, and 8-12 parts of gelatin. The lotus stem extract is combined with an oil phase encapsulation to strengthen the percutaneous absorption of effective ingredients and improve drug efficacy. Traditional Chinese medicine and algae are used as raw materials, fermented by microorganisms, enhanced drug efficacy, and reduced toxic and side effects. The present invention prepares a lotus stem fiber carrier, which is rich in a large number of pores, can load a large number of effective ingredients, prolong drug action time, and improve drug stability. The blood-activating and stasis-removing complex prepared by the present invention has good therapeutic effect, strong stability, long action time, and high safety.

Description

A blood-activating and stasis-removing compound containing lotus stem extract and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, and in particular to a blood-activating and blood-stasis-removing complex containing a lotus stem extract and a preparation method thereof.

Background Art

Different parts of lotus have certain functionality, such as lotus extract for whitening, lotus seed extract for anti-oxidation, etc. In comparison, the utilization of lotus stems is slightly insufficient. Literature research has found that lotus stems have certain effects of relaxing meridians and activating collaterals, enter the liver meridian, and can promote blood microcirculation. Therefore, lotus stems are more suitable for the preparation of blood-activating and stasis-removing preparations. Existing blood-activating and stasis-removing preparations have a strong smell of Chinese herbal medicine, which is easy to smell at social distance, and some patients are more concerned. On this basis, the present invention provides a blood-activating and stasis-removing preparation containing lotus stem extract and a preparation method thereof. The lotus stems are soaked and boiled to obtain supernatant, which is used as the water phase and combined with the oil phase for encapsulation and freeze-drying to obtain nano powder, thereby obtaining functional ingredients with the effects of promoting blood circulation and removing blood stasis. Combined with the utilization and extraction of other Chinese medicinal materials and microbial fermentation, microsphere particles with synergistic effects are finally obtained. The solid matter after the lotus stems are soaked and boiled contains a large amount of cellulose and other insoluble impurities, which are made into a carrier, and the microsphere particles are distributed in the lotus stem fiber carrier to obtain the final blood circulation and blood stasis complex, which is easy to use, has a light odor, and after testing, can significantly improve pain and bruising.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a blood-activating and stasis-removing compound containing lotus stem extract and a preparation method thereof.

The present invention is achieved through the following technical solutions:

A blood-activating and stasis-removing compound containing lotus stem extract comprises the following raw materials in parts by weight: 10-15 parts of lotus stem extract preparation, 8-12 parts of traditional Chinese medicine compound preparation, 60-90 parts of lotus stem fiber carrier, 20-30 parts of myrrh oil and 8-12 parts of gelatin.

Furthermore, the preparation method of the lotus stem extract preparation comprises the following steps:

(1) Cut the lotus stem into 1-2 cm in length, add it into distilled water, heat and boil it for 2-3 h, and filter it to obtain the supernatant and solid matter;

(2) adding dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate to anhydrous ethanol and mixing them evenly to obtain a mixed solution, adding the supernatant obtained in step (1) to the mixed solution, ultrasonicating at 100 W for 20-30 min, and rotary evaporating at 60° C. for 1-2 h to obtain a reaction mixture A;

(3) The reaction mixture A obtained in step (2) was added with a phosphate buffer solution of pH 7.4, heated at 80°C for 20 min, filtered through a 0.22 μm filter, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a lotus stem extract preparation.

Furthermore, in step (1), the ratio of lotus stem to distilled water is 1 g:25 mL.

Furthermore, in step (2), the dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, methyl salicylate and anhydrous ethanol is 5 g:5 g:4 g:50 mL.

Furthermore, the volume ratio of the supernatant to the mixed solution in step (2) is 1:10.

Furthermore, in step (3), the mass ratio of the reaction mixture A to the phosphate buffer is 1:5.

Furthermore, the preparation method of the Chinese medicine composite preparation comprises the following steps:

(a) Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima are mixed, dried at 100°C, crushed through an 80-mesh sieve to obtain a Chinese medicine composite powder, mixed evenly with deionized water, and sterilized at 121°C for 15 min to obtain a fermentation medium;

(b) inoculating the activated Bacillus subtilis and Lactobacillus plantarum into the fermentation medium obtained in step (a) at a volume ratio of 3%, and culturing at 37° C. and 150-200 r/min for 96 h in a shaking incubator to obtain a fermentation broth;

(c) The fermentation broth obtained in step (b) was centrifuged at 10,000 r/min for 20 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a Chinese medicine composite preparation.

Furthermore, the mass ratio of Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima in step (a) is 2:1:1.5:0.8.

Furthermore, the dosage ratio of the traditional Chinese medicine composite powder to deionized water in step (a) is 1 g:50 mL.

Furthermore, the Bacillus subtilis and Lactobacillus plantarum in step (b) are purchased from China General Microbiological Culture Collection Center, and are numbered CGMCC 1.12939 and CGMCC 1.12934, respectively.

Furthermore, the preparation method of the lotus stem fiber carrier comprises the following steps:

(i) drying the solid material obtained in step (1) at 60° C. to constant weight, crushing it through a 70-mesh sieve to obtain lotus stem powder, adding it to a mixed enzyme solution of pectin lyase and xylanase, adjusting the pH value to 3.8-4.2 with citric acid, reacting at 50° C. for 24 h, and centrifuging at 8000 rpm for 30 min to obtain a precipitate;

(ii) Cinnamaldehyde and curcumin were added to anhydrous ethanol and mixed evenly, and the precipitate obtained in step (i) was added, ultrasonicated at 150 W for 1 h, placed in an oven at 100 °C for 12 h, washed with deionized water, and dried at 55 °C to obtain a lotus stem fiber carrier.

Furthermore, in step (i), the ratio of the lotus stem powder to the mixed enzyme solution is 1 g:30 mL.

Furthermore, the mass ratio of pectin lyase, xylanase and water in the mixed enzyme solution of step (i) is 1.5:1:150.

Furthermore, in step (ii), the dosage ratio of cinnamaldehyde, curcumin, anhydrous ethanol and precipitate is 5 mg:10 mg:20 mL:1.5 g.

The present invention also provides a method for preparing the blood-activating and blood-stasis-removing complex containing lotus stem extract, comprising the following steps:

S1: Add the lotus stem extract preparation and the traditional Chinese medicine compound preparation into deionized water, stir at 180 rpm for 30 min, add the lotus stem fiber carrier, continue stirring for 5-6 h, and filter to obtain a filter cake;

S2: Mix myrrh oil and gelatin at 100 rpm for 2 h to obtain a myrrh oil-gelatin mixture, and mix it evenly with the filter cake obtained in step S1 to obtain a blood-activating and blood-stasis-removing complex containing lotus stem extract.

Furthermore, in step S1, the ratio of the lotus stem extract to deionized water is 1 g:30 mL.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention prepares a lotus stem extract preparation, and the lotus stem is soaked and boiled to obtain a supernatant, and the supernatant is used as an aqueous phase, combined with an oil phase encapsulation, and freeze-dried to obtain a nano powder. The present invention uses dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate as an oil phase to prepare liposomes, which can enhance the percutaneous absorption of effective ingredients and improve the efficacy. No antioxidant is needed to be added, and the stability of the active substance can be effectively protected. At the same time, it can be slowly released, thereby prolonging the drug action time. The present invention uses three Chinese medicinal materials, namely, Panax notoginseng, Angelica sinensis, and Poria cocos, and an algae, Spirulina maxima, as raw materials, and has the effects of promoting blood circulation, promoting blood circulation and removing blood stasis. It is fermented by Bacillus subtilis and Lactobacillus plantarum. After the action of microorganisms, substances such as cellulose and lignin in the cell wall of the Chinese medicine are degraded, and the active ingredients are released; the active ingredients of the Chinese medicine and the algae are enzymatically hydrolyzed into small molecular substances, which are beneficial to the body's absorption and enhance the efficacy. At the same time, after microbial fermentation, the toxicity of the Chinese medicine is reduced, and the toxic and side effects are reduced. At the same time, the cellulose, protein and other components in Chinese medicine and algae can be used by microorganisms to promote the growth and reproduction of microorganisms. Chinese medicine and microorganisms work synergistically and complement each other. The fermentation of Chinese medicine and algae through the growth metabolism and biotransformation of microorganisms can improve the efficacy of medicine, reduce the toxicity and toxic side effects of Chinese medicine, and the fermentation product shows a higher efficacy than the sum of the medicinal matrix. The present invention prepares a lotus stem fiber carrier. The solid matter after the lotus stem is soaked and boiled contains a large amount of cellulose and other insoluble impurities. It is made into a carrier, which is rich in a large number of pores and can load a large number of effective ingredients. It can release the drug slowly, prolong the drug action time, help protect the effective ingredients, and improve the drug stability. At the same time, the porous structure can make the drug more evenly distributed, reduce the irritation of the drug directly contacting the skin, and the porous material can enhance the air permeability. The lotus stem fiber carrier prepared by the present invention has the effect of sterilization and anti-inflammatory, and synergizes with the drug loaded thereon to promote blood circulation and blood stasis. The present invention adopts myrrh oil and gelatin to make a gel, which is easy to use, can fix the drug, prevent the drug from shifting, and ensure that the drug can better act on the target site. At the same time, it can provide a physical barrier, protect the skin, and reduce the risk of infection. At the same time, myrrh oil has the effects of promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, and anti-inflammatory and antibacterial. The blood circulation and blood stasis-removing complex containing lotus stem extract prepared by the present invention can effectively promote blood circulation and remove blood stasis, has good therapeutic effect, good lasting effect, less administration times, natural raw materials, and high safety.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a scanning electron micrograph of the lotus stem extract preparation (A) and the lotus stem fiber carrier (B) described in Example 1 of the present invention;

FIG2 is a graph showing the cytotoxicity test of the blood-activating and blood-stasis-removing composites of Examples 1-3 and Comparative Examples 1-3 of the present invention;

FIG3 is a test diagram showing the effect of the blood-activating and stasis-removing composites described in Examples 1-3 and Comparative Examples 1-3 on the caliber of the auricle blood vessels in mice;

FIG4 is a test diagram showing the effect of the blood-activating and stasis-removing complexes described in Examples 1-3 and Comparative Examples 1-3 on the number of open capillaries in the auricle of mice;

FIG. 5 is a graph showing the efficacy of the blood-activating and stasis-removing composites of Examples 1-3 and Comparative Examples 1-3 of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

Example 1: This example provides a blood-activating and stasis-removing complex containing lotus stem extract, comprising the following raw materials in parts by weight: 15 parts of lotus stem extract preparation, 12 parts of Chinese medicine compound preparation, 90 parts of lotus stem fiber carrier, 30 parts of myrrh oil, and 12 parts of gelatin.

The preparation method of the lotus stem extract preparation comprises the following steps:

(1) Cut the lotus stem into 2 cm in length, add it into distilled water, the ratio of lotus stem to distilled water is 1 g:25 mL, heat and boil for 3 h, filter and obtain the supernatant and solid matter;

(2) adding dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate to anhydrous ethanol and mixing them evenly to obtain a mixed solution, wherein the amount ratio of dipalmitoylphosphatidylcholine, cholesterol, methyl salicylate and anhydrous ethanol is 5 g:5 g:4 g:50 mL, adding the supernatant obtained in step (1) to the mixed solution, the volume ratio of the supernatant to the mixed solution is 1:10, ultrasonicating at 100 W for 30 min, and rotary evaporating at 60° C. for 2 h to obtain a reaction mixture A;

(3) The reaction mixture A obtained in step (2) was added to a phosphate buffer solution of pH 7.4, with a mass ratio of the reaction mixture A to the phosphate buffer solution being 1:5. The mixture was heated at 80°C for 20 min, filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a lotus stem extract preparation. The scanning electron micrograph is shown in FIG1A , which is nanospheres.

The preparation method of the Chinese medicine composite preparation comprises the following steps:

(a) Mixing Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima in a mass ratio of 2:1:1.5:0.8, drying at 100°C, crushing through an 80-mesh sieve to obtain a Chinese medicine composite powder, and mixing the mixture with deionized water in a ratio of 1 g:50 mL. After sterilization at 121°C for 15 min, a fermentation medium was obtained.

(b) inoculating the activated Bacillus subtilis and Lactobacillus plantarum into the fermentation medium obtained in step (a), with the inoculation amount being 3% by volume, and culturing in a shaking incubator at 37° C. and 200 rpm for 96 h to obtain a fermentation broth;

(c) The fermentation broth obtained in step (b) was centrifuged at 10,000 r/min for 20 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a Chinese medicine composite preparation.

The preparation method of the lotus stem fiber carrier comprises the following steps:

(i) drying the solid material obtained in step (1) at 60° C. to constant weight, crushing it through a 70-mesh sieve to obtain lotus stem powder, adding it to a mixed enzyme solution of pectin lyase and xylanase, wherein the mass ratio of pectin lyase, xylanase and water in the mixed enzyme solution is 1.5:1:150, and the amount ratio of lotus stem powder to the mixed enzyme solution is 1 g:30 mL, adjusting the pH value to 4.2 with citric acid, reacting at 50° C. for 24 h, and centrifuging at 8000 rpm for 30 min to obtain a precipitate;

(ii) Cinnamaldehyde and curcumin were added to anhydrous ethanol and mixed evenly, and the precipitate obtained in step (i) was added, the amount ratio of cinnamaldehyde, curcumin, anhydrous ethanol and precipitate was 5 mg:10 mg:20 mL:1.5 g, and ultrasonic treatment was performed at 150 W for 1 h. The mixture was placed in an oven at 100°C for 12 h, washed with deionized water, and dried at 55°C to obtain a lotus stem fiber carrier. The scanning electron microscope image is shown in Figure 1B, which contains a large number of pores and can load effective ingredients.

This embodiment also provides a method for preparing the blood-activating and stasis-removing complex containing lotus stem extract, comprising the following steps:

S1: Add the lotus stem extract preparation and the traditional Chinese medicine compound preparation into deionized water, the ratio of lotus stem extract to deionized water is 1 g:30 mL, stir at 180 rpm for 30 min, add the lotus stem fiber carrier, continue stirring for 5-6 h, filter and obtain the filter cake;

S2: Mix myrrh oil and gelatin at 100 rpm for 2 h to obtain a myrrh oil-gelatin mixture, and mix it evenly with the filter cake obtained in step S1 to obtain a blood-activating and blood-stasis-removing complex containing lotus stem extract.

Example 2: The example provides a blood-activating and stasis-removing complex containing lotus stem extract, comprising the following raw materials in parts by weight: 10 parts of lotus stem extract preparation, 8 parts of traditional Chinese medicine compound preparation, 60 parts of lotus stem fiber carrier, 20 parts of myrrh oil, and 8 parts of gelatin.

The preparation method of the lotus stem extract preparation comprises the following steps:

(1) Cut the lotus stem into 1 cm in length, add it into distilled water, the ratio of lotus stem to distilled water is 1 g:25 mL, heat and boil for 2 h, filter and obtain the supernatant and solid matter;

(2) adding dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate to anhydrous ethanol and mixing them uniformly to obtain a mixed solution, wherein the amount ratio of dipalmitoylphosphatidylcholine, cholesterol, methyl salicylate and anhydrous ethanol is 5 g:5 g:4 g:50 mL, adding the supernatant obtained in step (1) to the mixed solution, the volume ratio of the supernatant to the mixed solution is 1:10, ultrasonicating at 100 W for 20 min, and rotary evaporating at 60° C. for 1 h to obtain a reaction mixture A;

(3) The reaction mixture A obtained in step (2) was added with a phosphate buffer having a pH value of 7.4, the mass ratio of the reaction mixture A to the phosphate buffer being 1:5, heated at 80° C. for 20 min, filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80° C. for 24 h to obtain a lotus stem extract preparation.

The preparation method of the Chinese medicine composite preparation comprises the following steps:

(a) Mixing Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima in a mass ratio of 2:1:1.5:0.8, drying at 100°C, crushing through an 80-mesh sieve to obtain a Chinese medicine composite powder, and mixing the mixture with deionized water in a ratio of 1 g:50 mL. After sterilization at 121°C for 15 min, a fermentation medium was obtained.

(b) inoculating the activated Bacillus subtilis and Lactobacillus plantarum into the fermentation medium obtained in step (a), with the inoculation amount being 3% by volume, and culturing in a shaking incubator at 37° C. and 150 rpm for 96 h to obtain a fermentation broth;

(c) The fermentation broth obtained in step (b) was centrifuged at 10,000 r/min for 20 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a Chinese medicine composite preparation.

The preparation method of the lotus stem fiber carrier comprises the following steps:

(i) drying the solid material obtained in step (1) at 60° C. to constant weight, crushing it through a 70-mesh sieve to obtain lotus stem powder, adding it to a mixed enzyme solution of pectin lyase and xylanase, wherein the mass ratio of pectin lyase, xylanase and water in the mixed enzyme solution is 1.5:1:150, and the amount ratio of lotus stem powder to the mixed enzyme solution is 1 g:30 mL, adjusting the pH value to 3.8 with citric acid, reacting at 50° C. for 24 h, and centrifuging at 8000 rpm for 30 min to obtain a precipitate;

(ii) Cinnamaldehyde and curcumin were added to anhydrous ethanol and mixed evenly, and the precipitate obtained in step (i) was added, wherein the amount ratio of cinnamaldehyde, curcumin, anhydrous ethanol and precipitate was 5 mg:10 mg:20 mL:1.5 g. The mixture was ultrasonically treated at 150 W for 1 h, placed in an oven at 100 °C for 12 h, washed with deionized water, and dried at 55 °C to obtain a lotus stem fiber carrier.

This embodiment also provides a method for preparing the blood-activating and blood-stasis-removing composite containing lotus stem extract, comprising the following steps:

S1: Add the lotus stem extract preparation and the traditional Chinese medicine compound preparation into deionized water, the ratio of lotus stem extract to deionized water is 1 g:30 mL, stir at 180 rpm for 30 min, add the lotus stem fiber carrier, continue stirring for 5 h, filter and obtain the filter cake;

S2: Mix myrrh oil and gelatin at 100 rpm for 2 h to obtain a myrrh oil-gelatin mixture, and mix it evenly with the filter cake obtained in step S1 to obtain a blood-activating and blood-stasis-removing complex containing lotus stem extract.

Example 3: The example provides a blood-activating and stasis-removing complex containing lotus stem extract, comprising the following raw materials in parts by weight: 12 parts of lotus stem extract preparation, 10 parts of Chinese medicine compound preparation, 72 parts of lotus stem fiber carrier, 24 parts of myrrh oil, and 10 parts of gelatin.

The preparation method of the lotus stem extract preparation comprises the following steps:

(1) Cut the lotus stem into 1.5 cm in length, add it to distilled water, the ratio of lotus stem to distilled water is 1 g:25 mL, heat and boil for 2.5 h, filter and obtain the supernatant and solid matter;

(2) adding dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate to anhydrous ethanol and mixing them evenly to obtain a mixed solution, wherein the amount ratio of dipalmitoylphosphatidylcholine, cholesterol, methyl salicylate and anhydrous ethanol is 5 g:5 g:4 g:50 mL, adding the supernatant obtained in step (1) to the mixed solution, the volume ratio of the supernatant to the mixed solution is 1:10, ultrasonicating at 100 W for 25 min, and rotary evaporating at 60° C. for 1.5 h to obtain a reaction mixture A;

(3) The reaction mixture A obtained in step (2) was added with a phosphate buffer having a pH value of 7.4, the mass ratio of the reaction mixture A to the phosphate buffer being 1:5, heated at 80° C. for 20 min, filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80° C. for 24 h to obtain a lotus stem extract preparation.

The preparation method of the Chinese medicine composite preparation comprises the following steps:

(a) Mixing Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima in a mass ratio of 2:1:1.5:0.8, drying at 100°C, crushing through an 80-mesh sieve to obtain a Chinese medicine composite powder, and mixing the mixture with deionized water in a ratio of 1 g:50 mL. After sterilization at 121°C for 15 min, a fermentation medium was obtained.

(b) inoculating the activated Bacillus subtilis and Lactobacillus plantarum into the fermentation medium obtained in step (a), with the inoculation amount being 3% by volume, and culturing in a shaking incubator at 37° C. and 180 rpm for 96 h to obtain a fermentation broth;

(c) The fermentation broth obtained in step (b) was centrifuged at 10,000 r/min for 20 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h to obtain a Chinese medicine composite preparation.

The preparation method of the lotus stem fiber carrier comprises the following steps:

(i) drying the solid material obtained in step (1) at 60° C. to constant weight, crushing it through a 70-mesh sieve to obtain lotus stem powder, adding it to a mixed enzyme solution of pectin lyase and xylanase, wherein the mass ratio of pectin lyase, xylanase and water in the mixed enzyme solution is 1.5:1:150, and the amount ratio of lotus stem powder to the mixed enzyme solution is 1 g:30 mL, adjusting the pH value to 4.0 with citric acid, reacting at 50° C. for 24 h, and centrifuging at 8000 rpm for 30 min to obtain a precipitate;

(ii) Cinnamaldehyde and curcumin were added to anhydrous ethanol and mixed evenly, and the precipitate obtained in step (i) was added, wherein the amount ratio of cinnamaldehyde, curcumin, anhydrous ethanol and precipitate was 5 mg:10 mg:20 mL:1.5 g. The mixture was ultrasonically treated at 150 W for 1 h, placed in an oven at 100 °C for 12 h, washed with deionized water, and dried at 55 °C to obtain a lotus stem fiber carrier.

This embodiment also provides a method for preparing the blood-activating and stasis-removing complex containing lotus stem extract, comprising the following steps:

S1: Add the lotus stem extract preparation and the traditional Chinese medicine compound preparation into deionized water, the dosage ratio of lotus stem extract to deionized water is 1 g:30 mL, stir at 180 rpm for 30 min, add the lotus stem fiber carrier, continue stirring for 5-6 h, filter and obtain the filter cake;

S2: Mix myrrh oil and gelatin at 100 rpm for 2 h to obtain a myrrh oil-gelatin mixture, and mix it evenly with the filter cake obtained in step S1 to obtain a blood-activating and blood-stasis-removing complex containing lotus stem extract.

The difference between Comparative Example 1 and Example 1 is that the supernatant obtained in step (1) is filtered through a 0.22 μm filter membrane, freeze-dried at 0.02 Mbar and -80°C for 24 h, and the obtained product is used to replace the lotus stem extract preparation.

The difference between Comparative Example 2 and Example 1 is that Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima are mixed, the mass ratio of Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima is 2:1:1.5:0.8, dried at 100°C, crushed through an 80-mesh sieve to obtain a Chinese medicine composite powder, and mixed evenly with deionized water, the amount ratio of the Chinese medicine composite powder to deionized water is 1 g:50 mL, heated and boiled for 1 h, centrifuged at 10000 r/min for 20 min, the supernatant is filtered through a 0.22 μm filter membrane, and freeze-dried at 0.02 Mbar and -80°C for 24 h, and the resulting product is used to replace the Chinese medicine composite preparation.

The only difference between Comparative Example 3 and Example 1 is that the lotus stem fiber carrier is replaced by myrrh oil and gelatin in a mass ratio of 5:2.

Experimental Example 1: The cytotoxicity of the complexes of Examples 1-3 and Comparative Examples 1-3 was determined by the MTT method. According to the ratio of m complex/v complete medium = 50 mg/mL, the complex that was fully sterilized by ultraviolet for 1 h was completely immersed in the complete medium, and extracted in a 37°C water bath for 24 h to obtain an extract. Take L-929 in the logarithmic growth phase, adjust the concentration of L-929 to 2×10 ⁴ cells/mL after digestion, inoculate 100 μL per well into a 96-well plate, place it at 37°C, 5% CO ₂ , and after the cells adhere to the wall, add 100 μL of extract to each well, set a well with only 200 μL of complete medium as a blank control group, and set a well with 100 μL of complete medium + 100 μL of cells as a negative control. After adding the extract, continue to culture for 5 days, add 20 μL MTT (5 mg/mL in PBS) to each well in the dark every day, and add MTT on the last day and continue to culture for 4 h. After removing all the liquid in the well, add 150 μL DMSO solution to each well, shake at low speed for 15 min in the microplate reader, and measure the absorbance at λ=490 nm. The cytotoxicity is determined by calculating the relative growth rate (RGR, %): RGR=(OD experimental group-OD blank group)/(OD negative control group-OD blank group). The specific toxicity classification standard is: 0: RGR≥100%; 1: 99%≥RGR≥75; 2: 74≥RGR≥50; 3: 49≥RGR≥25; 4: 24≥RGR≥1; 5=0. Among them, 2 or above can be regarded as cytotoxic reaction. The results are shown in Figure 2.

The results in Figure 2 show that the RGR of Examples 1-3 is smaller than that of Comparative Example 1, but the toxicity levels of the complexes prepared in Examples 1-3 and Comparative Example 1-3 are both 0-1, indicating that the blood-activating and stasis-removing complex containing lotus stem extract of the present invention is non-cytotoxic, has high biosafety, and is safe and reliable.

Experimental Example 2: 72 SD rats were selected and randomly divided into 36 intact skin groups and 36 damaged skin groups. Preparation of intact skin: 24 hours before administration, the hair on both sides of the back spine was shaved, and the hair removal area was about 4×6 cm ^2. Preparation of damaged skin: The hair removal method was the same as that of intact skin preparation. After 24 hours of hair removal, the skin was disinfected and scratched with a needle to puncture the epidermis, not injure the dermis, and cause slight bleeding. The rats in the intact skin group and the damaged skin group were randomly divided into 6 groups, 6 rats in each group, and the composites of Examples 1-3 and Comparative Examples 1-3 were evenly applied, with an application amount of 0.2 g/cm ² , washed off after 24 hours, and observed for 7 consecutive days. Within 7 days, there was no death or poisoning reaction in the rats, no irritation and allergic reaction such as erythema, swelling and damage, and no skin allergy. After continuous administration for 14 days, the mixture was covered for 24 hours every day and washed off 6 hours after the last administration. The animals in each group had normal diet and activities, no irritability, good mental state, no systemic poisoning symptoms, and no skin allergic reaction. This shows that the blood-activating and stasis-removing composite containing lotus stem extract of the present invention has no skin irritation and high safety.

Experimental Example 3: 60 SPF Kunming mice were randomly divided into 6 groups, 10 mice in each group, and each mouse was intraperitoneally injected with 25 mg/kg of sodium pentobarbital. After anesthesia, the ear hair was trimmed with ophthalmic scissors, the mouse was fixed, the ear was flattened and a little cedar oil was applied to the surface, and it was placed on the microscope stage. The microcirculation of the mouse ear before administration was observed under a microscope, and the caliber of the auricular arteriole (A) and the venule (V) of each mouse ear, and the amount of capillary opening. Then 0.1 g of the composite of Example 1-3 and Comparative Example 1-3 was evenly applied on both sides of the left ear. After 10 min, 20 min, 40 min, 80 min and 120 min of administration, the caliber of the auricular arteriole (A) and the venule (V) and the amount of capillary opening were measured again, and the results are shown in Figures 3 and 4.

The results in Figures 3 and 4 show that the opening volume of the auricular arterioles, venules and capillaries of each group of mice increased to varying degrees after administration, among which the opening volume of the auricular arterioles, venules and capillaries of Comparative Examples 1 and 3 began to decrease at 40 minutes, while that of Examples 1-3 and Comparative Example 2 began to decrease after 80 minutes, and the effect of Examples 1-3 was better than that of Comparative Example 2, indicating that the blood-activating and stasis-removing complex containing lotus stem extract of the present invention has a sustained release effect, prolongs the drug action time, promotes the percutaneous absorption of the drug, improves the drug efficacy, and can effectively promote blood circulation and activate blood circulation and remove blood stasis.

Experimental Example 4: 60 SPF Kunming mice were randomly divided into 6 groups, 10 mice in each group, and 40 μL of 0.5% carrageenan suspension was subcutaneously injected into the right hind toe of each mouse to induce inflammation. Each group of mice was smeared with 0.2 g of the complex of Example 1-3 and Comparative Example 1-3 on the right hind toe, and the administration was continued for 3 days, once a day. The mice were killed 4 hours after the last administration, and the equivalent parts of the hind feet were taken and accurately weighed. The weight difference between the left and right feet was used as the swelling degree. The results are shown in Table 1.

Table 1: Effects of the complex on carrageenan-induced inflammation in the toes of mice

The results in Table 1 show that the swelling degree of Example 1-3 group is lower than that of Comparative Example 1-3, indicating that the blood-activating and stasis-removing complex drug containing lotus stem extract of the present invention has good transdermal absorption effect, long action time, can effectively resist inflammation and reduce swelling, activate blood circulation and remove blood stasis, and has good efficacy.

Experimental Example 5: 60 mice were randomly divided into 6 groups, 10 mice in each group, and the pain threshold of each mouse was measured by the hot plate method before medication. 0.2 g of the complex of Example 1-3 and Comparative Example 1-3 was applied to the two hind limbs of each mouse, and the pain threshold of each mouse (the time of starting to lick the hind foot) was measured by the hot plate method 2 hours after medication, and the pain threshold increase rate was calculated, pain threshold increase rate (%) = [(pain threshold after medication - pain threshold before medication) / pain threshold before medication] × 100%. The results are shown in Table 2.

Table 2: Test of analgesic effect of the compound

The results in Table 2 show that the pain threshold improvement rates of Example 1-3 groups are higher than those of Comparative Examples 1-3, indicating that the blood-activating and stasis-removing complex containing lotus stem extract of the present invention can effectively remove blood stasis and relieve pain, has a long drug action time, can reduce the number of medications, and has significant efficacy.

Experimental Example 6: Patients with local pain, swelling, and bruises caused by traumatic injuries, or patients with joint dysfunction such as rheumatism, gout, and arthritis, or patients with subcutaneous bruises and ecchymoses caused by other reasons were selected. The male-female ratio, average age, and injury type of each group of patients were the same. The affected area was washed and dried with warm water, and the composites of Example 1-3 and Comparative Example 1-3 were evenly applied to the affected area with a sterile cotton ball or cotton swab, once a day, for 8 hours a day, with an application amount of 20 mg/ ^cm2 , and the medication was continued for 7 days. The number and ratio of cured, markedly effective, effective, and ineffective patients were counted, and the results are shown in Table 3.

Table 3: Compound efficacy test

The results in Table 3 show that the cure rate of the above cases treated with the composites of Examples 1-3 can reach 90%, with significant therapeutic effects, which are better than those of Comparative Examples 1-3. No irritation reaction or skin allergic reaction was observed on normal and damaged skin, and the composites are safe to use.

Experimental Example 7: Prepare tryptic soy peptone medium, dilute the bacterial solution with purified water on a clean bench, dilute 1000 times when the OD630 of Escherichia coli is 0.8, dilute 500 times when the OD630 of Staphylococcus aureus is 0.7, take 100 μL of bacterial solution respectively, mix, and apply to the plate until the surface of the medium is completely dry. 0.1 g of the composite of Example 1-3 and Comparative Example 1-3 was added to the surface of the medium, placed upright for 30 min, and then inverted at 37°C for 24 h, and the size of the inhibition zone was measured. The results are shown in Table 4.

Table 4: Size of inhibition zone

The results in Table 4 show that the antibacterial zone formed by the complexes of Examples 1-3 is larger than that of Comparative Examples 1-3, indicating that the mixed blood-dissolving complex containing lotus stem extract of the present invention has a good antibacterial effect, can prevent infection, and inhibit bacteria and inflammation.

Experimental Example 8: Rat bruise model was established. The rats were fixed on a striking table, and the same part of the rats was struck at the same height with a blunt iron pestle for 4 consecutive times. The striking area was 1 cm ² . The striking area was swollen, with scattered bleeding spots but no skin damage, and no fractures or dislocations were touched. After 24 hours, the rats were randomly divided into 6 groups, 10 rats in each group, and the composites of Examples 1-3 and Comparative Examples 1-3 were applied to the parts with swelling and bruises, once a day, with a coverage time of 8 hours a day, and the application amount was 20 mg/cm ^2. The healing time was recorded. The results are shown in Figure 5.

The results in FIG5 show that the recovery time of Examples 1-3 is shorter than that of Comparative Examples 1-3, indicating that the blood-activating and stasis-removing complex containing lotus stem extract of the present invention has good efficacy and can effectively activate blood circulation, remove blood stasis, reduce swelling and relieve pain.

The composites obtained in Examples 1-3 and Comparative Examples 1-3 were sealed in glass bottles and placed in an environment of 40° C. and 75% humidity for 6 months. After being taken out, the rat bruise model was established again in the same manner, and the number of days to recovery was recorded. The results are shown in Table 5.

Table 5: Efficacy of the complex after storage

Comparing the results in Table 5 with the results in FIG. 5 , the changes in Examples 1-3 and Comparative Example 2 are relatively low, while the changes in Comparative Examples 1 and 3 are relatively large, indicating that the blood-activating and stasis-removing composite containing lotus stem extract of the present invention has strong stability and is not easily reduced in efficacy due to changes in the external environment.

Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (9)

1. A blood-activating and stasis-removing compound containing lotus stem extract, characterized in that it comprises the following raw materials in parts by weight: 10-15 parts of lotus stem extract preparation, 8-12 parts of traditional Chinese medicine compound preparation, 60-90 parts of lotus stem fiber carrier, 20-30 parts of myrrh oil, and 8-12 parts of gelatin; The preparation method of the lotus stem extract preparation comprises the following steps: (1) Cut the lotus stem into 1-2 cm in length, add it into distilled water, heat and boil it, and filter it to obtain the supernatant and solid matter; (2) adding dipalmitoylphosphatidylcholine, cholesterol and methyl salicylate to anhydrous ethanol and mixing them evenly to obtain a mixed solution, adding the supernatant obtained in step (1) to the mixed solution, sonicating, and rotary evaporating at 60° C. to obtain a reaction mixture A; (3) adding the reaction mixture A obtained in step (2) to a phosphate buffer solution of pH 7.4, heating the mixture to 80° C., filtering the mixture through a 0.22 μm filter membrane, and freeze-drying the mixture to obtain a lotus stem extract preparation; The preparation method of the Chinese medicine composite preparation comprises the following steps: (a) Mixing Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima, drying at 100°C, crushing through an 80-mesh sieve to obtain a Chinese medicine composite powder, mixing it evenly with deionized water, and sterilizing it at 121°C to obtain a fermentation medium; (b) inoculating the activated Bacillus subtilis and Lactobacillus plantarum into the fermentation medium obtained in step (a) at a volume ratio of 3%, and culturing at 37° C. and 150-200 r/min for 96 h in a shaking incubator to obtain a fermentation broth; (c) centrifuging the fermentation broth obtained in step (b), filtering the supernatant through a 0.22 μm filter membrane, and freeze-drying to obtain a Chinese medicine composite preparation; The preparation method of the lotus stem fiber carrier comprises the following steps: (i) drying the solid material obtained in step (1) at 60° C. to constant weight, crushing it through a 70-mesh sieve to obtain lotus stem powder, adding it to a mixed enzyme solution of pectin lyase and xylanase, adjusting the pH value to 3.8-4.2 with citric acid, reacting at 50° C. for 24 h, and centrifuging to obtain a precipitate; (ii) Cinnamaldehyde and curcumin were added to anhydrous ethanol and mixed evenly, and the precipitate obtained in step (i) was added, ultrasonicated, placed in an oven at 100° C. for 12 h, washed with deionized water, and dried at 55° C. to obtain a lotus stem fiber carrier. 2. The blood-activating and stasis-removing composite containing lotus stem extract according to claim 1, characterized in that the ratio of the lotus stem to distilled water in step (1) is 1 g:25 mL. 3. The blood-activating and stasis-removing composite containing lotus stem extract according to claim 2, characterized in that the dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, methyl salicylate and anhydrous ethanol in step (2) is 5 g:5 g:4 g:50 mL; and the volume ratio of the supernatant to the mixed solution in step (2) is 1:10. 4. The blood-activating and stasis-removing complex containing lotus stem extract according to claim 3, characterized in that the mass ratio of the reaction mixture A to the phosphate buffer in step (3) is 1:5. 5. The blood-activating and stasis-removing composite containing lotus stem extract according to claim 4, characterized in that the mass ratio of Panax notoginseng, Angelica sinensis, Poria cocos and Spirulina maxima in step (a) is 2:1:1.5:0.8; and the dosage ratio of the traditional Chinese medicine composite powder to deionized water in step (a) is 1 g:50 mL. 6. The blood-activating and stasis-removing complex containing lotus stem extract according to claim 5, characterized in that the dosage ratio of the lotus stem powder to the mixed enzyme solution in step (i) is 1 g:30 mL; and the mass ratio of pectin lyase, xylanase and water in the mixed enzyme solution in step (i) is 1.5:1:150. 7. The blood-activating and stasis-removing composite containing lotus stem extract according to claim 6, characterized in that the amount ratio of cinnamaldehyde, curcumin, anhydrous ethanol and precipitate in step (ii) is 5 mg:10 mg:20 mL:1.5 g. 8. The method for preparing the blood-activating and stasis-removing composite containing lotus stem extract according to any one of claims 1 to 7, characterized in that it comprises the following steps: S1: adding the lotus stem extract preparation and the traditional Chinese medicine compound preparation into deionized water, stirring, adding the lotus stem fiber carrier, continuing to stir, and filtering to obtain a filter cake; S2: Mixing myrrh oil and gelatin to obtain a myrrh oil-gelatin mixture, and uniformly mixing the mixture with the filter cake obtained in step S1 to obtain a blood-activating and blood-stasis-removing complex containing lotus stem extract. 9. The method for preparing a blood-activating and stasis-removing composite containing lotus stem extract according to claim 8, characterized in that in step S1, the ratio of the lotus stem extract to deionized water is 1 g:30 mL.