Li medicine containing chaenomeles cathayensis seed oil cyclodextrin inclusion compound and preparation method of Li medicine containing chaenomeles cathayensis seed oil cyclodextrin inclusion compound

Abstract

The invention discloses a Li medicine containing a chaenomeles speciosa seed oil cyclodextrin inclusion compound and a preparation method of the Li medicine. According to the method, on the basis of an original traditional Li medicine, a chaenomeles cathayensis seed oil cyclodextrin inclusion compound is added, so that the Li medicine is developed. A mouse auricle swelling anti-inflammatory test, wound healing rate comparison, wound colony count comparison and wound tissue pathological change analysis are carried out on the Li medicine, and the results show that the Taiwan crabapple seed oil cyclodextrin inclusion compound, the traditional Li medicine and the Li medicine containing the Taiwan crabapple seed oil cyclodextrin inclusion compound have remarkable anti-inflammatory and antibacterial effects. In addition, the anti-inflammatory and antibacterial effects of the Li medicine are better than those of the single use of the tamanu seed oil cyclodextrin inclusion compound or the traditional Li medicine, so that the Li medicine can be used for preparing the anti-inflammatory and antibacterial medicine.

Description

Technical Field

[0001] The present invention relates to a Li medicine containing calophyllum inophyllum seed oil cyclodextrin inclusion compound and a preparation method thereof, belonging to the field of ethnic medicine. Background Art

[0002] The Hainan Li ethnic group has no written language of its own, which poses many challenges to the Li medicine passed down orally for thousands of years. There are problems such as the lack of a systematic Li medicine system, a complete pharmacopoeia, and modern medical practice qualifications. These problems have led to the gradual lack of successors for Li medicine and its endangerment. Therefore, it is of great significance to explore, sort out, and inherit the Li medicine prescriptions, as well as to conduct secondary innovation and protection of the single prescriptions, empirical prescriptions, and secret recipes of Li medicine.

[0003] The traditional usage of Li medicine among the people is to crush it into powder, or mix the powder with common dressings such as oil to make simple dosage forms for repairing wounds and surfaces, such as sword wounds and burns and scalds, with remarkable curative effects. However, the usage of Li medicine is simple and rough, the effective parts are unclear, the action mechanism is not clear, and there is a lack of quality standards, which limit its further development and utilization.

[0004] An anti-inflammatory and antibacterial traditional Li medicine is composed of five Li medicinal materials: Callicarpa nudiflora Hook.et Arn, Stemona tuberosa Lour, Melastoma penicillatum Wall., Sarcococca hainanensis Stapf, and Pteris multifida Poir., and is mainly used for treating trauma, anti-inflammatory and antibacterial, and skin infections and other diseases.

[0005] The dry leaves of Callicarpa nudiflora Hook.et Arn, a plant of the genus Callicarpa in the Verbenaceae family, is a Hainan Li medicine, mainly used for treating traumatic bleeding, burns, scalds, and suppurative inflammation. The chemical components in Callicarpa nudiflora mainly include phenylpropanoids, flavonoids, triterpenoids, diterpenoids, etc. Modern pharmacological activity studies have shown that it has the effects of hemostasis, anti-inflammatory, antibacterial, and enhancing immunity.

[0006] The dry roots of Stemona tuberosa Lour, a plant of the family Stemonaceae, are distributed in Hainan, Guangdong and other places. It is bitter, slightly sweet, and slightly warm in nature. Its functions and indications are moistening the lungs to relieve cough, killing insects and lice, and treating oxyuriasis and tinea. It has strong antibacterial activity against Streptococcus pneumoniae, group B hemolytic streptococcus, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Neisseria gonorrhoeae, Bacillus anthracis, and Mycobacterium tuberculosis hominis. Its chemical components are mainly tuberostemonine, isotuberostemonine, pretuberostemonine, oxy-tuberostemonine, stemotuberol, stemotubone, etc.

[0007] Melastoma penicillatum Naud is a plant of the genus Melastoma in the family Melastomataceae and is endemic to Hainan. It has the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and has antibacterial activity against Propionibacterium acnes, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. Its chemical constituents include gallic acid, ellagic acid, chlorogenic acid, and casuarine.

[0008] The dried leaves of Sarcococca vagans Stapf, a plant of the genus Sarcococca in the family Buxaceae, are mainly distributed in Hainan and Yunnan. In Hainan folk medicine, it is used for promoting blood circulation and removing blood stasis, as well as anti-inflammatory and analgesic effects, and is often used for stopping bleeding and removing blood stasis after traumatic injuries, relieving sore throats, and treating wind-heat colds. Its main chemical constituents include pachysamine A, yedamosine F, sarcococca alkaloids A-D, etc.

[0009] Pteris multifida Poir belongs to the dried whole herb of the fern family Pteridaceae, and has the effects of clearing heat and promoting diuresis, detumescence and detoxification, and cooling blood to stop bleeding. It is mainly used for treating dysentery, furuncles, and other symptoms. It has inhibitory effects on Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, and Mycobacterium tuberculosis. Its chemical constituents include flavonoids, terpenoids, and sterols, etc.

[0010] Calophyllum inophyllum seed oil is derived from the seeds of the evergreen tree Calophyllum inophyllum Linn of the genus Calophyllum in the family Clusiaceae and is prepared by pressing. Calophyllum inophyllum is mainly distributed in Hainan and Guangdong regions. As one of the characteristic oils in Hainan, Calophyllum inophyllum seed oil is rich in chemical constituents such as tocopherols, phenols, flavonoids, coumarins, and alkaloids, and has significant medicinal value. In Hainan folk medicine, Calophyllum inophyllum seed oil has a long application history and is often used as an antiseptic, antibacterial agent, astringent, and analgesic, for treating traumatic injuries, external bleeding, and relieving joint pain and neuralgia. In addition, Calophyllum inophyllum seed oil has antibacterial and hemostatic pharmacological activities and shows strong antibacterial effects against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Therefore, it is of great value to compound Calophyllum inophyllum seed oil with traditional Li medicine prescriptions with anti-inflammatory and antibacterial effects to develop an anti-inflammatory and antibacterial Li medicine preparation.

[0011] However, Tamanu oil is unstable, prone to oxidation and insoluble in water, which limits its application in the development of drug formulations. To improve its stability and bioavailability, it needs to be processed through inclusion technology. By changing the physical state, liquid Tamanu oil is made into powder through inclusion technology for easy use, and made into gels and sprays. These dosage forms have the characteristics of easy absorption, rapid onset, simple production process, convenient carrying and use. Therefore, it is very necessary to compound the cyclodextrin inclusion complex containing Tamanu oil with traditional Li medicines with anti-inflammatory and antibacterial effects to prepare a topical gel. After literature search, there is currently no report on the cyclodextrin inclusion complex of Tamanu oil in the medical field. Summary of the Invention

[0012] In view of this, the purpose of the present invention is to provide a Li medicine containing the cyclodextrin inclusion complex of Tamanu oil and its preparation method, so as to provide a research basis for the development of Tamanu oil drugs.

[0013] The technology of the present invention is realized as follows:

[0014] A Li medicine containing the cyclodextrin inclusion complex of Tamanu oil and its preparation method, comprising the following steps:

[0015] 1. Preparation of the extract of Callicarpa nudiflora: Take 100 g of dried leaves of Callicarpa nudiflora and crush them using a pulverizer. Place the crushed medicinal materials in a reflux extraction tank, and add 60% (V / V) ethanol for reflux extraction at 75 °C for a total of 3 times, with each reflux for 1 h. Add 550 mL of ethanol for the first time, 350 mL for the second time, and 250 mL for the third time. After combining the three extraction solutions, distill and recover ethanol under reduced pressure, concentrate the extraction solution, and finally freeze-dry the extraction solution to obtain the extract.

[0016] 2. Preparation of the extract of Stemona tuberosa: Remove the fine roots and soil from the collected tuberous roots of Stemona tuberosa, take them out and dry them in the sun when they are just cooked through in boiling water. Use a pulverizer to crush the dried tuberous roots, and put the obtained powder into a sealed bag and store it in a dark place for later use. Weigh 100 g of the sample and place it in a reflux extraction tank, and use 60% (V / V) ethanol for reflux extraction at 75 °C for a total of three times, with each extraction for 1 h. Add 550 mL of ethanol for the first time, 350 mL for the second time, and 250 mL for the third time. Combine the three extraction solutions, distill and recover ethanol under reduced pressure and concentrate the extraction solution, and finally obtain the extract through freeze-drying.

[0017] 3. Preparation of Melastoma penicillatum extract: Weigh the dried leaves of Melastoma penicillatum and crush them. Take 100 g of the obtained powder and place it in a reflux extraction tank. Under the condition of 75 °C, use 60% (V / V) ethanol for reflux extraction for a total of 3 times, with each reflux lasting 1 h. Add 550 mL of ethanol for the first time, 350 mL for the second time, and 250 mL for the third time. Combine the extraction solutions from the three extractions, distill and recover ethanol under reduced pressure and concentrate the extraction solution. Finally, freeze-dry the extraction solution to obtain the extract.

[0018] 4. Preparation of Sarcococca hainanensis extract: Take 100 g of the dried leaves of Sarcococca hainanensis, crush them and place them in a reflux extraction tank. Under the condition of 75 °C, use 60% (V / V) ethanol for reflux extraction for a total of 3 times, with each extraction lasting 1 hour. Add 550 mL of ethanol for the first time, 350 mL for the second time, and 250 mL for the third time. Combine the extraction solutions from the three extractions, distill and recover ethanol under reduced pressure and concentrate the extraction solution. Finally, freeze-dry the extraction solution to obtain the extract.

[0019] 5. Preparation of Pteris multifida extract: Weigh the dried whole herb of Pteris multifida and crush it. Take 100 g of the obtained powder and place it in a reflux extraction tank. Under the condition of 75 °C, add 60% (V / V) ethanol for reflux extraction for a total of 3 times, with each reflux lasting 1 hour. Add 550 mL of ethanol for the first time, 350 mL for the second time, and 250 mL for the third time. Combine the extraction solutions from the three extractions, distill and recover ethanol under reduced pressure, concentrate the extraction solution, and finally freeze-dry the extraction solution to obtain the extract.

[0020] 6. Preparation of Calophyllum inophyllum seed oil cyclodextrin inclusion complex: Dry sulfobutyl-β-cyclodextrin at 50 °C until constant weight. Take 16 g of the dried sulfobutyl-β-cyclodextrin and place it in a 250 mL conical flask, add 80 mL of distilled water. Place the conical flask on a magnetic stirrer and stir until completely dissolved. After the substances in the flask are completely dissolved, raise the temperature to 60 °C and slowly add 1 g of Calophyllum inophyllum seed oil (diluted with 3 mL of absolute ethanol). Stir for a certain time, mix evenly, filter through a 0.45 μm microporous filter membrane, and then perform freeze-drying to obtain the inclusion complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Appendix Figure 1: Anti-inflammatory experiment on xylene-induced auricular swelling in mice (Group A is the blank group, Group B is the positive control, Group C is the experimental group containing the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil, Group D is the traditional Li medicine group, and Group E is the Li medicine group)

[0023] Appendix Figure 2 : Pictures of the wound healing area of mice in each group. Group A is the model group, the wound healing area on the 3rd day is A1, the wound healing area on the 7th day is A2, and the wound healing area on the 14th day is A3. Group B is the positive control group, the wound healing area on the 3rd day is B1, the wound healing on the 7th day is B2, and the wound healing area on the 14th day is B3. Group C is the experimental group containing the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil, the wound healing on the 3rd day is C1, the wound healing on the 7th day is C2, and the wound healing on the 14th day is C3. Group D is the traditional Li medicine group, the wound healing on the 3rd day is D1, the wound healing on the 7th day is D2, and the wound healing on the 14th day is D3. Group E is the Li medicine group, the wound healing on the 3rd day is E1, the wound healing on the 7th day is E2, and the wound healing on the 14th day is E3.

[0024] Appendix Figure 3 : Histopathological pictures of the wound tissues of mice in each group (HE staining ×200). Group A is the model group, A1 is × 20, A2 is × 200, A3 is × 200, A4 is × 200. Group B is the positive control group, B1 is × 20, B2 is × 200, B3 is × 200. Group C is the experimental group containing the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil, C1 is × 20, C2 is × 200, C3 is × 200. Group D is the traditional Li medicine group, D1 is ×20, D2 is ×200, D3 is ×200. Group E is the experimental group of Li medicine, E1 is ×20, E2 is ×200, and E3 is × 200. Detailed implementation methods

[0025] Example 1: Preparation of the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil

[0026] 1. Influence of the dropping rate of Calophyllum inophyllum seed oil on the antibacterial effect

[0027] Accurately weigh 5 portions of Calophyllum inophyllum seed oil. Place 16 g of sulfobutyl-β-cyclodextrin in a beaker and add 80 mL of distilled water. Place the beaker on a magnetic stirrer and stir to dissolve, and raise the temperature to 60 °C.

[0028] The Jatropha curcas L. seed oil was dropped into beakers at the dropping rates of 0.5 mL / min, 1.0 mL / min, 1.5 mL / min, 2.0 mL / min and 2.5 mL / min (the ethanol addition amount was 3 times the amount of Jatropha curcas L. seed oil used, w / w), respectively. After stirring for 1 h, the mixed solutions were transferred to separating funnels respectively, and the uncomplexed Jatropha curcas L. seed oil in the upper layer was extracted with petroleum ether, then filtered through a 0.45 μm microporous membrane, and analyzed after freeze-drying.

[0029] Table 1 Effect of the dropping rate of Jatropha curcas L. seed oil (mL / min) on the antibacterial effect

[0030]

[0031] According to the above data, taking the antibacterial effect of the clathrate as the index, when the dropping rate of Jatropha curcas L. seed oil was 1 mL / min, the antibacterial effect of the clathrate was better than that of other dropping rates. Therefore, under the condition of keeping the dropping rate of Jatropha curcas L. seed oil at 1 mL / min, three factors, namely the inclusion temperature, inclusion time, and the ratio of sulfobutyl-β-cyclodextrin to distilled water, were investigated.

[0032] 2. Effect of the clathrate at different temperatures on the antibacterial effect

[0033] Accurately weigh 5 portions of sulfobutyl-β-cyclodextrin, 16 g for each portion, and place them in beakers respectively. Add 80 mL of distilled water to each beaker, fully dissolve it, and then place the beaker on a magnetic stirrer for stirring and dissolving. Subsequently, raise the solution temperature to 30 °C, 40 °C, 50 °C, 60 °C and 70 °C respectively. During the stirring process, drop Jatropha curcas L. seed oil at a rate of 1 mL / min, and keep the inclusion time for 1 hour. After the inclusion is completed, transfer the mixed solutions to separating funnels respectively, and extract the uncomplexed Jatropha curcas L. seed oil in the upper layer with petroleum ether. The extracted solution is filtered through a 0.45 μm microporous membrane, and finally analyzed after freeze-drying.

[0034] Table 2 Effect of the clathrate at different temperatures on the antibacterial effect

[0035]

[0036] According to the above data, taking the antibacterial effect of the clathrate as the index, when the inclusion temperature was 60 °C, its antibacterial effect was better than that of other temperatures. Therefore, 60 °C was used as the inclusion temperature for subsequent experiments. After 1 h of inclusion time, transfer the mixed solution to a separating funnel, extract the uncomplexed Jatropha curcas L. seed oil in the upper layer with petroleum ether, then filter it through a 0.45 μm microporous membrane, and analyze it after freeze-drying.

[0037] 3. Effect of the clathrate with different stirring times on the antibacterial effect

[0038] Accurately weigh 5 portions of sulfobutyl-β-cyclodextrin, 16 g for each portion, and place them in beakers respectively. Add 80 mL of distilled water to each portion, and place them on a magnetic stirrer to stir and dissolve. At the same time, raise the temperature to 60 °C. Stir the 5 samples respectively, and the stirring times are 20 min, 30 min, 40 min, 50 min and 60 min respectively. Subsequently, add tamanu oil dropwise at a rate of 1 mL / min. Transfer the inclusion complex to a separating funnel, and extract the uncomplexed tamanu oil in the upper layer with petroleum ether. Finally, filter with a 0.45 μm microporous membrane and analyze after freeze-drying.

[0039] Table 3. Influence of inclusion complexes with different stirring times on antibacterial effect

[0040]

[0041] Based on the above data, taking the antibacterial effect of the inclusion complex as the evaluation index, the stirring time set in the preliminary experiment was 60 min. However, the results of the single-factor experiment showed that an ideal effect could be achieved in 40 min. This discovery solved the key energy-saving technology problem in large-scale production. In the production process flow, shortening the stirring time from 60 min to 40 min not only achieved the energy-saving goal, but also its antibacterial effect was better than other stirring times. Therefore, in subsequent inclusion experiments, 40 min was used as the stirring time.

[0042] 4. Influence of inclusion complexes of sulfobutyl-β-cyclodextrin and distilled water in different ratios on antibacterial effect

[0043] Accurately weigh 5 portions of sulfobutyl-β-cyclodextrin and mix them with distilled water in the following ratios respectively: 10:80, 12:80, 14:80, 16:80, 18:80. Place the mixture on a magnetic stirrer to stir and dissolve, the inclusion temperature is 60 °C, and the stirring time is 40 min. Add tamanu oil dropwise at a rate of 1 mL / min. Transfer the inclusion complexes to separating funnels respectively, extract the uncomplexed tamanu oil with petroleum ether, filter with a 0.45 μm microporous membrane, and finally analyze after freeze-drying.

[0044] Table 4. Influence of inclusion complexes of sulfobutyl-β-cyclodextrin and distilled water in different ratios on antibacterial effect

[0045]

[0046] In summary, according to the experimental data, the ratio of sulfobutyl-β-cyclodextrin to distilled water was 16:80 during the preliminary experiment, and the single-factor experiment results showed that the optimal ratio was 12:80. This ratio saved costs during large-scale production. Therefore, when the ratio of sulfobutyl-β-cyclodextrin to distilled water was 12:80, the inclusion temperature was 60 °C, the stirring time was 40 min, and the dropping rate of Calophyllum inophyllum seed oil was 1 mL / min, the antibacterial effect of the inclusion complex was significant.

[0047] Example 2: Antibacterial experiment of Calophyllum inophyllum seed oil cyclodextrin inclusion complex against Staphylococcus aureus

[0048] The powder containing Calophyllum inophyllum seed oil cyclodextrin inclusion complex was formulated into samples with concentrations of 2.5 mg / mL, 5.0 mg / mL, 10.0 mg / mL, and 25.0 mg / mL according to a certain ratio and stored in a refrigerator at 0-4 °C for later use. Filter paper discs with a diameter of 6 mm were soaked with the prepared liquid of different concentrations. After natural drying, they were placed in a common nutrient culture dish inoculated with bacteria. One filter paper disc containing Calophyllum inophyllum seed oil cyclodextrin inclusion complex was placed in each culture dish, and a blank filter paper disc dipped in sterile water was also placed as a control. Through diffusion, the drug in the filter paper disc dissolved into the culture medium. After the bacteria were cultured at 37 °C for 18 hours, it was observed whether an antibacterial zone appeared in the culture dish. If there was an antibacterial zone, a vernier caliper or computer was used to measure its size, record it, and take a photo. The experimental results of the antibacterial effect of Calophyllum inophyllum seed oil cyclodextrin inclusion complex against Staphylococcus aureus are shown in Table 5.

[0049] Table 5 Antibacterial effect of Calophyllum inophyllum seed oil cyclodextrin inclusion complex against Staphylococcus aureus

[0050]

[0051] The minimum inhibitory concentration of Calophyllum inophyllum seed oil cyclodextrin inclusion complex against Staphylococcus aureus was 2.5 mg / mL.

[0052] Example 3: Antibacterial test of Calophyllum inophyllum seed oil cyclodextrin inclusion complex against Escherichia coli

[0053] The powder containing the tocopheryl acetate - cyclodextrin clathrate was formulated into samples with concentrations of 2.5 mg / mL, 5.0 mg / mL, 10.0 mg / mL, and 25.0 mg / mL according to a certain ratio, and stored in a refrigerator (0 - 4 °C) for later use. The prepared liquid with different concentrations was used to soak filter paper disks (diameter 6 mm). After they were naturally air - dried, they were placed into ordinary nutrient culture dishes inoculated with bacteria. One filter paper disk containing the tocopheryl acetate - cyclodextrin clathrate was placed in each culture dish, and at the same time, a blank filter paper disk (dipped in sterile water) was placed as a control. Through diffusion, the drug in the filter paper disk dissolved into the culture medium. After the bacteria were cultured at 37 °C for 18 hours, it was observed whether an inhibition zone appeared in the culture dish. For the samples with an inhibition zone, a vernier caliper or computer was used to measure the size of the inhibition zone, and records and photos were taken. The experimental results of the antibacterial effect of the tocopheryl acetate - cyclodextrin clathrate on Escherichia coli are shown in Table 6.

[0054] Table 6 Inhibitory effect of tocopheryl acetate - cyclodextrin clathrate on Escherichia coli

[0055]

[0056]

[0057] The minimum inhibitory concentration of the tocopheryl acetate - cyclodextrin clathrate against Escherichia coli is 5.0 mg / mL.

[0058] Example 4: Antibacterial test of tocopheryl acetate - cyclodextrin clathrate against Candida albicans The powder containing the tocopheryl acetate - cyclodextrin clathrate was formulated into samples with concentrations of 2.5 mg / mL, 5.0 mg / mL, 10.0 mg / mL, and 25.0 mg / mL according to a certain ratio, and stored in a refrigerator at 0 - 4 °C for later use. The prepared liquid with different concentrations was used to soak filter paper disks with a diameter of 6 mm. After they were naturally air - dried, they were placed into Sabouraud culture dishes inoculated with the strain. One filter paper disk containing the tocopheryl acetate - cyclodextrin clathrate was placed in each culture dish, and at the same time, a blank filter paper disk dipped in sterile water was placed as a control. Through diffusion, the drug in the filter paper disk dissolved into the culture medium. After the culture dishes were cultured at 37 °C for 18 h, it was observed whether an inhibition zone was formed. For the samples with an inhibition zone, a vernier caliper or computer was used to measure the size of the inhibition zone, and records and photos were taken. The experimental results of the antibacterial effect of the tocopheryl acetate - cyclodextrin clathrate on Candida albicans are shown in Table 7.

[0059] Table 7 Inhibitory effect of tocopheryl acetate - cyclodextrin clathrate on Candida albicans

[0060]

[0061] The minimum inhibitory concentration of the calophyllum inophyllum seed oil cyclodextrin inclusion complex against candida albicans is 10.0 mg / mL.

[0062] Example 5: Preparation of a Li medicine gel containing calophyllum inophyllum seed oil cyclodextrin inclusion complex

[0063] Containing 55 g of calophyllum inophyllum seed oil cyclodextrin inclusion complex, 10 g of callicarpa nudiflora extract, 10 g of stemona tuberosa extract, 10 g of melastoma sanguineum extract, 10 g of sarcococca hainanensis extract, and 5 g of pteris multifida extract.

[0064] The total weight reaches 100 g. Grind the above drugs finely, mix them evenly to make a gel, and dispense it into small tubes, with each tube weighing 20 g net. Apply an appropriate amount of the gel externally to the affected area 3 times a day.

[0065] Example 6: Preparation of a Li medicine spray containing calophyllum inophyllum seed oil cyclodextrin inclusion complex

[0066] Containing 55 g of calophyllum inophyllum seed oil cyclodextrin inclusion complex, 10 g of callicarpa nudiflora extract, 10 g of stemona tuberosa extract, 10 g of melastoma sanguineum extract, 10 g of sarcococca hainanensis extract, and 5 g of pteris multifida

[0067] The total weight reaches 100 g. Grind the above drugs finely, soak them in rice wine for 1 month, filter, and dispense them into a spray, with each bottle being 20 mL. Except for open wounds, the patient sprays the affected area 2 - 3 times a day.

[0068] Example 7: Anti - inflammatory experiment on xylene - induced auricular swelling in mice

[0069] 1. Drug preparation

[0070] (1) Sterile distilled water

[0071] (2) Indomethacin: Take 5 tablets (5×25 mg), crush them and place them in a 100 mL volumetric flask, add pure water to dissolve and make up the volume to the mark.

[0072] (3) Calophyllum inophyllum seed oil cyclodextrin inclusion complex: Accurately weigh 0.6 g and place it in a 100 mL volumetric flask, add pure water to make up the volume to the mark.

[0073] (4) Traditional Li medicine: Accurately weigh 0.6 g and place it in a 100 mL volumetric flask, add pure water to make up the volume to the mark.

[0074] (5) Li medicine: Accurately weigh 0.6 g and place it in a 100 mL volumetric flask, add pure water to make up the volume to the mark.

[0075] (6) The strains of staphylococcus aureus, escherichia coli, and candida albicans are provided by the School of Pharmacy, Hainan Medical University.

[0076] (7) The Calophyllum inophyllum seeds were collected from Sanya Bay, Sanya City, and the Calophyllum inophyllum seed oil was prepared in the laboratory.

[0077] (8) The batch number of the Lidandan herbal bacteriostatic gel: HM24-03Y0201, provided by Hainan Qixing Biotechnology Co., Ltd.

[0078] 2. Experimental animals

[0079] ICR mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., SPF level, and the license number is SYXk(Qiong)2022-0013.

[0080] 3. Anti-inflammatory experiment on xylene-induced auricular swelling in mice

[0081] Grouping of animal experiments

[0082] Take 50 male mice with a body weight between 18 - 22 g and divide them into 5 groups, with 10 mice in each group. The specific experimental methods are as follows:

[0083] Group A: Blank group, gavaged. Each mouse was gavaged with 0.2 mL of distilled water at 10 mL / kg body weight for 6 consecutive days.

[0084] Group B: Positive control group, indomethacin was gavaged. Each mouse was gavaged with 0.2 mL (containing 0.25 mg of the drug) every day for 6 consecutive days.

[0085] Group C: Containing the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil, each mouse was gavaged with 0.2 mL (containing 1.2 mg of the drug) every day for 6 consecutive days.

[0086] Group D: Traditional Li medicine, each mouse was gavaged with 0.2 mL (containing 1.2 mg of the drug) every day for 6 consecutive days.

[0087] Group E: Li medicine, each mouse was gavaged with 0.2 mL (containing 1.2 mg of the drug) every day for 6 consecutive days.

[0088] 1 h after the last administration, 0.2 mL of xylene was applied to the front and back of the right ear of each mouse, and the left ear was used as a blank control. At the same time, the degree of swelling of the right ear of each group of mice was observed visually. After 25 min, the mice were sacrificed by cervical dislocation, and the two ears were cut along the auricular baseline. Circular ear pieces were punched at the same position on both ears with a punch with a diameter of 8 mm, and the weights of the left and right ear pieces were weighed. The difference in the weights of the left and right ear pieces was used as the swelling degree, and the swelling degree and inhibition rate were calculated.

[0089] Degree of ear piece swelling: Weight of the inflamed side ear piece (right ear) - Weight of the control side ear piece (left ear).

[0090] Swelling inhibition rate = (Swelling rate of the control group - Swelling rate of the drug administration group) / Swelling rate of the control group × 100%.

[0091] Result analysis: See the mouse acute auricular swelling experiment in (AppendixFigure 1 )。After applying xylene to both the front and back sides of the right ear of the mice in Group A, obvious swelling was observed with the naked eye (Blank Group A). After applying xylene to both the front and back sides of the right ear of the mice in Group B, slight swelling of the mouse ear was observed with the naked eye (Positive Control Group B). After applying xylene to both the front and back sides of the right ear of the mice in Group C, the degree of swelling observed with the naked eye was mild (Inclusion Complex Group C). After applying xylene to both the front and back sides of the right ear of the mice in Group D, the degree of swelling observed with the naked eye was less than that of Group C (Traditional Li Medicine Group D). After applying xylene to both the front and back sides of the right ear of the mice in Group E, the degree of swelling observed with the naked eye was relatively mild and close to that of Group B (Li Medicine Group E).

[0092] Table 8 Anti-inflammatory Experiment Results of Auricular Swelling in Mice Induced by Xylene

[0093]

[0094]

[0095] Note: Compared with the blank control group, *P<0.01, *P<0.01

[0096] The results indicate that the inclusion complex of calophyllum inophyllum seed oil with cyclodextrin experimental group, traditional Li medicine control group, and Li medicine experimental group all have anti-inflammatory effects. The anti-inflammatory effect of the Li medicine in Group E is superior to that of Group C and Group D, but lower than that of Group B.

[0097] Example 8: Comparison of Wound Healing Rates of Mice in Each Group

[0098] 1. Establishment of Animal Model

[0099] Sixty male mice aged 6 - 8 weeks with a body weight of 19 - 22 g were taken. They were anesthetized by intraperitoneal injection of 1% chloral hydrate (0.006 mL / g). After anesthesia, the hair on the back of the mice was removed, and then the local area was disinfected with 75% alcohol. Then, a circular mark with a diameter of about 2 cm was made in the depilated area, and the skin was cut along the edge. 0.1 mL of Staphylococcus aureus bacterial suspension (bacterial concentration 6×10 8 CFU / mL) was inoculated at the skin lesion of the mice. After 24 hours of Staphylococcus aureus infection, when the clinical manifestations of the skin lesions of the mice were erythema, thickening, and yellow crusting, it was determined that the model was successfully established.

[0100] 2. Administration Method

[0101] To verify the efficacy of the Li medicine, 10 mice with unsuccessful model establishment were excluded. Fifty mice with successful model establishment were taken, and 5 groups of experiments were designed:

[0102] Model Group: Apply sterile distilled water

[0103] Positive Control Group: Apply Lidanlan Herbal Bacteriostatic Gel.

[0104] Experimental group with Tamanu oil cyclodextrin inclusion complex: Apply the Tamanu oil cyclodextrin inclusion complex

[0105] complex

[0106] Traditional Li medicine: Apply traditional Li medicine

[0107] Experimental group with Li medicine: Apply Li medicine

[0108] For each of the above groups, apply twice a day for 14 consecutive days. For the calculation of the wound healing rate, calculate the wound healing rate of each group of mice on the 3rd, 7th, and 14th days of administration. The wound healing rate = (wound area before administration - wound area after administration) / wound area before administration × 100%.

[0109] Table 9 Comparison of wound healing rates of each group of mice

[0110]

[0111] Note: Compared with the model group, a P < 0.01; compared with the positive control group, b P < 0.01; compared with the experimental group with Tamanu oil cyclodextrin inclusion complex, c P < 0.01; compared with the traditional Li medicine control group, d P < 0.01; compared with the experimental group with Li medicine, e P < 0.01.

[0112] For the comparison of the wound healing rates of each group of mice, on the 3rd, 7th, and 14th days of administration, the wound healing rates of the mice in each treatment group were higher than those in the model group (all P < 0.01). Among them, the experimental group with Tamanu oil cyclodextrin inclusion complex, the traditional Li medicine control group, and the experimental group with Li medicine had lower healing rates than the positive control group (all P < 0.01), while there was no significant difference between the experimental group with Li medicine and the positive control group (all P < 0.01). The wound healing rate of the experimental group with Li medicine was gradually increasing (all P < 0.01) (see attachment Figure 2 ).

[0113] Example 9: Comparison of the number of wound colonies of each group of mice

[0114] 1. Establishment of animal model

[0115] Take 60 male mice aged 6 - 8 weeks with a body weight of 19 - 22 g. Anesthetize them by intraperitoneal injection of 1% chloral hydrate according to (0.006 mL / g). After anesthesia, depilate the back of the mice, then disinfect the local area with 75% alcohol, and then make a circular mark with a diameter of about 2 cm in the depilated area. Cut the skin along the edge, and inoculate 0.1 mL of Staphylococcus aureus suspension (bacterial concentration 6×10 8(CFU / mL). After 24 hours of Staphylococcus aureus infection, when the clinical manifestations of the skin lesions in mice were erythema, thickening, and yellow crusts, the modeling was considered successful.

[0116] 2. Administration method

[0117] To verify the efficacy of the Li medicines, 10 mice with unsuccessful modeling were excluded, and 50 successfully modeled mice were selected. Five groups of experiments were designed:

[0118] Model group: Apply sterile distilled water

[0119] Positive control group: Apply Lidanlan herbal bacteriostatic gel.

[0120] Experimental group containing calophyllum inophyllum seed oil cyclodextrin inclusion complex: Apply the group containing calophyllum inophyllum seed oil cyclodextrin

[0121] inclusion complex

[0122] Traditional Li medicine group: Apply traditional Li medicine

[0123] Experimental group of Li medicine: Apply Li medicine

[0124] For the comparison test of the number of colonies on the wound surface of mice, on the 3rd, 7th, and 14th days after administration, take the wound tissue of mice, cut it into pieces, mix it with 1.2 mL of sterile PBS, then homogenize, centrifuge, and dilute. Take 10 μL of the bacterial solution and inoculate it on the MRSA chromogenic plate and MH blood plate respectively. After culturing at 37 °C for 48 h, calculate and take pictures.

[0125] Table 10 Comparison of the number of colonies on the wound surface of mice in each group

[0126]

[0127]

[0128] Note: Compared with the model group, a P < 0.01; compared with the positive control group, b P < 0.01; compared with the experimental group containing calophyllum inophyllum seed oil cyclodextrin inclusion complex, c P < 0.01; compared with the traditional Li medicine group, d P < 0.01; compared with the Li medicine group e P < 0.01.

[0129] For the comparison of the number of colonies on the wound surface of mice in each group, with the prolongation of the administration time, the number of colonies on the wound surface of mice in the model group increased (sterile distilled water was applied after modeling), while that in other groups decreased (all P < 0.01). However, the experimental group containing calophyllum inophyllum seed oil cyclodextrin inclusion complex and the traditional Li medicine control group were both higher than the positive control group (P < 0.01), and there was no significant difference in the number of colonies between the experimental group of Li medicine and the positive control group (P > 0.05).

[0130] Example 10: Analysis of pathological changes in the wound tissues of mice in each group (attached Figure 3 )

[0131] Group A (model group): A large number of free necrotic cell debris were present on the surface of the skin tissue (indicated by black arrows). The epidermal structure was incomplete, the skin layers were unclear, and small areas of necrosis in the epidermis and dermis were visible locally (marked by gray arrows), accompanied by a large amount of necrotic cell debris, unstructured eosinophilic substances, and granulocyte infiltration. The epidermal thickness was uneven, and mild to moderate acanthosis was observed locally (indicated by green arrows), with a significant increase in the number of spinous cells. The boundary between the dermis and subcutaneous tissue was blurred, and fibroconnective tissue hyperplasia was visible in a wide area (indicated by light blue arrows), which contained a large number of fibroblasts and collagen fibers, and mild bleeding was present locally (marked by red arrows). In addition, a small amount of new blood vessels (indicated by light green arrows) and vascular congestion (marked by purple arrows) were visible, accompanied by a large amount of lymphocyte, granulocyte, and macrophage infiltration (indicated by blue arrows), and significant neutrophil infiltration (marked by yellow arrows). The skin appendages (including hair follicles and sebaceous glands) were unevenly distributed but were numerous. The black square was used to mark the magnified field area.

[0132] Group B (positive control group): The epidermis of the skin tissue was intact and thin, and hyperkeratosis of the stratum corneum was visible in local areas (black arrows). The structures of the dermis and subcutaneous tissue were widely blurred, and fibroconnective tissue hyperplasia was observed in local areas (light blue arrows), accompanied by a large number of fibroblasts, a small amount of collagen fibers, and new blood vessels (light green arrows). In addition, a small amount of vascular congestion (purple arrows) was visible, as well as a large amount of granulocyte, lymphocyte, and macrophage infiltration (blue arrows), occasional neutrophil infiltration (yellow arrows), and less necrotic cell debris (gray arrows). The skin appendages such as hair follicles and sebaceous glands were unevenly distributed (orange arrows) and were numerous. The black square marked the position of the magnified field.

[0133] Group C (experimental group containing Calophyllum inophyllum seed oil cyclodextrin inclusion complex): The epidermis of the skin tissue was visible to be intact, with uneven thickness, moderate hyperkeratosis of the stratum corneum in a moderate range (black arrows), and mild to moderate acanthosis in a moderate range (green arrows), with an increase in the number of spinous cells. The structures of the dermis and subcutaneous tissue were unclear in a relatively large area, and relatively large fibroconnective tissue hyperplasia was visible (light blue arrows), a large number of fibroblasts and a relatively large amount of collagen fibers were visible, a small amount of new blood vessels were visible (light green arrows), less necrotic cell debris was visible (gray arrows), accompanied by a large amount of granulocyte, lymphocyte, and macrophage infiltration (blue arrows), and a small amount of neutrophil infiltration was visible (yellow arrows). The skin appendages such as hair follicles and sebaceous glands were unevenly distributed (orange arrows), the number of hair follicles and sebaceous glands was numerous, and occasional hair follicle dilation was visible (brown arrows), and a large amount of keratinized substances were visible in the lumen. The black square indicated the position of the magnified field.

[0134] D Traditional Li medicine group: The skin tissue showed a complete epidermis, but the thickness was uneven. There was extensive hyperkeratosis of the stratum corneum (black arrow) and mild hypertrophy of the stratum spinosum in a large area (green arrow), and the number of spinous cells increased. The large-scale structure of the dermis and subcutaneous tissue was unclear. Extensive proliferation of fibrous connective tissue (light blue arrow) was visible, accompanied by a large number of fibroblasts and collagen fibers. In addition, a small amount of newly formed blood vessels (light green arrow) were observed, as well as a large number of granulocytes, lymphocytes and macrophages infiltration (blue arrow), accompanied by a small amount of neutrophil infiltration (yellow arrow). The skin appendages such as hair follicles and sebaceous glands were unevenly distributed (orange arrow), and the number of hair follicles and sebaceous glands was relatively large. The black square indicates the position of the magnified view.

[0135] E Experimental group of Li medicine: The skin tissue showed a complete epidermis, but the thickness was uneven. Extensive hyperkeratosis of the stratum corneum was presented (black arrow), and mild hypertrophy of the stratum spinosum occurred (green arrow), and the number of spinous cells increased. The large-scale structure of the dermis and subcutaneous tissue was unclear. Proliferation of fibrous connective tissue was visible (light blue arrow), accompanied by more fibroblasts and a large number of collagen fibers. There were more newly formed blood vessels (light green arrow), a small amount of vascular congestion (purple arrow), and occasional petechial hemorrhage (red arrow). In addition, more granulocytes and lymphocytes infiltration were visible (blue arrow), as well as a small amount of neutrophil infiltration (yellow arrow). The skin appendages such as hair follicles and sebaceous glands were unevenly distributed, with a relatively large number. Occasional hair follicle dilation was observed (brown arrow), and a large amount of keratin was visible in the hair follicles. The black square indicates the position of the magnified view.

Claims

1. A Li medicine containing the cyclodextrin inclusion complex of Calophyllum inophyllum seed oil, characterized in that, It is composed of the following: 55% of calophyllum inophyllum seed oil cyclodextrin clathrate, 10% of callicarpa nudiflora extract, 10% of stemona tuberosa extract, 10% of melastoma sanguineum extract, 10% of sarcococca hainanensis extract, and 5% of pteris multifida extract. Among them, callicarpa nudiflora, stemona tuberosa, melastoma sanguineum, sarcococca hainanensis, and pteris multifida are all ethanol extracts.

2. The preparation method of the Li medicine according to claim 1, characterized in that, The extract includes callicarpa nudiflora, stemona tuberosa, melastoma sanguineum, sarcococca hainanensis, and pteris multifida. The ethanol extract is obtained by reflux extraction with an ethanol solution under heating conditions, and then the ethanol is recovered and freeze-dried.

3. The Li medicine according to claim 1 or the Li medicine prepared by the preparation method according to claim 2, characterized in that, The Li medicine can be made into gels and sprays.

4. Uses and applications: A Li medicine containing calophyllum inophyllum seed oil cyclodextrin clathrate can be made into gels and sprays for use as an external preparation for anti-inflammatory and antibacterial purposes.

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