A vaginal administration preparation of Artemisia annua volatile oil and its preparation method and application

Abstract

The present invention provides an artemisia annua volatile oil vaginal administration preparation and a preparation method and application thereof, belonging to the field of biomedical technology. The preparation method of the artemisia annua volatile oil vaginal administration preparation comprises the following steps: uniformly mixing poloxamer 188 and poloxamer 407 with water, refrigerating, and obtaining a thermosensitive gel solution; mixing glycerol, artemisia annua volatile oil, anhydrous ethanol and water to obtain a stable artemisia annua volatile oil solution; mixing methyl paraben with water to obtain a methyl paraben solution; uniformly mixing the thermosensitive gel solution, the stable artemisia annua volatile oil solution and the methyl paraben solution, and then adding water to mix to obtain the artemisia annua volatile oil vaginal administration preparation. The research results of the present invention show that the artemisia annua volatile oil vaginal administration preparation can effectively treat vaginitis, especially vaginitis caused by one or more infections of Staphylococcus aureus, Escherichia coli and Candida albicans. The present invention provides a choice for drugs for clinical treatment of vaginitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an artemisia annua volatile oil vaginal administration preparation and a preparation method and application thereof. Background Art

[0002] Gynecological inflammation is a common disease among women, and its pathological mechanism is relatively complex. Patients often experience symptoms such as low back and abdominal pain or heavy pain, abnormal leucorrhea, cervical erosion, and menstrual disorders. Symptomatic treatment and management are needed as soon as possible to avoid affecting the patient's physical and mental health, or even causing problems such as infertility. Gynecological inflammation specifically includes female vulvitis, vaginitis, cervicitis, and pelvic inflammatory disease. Common symptoms are mainly vulvar and vaginal itching, and some local erosions occur, accompanied by burning pain, urinary pain, frequent urination, increased vaginal discharge, redness and swelling, etc. Therefore, the treatment of vaginal inflammation is of great significance to protecting the health of the female reproductive tract and the prevention and control of cervical cancer.

[0003] There are abundant capillaries and lymphatic vessels in the vagina, but no obvious nerve endings. Therefore, local vaginal administration can not only effectively reduce the pain and irritation of administration, but also improve the effective bioavailability of drugs, avoid first-pass metabolism, and achieve higher local drug concentrations. In recent years, commonly used vaginal drug preparations include hydrogels, tablets, suppositories, creams and capsules, but due to the self-cleaning effect of the vagina, traditional vaginal drug preparations have limitations such as poor drug distribution, poor adhesion, and short retention time. Suppositories and tablets are inconvenient to use and are prone to cause vaginal damage. Suppositories are easy to flow out and contaminate clothing.

[0004] Gels are one of the most active drug dosage forms in recent years and a hot topic in the research and development of topical preparations in recent years. They have the advantages of smooth, transparent and delicate appearance; non-irritating, not interfering with the normal function of the skin; suitable consistency and viscosity, easy to apply and use, extremely convenient to carry, use, store and transport, and good stability. However, gels are vaginally administered, and such drugs tend to be quickly discharged from the vagina under the action of vaginal secretions, resulting in a short retention time of the gel in the vagina, which greatly affects the therapeutic effect. Therefore, it is crucial to find a gel that can prolong the retention time of the drug in the body and improve the bioavailability of the drug.

[0005] Volatile oil is widely present in many Chinese medicinal materials such as antipyretic drugs, qi-promoting and blood-activating drugs, aromatic and damp-removing drugs, and its plant sources are very wide. The chemical composition of volatile oil is complex, mainly including terpenes, semiterpenes and other aromatic aliphatic components. It has multiple biological activities such as anti-inflammatory and antibacterial, anti-allergic, antiviral, and anti-tumor, and is widely used in medicine, food, health, cosmetics and other fields. Artemisia annua L., scientific name of Artemisia annua L., belongs to the genus Artemisia of the family Asteraceae, and is a traditional Chinese medicine with high medicinal value. However, there is no report on the vaginal administration preparation of Artemisia annua volatile oil in the treatment of vaginitis. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an Artemisia annua volatile oil vaginal administration preparation and a preparation method and application thereof, wherein the Artemisia annua volatile oil vaginal administration preparation can effectively treat vaginitis.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing an Artemisia annua volatile oil vaginal administration preparation, comprising the following steps:

[0009] Mix poloxamer 188, poloxamer 407 and water evenly, and refrigerate to obtain a thermosensitive gel solution;

[0010] Mixing glycerin, Artemisia annua volatile oil, anhydrous ethanol and water to obtain a stable Artemisia annua volatile oil solution;

[0011] mixing methylparaben with water to obtain a methylparaben solution;

[0012] The thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methyl paraben solution are mixed evenly, and then water is added and mixed evenly to obtain an Artemisia annua volatile oil vaginal administration preparation.

[0013] Preferably, the mass volume ratio of poloxamer 188, poloxamer 407 and water is 1-7 g:20-26 g:30-50 mL.

[0014] Preferably, the refrigeration temperature is 2-6°C, and the refrigeration time is more than 12 hours.

[0015] Preferably, when glycerol, Artemisia annua volatile oil, anhydrous ethanol and water are mixed, the mass ratio of glycerol, Artemisia annua volatile oil, anhydrous ethanol and water is 1-3:0.2-1:0.5-1.5:5-7;

[0016] When methylparaben is mixed with water, the mass ratio of methylparaben to water is 0.01 to 0.03:5;

[0017] After the thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methyl paraben solution are uniformly mixed, water is added and mixed, and the mass ratio of the methyl paraben solution to the water is 5.01-5.03:20-30.

[0018] Preferably, the method for preparing the Artemisia annua volatile oil comprises the following steps:

[0019] After drying the effective part of Artemisia annua, pulverizing and sieving to obtain Artemisia annua powder; mixing the Artemisia annua powder with water and soaking it to obtain an Artemisia annua solution, distilling and refluxing the Artemisia annua solution to obtain a liquid oil-water mixture, collecting the oil layer of the liquid oil-water mixture; drying the oil layer to obtain Artemisia annua volatile oil.

[0020] Preferably, the method for drying the effective part of Artemisia annua comprises drying at 20-30°C for 6-8 days or drying at 35-45°C for 2-13 hours; the mass volume ratio of the Artemisia annua powder to water is 1g:6-12mL; the soaking time is 6-48h; the extraction time is 4.5-6.5h; the method for drying the oil layer comprises mixing the oil layer with anhydrous calcium chloride and letting it stand, centrifuging and collecting the supernatant to obtain Artemisia annua volatile oil; the effective part of Artemisia annua is Artemisia annua leaf; the Artemisia annua comes from Luocheng Mulao Autonomous County of Hechi City or Shuangqiao Village of Youyang, Chongqing.

[0021] The invention provides an artemisia annua volatile oil vaginal administration preparation prepared by the preparation method.

[0022] The present invention provides an application of the above-mentioned artemisia annua volatile oil vaginal administration preparation in the preparation of a medicine for treating vaginitis.

[0023] Preferably, the vaginitis includes one of bacterial vaginitis, fungal vaginitis, and vaginitis caused by bacterial and fungal co-infection.

[0024] Preferably, the vaginitis includes one or more of Staphylococcus aureus, Escherichia coli and Candida albicans.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a vaginal administration preparation of Artemisia annua volatile oil and a preparation method and application thereof. The research results of the present invention show that the vaginal administration preparation of Artemisia annua volatile oil can effectively treat vaginitis, especially vaginitis caused by one or more of Staphylococcus aureus, Escherichia coli and Candida albicans infection. The present invention provides a new choice for drugs for clinical treatment of vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The gel temperature test results of Poloxamer 407 with different mass volume concentrations;

[0028] Figure 2 is the gelation temperature of the thermosensitive gel precursor gel preparation after mixing poloxamer 407 and poloxamer 188 at different mass volume concentrations;

[0029] Figure 3 are the viscosity values ​​of different mass concentration ratios of poloxamer 407 and poloxamer 188 at 20, 25 and 30°C;

[0030] Figure 4 Staining the vaginal secretion smears of rats in the blank control group and the model control group of Example 18;

[0031] Figure 5 HE staining of the rat vagina in the blank control group and the model control group of Example 18;

[0032] Figure 6 The HE staining images of the vagina of rats in different groups, wherein A is the HE staining image of the vagina of rats after being treated with the blank group, B is the HE staining image of the vagina of rats after being treated with the model group, C is the HE staining image of the vagina of rats after being treated with the positive drug group, D is the HE staining image of the vagina of rats after being treated with the low-dose vaginal preparation of Artemisia annua volatile oil, E is the HE staining image of the vagina of rats after being treated with the medium-dose vaginal preparation of Artemisia annua volatile oil, and F is the HE staining image of the vagina of rats after being treated with the high-dose vaginal preparation of Artemisia annua volatile oil;

[0033] Figure 7 The results of IL-1β content detection in the serum of rats in different groups;

[0034] Figure 8 The results of IL-6 content detection in the serum of rats in different groups. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing an Artemisia annua volatile oil vaginal administration preparation, comprising the following steps:

[0036] Mix poloxamer 188, poloxamer 407 and water evenly, and refrigerate to obtain a thermosensitive gel solution;

[0037] Mixing glycerin, Artemisia annua volatile oil, anhydrous ethanol and water to obtain a stable Artemisia annua volatile oil solution;

[0038] mixing methylparaben with water to obtain a methylparaben solution;

[0039] The thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methyl paraben solution are mixed evenly, and then water is added and mixed evenly to obtain an Artemisia annua volatile oil vaginal administration preparation.

[0040] In the present invention, poloxamer 188, poloxamer 407 and water are mixed evenly and refrigerated to obtain a thermosensitive gel solution. The mass volume ratio of poloxamer 188, poloxamer 407 and water is preferably 1-7g:20-26g:30-50mL, more preferably 2-6g:21-25g:35-45mL, and more preferably 2g:23g:40mL. The gel temperature of the thermosensitive gel solution obtained by compounding poloxamer 188 and poloxamer 407 with the above-mentioned mass volume ratio is 28-35°C, which is suitable as a gel carrier. The refrigeration temperature is preferably 2-6°C, more preferably 3-5°C, and more preferably 4°C; the refrigeration time is preferably more than 12h. The present invention can fully swell and dissolve poloxamer 188 and poloxamer 407 by refrigeration. The present invention has no special limitation on the sources of poloxamer 188 and poloxamer 407, and they can be prepared by conventional preparation methods in the art and commercial products.

[0041] Glycerol, Artemisia annua volatile oil, anhydrous ethanol and water are mixed to obtain a stable Artemisia annua volatile oil solution. The mass ratio of the glycerol, Artemisia annua volatile oil, anhydrous ethanol and water is preferably 1-3:0.2-1:0.5-1.5:5-7, more preferably 1.5-2.5:0.3-0.7:0.7-1.2:5.5-6.5, and more preferably 2:0.5:1:6. The Artemisia annua volatile oil vaginal administration preparation prepared by the present invention has a gel temperature of 28-35°C, low viscosity, and is easy for vaginal administration.

[0042] Methyl paraben is mixed with water to obtain a methyl paraben solution, wherein the mass ratio of the methyl paraben to water is preferably 0.01 to 0.03:5, and more preferably 0.02:5.

[0043] In the present invention, there is no particular order for preparing the thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methylparaben solution.

[0044] After respectively preparing a thermosensitive gel solution, a stable Artemisia annua volatile oil solution and a methylparaben solution, the thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methylparaben solution are uniformly mixed, and then water is added to mix well to obtain an Artemisia annua volatile oil vaginal administration preparation. The mass ratio of the methylparaben solution to the water is preferably 5.01-5.03:20-30, and more preferably 5.02:25.48.

[0045] The artemisia annua volatile oil vaginal administration preparation of the present invention is a temperature-sensitive in-situ gel, which is characterized by having a critical phase transition temperature. When the temperature reaches the critical phase transition temperature, the preparation will change from a liquid state to a semi-solid state, forming a gel film that adheres to the biological membrane, prolonging the retention time of the drug in the body, thereby improving the bioavailability of the drug and improving the efficacy.

[0046] In the present invention, the method for preparing the Artemisia annua volatile oil comprises the following steps:

[0047] After drying the effective part of Artemisia annua, pulverizing and sieving to obtain Artemisia annua powder; mixing the Artemisia annua powder with water and soaking it to obtain an Artemisia annua solution, distilling and refluxing the Artemisia annua solution to obtain a liquid oil-water mixture, collecting the oil layer of the liquid oil-water mixture; drying the oil layer to obtain Artemisia annua volatile oil.

[0048] In the present invention, after the effective part of Artemisia annua is dried, it is powdered and sieved to obtain Artemisia annua powder. The present invention optimizes the extraction of effective parts of Artemisia annua, and the effective part of Artemisia annua is preferably Artemisia annua leaf or the aerial part of Artemisia annua, and more preferably Artemisia annua leaf. The present invention also optimizes the method of drying the effective part of Artemisia annua, and the method of drying Artemisia annua preferably includes drying at 20-30°C for 6-8 days or drying at 35-45°C for 2-13h, more preferably drying at 22-27°C for 6.5-7.5 days or drying at 37-42°C for 3-12h, more preferably drying at 25°C for 7 days, drying at 40°C for 3h, drying at 40°C for 6h or drying at 40°C for 12h, and most preferably drying at 25°C for 7 days. The mesh number of the sieve is preferably 35-45 mesh, and more preferably 40 mesh. The present invention can effectively improve the yield of Artemisia annua volatile oil by optimizing the extraction of effective parts of Artemisia annua and the method of drying Artemisia annua.

[0049] After obtaining the artemisia annua powder, the artemisia annua powder is mixed with water and soaked to obtain an artemisia annua solution. The mass volume ratio of the artemisia annua powder to water is preferably 1g:6-12mL, more preferably 1g:8-10mL, and more preferably 1g:10mL; the soaking time is preferably 6-48h, more preferably 12-36h, and more preferably 24h. The present invention significantly improves the yield of artemisia annua volatile oil by optimizing the solid-liquid ratio of artemisia annua powder to water.

[0050] After obtaining the soaked Artemisia annua solution, the soaked Artemisia annua solution is extracted by distillation and reflux to obtain a liquid oil-water mixture, and the oil layer of the liquid oil-water mixture is collected. The distillation temperature is preferably 100°C, and the extraction time is preferably 4.5 to 6.5 hours, more preferably 5 to 6 hours, and more preferably 6 hours. The present invention effectively improves the yield of Artemisia annua volatile oil by optimizing the extraction time. In the present invention, since the density of water is slightly higher than that of Artemisia annua volatile oil, the oil and water in the measuring device containing the liquid oil-water mixture will naturally separate, with water at the bottom and volatile oil at the top, which is light yellow-green. The water at the bottom is slowly released to collect the oil layer of the liquid oil-water mixture.

[0051] After obtaining the oil layer of the collected liquid oil-water mixture, the oil layer is dried to obtain Artemisia annua volatile oil. The method of drying the oil layer preselected includes mixing the oil layer with anhydrous calcium chloride and letting it stand, centrifuging and collecting the supernatant to obtain Artemisia annua volatile oil. The standing time is preferably 4 to 6 minutes, more preferably 4.5 to 5.5 minutes, and more preferably 5 minutes. The centrifugal method is preferably centrifuged at 1000 to 2000 rpm for 3 to 7 minutes, more preferably centrifuged at 1200 to 1800 rpm for 4 to 6 minutes, and more preferably centrifuged at 1500 rpm for 5 minutes. The present invention dries Artemisia annua volatile oil by anhydrous calcium chloride, which can effectively ensure a high yield of Artemisia annua volatile oil.

[0052] The invention provides an artemisia annua volatile oil vaginal administration preparation prepared by the preparation method.

[0053] The present invention provides an application of the above-mentioned artemisia annua volatile oil vaginal administration preparation in the preparation of a medicine for treating vaginitis.

[0054] In the present invention, the vaginitis preferably includes bacterial vaginitis or fungal vaginitis. The bacterial vaginitis preferably includes one or both of Staphylococcus aureus and Escherichia coli, and the fungal vaginitis preferably includes Candida albicans. The Staphylococcus aureus preferably includes Staphylococcus aureus CMCC (B) 26003. The Escherichia coli preferably includes Escherichia coli CMCC (B) 44102. The Candida albicans preferably includes Candida albicans CMCC (F) 98001. The drug also includes pharmaceutically acceptable excipients, and the excipients include excipients, flavoring agents, solvents or surfactants. The Artemisia annua volatile oil or Artemisia annua volatile oil fraction accounts for 30% to 90% of the weight of the drug. The drug can use the Artemisia annua volatile oil vaginal administration preparation as an active ingredient in combination with other active ingredients for treating vaginitis, or can use the Artemisia annua volatile oil vaginal administration preparation as the only active ingredient to treat vaginitis.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1

[0057] A method for preparing artemisia annua volatile oil, comprising the following steps:

[0058] (1) Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing were dried at 25° C. for 7 days, cut into small pieces of 2-3 cm, ground into powder, and passed through a 40-mesh sieve to obtain Artemisia annua powder;

[0059] (2) accurately weighing 40 g of the above-mentioned Artemisia annua powder and mixing it with 400 mL of water and soaking it for 24 hours to obtain an Artemisia annua solution, distilling and refluxing the Artemisia annua solution at 100° C. for 6 hours to obtain a liquid oil-water mixture, drying it to room temperature, and collecting the oil layer of the liquid oil-water mixture;

[0060] 1 mL of the above oil layer was mixed with 50 mg of anhydrous calcium chloride, and then allowed to stand for 5 min. The mixture was centrifuged at 1500 rpm for 5 min, and the supernatant was collected to obtain the dried volatile oil, namely, Artemisia annua volatile oil, which was named Artemisia annua volatile oil B1.

[0061] Example 2

[0062] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 1 is that the step (1) of the preparation of the Artemisia annua volatile oil of this embodiment is to dry the Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 3h, cut them into small pieces of 2-3cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder, and the remaining steps are the same as those of the preparation method of Example 1. The Artemisia annua volatile oil prepared in this embodiment is named Artemisia annua volatile oil B2.

[0063] Example 3

[0064] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 1 is that the step (1) of the preparation of the Artemisia annua volatile oil of this embodiment is to dry the Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 6 hours, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder, and the remaining steps are the same as those of the preparation method of Example 1. The Artemisia annua volatile oil prepared in this embodiment is named Artemisia annua volatile oil B3.

[0065] Example 4

[0066] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 1 is that the step (1) of the preparation of the Artemisia annua volatile oil of this embodiment is to dry the Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 12 hours, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder, and the remaining steps are the same as those of the preparation method of Example 1. The Artemisia annua volatile oil prepared in this embodiment is named Artemisia annua volatile oil B4.

[0067] Example 5

[0068] The difference between the method for preparing the Artemisia annua volatile oil of this embodiment and that of Example 1 is that the Artemisia annua volatile oil of this embodiment uses Artemisia annua leaves purchased from Tianhe Town, Luocheng Mulao Autonomous County, Hechi City, and the remaining steps are the same as those of Example 1.

[0069] Example 6

[0070] The difference between the method for preparing the Artemisia annua volatile oil of this embodiment and that of Example 1 is that the Artemisia annua volatile oil of this embodiment uses Artemisia annua leaves purchased from Siba Town, Luocheng Mulao Autonomous County, Hechi City, and the remaining steps are the same as those of Example 1.

[0071] Example 7 Optimization of the preparation method of Artemisia annua volatile oil

[0072] 1. Optimization of material-liquid ratio and distillation time

[0073] S1. A method for preparing Artemisia annua volatile oil 1, comprising the following steps:

[0074] (1) The above-ground parts of wild Artemisia annua plants collected from Shuangqiao Village, Youyang, Chongqing were completely dried in an oven at 60°C, cut into small pieces of 2-3 cm, ground, and passed through a 40-mesh sieve to obtain Artemisia annua powder;

[0075] (2) accurately weighing 100 g of the above-mentioned Artemisia annua powder and mixing it with 600 mL of water, distilling and refluxing for 4 h at 100° C. to obtain a liquid oil-water mixture, drying it to room temperature, and collecting the oil layer of the liquid oil-water mixture;

[0076] (3) 1 mL of the above oil layer was mixed with 50 mg of anhydrous calcium chloride, and the mixture was allowed to stand for 5 min. The mixture was centrifuged at 1500 rpm for 5 min to obtain the dried volatile oil, i.e., Artemisia annua volatile oil, which was numbered as Artemisia annua volatile oil 1.

[0077] S2. A method for preparing Artemisia annua volatile oil 2. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 2 is to accurately weigh 100 g of the above-mentioned Artemisia annua powder and mix it with 800 mL of water, and distill and reflux for extraction at 100°C for 5 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0078] S3. A method for preparing Artemisia annua volatile oil 3. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 3 is to accurately weigh 100 g of the above-mentioned Artemisia annua powder and mix it with 1000 mL of water, and distill and reflux for extraction at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0079] S4. A method for preparing Artemisia annua volatile oil 4. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 4 is to accurately weigh 100 g of the above Artemisia annua powder and mix it with 600 mL of water and soak it for 0.5 h to obtain an Artemisia annua solution after soaking, and distill and reflux extract the Artemisia annua solution at 100°C for 5 h to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0080] S5. A method for preparing Artemisia annua volatile oil 5. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 5 is to accurately weigh 100 g of the above Artemisia annua powder and mix it with 800 mL of water and soak it for 0.5 h to obtain an Artemisia annua solution after soaking, and then distill and reflux extract the Artemisia annua solution at 100°C for 6 h to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0081] S6. A method for preparing Artemisia annua volatile oil 6. The difference between the preparation method and the preparation method of Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 6 is to accurately weigh 100g of the above-mentioned Artemisia annua powder and mix it with 1000mL of water and soak it for 0.5h to obtain the soaked Artemisia annua solution, and distill and reflux extract the soaked Artemisia annua solution at 100°C for 4h to obtain a liquid oil-water mixture. The remaining steps are the same as the preparation method of Artemisia annua volatile oil 1.

[0082] S7. A method for preparing Artemisia annua volatile oil 7. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 7 is to accurately weigh 100 g of the above Artemisia annua powder and mix it with 600 mL of water and soak it for 1 hour to obtain an Artemisia annua solution after soaking, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0083] S8. A method for preparing Artemisia annua volatile oil 8. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 8 is to accurately weigh 100 g of the above Artemisia annua powder and mix it with 800 mL of water and soak it for 1 hour to obtain an Artemisia annua solution after soaking, and distill and reflux extract the Artemisia annua solution at 100°C for 4 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the method for preparing Artemisia annua volatile oil 1.

[0084] S9. A method for preparing Artemisia annua volatile oil 9. The difference between the method for preparing Artemisia annua volatile oil 1 is that step (2) of Artemisia annua volatile oil 9 is to accurately weigh 100 g of the above Artemisia annua powder and mix it with 1000 mL of water and soak it for 1 hour to obtain an Artemisia annua solution after soaking, and then distill and reflux the soaked Artemisia annua solution at 100°C for 5 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil 1.

[0085] This example evaluates the preparation method by the yield of volatile oil, and calculates the yield of volatile oil of Artemisia annua volatile oil 1 to 9.

[0086] Volatile oil yield (%) = oil yield (mL) ÷ test sample weight (g) × 100% (Formula I).

[0087] The yields of volatile oils 1 to 9 from Artemisia annua volatile oils are shown in Table 1.

[0088] Table 1 Yield of volatile oil from Artemisia annua under different conditions in orthogonal experiment

[0089]

[0090]

[0091] The results in Table 1 show that the best conditions for extracting volatile oil are distillation for 6 hours and a water addition ratio of 10 times the material ratio, while the conditions for extracting volatile oil are distillation for 5 to 6 hours and a material-liquid ratio of 8 to 10 g / mL. The volatile oil yield is relatively high, at more than 0.35%.

[0092] 2. Optimization of soaking time

[0093] ① A method for preparing Artemisia annua volatile oil A1. The difference between the preparation method of the Artemisia annua volatile oil 9 is that step (2) of Artemisia annua volatile oil A1 is to accurately weigh 40g of the above-mentioned Artemisia annua powder and mix it with 400mL of water and soak it for 3h to obtain an Artemisia annua solution after soaking, and distill and reflux extract the Artemisia annua solution at 100°C for 6h to obtain a liquid oil-water mixture. The remaining steps are the same as the preparation method of Artemisia annua volatile oil 9.

[0094] ② A method for preparing Artemisia annua volatile oil A2, which differs from the method for preparing Artemisia annua volatile oil A1 in that step (2) of Artemisia annua volatile oil A2 is to accurately weigh 40 g of the Artemisia annua powder and mix it with 400 mL of water and soak it for 6 hours to obtain an Artemisia annua solution after soaking, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A1.

[0095] ③ A method for preparing Artemisia annua volatile oil A3, which differs from the method for preparing Artemisia annua volatile oil A1 in that step (2) of Artemisia annua volatile oil A3 is to accurately weigh 40 g of the Artemisia annua powder and mix it with 400 mL of water and soak it for 12 hours to obtain an Artemisia annua solution after soaking, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A1.

[0096] ④ A method for preparing Artemisia annua volatile oil A4, which differs from the method for preparing Artemisia annua volatile oil A1 in that step (2) of Artemisia annua volatile oil A4 is to accurately weigh 40 g of the Artemisia annua powder and mix it with 400 mL of water and soak it for 24 hours to obtain an Artemisia annua solution after soaking, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A1.

[0097] ⑤ A method for preparing Artemisia annua volatile oil A5, which differs from the method for preparing Artemisia annua volatile oil A1 in that step (2) of Artemisia annua volatile oil A5 is to accurately weigh 40 g of the Artemisia annua powder and mix it with 400 mL of water and soak it for 36 hours to obtain an Artemisia annua solution, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A1.

[0098] ⑥ A method for preparing Artemisia annua volatile oil A6, which differs from the method for preparing Artemisia annua volatile oil A1 in that step (2) of Artemisia annua volatile oil A6 is to accurately weigh 40 g of the Artemisia annua powder and mix it with 400 mL of water and soak it for 48 hours to obtain an Artemisia annua solution, and then distill and reflux the soaked Artemisia annua solution at 100°C for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A1.

[0099] In this example, the volatile oil yields of Artemisia annua volatile oils A1 to A6 were calculated respectively. The calculation of the volatile oil yields is shown in Formula I.

[0100] The yield of volatile oil from Artemisia annua at different soaking times is shown in Table 2.

[0101] Table 2 Volatile oil yield at different soaking times

[0102]

[0103] The results in Table 2 show that the immersion time of Artemisia annua powder is 6 to 48 hours, the yield of volatile oil of Artemisia annua powder is relatively high, above 0.43%, and when the immersion time of Artemisia annua powder is 24 hours, the yield of volatile oil tends to be stable, so 24 hours is subsequently used as the optimal immersion time of Artemisia annua.

[0104] 3. Selection of effective parts of Artemisia annua

[0105] In order to obtain a relatively efficient oil-producing part when extracting the Artemisia annua volatile oil, this example investigated the difference in the yield of Artemisia annua volatile oil when the aerial part of Artemisia annua and the Artemisia annua leaves were used as raw materials.

[0106] The difference between the preparation method of Artemisia annua volatile oil A1 and Artemisia annua volatile oil A4 is that step (1) of Artemisia annua volatile oil A1 is to completely dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing in an oven at 60°C, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the preparation method of Artemisia annua volatile oil A4.

[0107] The yields of volatile oil extracted from different parts are shown in Table 3, and the calculation of volatile oil yield is shown in Formula Ⅰ.

[0108] Table 3 Volatile oil yields from different parts of Artemisia annua

[0109]

[0110] The results in Table 3 show that the yield of volatile oil from Artemisia annua leaves is higher than that from the aboveground parts of Artemisia annua plants.

[0111] 4. Optimization of raw material pretreatment methods

[0112] The extraction of Artemisia annua volatile oil requires pretreatment of drying and powdering. In order to obtain the best sample pretreatment method, the single factor variable method was used to explore the drying method of Artemisia annua samples.

[0113] (I) A method for preparing Artemisia annua volatile oil B1. The method for preparing Artemisia annua volatile oil B1 differs from the method for preparing Artemisia annua volatile oil A4 in that step (1) of Artemisia annua volatile oil B1 is to dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 25°C for 7 days, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil A4.

[0114] (II) A method for preparing Artemisia annua volatile oil B2. The method for preparing Artemisia annua volatile oil B2 differs from the method for preparing Artemisia annua volatile oil B1 in that step (1) of preparing Artemisia annua volatile oil B2 is to dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 3 hours, cut the leaves into small pieces of 2-3 cm, grind the pieces, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil B1.

[0115] (III) A method for preparing Artemisia annua volatile oil B3. The method for preparing Artemisia annua volatile oil B3 differs from the method for preparing Artemisia annua volatile oil B1 in that step (1) of preparing Artemisia annua volatile oil B3 is to dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 6 hours, cut the leaves into small pieces of 2-3 cm, grind the pieces, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil B1.

[0116] (IV) A method for preparing Artemisia annua volatile oil B4. The method for preparing Artemisia annua volatile oil B4 differs from the method for preparing Artemisia annua volatile oil B1 in that step (1) of Artemisia annua volatile oil B4 is to dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 40°C for 12 hours, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil B1.

[0117] (V) A method for preparing Artemisia annua volatile oil B5. The method for preparing Artemisia annua volatile oil B5 differs from the method for preparing Artemisia annua volatile oil B1 in that step (1) of preparing Artemisia annua volatile oil B5 is to directly dry Artemisia annua leaves collected from wild Artemisia annua plants in Shuangqiao Village, Youyang, Chongqing at 60°C, cut them into small pieces of 2-3 cm, grind them into powder, and pass them through a 40-mesh sieve to obtain Artemisia annua powder. The remaining steps are the same as those of the method for preparing Artemisia annua volatile oil B1.

[0118] The volatile oil yields of Artemisia annua volatile oils B1 to B5 were calculated respectively. The calculation of the volatile oil yields is shown in Formula Ⅰ.

[0119] The yields of volatile oil after drying by different methods are shown in Table 4.

[0120] Table 4 Volatile oil yield after drying by different methods

[0121]

[0122]

[0123] It can be seen from Table 4 that the pretreatment method of Artemisia annua has a significant effect on the yield of volatile oil. Among them, the pretreatment method of Artemisia annua drying at 25°C for 7 days, drying at 40°C for 3h, drying at 40°C for 6h or drying at 40°C for 12h has a higher yield of volatile oil, which is above 0.95%. Relatively speaking, drying at 25°C for 7 days is the best treatment method for extracting Artemisia annua volatile oil.

[0124] Example 8

[0125] The yield of Artemisia annua volatile oil from Luocheng, Guangxi prepared in Example 5 and Example 6 was compared with that of Artemisia annua volatile oil from Chongqing

[0126] In order to evaluate the stability of the volatile oil yield of the Artemisia annua raw material, the volatile oil of Artemisia annua was extracted according to the preparation method described in Example 5 and Example 6, and the extraction was repeated three times. The volatile oil yield was calculated. The calculation of the volatile oil yield is shown in Formula I. The experimental results are shown in Table 5.

[0127] Table 5 Volatile oil yields of different Artemisia annua samples

[0128]

[0129] The results in Table 5 show that the yields of Artemisia annua volatile oil prepared from different origins are significantly different, among which the yield of Artemisia annua volatile oil prepared in Luocheng, Guangxi is high, so Luocheng, Guangxi is used as the raw material collection site for the preparation of Artemisia annua volatile oil.

[0130] Example 9

[0131] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that the Artemisia annua volatile oil of this embodiment uses the aerial parts of Artemisia annua purchased from Tianhe Town, Luocheng Mulao Autonomous County, Hechi City, and the remaining steps are the same as those of Example 5.

[0132] Example 10

[0133] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation method of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above-mentioned Artemisia annua powder and mix it with 320 mL of water and soak it for 24 hours to obtain an Artemisia annua solution after soaking, and distill and reflux extract the Artemisia annua solution at 100° C. for 6 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the preparation method of Example 5.

[0134] Embodiment 11

[0135] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation method of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above-mentioned Artemisia annua powder and mix it with 400 mL of water and soak it for 24 hours to obtain an Artemisia annua solution after soaking, and distill and reflux extract the Artemisia annua solution after soaking at 100° C. for 5 hours to obtain a liquid oil-water mixture. The remaining steps are the same as the preparation method of Example 5.

[0136] Example 12

[0137] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above Artemisia annua powder and mix it with 400 mL of water and soak it for 12 hours, and the remaining steps are the same as those of Example 5.

[0138] Embodiment 13

[0139] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above Artemisia annua powder and mix it with 400 mL of water and soak it for 36 hours, and the remaining steps are the same as those of Example 5.

[0140] Embodiment 14

[0141] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above Artemisia annua powder and mix it with 400 mL of water and soak it for 48 hours, and the remaining steps are the same as those of Example 5.

[0142] Embodiment 15

[0143] The difference between the preparation method of the Artemisia annua volatile oil of this embodiment and that of Example 5 is that step (2) of the preparation of the Artemisia annua volatile oil of this embodiment is to accurately weigh 40 g of the above Artemisia annua powder and mix it with 400 mL of water and soak it for 6 hours, and the remaining steps are the same as those of Example 5.

[0144] Example 16

[0145] Preparation and process optimization of gel preparations

[0146] 1.1 Determination of gel temperature based on poloxamer 407

[0147] 1.1.1 Experimental methods

[0148] The preparation method of poloxamer 407 with different mass volume concentrations is to mix 18g, 19g, 20g, 21g, 22g, 23g, 24g, and 25g of poloxamer 407 with 100mL of water, respectively, to obtain poloxamer 407 gel base with mass volume concentrations of 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0149] In this example, the tilting method was used to take about 5 mL of the above-mentioned poloxamer 407 gel base of different mass volume concentrations into a stoppered test tube, and a water bath was turned on to set the water bath at 1°C·min. -1 Continue to slowly increase the temperature, keep the temperature constant for 5 minutes at every 0.5℃ increase, tilt the test tube every 30 seconds, and the tilt angle is about 90℃. When the gel stops flowing, insert an alcohol thermometer with an accuracy of 0.1℃ and record the reading of the thermometer, which is the gelation temperature. Repeat the measurement 3 times for each sample and take the average value.

[0150] 1.1.2 Experimental Results

[0151] like Figure 1 As shown in the figure, with the increase of the proportion of poloxamer 407, the gel temperature gradually decreases, but the fluidity of the gel weakens, which is relatively more suitable as a drug carrier. Therefore, it is necessary to further optimize the composition of the gel to obtain a gel preparation carrier with a higher poloxamer 407 content and a higher gel temperature.

[0152] 1.2 Preparation of compound thermosensitive gel and determination of gel temperature

[0153] 1.2.1 Experimental methods

[0154] A good thermosensitive gel should be in a free-flowing liquid state at room temperature, and respond to temperature changes after administration, and can quickly gel in a short time, thereby increasing the retention time of the drug in the vagina. If the gelation is too fast, the liquid gel may gel in the applicator, which is not conducive to administration in liquid state. If the gelation is too slow, the gel is easy to flow out, causing drug leakage. Therefore, the gelation temperature of an ideal thermosensitive gel should be higher than room temperature (25°C) and lower than the temperature in the vagina (37°C), and should have a relatively suitable gelation time. Poloxamer 188 is one of the commonly used thermosensitive gel bases, so this embodiment intends to compound poloxamer 407 and poloxamer 188 to obtain a gel base with a suitable gel temperature.

[0155] The preparation method of poloxamer 188 gel base with different mass volume concentrations is as follows: 2 g, 4 g and 6 g of poloxamer 188 are mixed with 100 mL of water respectively to obtain poloxamer 188 with mass volume concentrations of 2%, 4% and 6%, respectively.

[0156] Based on this solution, a composite gel system was prepared by cold dissolution method. The specific operation is as follows:

[0157] Preparation of a thermosensitive gel precursor preparation: Accurately weigh 2 g of poloxamer 188 and 18, 19, 20, 21, 22, 23, 24, and 25 g of poloxamer 407, respectively, add 100 mL of pure water, stir with a glass rod to make it evenly dispersed, and refrigerate in a 4°C refrigerator for more than 12 hours until it is fully swollen and dissolved. The resulting solution is clear, has no lumps, and is evenly dispersed, which is the thermosensitive gel precursor preparation a1~a8.

[0158] Preparation of a thermosensitive gel precursor preparation: Accurately weigh 4 g of poloxamer 188 and 18, 19, 20, 21, 22, 23, 24, and 25 g of poloxamer 407, respectively, add 100 mL of pure water, stir with a glass rod to make it evenly dispersed, and refrigerate in a 4°C refrigerator for more than 12 hours until it is fully swollen and dissolved. The resulting solution is clear, free of lumps, and evenly dispersed, which is the thermosensitive gel precursor preparation b1~b8.

[0159] Preparation of a thermosensitive gel precursor preparation: Accurately weigh 6 g of poloxamer 188 and 18, 19, 20, 21, 22, 23, 24, and 25 g of poloxamer 407, respectively, add 100 mL of pure water, stir with a glass rod to make it evenly dispersed, and refrigerate in a 4°C refrigerator for more than 12 hours until it is fully swollen and dissolved. The resulting solution is clear, has no lumps, and is evenly dispersed, which is the thermosensitive gel precursor preparation c1 to c8.

[0160] The temperature of the thermosensitive gel precursor preparations a1 to a8, the thermosensitive gel precursor preparations b1 to b8 and the thermosensitive gel precursor preparations c1 to c8 was measured according to the method in section 1.1.1.

[0161] 1.2.2 Experimental results

[0162] Figure 2 The figure shows the gelation temperature of the gel obtained after mixing different volumes of poloxamer 407 and poloxamer 188. It can be seen from the figure that the addition of poloxamer 188 can significantly increase the gelation temperature of the thermosensitive gel precursor preparation, and under the condition of a fixed volume concentration of poloxamer 407, the gelation temperature will increase with the increase in the amount of poloxamer 188 added. The gelation temperature corresponding to the gel ratio within the dotted line range in the figure is 28-35°C, which is more suitable as a gel carrier.

[0163] The gelation temperatures of thermosensitive gel precursor preparations prepared with different volume concentrations of poloxamer 407 and poloxamer 188 are shown in Table 6.

[0164] Table 6 Gel temperature of thermosensitive gel precursor preparations prepared with different mass volumes of poloxamer 407 and poloxamer 188

[0165] Poloxamer 407(g) 2g-Poloxamer 188 4g-Poloxamer 188 6g Poloxamer 188 18 50.70℃ 52.57℃ 56.73℃ 19 41.63℃ 45.57℃ 48.43℃ 20 37.67℃ 40.57℃ 44.83℃ 21 34.60℃ 38.20℃ 42.00℃ 22 32.73℃ 34.97℃ 37.67℃ 23 30.33℃ 33.60℃ 35.60℃ 24 29.33℃ 32.53℃ 34.50℃ 25 28.43℃ 30.93℃ 33.63℃

[0166] It can be seen from Table 6 that when the amount of water added to the system is 100 g and the amount of poloxamer 188 added is 2 g, the added mass of poloxamer 407 is 21 g, 22 g, 23 g, 24 g or 25 g respectively; when the added amount of poloxamer 188 and is 4 g, the added mass of poloxamer 407 is 22 g, 23 g, 24 g or 25 g respectively; when the added amount of poloxamer 188 is 6 g, the added mass of poloxamer 407 is 24 g or 25 g, and the gel temperature of the obtained thermosensitive gel precursor preparation is between 28 and 35 ° C, which is suitable as a gel carrier.

[0167] 1.3 Optimal viscosity of thermosensitive gel

[0168] The viscosity of a gel refers to its ability to resist disturbance, that is, the degree of resistance of the liquid molecules inside it to external forces. Generally speaking, the higher the viscosity of a gel, the greater its viscosity, which means that a higher viscosity means that the gel is more resistant to external stress and is more difficult to deform. It can be seen that viscosity is also an important parameter for evaluating gels. Therefore, a gel base is made with the preferred ratio of 1.2 and is used as an object of investigation to evaluate its viscosity at different temperatures.

[0169] 1.3.1 Experimental methods

[0170] Table 7 Mass ratio of poloxamer 407 and poloxamer 188

[0171] <![CDATA[M P407 (g)]]> <![CDATA[M P188 (g)]]> <![CDATA[M P407 / M P188 ]]> 23 2 11.50 24 2 12.00 24 4 6.00 25 2 12.50 25 4 6.25

[0172] Thermosensitive gel precursor preparations 1 to 5 were prepared according to the volume concentrations of poloxamer 407 and poloxamer 188 as described in Table 7:

[0173] Accurately weigh 2 g of poloxamer 188 and 23 g, 24 g, and 25 g of poloxamer 407, respectively, add 100 mL of pure water, stir with a glass rod to make it evenly dispersed, and refrigerate in a 4°C refrigerator for more than 12 h until it is fully swollen and dissolved. The resulting solution is clear, free of lumps, and evenly dispersed, thereby obtaining thermosensitive gel precursor preparations 1 to 3, respectively.

[0174] Accurately weigh 4 g of poloxamer 188 and 24 g and 25 g of poloxamer 407, respectively, add 100 mL of pure water, stir with a glass rod to make it evenly dispersed, and refrigerate in a 4°C refrigerator for more than 12 h until it is fully swollen and dissolved. The resulting solution is clear, free of lumps, and evenly dispersed, thereby obtaining thermosensitive gel precursor preparations 4 to 5, respectively.

[0175] Take 30 mL of the thermosensitive gel precursor preparations 1 to 5 to be tested in a 50 mL beaker, place it in a constant temperature water bath, use an NDJ-5S digital viscometer to select a suitable rotor and speed, test the viscosity of the sample at 20°C, 25°C, and 30°C, measure it three times in parallel, and take the average value.

[0176] 1.3.2 Experimental Results

[0177] Figure 3 is the viscosity value at the corresponding temperature for different volume ratios. It can be seen that as M P407 / M P188 The viscosity of the gel decreases with the increase of P407 / M P188 It is 11.5, which means it is easier to use an applicator for vaginal administration.

[0178] Embodiment 17

[0179] A method for preparing an Artemisia annua volatile oil vaginal administration preparation, comprising the following steps:

[0180] (1) Weigh 40 g of deionized water, add 1881.6 g of poloxamer, and after dissolving evenly, add 40718.4 g of poloxamer, fully moisten, and place in a refrigerator at 4°C overnight to fully swell, to prepare a thermosensitive gel solution;

[0181] (2) 2 g of glycerol, 0.5 g of the Artemisia annua volatile oil prepared in Example 5, and 1 g of anhydrous ethanol were dispersed in 6 g of water to prepare a stable Artemisia annua volatile oil solution;

[0182] (3) dispersing 0.02 g of methylparaben in 5 g of water to obtain a methylparaben solution;

[0183] (4) The thermosensitive gel solution, the stabilized Artemisia annua volatile oil solution and the methylparaben solution were mixed and stirred evenly, and then 25.48 g of water was added and mixed evenly to obtain the Artemisia annua volatile oil vaginal administration preparation.

[0184] Example 18 Pharmacodynamic effects of the vaginal administration preparation of Artemisia annua volatile oil prepared in Example 17 on rats infected with vaginitis

[0185] 1.1 Preparation of mixed infectious vaginitis model in rats

[0186] Sixty female SD rats were subjected to adaptive feeding for 3 days and then randomly divided into 6 groups using the randomized block method, with 10 rats in each group, namely, a blank control group (also known as the blank group), a model control group (also known as the model group), a positive drug group, and high-, medium-, and low-dose groups of the vaginal administration preparation of Artemisia annua volatile oil (also known as the low-dose volatile oil group, the medium-dose volatile oil group, and the high-dose volatile oil group, respectively).

[0187] Except for the blank control group, the mixed infectious vaginitis model was established in the rats of the other groups. Each rat was subcutaneously injected with 2 mg / kg estradiol benzoate injection. -1 , 2 days / time, 3 times in a row, to induce pseudoestrus in rats. The day after injection of estradiol benzoate, each rat was subcutaneously injected with 60 mg·kg hydrocortisone -1 , once a day for 5 consecutive days to induce low immune function in rats. The next day, the prepared Escherichia coli CMCC (B) 44102, Staphylococcus aureus CMCC (B) 26003 and Candida albicans CMCC (F) 98001 suspensions were mixed in a ratio of 1:1:1, and a 1 mL syringe was connected to the gavage needle to draw 0.2 mL of the bacterial suspension per rat and injected into the vagina once every other day for a total of 5 times. After the bacteria were injected, a cotton ball was inserted into the vaginal opening to prevent the bacterial liquid from flowing out.

[0188] 1.2 Treatment of rats with vaginitis

[0189] The blank control group and the model control group were given normal saline by vaginal injection once a day for 15 consecutive days; the positive drug group was given 0.5mL metronidazole gel by injection once a day for 15 consecutive days; the high, medium and low dose groups of Artemisia annua volatile oil vaginal preparation were treated with 0.4mL, 0.2mL and 0.1mL of Artemisia annua volatile oil vaginal preparation by vaginal injection respectively, once a day for 15 consecutive days; the blank group was given normal saline by vaginal injection at a dose of 0.2mL / mouse for 15 consecutive days; the gel preparation group was given blank gel base by vaginal injection once a day for 15 consecutive days.

[0190] The preparation method of the blank gel substrate is as follows: (1) weighing 40 g of deionized water, adding 1881.6 g of poloxamer, and after uniform dissolution, adding 40718.4 g of poloxamer, fully moistening, placing in a refrigerator at 4°C overnight, fully swelling, and preparing a thermosensitive gel solution;

[0191] (2) 2 g of glycerol and 1 g of anhydrous ethanol were dispersed in 6 g of water to prepare a stable solution;

[0192] (3) dispersing 0.02 g of methylparaben in 5 g of water to obtain a methylparaben solution;

[0193] (4) The above-mentioned thermosensitive gel solution, stable solution and methyl paraben solution were mixed and stirred evenly, and then 25.98 g of water was added and mixed evenly to obtain a blank gel base.

[0194] 1.3 Experimental Results

[0195] After modeling, vaginal secretions were dipped into physiological saline with a sterile cotton swab, and 20 μL of the soaking solution was further drawn with a pipette to prepare a smear of vaginal secretions of rats. The smear of the model control group showed a characteristic color under microscopic examination using Gram staining and PAS staining (see Figure 4 ), in addition, HE staining of the vagina in the model control group showed swelling and mucosal inflammatory infiltration (see Figure 5 ), all of which can prove that the rat vaginitis infection was successful.

[0196] After 15 days of continuous treatment, vaginal tissues of rats in each group were obtained and the tissue morphology was evaluated using HE staining.

[0197] Figure 6 The results showed that Artemisia annua volatile oil has a certain improvement and treatment effect on vaginitis caused by mixed suspension of Staphylococcus aureus, Escherichia coli and Candida albicans, and this experiment provides experimental basis and data guidance for the subsequent in vivo "anti-inflammatory" effect of Artemisia annua volatile oil. The effect of the treatment group of medium-dose Artemisia annua volatile oil vaginal administration preparation is more obvious, which can effectively relieve the damage and inflammatory lesions of the vaginal mucosa.

[0198] The blood of rats in each group was extracted respectively, and the levels of IL-1β and IL-6 in rat serum were detected by Elisa kit method.

[0199] Figure 7-Figure 8 The results showed that compared with the model group, the levels of IL-1β and IL-6 in the serum were decreased after treatment with the vaginal administration preparation of Artemisia annua volatile oil. Therefore, the vaginal administration preparation of Artemisia annua volatile oil can effectively inhibit inflammation.

[0200] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a vaginal preparation of Artemisia annua volatile oil, characterized in that: The following steps are involved: Mix poloxamer 188, poloxamer 407 and water evenly, and refrigerate to obtain a thermosensitive gel solution; Mixing glycerin, Artemisia annua volatile oil, anhydrous ethanol and water to obtain a stable Artemisia annua volatile oil solution; mixing methylparaben with water to obtain a methylparaben solution; The thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methylparaben solution are uniformly mixed, and then water is added to mix well to obtain an Artemisia annua volatile oil vaginal administration preparation; The mass ratio of poloxamer 188 to poloxamer 407 is 2:21-25, 4:22-25 or 6:24-25.

2. The preparation method according to claim 1, characterized in that: The refrigeration temperature is 2-6° C., and the refrigeration time is more than 12 hours.

3. The preparation method according to claim 1, characterized in that: When glycerol, Artemisia annua volatile oil, anhydrous ethanol and water are mixed, the mass ratio of glycerol, Artemisia annua volatile oil, anhydrous ethanol and water is 1-3:0.2-1:0.5-1.5:5-7; When methylparaben is mixed with water, the mass ratio of methylparaben to water is 0.01 to 0.03:5; After the thermosensitive gel solution, the stable Artemisia annua volatile oil solution and the methyl paraben solution are uniformly mixed, water is added and mixed, and the mass ratio of the methyl paraben solution to the water is 5.01-5.03:20-30.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The preparation method of the Artemisia annua volatile oil comprises the following steps: After drying the effective part of Artemisia annua, pulverizing and sieving to obtain Artemisia annua powder; mixing the Artemisia annua powder with water and soaking it to obtain an Artemisia annua solution, distilling and refluxing the Artemisia annua solution to obtain a liquid oil-water mixture, collecting the oil layer of the liquid oil-water mixture; drying the oil layer to obtain Artemisia annua volatile oil.

5. The preparation method according to claim 4, characterized in that: The method for drying the effective part of Artemisia annua comprises placing it in a drying position at 20-30° C. for 6-8 days or drying it at 35-45° C. for 2-13 hours; the mass volume ratio of the Artemisia annua powder to water is 1:6-12 g / mL; the soaking time is 6-48 hours; the extraction time is 4.5-6.5 hours; the method for drying the oil layer comprises mixing the oil layer with anhydrous calcium chloride and letting it stand, collecting the supernatant by centrifugation, and obtaining Artemisia annua volatile oil; the effective part of Artemisia annua is Artemisia annua leaf; the Artemisia annua comes from Luocheng Mulao Autonomous County of Hechi City or Shuangqiao Village of Youyang, Chongqing.

6. The vaginal administration preparation of Artemisia annua volatile oil prepared according to any one of the preparation methods of claims 1 to 5.

7. Use of the vaginal administration preparation of Artemisia annua volatile oil according to claim 6 in the preparation of a drug for treating vaginitis.

8. The use according to claim 7, characterized in that: The vaginitis includes bacterial vaginitis, fungal vaginitis, and vaginitis caused by bacterial and fungal co-infection.

9. The use according to claim 7, characterized in that: The vaginitis includes one or more of Staphylococcus aureus, Escherichia coli and Candida albicans.

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