A traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, its preparation method and application.

Abstract

This invention relates to the field of traditional Chinese medicine compound preparations, specifically to a traditional Chinese medicine compound nasal spray with both rapid-release and sustained-release effects, its preparation method, and its application. The preparation method of the traditional Chinese medicine compound nasal spray with both rapid-release and sustained-release effects includes the following steps: Step S1: Preparing the traditional Chinese medicine compound composition into a microemulsion solution; the traditional Chinese medicine compound composition contains volatile oils; Step S2: Making the microemulsion solution to volume with distilled water, then adjusting the osmotic pressure with sodium chloride solution, and adjusting the pH with hydrochloric acid solution. This invention combines rapid and long-lasting effects. One-fifth of the traditional Chinese medicine compound composition in the nasal spray has a rapid-release effect, and its rapid-release mechanism can produce a protective effect the moment the virus invades the nasal cavity; while the four-fifths of the drug sustained-release microspheres in the spray have a long-lasting mechanism, which can prolong the drug's action time, fully adapting to the "time-limited characteristic" of influenza virus transmission up to 48 hours, filling the gap in current similar nasal sprays that lack sustained-release effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, its preparation method, and its application. Background Technology

[0002] Influenza is an acute, viral respiratory infectious disease that seriously endangers human health. It is primarily transmitted through close-range airborne transmission, close contact, or contact with the respiratory secretions of infected individuals, and is highly contagious. To date, the main global measures to combat influenza viruses remain vaccination and antiviral chemotherapy. However, current antiviral chemotherapy drugs, such as interferon-based drugs and antisense nucleic acid drugs, may interfere with the RNA of other normal cells in addition to modifying the influenza virus. Compared to vaccination and antiviral chemotherapy, traditional Chinese medicine (TCM) compound prescriptions, due to their diverse compositions, possess a more significant "broad-spectrum" antiviral effect, and hold promise for better addressing the complex problems caused by influenza viruses.

[0003] Influenza-like diseases, such as atypical pneumonia, fall under the category of "febrile diseases" or "epidemic febrile diseases" in Traditional Chinese Medicine (TCM), often caused by exposure to the six pathogenic factors (wind, cold, dampness, heat, dryness, fire, and fire) or heat toxins. Due to their rapid onset, severe illness, and high infectivity, prevention is extremely important. TCM has a long history of treating acute febrile diseases, leaving behind many famous prescriptions and effective remedies still in use today, and accumulating rich theories and experience in prevention and treatment through long-term clinical practice. TCM has a profound understanding of epidemic prevention and has created many preventative methods that are both uniquely Chinese and effective, playing a vital role in the prosperity and continuation of the Chinese nation. As early as the *Suwen* section of the *Huangdi Neijing* (Inner Canon of the Yellow Emperor), there are records of using small golden pills to prevent epidemics. Ancient books such as the *Zhouhou Fang* (Elbow Formulas for Emergencies) of the Jin Dynasty and the *Qianjin Yaofang* (Essential Formulas Worth a Thousand Gold Pieces) of the Tang Dynasty also list prescriptions for preventing febrile diseases, using medication to prevent their occurrence. For example, the *Qianjin Yaofang* (Essential Prescriptions Worth a Thousand Gold Pieces) includes formulas such as Realgar Pills, Red Powder, Taiyi Liujin Powder, Realgar Powder, Ghost-Slaying Burning Medicine, and Golden Tooth Powder, which respectively employ methods such as wearing medicine pouches, fumigation, internal administration, or application to the body surface to prevent the occurrence of febrile diseases. During the Yuan Dynasty, Hua Shou advocated using Disinfecting Infant-Protecting Pills and Substitute Heaven Propagation Pills to prevent disease during measles epidemics.

[0004] Therefore, providing a traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, along with its preparation method and application, is of great significance. Summary of the Invention

[0005] In view of this, it is necessary to provide a traditional Chinese medicine compound nasal spray that has both immediate and sustained release effects, as well as its preparation method and application.

[0006] In a first aspect, the present invention provides a method for preparing a traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, comprising the following steps:

[0007] Step S1: Prepare a microemulsion solution from the traditional Chinese medicine compound composition; the traditional Chinese medicine compound composition contains volatile oils;

[0008] Step S2: Dilute the microemulsion solution to volume with distilled water, then adjust the osmotic pressure with sodium chloride solution and adjust the pH with hydrochloric acid solution.

[0009] Furthermore, the microemulsion solution in step S1 is prepared in the following manner:

[0010] Step A1: Prepare a traditional Chinese medicine compound composition, and prepare four-fifths of the traditional Chinese medicine compound composition into drug sustained-release microspheres;

[0011] Step A2: Using one-fifth of the traditional Chinese medicine compound composition as the oil phase, add emulsifiers, co-emulsifiers and drug sustained-release microspheres, and shake to form a stable and dispersed microemulsion solution.

[0012] Furthermore, the preparation method of the drug sustained-release microspheres in step A1 includes the following steps:

[0013] Step B1: Membrane emulsification: An aqueous phase containing emulsifier is placed in a continuous phase container and circulated inside the membrane emulsification device by a peristaltic pump; the traditional Chinese medicine compound composition and PLGA are dissolved in dichloromethane to prepare a membrane permeate, which is then loaded into a dispersed phase container;

[0014] Step B2: Nitrogen gas is introduced, and the membrane permeate naturally passes through the membrane under the pressure of nitrogen gas. The permeate after passing through the membrane is solidified and the organic solvent is evaporated. Then, centrifugation is performed to precipitate drug-release microspheres.

[0015] Step B3: Remove the emulsifier from the drug-release microspheres and freeze-dry.

[0016] Furthermore, the device used in the membrane emulsification method in step B1 is a modular membrane emulsification device, which includes a nitrogen cylinder, a dispersed phase container, a membrane module, a peristaltic pump, and a continuous phase container connected in sequence by pipelines; the membrane module is also connected to the continuous phase container by pipelines.

[0017] Furthermore, the pipeline between the nitrogen cylinder and the dispersed phase container is also equipped with...

[0018] Furthermore, the emulsifying medium used in the membrane emulsification method described in step B1 is a PVDF membrane; wherein the membrane pore size is 0.36±0.15μm; and the membrane contact angle is 86 / 71°.

[0019] Furthermore, in step B1, the concentration of PLGA in dichloromethane is 60 mg / mL, and the concentration ratio of the traditional Chinese medicine compound composition to PLGA is 1:6.

[0020] Furthermore, the emulsifier mentioned in step B1 is gelatin, PVA1788, Tween-80, or Tween-20.

[0021] Preferably, the emulsifier is one of 0.5% gelatin and 0.5% PVA1788, 0.5% Tween-80 and 0.5% PVA1788, or 0.5% Tween-20 and 0.5% PVA1788.

[0022] Most preferably, the emulsifier is 0.5% Tween-80 and 0.5% PVA178; the emulsification pressure is 0.02 MPa.

[0023] Furthermore, the centrifugation process parameters for the drug sustained-release microsphere precipitation in step B2 are: centrifugation speed of 10000 r / min and centrifugation time of 30 min.

[0024] Furthermore, the method for removing the emulsifier in step B3 is to wash the precipitate three times with an appropriate amount of distilled water and then filter it.

[0025] Furthermore, the traditional Chinese medicine compound composition in step S1 is prepared in the following manner:

[0026] Step C1: The volatile oils and residues after extracting the volatile oils from patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and peppermint were obtained by steam distillation and membrane enrichment technology.

[0027] Step C2: The residue after extracting the volatile oil is refluxed with ethanol and purified with macroporous adsorption resin to obtain purified solution A; Dryopteris crassirhizoma is extracted with ethanol, and the resulting extract is purified with ceramic ultrafiltration membrane to obtain purified solution B; Purified solutions A and B are combined, concentrated with reverse osmosis membrane and then freeze-dried to obtain small molecule active substances.

[0028] Step C3: Mix the volatile oil from step C1, the small molecule active substance from step C2, and borneol, and stir until homogeneous to obtain the final product.

[0029] Furthermore, by weight, the traditional Chinese medicine compound composition includes 13-17 parts of patchouli, 13-17 parts of wild chrysanthemum, 8-12 parts of agastache, 8-12 parts of mugwort, 8-12 parts of schizonepeta, 3-6 parts of peppermint, 1-3 parts of borneol, and 7-13 parts of Dryopteris crassirhizoma.

[0030] Preferably, the traditional Chinese medicine compound composition, by weight, includes 15g of patchouli, 15g of wild chrysanthemum, 10g of agastache rugosa, 10g of mugwort, 10g of schizonepeta tenuifolia, 5g of peppermint, 1g of borneol, and 10g of Dryopteris crassirhizoma.

[0031] Furthermore, the specific method of the steam distillation extraction involves pulverizing patchouli, wild chrysanthemum, agastache rugosa, artemisia argyi, schizonepeta tenuifolia, and mint, passing them through a 20-80 mesh sieve, and then steam distilling them using a boiler steam pressure of 2-6 kg / cm². 2 The extraction time is 1-8 hours.

[0032] Preferably, patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and mint are pulverized and passed through a 40-mesh sieve, then distilled with steam at a boiler steam pressure of 4 kg / cm². 2 The extraction time is 5 hours.

[0033] Furthermore, the membrane enrichment technology involves passing an oil-water mixture obtained after steam distillation through an ultrafiltration membrane oil-water separator for oil-water separation; the separated oil is the volatile oil.

[0034] Preferably, the membrane material of the ultrafiltration membrane oil-water separator is polysulfone with a pore size of 140 kDa.

[0035] Furthermore, the ethanol reflux method is as follows: add 12 times the amount of 70% ethanol to the residue after extracting the volatile oil, reflux for 1 hour, and filter to obtain filtrate one and residue one; add 10 times the amount of 70% ethanol to residue one, reflux for 1 hour, and filter to obtain filtrate two and residue two; combine filtrate one and filtrate two, and recover ethanol under reduced pressure until there is no alcohol odor to obtain the ethanol extract of the residue.

[0036] Furthermore, the macroporous adsorption resin is purified by using AB-8, D101 macroporous resin or NAK-9 resin to purify the ethanol extract of the pharmaceutical residue. The eluent is 1-5 times the volume of 70%-90% ethanol, the pH of the solution is 3.5-6.5, and the flow rate is 1-5 BV / h. The ethanol eluent is collected and the ethanol is recovered under reduced pressure to obtain purified solution A.

[0037] Preferably, the ethanol extract of the pharmaceutical residue is refined using D101 macroporous resin, the pH of the extract is 3.5, and it is eluted with 5 times the volume of 70% ethanol at a flow rate of 3 BV / h.

[0038] Furthermore, the specific preparation method of the refined liquid B is as follows: weigh the dried medicinal material of Dryopteris crassirhizoma and crush it into coarse powder; extract it twice with 12 times the volume of 70% ethanol, each time for 1 hour; combine the two extracts and concentrate them under reduced pressure to recover the ethanol until there is no alcohol taste; refine the alcohol-free extract using a ceramic ultrafiltration membrane with a pore size of 50 nm and a material of Al2O3.

[0039] Furthermore, the reverse osmosis membrane concentration method involves concentrating the mixture of purified solution A and purified solution B using a reverse osmosis membrane until the relative density is 1.08 to 1.10; the process parameters for reverse osmosis concentration are an operating pressure of 0.3-2.0 MPa and a flow rate of 6-8 L / min.

[0040] Furthermore, step C3 also includes adding a surfactant for mixing.

[0041] Preferably, the surfactant in step C3 is Tween 80.

[0042] In the above-mentioned compound Chinese medicine formula, patchouli is pungent and slightly warm, and enters the spleen, kidney, and lung meridians. It aromatically transforms dampness, harmonizes the stomach and stops vomiting, and releases exterior pathogens to relieve summer heat. Chrysanthemum indicum is bitter, pungent, and slightly cold, and enters the liver and heart meridians. It clears heat and detoxifies, drains fire and calms the liver. Both herbs aromatically transform dampness and clear heat and detoxify, serving as the principal herbs. Eupatorium fortunei is pungent and neutral, and enters the spleen, stomach, and lung meridians. It aromatically transforms dampness, invigorates the spleen and stomach, releases exterior pathogens and relieves summer heat. Schizonepeta tenuifolia is pungent and slightly warm, and enters the lung and liver meridians. It releases exterior pathogens and disperses wind, promotes rash eruption, and eliminates sores. Mentha haplocalyx is pungent and cool, and enters the lung and liver meridians. It disperses wind-heat, clears the head and eyes, benefits the throat and promotes rash eruption, and soothes the liver and regulates qi. Artemisia argyi... The herbs are pungent, bitter, and warm, and enter the liver, spleen, and kidney meridians. They warm the meridians to stop bleeding, dispel cold, and relieve pain. These four herbs disperse wind-heat, dispel dampness, warm the meridians to dispel cold, and support the body's resistance to pathogens, acting as the assistant herbs. Dryopteris crassirhizoma, bitter, slightly cold, and slightly toxic, enters the liver and stomach meridians. It expels parasites, stops bleeding, clears heat, and detoxifies, serving as the adjuvant herb. Borneol, pungent, bitter, and slightly cold, enters the heart, spleen, and lung meridians. It opens the orifices, awakens the mind, clears heat, and relieves pain, serving as the guiding herb. In the above compound herbal formula, five herbs enter the lung meridian, and the medicinal power mainly targets symptoms occurring along the lung meridian, such as cough, wheezing, shortness of breath, chest tightness, and sore throat. The combined herbs work synergistically to dispel foul odors, clear heat and detoxify, nourish lung qi, and expel latent pathogens, exhibiting preventative and direct inhibitory effects against various influenza viruses.

[0043] The main sources and chemical compositions of the above-mentioned traditional Chinese medicine compound composition are as follows:

[0044] Patchouli is the dried aerial part of *Pogostemon cablin* (Blanco) Benth., a plant in the Lamiaceae family. Its antiviral active substances are volatile oils and flavonoids, which can inhibit and eliminate upper respiratory tract pathogens and have a significant effect on resisting the reproduction and growth of rhinoviruses.

[0045] Wild chrysanthemum, the capitulum of *Chrysanthemum indicum* L. (family Asteraceae), possesses the effects of dispelling wind and clearing heat, reducing swelling and detoxifying, and is widely used clinically. Pharmacological studies have shown that wild chrysanthemum has a certain killing effect on influenza virus and hepatitis B virus. Its antiviral active substances are mainly volatile oils and flavonoids.

[0046] Eupatorium fortunei Turcz, a plant in the Asteraceae family, is a dried aerial part. The volatile oil of Eupatorium fortunei has an inhibitory effect on influenza viruses.

[0047] Artemisia argyi LevL et Vant., a plant in the Asteraceae family, is a dried leaf. The volatile oil in Artemisia argyi has inhibitory effects on pathogens such as adenovirus, measles virus, and influenza virus.

[0048] Nepeta tenuifolia Briq., a plant in the Lamiaceae family, is the dried aerial part of the plant. Mentha haplocalyx Briq., a plant in the Lamiaceae family, is the dried aerial part of the plant.

[0049] Serum containing different combinations of peppermint and catnip can inhibit the intracellular proliferation of influenza virus in vitro, with the combination of peppermint-catnip volatile oil and peppermint-catnip flavonoids showing the most significant inhibitory effect on virus proliferation.

[0050] Borneol (synthetic borneol) is a colorless, transparent or white, translucent, brittle, flaky crystal with a fragrant aroma and a pungent taste. It is volatile. It promotes the transdermal absorption of other drugs and is often used as an adjuvant or guiding agent in compound traditional Chinese medicine.

[0051] Dryopteris crassirhizoma Nakai, also known as Guanzhong or Northeast Guanzhong, is the dried rhizome and petiole remnants of the Dryopteris crassirhizoma Nakai plant, belonging to the Dryopteris family. The active components of Dryopteris crassirhizoma are mainly phloroglucinol derivatives (a series of structurally similar homologues with the chemical formula C6H6O3, slightly soluble in water, and with a molecular weight of approximately 126 Da), exhibiting antibacterial, antiviral, antitumor, and antimalarial activities. Currently, 15 phloroglucinol monomeric compounds have been isolated from this plant, mainly including white phloroglucinol AP, white phloroglucinol PP, white phloroglucinol AA, dryopteris crassirhizoma ABBA, dryopteris acid ABA, as well as dryopteris acid BBB, dryopteris acid PBB, dryopteris acid PBP, yellow dryopteris acid AB, yellow dryopteris acid BB, and yellow dryopteris acid PB, etc.

[0052] Furthermore, in step A2, the emulsifier is poloxamer-108 and the co-emulsifier is ethanol; the mass ratio of poloxamer-108 to ethanol is 1:10.

[0053] Furthermore, in step S2, the volume is adjusted by distilling the microemulsion to 100 ml; the sodium chloride solution has a mass fraction of 0.7%; and the pH of the solution is adjusted to 3-8 with 0.1 mol / L hydrochloric acid.

[0054] Preferably, the pH of the hydrochloric acid solution is adjusted to 6.0~7.0.

[0055] Secondly, the present invention provides a product prepared by the preparation method of the above-mentioned compound nasal spray of traditional Chinese medicine with both immediate and sustained release effects.

[0056] Thirdly, the present invention provides the application of the above-mentioned compound nasal spray of traditional Chinese medicine with both immediate and sustained release effects and its preparation method in the prevention of influenza virus in medical devices.

[0057] The beneficial effects of this invention are as follows:

[0058] 1. In view of the large number of influenza viruses, their frequent mutations and complexities, this invention selects a traditional Chinese medicine compound with dual functions of antiviral and immune regulation, and solves the problem of complex and variable viruses by means of "broad-spectrum antiviral" and "diverse chemical composition".

[0059] 2. This invention creatively applies the traditional Chinese medicine theory of "the lung opens into the nose," which has been confirmed by modern science from the perspective of the "nasopulmonary reflex arc," to the prevention and control of influenza viruses. It heavily utilizes traditional Chinese medicine formulas that enter the lung meridian (62.5% of the formula consists of lung-meridian-related herbs). At the same time, it organically combines the "aromatic expulsion of evil" principle of famous traditional Chinese medicine prescriptions for treating epidemics with the theory and technology of "protein molecular structure of virus-cell membrane fusion" in the field of modern medical viral molecular biology. This invention has important theoretical and academic significance and practical application value for the research of traditional Chinese medicine in combating influenza viruses, which is based on the principle of "preserving the orthodox and innovating."

[0060] 3. This invention uses the "mechanical sieving" mechanism of membrane technology to separate and enrich volatile oils from steam distillation of traditional Chinese medicine compound compositions. It is energy-saving, efficient, simple, and free of organic solvent pollution.

[0061] 4. The herbal compound nasal spray of this invention is administered via nasal spray or oral administration, constructing the body's first line of defense against viruses. It prevents viruses from directly invading the rich capillary walls of the nasal cavity and throat, entering the bloodstream, and effectively preventing rapid viral self-replication. Simultaneously, nasal drops or nebulization allow for rapid drug absorption, directly entering the systemic circulation after absorption through the nasal capillaries, avoiding degradation by enzymes in the gastrointestinal tract.

[0062] 5. This invention utilizes the principle of micellar solubilization and water miscibility to obtain volatile oils and poorly soluble drugs. By using an appropriate ratio of emulsifiers and co-emulsifiers to form microemulsions with volatile oils and water, the solubility of volatile oils is increased, ensuring that the effective components of volatile oils are miscible with the water phase in the final molding system to form a clear solution.

[0063] 6. This invention combines rapid and long-lasting effects. One-fifth of the herbal compound nasal spray contains a herbal compound composition with a rapid release effect, and its rapid-acting mechanism can provide resistance the moment the virus invades the nasal cavity; while the four-fifths of the spray contains drug-release microspheres with a long-lasting mechanism, which can prolong the drug's action time and fully adapt to the "time-limited characteristic" of influenza virus transmission, which can last up to 48 hours, filling the gap in current nasal sprays that lack sustained-release effects. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of the modular membrane emulsification device of the present invention;

[0065] Figure 2 The freeze-drying curve of the traditional Chinese medicine compound composition in Example 4 is shown below.

[0066] Figure 3 The particle size distribution of microspheres prepared from the four types of PVDF membranes in Example 5 is shown.

[0067] Figure 4 The cumulative release curves of the drug sustained-release microspheres and microemulsion in Example 6 are shown. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described clearly and completely below in conjunction with the embodiments of this invention. It should be noted that the described embodiments are merely some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0069] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0070] A method for preparing a traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects includes the following steps:

[0071] Step S1: Prepare a microemulsion solution from the traditional Chinese medicine compound composition; the traditional Chinese medicine compound composition contains volatile oils;

[0072] Step S2: Dilute the microemulsion solution to volume with distilled water, then adjust the osmotic pressure with sodium chloride solution and adjust the pH with hydrochloric acid solution.

[0073] In some embodiments, the microemulsion solution in step S1 is prepared in the following manner:

[0074] Step A1: Prepare drug sustained-release microspheres from the traditional Chinese medicine compound composition;

[0075] Step A2: Using the traditional Chinese medicine compound composition as the oil phase, add emulsifiers, co-emulsifiers and drug sustained-release microspheres, and shake to form a stable and dispersed microemulsion solution.

[0076] In some embodiments, the method for preparing drug sustained-release microspheres in step A1 includes the following steps:

[0077] Step B1: Membrane emulsification: An aqueous phase containing emulsifier is placed in a continuous phase tank and circulated inside the membrane emulsification device by a peristaltic pump; the traditional Chinese medicine compound composition and PLGA are dissolved in dichloromethane to prepare a membrane permeate, which is then loaded into a dispersed phase container;

[0078] Step B2: Nitrogen gas is introduced, and the membrane permeate naturally passes through the membrane under the pressure of nitrogen gas. The permeate after passing through the membrane is solidified and the organic solvent is evaporated. Then, centrifugation is performed to precipitate drug-release microspheres.

[0079] Step B3: Remove the emulsifier from the drug-release microspheres and freeze-dry.

[0080] In some embodiments, the apparatus used in the membrane emulsification method in step B1 is a modular membrane emulsification apparatus, such as... Figure 1 As shown, it includes membrane modules, peristaltic pumps, dispersed phase containers, continuous phase containers, etc.

[0081] In some embodiments, the emulsifying medium used in the modular membrane emulsification device described in step B1 is a PVDF membrane; wherein the membrane pore size is 0.36±0.15μm and the membrane contact angle is 86 / 71°.

[0082] In some embodiments, the concentration of PLGA in dichloromethane in step B1 is 60 mg / mL, and the concentration ratio of the traditional Chinese medicine compound composition to PLGA is 1:6.

[0083] In some embodiments, the emulsifier in step B1 is gelatin, PVA1788, Tween-80, or Tween-20.

[0084] In some embodiments, the emulsifier is one of 0.5% gelatin and 0.5% PVA1788, 0.5% Tween-80 and 0.5% PVA1788, or 0.5% Tween-20 and 0.5% PVA1788.

[0085] In some embodiments, the emulsifier is 0.5% Tween-80 and 0.5% PVA178; the emulsification pressure is 0.02 MPa.

[0086] In some embodiments, the centrifugation process parameters for the drug sustained-release microsphere precipitation in step B2 are: centrifugation speed of 10000 r / min and centrifugation time of 30 min.

[0087] In some embodiments, the method for removing the emulsifier in step B3 is to wash the precipitate three times with an appropriate amount of distilled water and then filter it.

[0088] In some embodiments, the traditional Chinese medicine compound composition in step A1 is prepared in the following manner:

[0089] Step C1: The volatile oils and residues after extracting the volatile oils from patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and peppermint were obtained by steam distillation and membrane enrichment technology.

[0090] Step C2: The residue after extracting the volatile oil is refluxed with ethanol and purified with macroporous adsorption resin to obtain purified solution A; Dryopteris crassirhizoma is extracted with ethanol, and the resulting extract is purified with ceramic ultrafiltration membrane to obtain purified solution B; Purified solutions A and B are combined, concentrated with reverse osmosis membrane and then freeze-dried to obtain small molecule active substances.

[0091] Step C3: Mix the volatile oil from step C1, the small molecule active substance from step C2, and borneol, and stir until homogeneous to obtain the final product.

[0092] In some embodiments, the traditional Chinese medicine compound composition includes, by weight, 13-17 parts of patchouli, 13-17 parts of wild chrysanthemum, 8-12 parts of agastache, 8-12 parts of mugwort, 8-12 parts of schizonepeta, 3-6 parts of peppermint, 1-3 parts of borneol, and 7-13 parts of Dryopteris crassirhizoma.

[0093] In some embodiments, the traditional Chinese medicine compound composition includes, by weight, 15g parts of patchouli, 15g parts of wild chrysanthemum, 10 parts of agastache rugosa, 10 parts of mugwort, 10 parts of schizonepeta tenuifolia, 5 parts of peppermint, 1 part of borneol, and 10 parts of Dryopteris crassirhizoma.

[0094] In some embodiments, the steam distillation extraction method specifically involves pulverizing patchouli, wild chrysanthemum, agastache rugosa, artemisia argyi, schizonepeta tenuifolia, and mint, passing them through a 20-80 mesh sieve, and then performing steam distillation. The boiler steam pressure is 2-6 kg / cm². 2 The extraction time is 1-8 hours.

[0095] In some embodiments, patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and mint are pulverized through a 40-mesh sieve and distilled with steam at a boiler steam pressure of 4 kg / cm². 2 The extraction time is 5 hours.

[0096] In some embodiments, the membrane enrichment technology involves passing an oil-water mixture obtained after steam distillation through an ultrafiltration membrane oil-water separator to separate the oil and water; the separated oil is the volatile oil.

[0097] In some embodiments, the ultrafiltration membrane oil-water separator uses polysulfone as the membrane material and has a pore size of 140 kDa.

[0098] In some embodiments, the ethanol reflux is performed by adding 12 times the amount of 70% ethanol to the residue after extracting the volatile oil, refluxing for 1 hour, and filtering to obtain filtrate one and residue one; adding 10 times the amount of 70% ethanol to residue one, refluxing for 1 hour, and filtering to obtain filtrate two and residue two; combining filtrate one and filtrate two, and recovering ethanol under reduced pressure until there is no alcohol odor to obtain the ethanol extract of the residue.

[0099] In some embodiments, the macroporous adsorption resin is purified by using AB-8, D101 macroporous resin or NAK-9 resin to purify the ethanol extract of the pharmaceutical residue. The eluent is 1-5 times the volume of 70%-90% ethanol, the pH of the solution is 3.5-6.5, and the flow rate is 1-5 BV / h. The ethanol eluent is collected and the ethanol is recovered under reduced pressure to obtain purified solution A.

[0100] In some embodiments, the ethanol extract of pharmaceutical residue is purified using D101 macroporous resin, with a pH of 3.5, and eluted with 5 times the volume of 70% ethanol at a flow rate of 3 BV / h.

[0101] In some embodiments, the specific preparation method of the refined liquid B is as follows: weigh the dried medicinal material of Dryopteris crassirhizoma and crush it into coarse powder; extract it twice with 12 times the volume of 70% ethanol, each time for 1 hour; combine the two extracts and concentrate them under reduced pressure to recover the ethanol until there is no alcohol taste; refine the alcohol-free extract with a ceramic ultrafiltration membrane with a pore size of 50 nm and a material of Al2O3.

[0102] In some embodiments, the reverse osmosis membrane concentration method is to concentrate the mixture of purified solution A and purified solution B using a reverse osmosis membrane to a relative density of 1.08 to 1.10; the process parameters for reverse osmosis concentration are an operating pressure of 0.3-2.0 MPa and a flow rate of 6-8 L / min.

[0103] In some embodiments, step C3 further includes adding a surfactant for mixing.

[0104] In some embodiments, the surfactant in step C3 is Tween 80.

[0105] In some embodiments, the emulsifier in step A2 is poloxamer-108 and the co-emulsifier is ethanol; the mass ratio of poloxamer-108 to ethanol is 1:10.

[0106] In some embodiments, in step S2, the volume adjustment is performed by bringing the microemulsion to 100 ml with distilled water; the sodium chloride solution has a mass fraction of 0.7%; and the pH of the solution is adjusted to 3-8 with 0.1 mol / L hydrochloric acid.

[0107] In some embodiments, hydrochloric acid is used to adjust the pH of the solution to 6.0-7.0.

[0108] Example 1: Study on the volatile oil extraction process in step C1

[0109] Weigh out 1000g (three portions) of patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and mint according to the weight ratio, grind them through a 40-mesh sieve, and distill them with steam at a boiler steam pressure of 4 kg / cm². 2 The extraction times were 1, 2, 3, 4, 5, 6, 7, and 8 hours, respectively. The oil-water mixture obtained after steam distillation was passed through an ultrafiltration membrane oil-water separator for oil-water separation. The separated oil was the volatile oil.

[0110] The volatile oil content was determined using Appendix XD of the 2020 edition of the Chinese Pharmacopoeia, Part I, according to the method for determination of volatile oils. The results are shown in Table 1.

[0111] Table 1. Effect of extraction time on volatile oil extraction rate (laboratory scale, 500g, n=3)

[0112] Note: The volatile oil extraction rate is 100% after 8 hours.

[0113] Based on this, a scale-up experiment was conducted. 15 kg of patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and mint were taken according to the specified weight ratio, pulverized through a 40-mesh sieve, and placed in a 500L multi-functional extraction tank. Steam distillation was performed using a boiler steam pressure of 4 kg / cm². 2 The extraction times ranged from 1 to 8 hours. The oil-water mixture obtained after steam distillation was passed through an ultrafiltration membrane oil-water separator for oil-water separation. The separated oil was the volatile oil. The results are shown in Table 2.

[0114] Table 2 Effect of extraction time on volatile oil extraction rate (pilot-scale, 15 kg)

[0115] Note: The volatile oil extraction rate is 100% after 8 hours.

[0116] As shown in Tables 1 and 2, steam distillation for 5 hours is the optimal method for extracting volatile oils. Extending the extraction time not only results in a small increase in volatile oil yield but also increases heat consumption, hindering cost reduction. Furthermore, prolonged heating can lead to increased loss of other components.

[0117] Example 2: Study on the preparation process of purified liquid A

[0118] 2.1 Study on the ethanol reflux extraction process of the residue after extracting volatile oil in step C2

[0119] The study investigated the influencing factors of ethanol reflux extraction: ethanol concentration (A), ethanol dosage (B), and extraction time (C). Using the ethanol reflux method, with the yields of total flavonoids and extract as indicators, L9(3) was selected. 4An orthogonal array was used to comprehensively examine these three factors, as shown in Table 3, to study the optimal process conditions for ethanol reflux extraction.

[0120] Table 3 Factor Level Table

[0121] Orthogonal experiments were conducted according to the design levels of each factor (Table 3). After extraction, the alcohol extracts of each batch of medicinal materials were filtered and combined. The total flavonoid yield and extract yield were used as evaluation criteria. Based on the actual situation and according to the commonly used proportions, the weighting coefficients of the total flavonoid yield and extract yield were set to 0.8 and 0.2, respectively, to investigate the extraction process. The results of the orthogonal experiments are shown in Table 4, and the results of the analysis of variance are shown in Table 5.

[0122] Table 4 Orthogonal Experiment and Results

[0123] Table 5. Analysis of Variance Table

[0124] From the K values ​​in Table 4, it can be seen that when judging by comprehensive indicators, A2>A1>A3, B3>B2>B1, C2>C3>C1, and the influence of factors on the extraction process is A>C>B. Therefore, the optimal process is A1B3C3. The variance analysis results in Table 5 show that factor B has a significant difference in the experimental results (P<0.05), while A and C have no significant difference. The optimal extraction process for combining the 6 medicinal residues is determined to be A2B3C2. That is, the optimal process for ethanol reflux extraction of the medicinal residue after extracting the volatile oil in step C2 is to add 70% ethanol to the remaining medicinal residue and reflux twice. The first time, add 12 times the amount of 70% ethanol and reflux for 1 hour, then filter to obtain filtrate one and filter residue one. Add 10 times the amount of 70% ethanol to filter residue one and reflux for 1 hour. The first time, add 12 times the amount of ethanol and reflux for 1 hour, and the second time, add 10 times the amount of ethanol and reflux for 1 hour.

[0125] 2.2 Research on the Refining Process of Macroporous Adsorption Resin

[0126] We selected macroporous adsorption resins of different polarities (AB-8, D101, and NAK-9 models) to screen and study the refined flavonoids (the main active components of the residue after extracting volatile oils from six herbs, including patchouli, are flavonoids) using three different macroporous adsorption resins.

[0127] 2.2.1 Static adsorption-elution experiment of different resins on the decocted residue after extracting volatile oil in step C1

[0128] (1) Determination of static adsorption capacity and adsorption rate: Accurately weigh 0.5 g of dried resin and place it in a 250 ml conical flask. Add 40 ml of extract of the drug residue (hereinafter referred to as compound) of known concentration. Place the flask in a constant temperature (25℃) shaker with a vibration frequency of 140 times / min. After 24 h, determine the total flavonoids in the drug solution after adsorption by spectrophotometry to investigate the static adsorption effect of the resin. Calculate the adsorption rate according to the following formula. The adsorption rate is shown in Table 6.

[0129]

[0130] Table 6. Adsorption of total flavonoids in the compound by different adsorption resins

[0131] As shown in Table 6, for the adsorption of total flavonoids in the compound, D101 > NAK-9 > AB-8, therefore D101 resin should be selected.

[0132] (2) Determination of static desorption rate: After the resin adsorbs the total flavonoids of the compound, considering adsorption efficiency, energy saving, and time saving, alcohol is the best eluent. Take D101 resin and add 30 ml each of 60%, 70%, and 80% ethanol, respectively. Place it in a constant temperature shaker with a vibration frequency of 140 times / min. After 24 hours, use spectrophotometry and HPLC to determine the components in the adsorbed drug solution and examine the static desorption effect of the resin. The desorption rate is calculated according to the following formula, and the results are shown in Table 7.

[0133] Desorption rate (%) =

[0134] Table 7. Desorption rates of components in the compound preparation by ethanol at different concentrations

[0135] As shown in Table 7, for D101 resin, elution with 70% ethanol is the preferred method for eluting total flavonoids.

[0136] 2.2.2 Dynamic adsorption properties of D101 resin and its influencing factors

[0137] (1) Dynamic adsorption performance of D101 resin: 50 ml of AB8 resin and a suitable concentration of compound drug solution were wet-packed into a 150 ml (20 mm × 300 mm) glass column, and the pH was adjusted to 3.5. The drug solution was passed through the resin column at a flow rate of 3 BV / h, and a total of 250 ml of drug solution was treated. The effluent from each 20 ml treatment was collected separately, and the concentration of total flavonoids in the effluent was determined by HPLC and spectrophotometry. The results showed that D101 resin reached saturation for total flavonoids in the compound at approximately 5 BV volume.

[0138] (2) Effect of pH value of drug solution on adsorption: 100 ml of resin was packed into the column by wet method, and the drug solution was adjusted to different pH values ​​for adsorption experiments. The column was washed with 5 times the amount of water, and then eluted with 4 times the amount of 70% ethanol. The content of the eluent was measured. The results are shown in Table 8.

[0139] Table 8 Relationship between pH value and elution rate

[0140] The pH value of the solution has a significant impact on adsorption. pH affects the solubility of flavonoids in water; acidic compounds are readily adsorbed in acidic solutions, while alkaline compounds are readily adsorbed under alkaline conditions, thus affecting the resin's adsorption performance. Total flavonoids possess polyphenolic structures and glycosidic chains, exhibiting weak acidity; therefore, adsorption is optimal under acidic conditions. According to Table 8, a pH of 3.5 is suitable for the compound filtrate.

[0141] (3) Effect of drug concentration on adsorption: Adsorption experiments were conducted using compound filtrate of different concentrations. The results showed that the initial concentration of the compound drug solution was preferably 13 μg / ml with a total flavonoid content.

[0142] (4) Desorption curve: The total flavonoid concentration was determined by spectrophotometry using 70% ethanol at a flow rate of 3 BV / h. The results showed that the total flavonoid components in the compound could be basically desorbed using 5 times the volume of resin bed eluent.

[0143] Through the above experimental studies, the dynamic adsorption performance of the resin, the effect of pH value of the drug solution on adsorption, the effect of drug concentration on adsorption, and the desorption curve were investigated to determine the process for adsorbing flavonoid active ingredients in compound drug solutions using D101 macroporous adsorption resin. The optimal process is as follows: the total flavonoid concentration in the drug solution is 13 μg / ml, the pH value is 3.5, and elution is performed with 5 times the volume of 70% ethanol at a flow rate of 3 BV / h.

[0144] Example 3: Study on the preparation process of purified solution B

[0145] 3.1 Study on the extraction process of Dryopteris crassirhizoma by ethanol extraction

[0146] Given that the antiviral active substances in Dryopteris crassirhizoma are mainly phloroglucinol compounds, and that phloroglucinol is slightly soluble in water but soluble in ethanol, ethanol extraction of Dryopteris crassirhizoma was used. The ethanol extract was then purified using a ceramic ultrafiltration membrane to obtain purified solution B.

[0147] The content of phloroglucinol in Dryopteris crassirhizoma was determined by sodium hydroxide titration using a CF1Plus potentiometric titrator. The yield of phloroglucinol was used as the indicator, and L9(3) was selected. 4An orthogonal array was used to comprehensively investigate four factors: ethanol concentration, ethanol dosage, extraction time, and number of extractions (see Table 9, Factor Level Table) to explore the optimal extraction conditions for Dryopteris crassirhizoma. The results are shown in Table 10 (orthogonal experiment and results) and Table 11 (analysis of variance).

[0148] Table 9 Factor Level Table

[0149] Table 10 Orthogonal Experiment and Results

[0150] Table 11 Analysis of Variance Table

[0151] Tables 10 and 11 show that the order of influence of the four factors on the yield of phloroglucinol from *Dryopteris crassirhizoma* is D > B > A > C, and the optimal extraction process is A2B3C2D3. Analysis of variance, using factor C (with the smallest range) as the error term, shows that factors A, B, and D have no significant difference in their influence on the yield of phloroglucinol. Therefore, considering both the orthogonal experimental results and economic costs, the optimal extraction process is determined to be A2B3C1D2, i.e., two extractions using 12 times the volume of 70% ethanol under reflux, each lasting 1 hour.

[0152] 3.2 Research on Refined Ultrafiltration Membrane Materials

[0153] Two types of ceramic membranes (Al2O3 and ZrO2, both with a pore size of 50 nm) were used to purify the ethanol extract of Dryopteris crassirhizoma. The permeation flux and the retention rate of the indicative component phloroglucinol were used to evaluate the performance of the two different ceramic membrane materials. Operating conditions: pressure difference: 0.15 MPa, temperature: 40℃, feed flow rate: 3 m / s.

[0154] 3.2.1 The effect of membrane material on permeation flux:

[0155] Table 12 shows the fluxes of water extracts from membranes of different materials. As can be seen from Table 12, the initial fluxes of Al₂O₃ and ZrO₂ membranes, both with an average pore size of 50 nm, are similar, but their stable fluxes differ significantly: the stable flux of the 50 nm pore size Al₂O₃ membrane is greater than that of ZrO₂, and the decay is slower, with a flux decrease of 47.3%. In contrast, the flux decrease of the 50 nm pore size ZrO₂ membrane reaches 81.9%, with a stable flux of only 11 L / m³. 2 h.

[0156] Table 12 Flux of water extracts from membranes of different materials (all pore sizes are 50 nm)

[0157] Note: Flux decrease rate = (Initial flux - Steady flux) / Initial flux

[0158] 3.2.2 Effect of membrane material on the retention rate of phloroglucinol

[0159] The retention rates of phloroglucinol before and after ultrafiltration for different membrane materials are shown in Table 13.

[0160] Table 13 Comparison of phloroglucinol retention rates before and after ultrafiltration using different membrane materials (pore size: 50 nm).

[0161] Table 13 shows that the retention rate of the active ingredient phloroglucinol was relatively high at 87.3% for the Al2O3 membrane with a pore size of 50 nm; while the drug solution treated with the ZrO2 membrane with a pore size of 50 nm suffered a loss of 50.8% of the active ingredient. Since the two membranes have the same pore size, it is hypothesized that the difference in the properties of the membrane materials, such as hydrophilicity and electrochemical properties, may have caused the difference in the adsorption and retention performance of the components.

[0162] 3.3 Study on the pore size of ceramic membranes

[0163] Al₂O₃ membranes with pore sizes of 10 nm and 50 nm were used to purify the ethanol extract of *Dryopteris crassirhizoma*. The operating conditions were: temperature 40 °C, operating pressure difference 0.15 MPa, and membrane flow rate 3 m / s. -1 The effects of different pore sizes on ceramic membranes were investigated using the retention rate of the indicative component phloroglucinol and the membrane flux as indicators.

[0164] Table 14 shows the effect of membrane pore size on the retention rate of the indicator component phloroglucinol and the solids removal performance. As can be seen from Table 14, the phloroglucinol retention rate of the membrane with a 50 nm pore size is significantly higher than that of the membrane with a 10 nm pore size. The solids removal rate of the membrane with a 50 nm pore size is slightly higher than that of the 10 nm pore size. Therefore, among the two pore sizes of Al2O3 membranes, the 50 nm pore size is more suitable.

[0165] Table 14 Effect of membrane pore size on phloroglucinol transfer rate

[0166] Note: Retention rate (%) = (Component content in permeate / Component content in original solution) × 100

[0167] Example 4: Study on the concentration and freeze-drying process of a mixture of refined liquid A and refined liquid B.

[0168] 4.1 Study on the concentration process of refined solutions A and B

[0169] Combined purified solutions A and B, and took two 1000ml portions of the combined mixture. These were concentrated to a relative density of 1.08–1.10 (60℃) using vacuum concentration (0.09 MPa, 60℃) and reverse osmosis concentration (membrane material: cellulose acetate; process parameters: operating pressure 0.3–2.0 MPa, flow rate 6–8 L / min), respectively. The concentrates were then freeze-dried to obtain small-molecule active substances (mainly composed of flavonoids and phloroglucinols). The concentration effects of the two different technologies were evaluated using the content of the main active components, total flavonoids and phloroglucinols, as the evaluation indicators. The results are shown in Table 15.

[0170] Table 15 Effects of different concentration methods on the content of total flavonoids and phloroglucinol

[0171] As shown in Table 15, compared with vacuum concentration, reverse osmosis concentration can better retain the index components total flavonoids and phloroglucinol.

[0172] 4.2 Study on freeze-drying process of concentrated solutions A and B

[0173] 4.2.1 Study on drug concentration

[0174] Prepare concentrated solutions of purified solutions A and B with a concentration of 2g crude drug / ml. Divide the solutions into four equal portions and dilute them to 0.5g crude drug / ml, 1g crude drug / ml, and 1.5g crude drug / ml, respectively. Leave the fourth portion undiluted. Dispense the solutions into vials, each containing 3ml, and freeze-dry.

[0175] Table 16 Optimal concentration of drug solution in freeze-drying process

[0176] As shown in Table 16, the freeze-dried sample with a concentration of 1g crude drug / ml liquid has the best shape retention and a shorter rehydration time.

[0177] 4.2.2 Comparison of slow freezing and fast freezing methods

[0178] The concentrated solutions of purified solutions A and B, with a concentration of 1g crude drug / ml, were dispensed into vials, each containing 3ml, and divided into two groups, A and B. Group A was slowly frozen in a freeze dryer for 12 hours; group B was rapidly frozen in a refrigerator for 4 hours. Vacuum was then applied, and the solutions were freeze-dried for 2 days. The solutions were then removed and observed. The results are shown in Table 17.

[0179] Table 17 Comparison of slow freezing and fast freezing methods

[0180] As shown in Table 17, considering both rehydration time and appearance, the rapid freezing method is selected.

[0181] 4.2.3 Selection of freeze-drying excipients

[0182] A 3ml vial was dispensed into 1g crude drug / ml injection solution and divided into 4 groups: Group 1 with 5% glucose; Group 2 with 5% sodium chloride; Group 3 with 4% β-cyclodextrin; Group 4 as a blank. The solution was freeze-dried. The results were observed and are shown in Table 18.

[0183] Table 18 Selection of freeze-drying excipients

[0184] As shown in Table 18, although the blank color is slightly darker, it has good moldability, a better appearance, and faster rehydration. Therefore, it is best not to add any additives.

[0185] 4.2.4 Freeze-drying curve

[0186] Dispense 3ml of the injection solution (1g crude drug / ml) into vials. Freeze-dry the vials at a maximum pressure of 0.1mbar, recording the plate temperature and the sample temperature. Plot the freeze-drying curve. Figure 2 .Depend on Figure 2 The optimal freezing rate of the concentrates of refined solutions A and B can be determined to obtain high-quality products and shorter freeze-drying times.

[0187] Example 5: Study on emulsification process in the preparation of sustained-release drug microspheres from traditional Chinese medicine compound compositions.

[0188] 5.1 Preparation of drug sustained-release microspheres by membrane emulsification

[0189] Adopting such Figure 1 The membrane emulsification device shown involves adding an aqueous phase containing Tween-80 and PVA into a continuous phase tank, where a peristaltic pump circulates the aqueous phase within the device. A traditional Chinese medicine compound and PLGA are dissolved in a certain volume of dichloromethane to prepare a membrane permeate, which is then placed in a dispersed phase container. Nitrogen gas is introduced, and the permeate passes through the membrane naturally under nitrogen pressure. The permeate is then solidified to allow the organic solvent to fully evaporate. Centrifugation causes the drug-releasing microspheres to precipitate. The precipitate is then washed three times with distilled water to remove surfactants and dried using a freeze dryer to obtain the final product.

[0190] 5.1.1 Study on pore size and contact angle of membrane in membrane emulsification method

[0191] This invention independently designed and prepared four different asymmetric hydrophilic PVDF membranes. Water contact angle experiments and SEM scanning were performed on the four prepared PVDF membranes, and the results are shown in Table 19. Using these four membranes as media, membrane emulsification experiments were conducted under the same emulsification conditions. The resulting emulsions were then analyzed using SEM and their particle size distribution was statistically analyzed. The experimental results are as follows: Figure 3As shown, microspheres prepared with different membranes exhibit different particle sizes and size distributions. Statistical analysis of the particle size distribution of these particles shows that the average particle sizes of the microspheres prepared with the four membranes are 3.6 μm, 1.8 μm, 1.9 μm, and 473 nm, respectively. Among them, the microspheres prepared with membrane D have the most uniform particle size.

[0192] Table 19 Pore size and contact angle of four self-designed and prepared PVDF membranes

[0193] 5.1.2 Research on Emulsification Process in Membrane Emulsification

[0194] Based on the results of preliminary experiments, four main factors significantly influenced the microsphere preparation process, as shown in Table 20: A: PLGA concentration in dichloromethane (mg / mL); B: the concentration ratio of the traditional Chinese medicine compound to PLGA; C: the type of emulsifier; and D: emulsification pressure. Orthogonal design was used to optimize the preparation conditions. L3 was selected. 4 Experiments were conducted using orthogonal arrays, with encapsulation efficiency, drug loading rate, particle size distribution (Span value), and morphology as the four evaluation indicators. Scoring was performed using the following method:

[0195] Y1 = Drug loading rate (maximum score 1); Y2 = Encapsulation efficiency (maximum score 1); Y3 = Span value; Y4 = Morphology (round and smooth morphology is preferred, maximum score 1).

[0196] Total score (Y) = Y1 + Y2 - Y3 + Y4, and the larger the Y value, the better.

[0197] The factor level table is shown in Table 20, the results of the orthogonal design experiment are shown in Table 21, and the results of the analysis of variance are shown in Table 22.

[0198] Table 20 Orthogonal Design Factor Level Table

[0199] In Table 20, A: Concentration of PLGA in dichloromethane (unit, mg / mL), B: Concentration ratio of traditional Chinese medicine compound composition to PLGA, C: Type of emulsifier, C1 (aqueous solution of 0.5% gelatin and 0.5% PVA1788), C2 (aqueous solution of 0.5% Tween-80 and 0.5% PVA1788), C3 (aqueous solution of 0.5% Tween-20 and 0.5% PVA1788), D: Emulsification pressure, D1 (0.02 MPa), D2 (0.06 MPa), D3 (0.1 MPa).

[0200] Table 21 Results of Orthogonal Design Experiments

[0201] Table 22 Analysis of Variance Table

[0202] As shown in Tables 21 and 22, the optimal emulsification process is A3B3C2D1, which means that the concentration of PLGA in dichloromethane is 60 mg / mL, the concentration ratio of the traditional Chinese medicine compound composition to PLGA is 1:6, the emulsifier formula is 0.5% Tween-80 + 0.5% PVA1788, and the emulsification pressure is 0.02 MPa.

[0203] 5.2 Research on Emulsification Methods

[0204] Preparation of drug sustained-release microspheres by homogenization emulsification: Weigh an appropriate amount of traditional Chinese medicine compound composition and PLGA and dissolve them in a certain volume of dichloromethane (dispersed phase). The continuous phase is an aqueous solution containing an appropriate amount of PEG 4000 (emulsifier) ​​and PVA (stabilizer). Under a homogenizing emulsifier, the dispersed phase is slowly added to the continuous phase to emulsify and form an O / W type emulsion. The emulsion is then immediately placed at room temperature and stirred overnight. After the dichloromethane evaporates, PLGA gradually precipitates and solidifies into microspheres. After standing and separating into layers, the microspheres are centrifuged, washed with water three times, and then freeze-dried to obtain the final product.

[0205] The drug-release microspheres prepared by homogenization emulsification were compared with those prepared by membrane emulsification in section 5.1. The microspheres prepared by homogenization emulsification had an average particle size distribution of approximately 5 μm with poor uniformity, a total drug loading of 4.74%, and a total encapsulation efficiency of 41.48%. The microspheres prepared by membrane emulsification had a smooth, round, and non-adhesive surface, uniform particle size (average particle size 473 nm), a PDI index of 0.164, and a total drug loading of 7.85%.

[0206] Experimental results show that the drug sustained-release microspheres prepared by membrane emulsification are superior to those prepared by homogeneous emulsification in terms of particle size and distribution, drug loading, and encapsulation efficiency. This is because the membrane pores are more uniformly distributed, and the droplets are formed under the combined action of lower pressure and mild shear force. In contrast, homogeneous emulsification only involves shear force during the emulsification process, and heat is inevitably generated during shearing. The higher the system temperature, the more it affects the viscosity of the dispersed phase and the continuous phase. For example, dichloromethane has a boiling point of around 40°C and some of it will evaporate during the emulsification process, causing solute precipitation.

[0207] Example 6: In vitro release rate study

[0208] Using the total flavonoids in the traditional Chinese medicine compound composition as an indicator, the in vitro release rate of the drug-release microspheres and microemulsion was determined by dialysis. The cumulative release rate was plotted on the ordinate, and release time on the abscissa. The cumulative release curve of the drug-release microspheres is shown below. Figure 4 The curve is formed by the midpoints. Figure 4It can be seen that the sustained-release microspheres can release drugs stably within 48 hours, with a release rate of 27.6%; the microemulsion solution (i.e., the traditional Chinese medicine compound nasal spray) has both immediate and sustained-release effects (such as...). Figure 4 As shown in the curve of the Chinese center, it can take effect immediately and maintain its effect for more than 48 hours.

[0209] Example 7: Study on the effects of osmotic pressure and pH on nasal mucosa and mucociliary toxicity in step S2

[0210] 7.1 Study on the effects of different osmotic pressures on the toxicity of the mucociliary membrane of toad palate

[0211] The method for determining ciliary movement frequency was employed: Toads were fixed supine on a frog board, their mouths were opened with hemostatic forceps, and the palatal mucosa was separated using surgical scissors. A 3mm × 3mm piece of mucosa was taken, washed with physiological saline to remove blood clots and debris, and then laid flat on a glass slide. Sodium chloride solutions with different osmotic pressures (0.3%, 0.5%, 0.7%, and 0.9%) were added to the mucosa surface to allow it to soak, and a coverslip was placed on top. The movement of the cilia was observed under a 40x microscope. The slide was then placed in a chromatography tank saturated with a small amount of distilled water and sealed at room temperature (20-25℃). Specimens were removed at regular intervals and observed under a microscope until the cilia stopped moving, and the duration of continuous movement was recorded. In each experiment, a piece of palatal mucosa from the same toad was used for each group. The results showed that among solutions with different osmotic pressures, the 0.7% sodium chloride solution had the least effect on ciliary movement.

[0212] 7.2 Study on the effects of different pH values ​​on the toxicity of the mucociliary membrane of toad palate

[0213] The method for determining the duration of ciliary movement was employed: The toad was fixed supine on a frog board. The mouth was opened using hemostatic forceps, and the palatal mucosa was separated using surgical scissors. A 3mm × 3mm piece of mucosa was taken, washed with physiological saline to remove blood clots and debris, and then laid flat on a glass slide. 0.2 ml of sodium chloride solution with different pH values ​​(3, 4, 5, 6, 7, 8) was added to the mucosa surface to allow it to soak, and a coverslip was placed on top. The movement of the cilia was observed under a 40x microscope. The slide was then placed in a chromatography tank saturated with a small amount of distilled water, sealed, and placed at room temperature (20-25℃). The specimen was then removed at regular intervals and observed under a microscope until the cilia stopped moving. The duration of ciliary movement was recorded. In this experiment, three parallel samples of the palatal mucosa from the same toad were used for each group.

[0214] The results showed that the effect on ciliary movement time was relatively small at pH=6 and pH=7. Therefore, controlling the pH of the drug solution between 6 and 7 is advisable.

[0215] Example 8: In vitro inhibitory effect of traditional Chinese medicine compound nasal spray on H1N1FM1 during prophylactic administration.

[0216] The inoculum concentration in 96-well microplates was 1×10⁻⁶. 5 MDCK (made in-Darbycanine Kidney, commonly used for in vitro drug absorption studies) cells (100 μl / well, cells / ml) were cultured at 37°C in 5% CO2. After the cells grew into a monolayer, the supernatant was discarded, and 100 μl of different concentrations of traditional Chinese medicine compound nasal spray were added to each well (4 wells per concentration). The cells were pre-treated for 24 h, and after overnight treatment, the supernatant was discarded. 100 μl / well of virus containing 100 TCID50 (median lethal dose for tissue culture, i.e., cells) was added, and after 2 h of adsorption, maintenance medium containing different concentrations of traditional Chinese medicine compound nasal spray was added to each well (100 μl / well). The treatment was terminated after 4 days. A normal cell control and a positive drug group were included in the experiment. The antiviral effect was determined by chicken hemoglobin agglutination assay. The results showed that the traditional Chinese medicine compound nasal spray at 12.8 μg / ml had a good preventive effect against the mouse lung-adapted strain of influenza virus H1N1.

[0217] Example 9: Effect of a traditional Chinese medicine compound composition on the lung index of mice infected with influenza A virus

[0218] Experimental Procedure: Healthy KM mice, half male and half female, weighing 17–20 g, were randomly divided into four groups: a model group, a ribavirin positive drug group, and two groups (high and low doses) of a traditional Chinese medicine compound. The drugs were administered by gavage once daily. Two days after administration, the animals in each group were lightly anesthetized with ether and then infected nasally with 3 LD50 influenza virus (FM1) solution. After natural recovery, the administration was continued for four consecutive days (a total of six days). A blank control group was also included. On the fifth day, the mice were weighed, and their lungs were dissected and weighed. The lung index was calculated for each mouse, and the lung index inhibition rate was determined. A t-test was used to compare the results with the model group to determine the significance of the comparison.

[0219] Lung Index = Lung Weight (g) / Body Weight (g) × 10

[0220] ×100%

[0221] The experimental results showed that the traditional Chinese medicine compound composition at doses of 22.5 g / kg and 45 g / kg could significantly reduce the lung index value of mice infected with influenza A virus. After statistical processing, the difference was statistically significant compared with the model group (P < 0.01).

[0222] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, comprising the following steps: Step S1: Prepare the traditional Chinese medicine compound composition into a microemulsion solution; The traditional Chinese medicine compound composition contains volatile oils; Step S2: Dilute the microemulsion solution to volume with distilled water, then adjust the osmotic pressure with sodium chloride solution and adjust the pH with hydrochloric acid solution.

2. The preparation method of the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 1, characterized in that, The microemulsion solution in step S1 is prepared as follows: Step A1: Prepare drug sustained-release microspheres from the traditional Chinese medicine compound composition; Step A2: Using the traditional Chinese medicine compound composition as the oil phase, add emulsifiers, co-emulsifiers and drug sustained-release microspheres, and shake to form a stable and dispersed microemulsion solution.

3. The preparation method of the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 2, characterized in that, The preparation method of the drug sustained-release microspheres in step A1 includes the following steps: Step B1: Membrane emulsification: An aqueous phase containing emulsifier is placed in a continuous phase tank and circulated inside the membrane emulsification device by a peristaltic pump; the traditional Chinese medicine compound composition and PLGA are dissolved in dichloromethane to prepare a membrane permeate, which is then loaded into a dispersed phase container; Step B2: Nitrogen gas is introduced, and the membrane permeate naturally passes through the membrane under the pressure of nitrogen gas. The permeate after passing through the membrane is solidified and the organic solvent is evaporated. Then, centrifugation is performed to precipitate drug-release microspheres. Step B3: Remove the emulsifier from the drug-release microspheres and freeze-dry them.

4. The preparation method of the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 3, characterized in that, In step B1, the concentration of PLGA in dichloromethane is 60 mg / mL, and the concentration ratio of the traditional Chinese medicine compound composition to PLGA is 1:

6.

5. The method for preparing the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 1, characterized in that, The traditional Chinese medicine compound composition in step S1 is prepared in the following manner: Step C1: The volatile oils and residues after extracting the volatile oils from patchouli, wild chrysanthemum, agastache, artemisia, schizonepeta, and peppermint were obtained by steam distillation and membrane enrichment technology. Step C2: The residue after extracting the volatile oil is refluxed with ethanol and purified with macroporous adsorption resin to obtain purified solution A; Dryopteris crassirhizoma is extracted with ethanol, and the resulting extract is purified with ceramic ultrafiltration membrane to obtain purified solution B; Purified solutions A and B are combined, concentrated with reverse osmosis membrane and then freeze-dried to obtain small molecule active substances. Step C3: Mix the volatile oil from step C1, the small molecule active substance from step C2, and borneol, and stir until homogeneous to obtain the final product.

6. The method for preparing the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 5, characterized in that, The specific method of steam distillation extraction involves pulverizing patchouli, wild chrysanthemum, agastache rugosa, artemisia argyi, schizonepeta tenuifolia, and mint, passing them through a 20-80 mesh sieve, and then distilling them with steam at a boiler steam pressure of 2-6 kg / cm². 2 The extraction time is 1-8 hours; the membrane enrichment technology involves passing the oil-water mixture obtained after steam distillation through an ultrafiltration membrane oil-water separator for oil-water separation; the separated oil is the volatile oil.

7. The preparation method of the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 2, characterized in that, In step A2, the emulsifier is poloxamer-10 and the co-emulsifier is ethanol; the mass ratio of poloxamer-108 to ethanol is 1:

10.

8. The method for preparing the traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects according to claim 1, characterized in that, In step S2, the volume is adjusted by distilling the microemulsion to 100 ml; the sodium chloride solution has a mass fraction of 0.7%; and the pH of the solution is adjusted to 3-8 with 0.1 mol / L hydrochloric acid.

9. A traditional Chinese medicine compound nasal spray with both immediate and sustained-release effects, prepared by the preparation method described in any one of claims 1 to 8.

10. The application of the traditional Chinese medicine compound nasal spray with both immediate and sustained release effects as described in claim 9, or the preparation method of the traditional Chinese medicine compound nasal spray with both immediate and sustained release effects as described in any one of claims 1 to 8, in the medical device for the prevention of influenza virus.

Images