Capsule volatile oil composition capable of dispelling wind and detoxifying as well as preparation method and application of capsule volatile oil composition

Abstract

According to the wind-dispelling and detoxifying capsule volatile oil composition and the preparation method and application thereof, compared with the prior art, molecules in wind-dispelling and detoxifying capsule volatile oil are included in a cavity of cyclodextrin in an aqueous solution state, drying is further performed, and wind-dispelling and detoxifying capsule and volatile oil inclusion compound solid powder is obtained; the water solubility and the stability of the volatile oil of the wind-dispelling and detoxifying capsule are effectively improved, and in addition, the inclusion compound is solid powder, so that the mixing difficulty in the preparation process of the wind-dispelling and detoxifying capsule can be reduced. Meanwhile, through inclusion of cyclodextrin, the cytotoxic activity is reduced, the bioavailability is improved, the anti-influenza virus activity is also remarkably improved, and the compound has a very good application prospect and development potential.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a volatile oil composition of Shufeng Jiedu Capsule, a preparation method thereof, and an application thereof. Background Art

[0002] Shufeng Jiedu Capsule (SFJDC) evolved from "Qudu Powder" donated by the old traditional Chinese medicine doctor Xiang Chuxian in Hunan, and was later developed and registered by Anhui Jiren Pharmaceutical Co., Ltd. (National Medicine Approval Number: Z20090047). Shufeng Jiedu Capsule has the effects of dispelling wind and clearing heat, and detoxifying and relieving sore throat. Clinically, it is used for acute upper respiratory tract infection belonging to the wind-heat syndrome, including symptoms such as fever, aversion to wind, sore throat, headache, nasal congestion, runny turbid nasal discharge, cough, etc. According to the "Chinese Pharmacopoeia (2020 Edition)", the capsule is composed of eight Chinese herbal medicines, including polygonum cuspidatum, forsythia suspensa, isatis root, bupleurum chinense, poria cocos, verbena officinalis, phragmites australis root, and licorice. Among them, forsythia suspensa and bupleurum chinense need to be extracted by steam distillation method first to obtain the volatile oil of Shufeng Jiedu Capsule (SFJDC-EOs), and as one of the components of Shufeng Jiedu Capsule, it is then sprayed and mixed into other component Chinese medicine extracts.

[0003] Modern pharmacological studies have shown that Chinese medicine volatile oils have a wide range of biological activities, such as antiviral, antibacterial, antifungal, antioxidant, and anti-inflammatory effects, etc. However, due to its oily substance, small polarity, poor water solubility, it is extremely easy to cause uneven mixing during the preparation process of Chinese medicine compound preparations; in addition, volatile oils also have characteristics such as easy volatilization and easy degradation, which also bring difficulties to the stability and quality control of Shufeng Capsule.

[0004] The patent with the publication number CN 118845671A, which was published on October 29, 2024, discloses a Shufeng Jiedu Granule and a preparation method thereof. First, the volatile oil of chaiqiao is added to silica 350FCP, stirred and adsorbed, and then mixed with other components. This preparation process can improve the mixing uniformity, but since the volatile oil is adsorbed on the surface of silica, its characteristics such as easy volatilization and easy degradation have not been changed.

[0005] Influenza virus pneumonia is a respiratory disease caused by infection with the H1N1 subtype influenza virus. If not treated promptly and effectively, it may further cause complications such as respiratory distress syndrome and multiple organ function damage, endangering the life safety of patients. Clinical studies have shown that Shufeng Jiedu Capsule has a positive effect in the treatment of such diseases, so it has been included in the "Diagnosis and Treatment Plan for Influenza A (H1N1)" of the National Health Commission, as well as the "Diagnosis and Treatment Guidelines for Influenza (2011 Edition)", the "Diagnosis and Treatment Plan for Exogenous Fever (Upper Respiratory Tract Infection) (2017 Edition)" of the State Administration of Traditional Chinese Medicine, and the "Diagnosis and Treatment Plan for Seasonal Influenza (Influenza A (H1N1)) (2017 Edition)" many times. However, there is no report on the anti-influenza virus effect of volatile oil in the study of the pharmacodynamic material basis. Summary of the Invention

[0006] The object of the present invention is to provide a volatile oil composition of Shufeng Jiedu Capsule and a preparation method thereof. By using the cyclodextrin inclusion technology, the volatile oil of Shufeng Jiedu Capsule is encapsulated, and through technologies such as vacuum drying or freeze-drying, the stability and water solubility of the volatile oil of Shufeng Jiedu Capsule are effectively improved, and the volatility of the volatile oil of Shufeng Jiedu Capsule is effectively inhibited. At the same time, through the inclusion of cyclodextrin, the cytotoxic activity of the volatile oil of Shufeng Jiedu Capsule is reduced, and its anti-influenza virus activity is improved, having good development and application value.

[0007] Another object of the present invention is to provide an application of the volatile oil composition of Shufeng Jiedu Capsule for preparing a drug for anti-influenza virus.

[0008] The specific technical solution of the present invention is as follows:

[0009] A volatile oil composition of Shufeng Jiedu Capsule, comprising the volatile oil of Shufeng Jiedu Capsule and cyclodextrin;

[0010] The mass ratio (w / w) of the cyclodextrin to the volatile oil of Shufeng Jiedu Capsule is 5:1 to 200:1, preferably 10:1 to 100:1.

[0011] The cyclodextrin includes β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, α-cyclodextrin, γ-cyclodextrin, and is preferably β-cyclodextrin or methyl-β-cyclodextrin.

[0012] The volatile oil of Shufeng Jiedu Capsule is the volatile oil obtained by steam distillation of Bupleuri Radix and Forsythiae Fructus, and the preparation method is the preparation method of the volatile oil of Chaiqiao described in the invention patent of CN 118845671A.

[0013] The preparation method of a volatile oil composition of Shufeng Jiedu Capsule provided by the present invention is prepared by the saturated aqueous solution method (also known as the co-precipitation method), the grinding method or the freeze-drying method.

[0014] The method for preparing the volatile oil composition of Shufeng Jiedu Capsule by the saturated aqueous solution method is as follows:

[0015] (1) Add cyclodextrin to purified water, and under constant temperature conditions, stir until completely dissolved to obtain a saturated aqueous solution of cyclodextrin;

[0016] (2) Dropwise add the volatile oil of Shufeng Jiedu Capsule continuously drop by drop to the aqueous solution of cyclodextrin. After the addition is completed, at 20°C to 40°C, fill with N 2 Protect, stir under light-proof conditions to obtain an aqueous solution of the inclusion complex of the volatile oil of Shufeng Jiedu Capsule and cyclodextrin;

[0017] (3) Let the solution obtained in step (2) stand at low temperature, filter, and take the solid for freeze-drying or vacuum drying to obtain the volatile oil composition of Shufeng Jiedu Capsule.

[0018] In step (1), the cyclodextrin selected is β-cyclodextrin.

[0019] In step (1), the mass ratio of purified water to cyclodextrin is the mass ratio of β-cyclodextrin to water is 100:1 to 10:1 (w / w), preferably 50:1 to 20:1.

[0020] In step (1), stir at a constant temperature of 40°C to 60°C until completely dissolved;

[0021] In step (1), after complete dissolution, the temperature is lowered to 10°C to 30°C, preferably 15°C to 25°C.

[0022] In step (2), the essential oil of Shufeng Jiedu Capsules can be continuously added drop by drop to the cyclodextrin aqueous solution, which can also be replaced by: dissolving the essential oil of Shufeng Jiedu Capsules in an ethanol solution, and then continuously adding it drop by drop to the cyclodextrin aqueous solution;

[0023] In step (2), for the stirring, the stirring rate is 200 - 600 rpm, and the time is 0.5 h to 24 h, preferably 6 h to 12 h.

[0024] In step (3), the low-temperature standing means placing it at 4°C for 12 - 24 hours to precipitate solids;

[0025] In step (3), for the vacuum drying, the temperature is 40°C to 60°C, the vacuum degree is below 10 Pa, and it is dried to a powder state to obtain the essential oil composition powder of Shufeng Jiedu Capsules, that is, the essential oil composition of Shufeng Jiedu Capsules.

[0026] In step (3), for the freeze-drying, the freeze-drying conditions are: pre-freezing at (-40)°C to (-80)°C for 2 h to 6 h, then sublimation drying at a vacuum degree of 0.01 mbar to 0.1 mbar and -10°C to 10°C for 10 h to 20 h, and desorption drying at 10°C to 25°C for 4 h to 8 h to obtain the dried essential oil composition powder of Shufeng Jiedu Capsules, that is, the essential oil composition of Shufeng Jiedu Capsules.

[0027] The method for preparing the essential oil composition of Shufeng Jiedu Capsules by the grinding method is as follows:

[0028] S1: Add β-cyclodextrin and purified water to a mortar and grind well to form a β-cyclodextrin paste;

[0029] S2: Continuously add the essential oil of Shufeng Jiedu Capsules drop by drop to the β-cyclodextrin paste and grind to obtain an inclusion complex of the essential oil of Shufeng Jiedu Capsules and β-cyclodextrin;

[0030] S3: Freeze-dry or vacuum-dry the clathrate obtained in step S2 to obtain the volatile oil composition of Shufeng Jiedu Capsules.

[0031] In step S1, the mass ratio of purified water to cyclodextrin is 5:1 to 2:1 (w / w).

[0032] In step S2, the gradual and continuous dropwise addition of the volatile oil of Shufeng Jiedu Capsules to the β-cyclodextrin paste can also be replaced by: dissolving the volatile oil of Shufeng Jiedu Capsules in an ethanol solution and then gradually and continuously dropwise adding it to the β-cyclodextrin clathrate;

[0033] In step S2, the grinding time is 20 min to 120 min, preferably 40 min to 90 min.

[0034] In step S3, for the vacuum drying, the temperature is 40°C to 60°C, the vacuum degree is below 10 Pa, and it is dried to a powder state to obtain the powder of the volatile oil composition of Shufeng Jiedu Capsules, that is, the volatile oil composition of Shufeng Jiedu Capsules.

[0035] In step S3, for the freeze-drying, the freeze-drying conditions are: pre-freezing at (-40)°C to (-80)°C for 2 h to 6 h, then sublimation drying at a vacuum degree of 0.01 mbar to 0.1 mbar and -10°C to 10°C for 10 h to 20 h, and desorption drying at 10°C to 25°C for 4 h to 8 h to obtain the dried powder of the volatile oil composition of Shufeng Jiedu Capsules, that is, the volatile oil composition of Shufeng Jiedu Capsules.

[0036] The method for preparing the volatile oil composition of Shufeng Jiedu Capsules by the freeze-drying method is as follows:

[0037] A. Add cyclodextrin to purified water and stir until completely dissolved under constant temperature conditions to obtain an aqueous cyclodextrin solution;

[0038] B. Gradually and continuously dropwise add the volatile oil of Shufeng Jiedu Capsules to the aqueous cyclodextrin solution; or dissolve the volatile oil of Shufeng Jiedu Capsules in an ethanol solution and then gradually and continuously dropwise add it to the aqueous cyclodextrin solution;

[0039] C. After completing step B, fill with N 2 gas, and stir at 200 - 800 rpm / min for 0.5 h to 24 h under constant temperature and light-shielded conditions at 10°C to 30°C to obtain an aqueous solution of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and cyclodextrin;

[0040] D. Freeze-dry the solution obtained in step C to obtain the powder of the volatile oil composition of Shufeng Jiedu Capsules.

[0041] In step A, after stirring until completely dissolved under constant temperature conditions at 20°C to 80°C; then lower the temperature by 10°C to 40°C; preferably 15°C to 25°C.

[0042] In step A, if β-cyclodextrin is used, the mass ratio of purified water to cyclodextrin is 100:1 to 20:1 (w / w); preferably 50:1 to 20:1;

[0043] In step A, when methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, α-cyclodextrin or γ-cyclodextrin is used, the mass ratio of purified water to methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, α-cyclodextrin or γ-cyclodextrin is 50:1 to 2:1 (w / w), 20:1 to 2:1;

[0044] In step D, the conditions for freeze-drying are pre-freezing at (-40) °C to (-80) °C for 2 h to 6 h, then sublimation drying at a vacuum degree of 0.01 mbar to 0.1 mbar and -10 °C to 10 °C for 10 h to 20 h, and desorption drying at 10 °C to 25 °C for 4 h to 8 h to obtain the dried volatile oil composition powder of Shufeng Jiedu Capsules, namely the volatile oil composition of Shufeng Jiedu Capsules.

[0045] In the present invention, the saturated aqueous solution method is to dissolve cyclodextrin in purified water to form a saturated solution, then add the volatile oil of Shufeng Jiedu Capsules, promote inclusion by stirring, cool down to precipitate the inclusion complex, and obtain the product after filtration and drying. The principle of the saturated aqueous solution method is to utilize the solubility difference between the host molecule and the guest molecule in water to form a stable inclusion complex through physical actions (such as van der Waals force, hydrophobic interaction, hydrogen bond, etc.). The freeze-drying method is that cyclodextrin and the volatile oil of Shufeng Jiedu Capsules form an inclusion complex in solution, then the solution is quickly frozen into a solid state, and the ice crystals are removed by sublimation under vacuum conditions to obtain a dried porous product. The principle of the freeze-drying method is to remove water by sublimation at low temperature, retain the porous structure of the inclusion complex, and avoid thermal damage.

[0046] An application of the volatile oil composition of Shufeng Jiedu Capsules provided by the present invention is for preparing a drug against influenza virus. The present invention reduces the cytotoxic activity of the volatile oil of Shufeng Jiedu Capsules and improves its anti-influenza virus activity through the inclusion of cyclodextrin.

[0047] The inventor previously studied by gas chromatograph and found that the volatile oil contents among different batches of Shufeng Jiedu Capsules were inconsistent. Therefore, it is necessary to further optimize the volatile oil composition and preparation process in Shufeng Jiedu Capsules. In the present invention, inclusion is carried out in an aqueous solution state, and the volatile oil molecules increase the collision probability with cyclodextrin through stirring, and thus are included into the hydrophobic cavity of cyclodextrin. The hydrophobic cavity of cyclodextrin wraps the non-polar molecules (such as terpenoids and aromatic compounds) in the volatile oil, and its hydrophilic outer surface makes it soluble in water to form a stable inclusion complex for pharmaceutical use and improve its dispersion uniformity. The inclusion effect of the present invention isolates the volatile oil from contact with light, oxygen and heat and improves its stability.

[0048] Compared with the prior art, the present invention utilizes the cavity of cyclodextrin to include medium-sized molecules in the volatile oil of Shufeng Jiedu Capsule in an aqueous solution state, and further through drying, a solid powder of the inclusion compound of the volatile oil of Shufeng Jiedu Capsule is obtained, effectively improving the water solubility and stability of the volatile oil of Shufeng Jiedu Capsule. In addition, since the inclusion compound is a solid powder, the difficulty of mixing in the preparation process of Shufeng Jiedu Capsule can be reduced, and the uniformity of the volatile oil content among batches can be improved. The inclusion method adopted by the present invention can improve the water solubility, stability and bioavailability of the volatile oil of Shufeng Jiedu Capsule. At the same time, the cytotoxic activity is reduced through the inclusion of cyclodextrin, and the bioavailability is improved, and its anti-influenza virus activity is also significantly improved, having good application prospects and development potential. Description of the Drawings

[0049] Figure 1 Scanning electron micrograph (SEM) of Example 1;

[0050] Figure 2 Fourier transform infrared spectroscopy (FT-IR) of Example 1;

[0051] Figure 3 Thermogravimetric analysis (TGA) of Example 1;

[0052] Figure 4 1H nuclear magnetic resonance spectrum of the volatile oil of Shufeng Jiedu Capsule in Example 1 1 1H NMR);

[0053] Figure 5 1H nuclear magnetic resonance spectrum of the inclusion compound of the volatile oil of Shufeng Jiedu Capsule in Example 1 1 1H NMR);

[0054] Figure 6 Inhibitory effects of Examples 1, 3 and 4 on MDCK cells, (Mean±SD, n = 3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0055] Figure 7 Inhibitory effects of Examples 1, 3 and 4 on the H1N1 virus load in MDCK cells, (Mean±SD, n = 3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Embodiments

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will, in combination with the embodiments of the present invention, clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0057] The test materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0058] For those not specifying specific technologies or conditions in the embodiments, they can all be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions.

[0059] The volatile oil of Shufeng Jiedu Capsules used in the present invention is the volatile oil extracted from Bupleuri Radix and Forsythiae Fructus by steam distillation, and the preparation method is the preparation method of Chaiqiao volatile oil described in the invention patent of CN 118845671A.

[0060] Example 1

[0061] A preparation method of a volatile oil composition of Shufeng Jiedu Capsules, and the prescription is shown in Table 1.

[0062] Table 1 Prescription of Example 1

[0063] Component Dosage Volatile oil of Shufeng Jiedu Capsule 1.0g β-Cyclodextrin 25.9g Purified water 1000g

[0064] Specific preparation process:

[0065] (1) Take the prescribed amount of purified water, heat it to a temperature of 50°C to 60°C, take the prescribed amount of β-cyclodextrin, slowly add it to the purified water, and stir magnetically at 400 rpm until completely dissolved, then cool it to 15°C to 25°C. A saturated aqueous solution of β-cyclodextrin is obtained;

[0066] (2) Take the prescribed amount of the volatile oil of Shufeng Jiedu Capsules, weigh it precisely, and continuously dropwise add it drop by drop to the solution in step (1). After the dropping is completed, at 20°C to 30°C, under the protection of nitrogen gas and in the dark, continue to stir at 400 rpm for 12 h to obtain an aqueous solution of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and β-cyclodextrin.

[0067] (3) In the solution obtained in step (2), place it at a temperature of 4°C and let it stand overnight for 12 h. After the solid precipitates, filter it with a Buchner funnel, discard the filtrate, take the solid on the filter paper, place it in a freeze dryer for drying, pre-freeze it at (-60)°C for 4 h, then carry out sublimation drying at a vacuum degree of 0.05 mbar and -10°C for 15 h, and continue to dry at 15°C for 6 h to obtain the solid powder of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and β-cyclodextrin.

[0068] Example 2

[0069] A preparation method of a volatile oil composition of Shufeng Jiedu Capsules, and the prescription is shown in Table 2.

[0070] Table 2 Prescription of Example 2

[0071] Component Dosage Volatile oil of Shufeng Jiedu Capsule 1.0g β-Cyclodextrin 25.9g Absolute ethanol 5 mL Purified water 100g

[0072] Preparation process:

[0073] (1) Take the prescribed amount of purified water and β-cyclodextrin, and grind them in a mortar until a paste is formed;

[0074] (2) Take the prescribed amount of the volatile oil of Shufeng Jiedu Capsules, weigh it precisely, dissolve it with 5 ml of absolute ethanol, and then continuously dropwise add it to the β-cyclodextrin paste in step (1), and continue to grind for 60 min;

[0075] (3) Place the ground product obtained in step (2) in a vacuum drying oven, set the temperature to 40 °C for drying, and obtain the solid powder of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and β-cyclodextrin.

[0076] Example 3

[0077] A preparation method of a volatile oil composition of Shufeng Jiedu Capsules, and the prescription is shown in Table 3.

[0078] Table 3 Prescription of Example 3

[0079] Component Dosage Volatile oil of Shufeng Jiedu Capsule 1.0g Methyl-β-cyclodextrin 32.9g Add water for injection 100g

[0080] Preparation process:

[0081] (1) Take the prescribed amount of purified water, heat it to a temperature of 50 °C - 60 °C, take the prescribed amount of methyl-β-cyclodextrin, slowly add it to the purified water, stir until completely dissolved, and then cool it to 20 °C ± 5 °C;

[0082] (2) Take the prescribed amount of the volatile oil of Shufeng Jiedu Capsules, weigh it precisely, continuously dropwise add it to the solution in step (1), then fill it with nitrogen, seal it and keep it away from light, and stir at 30 °C ± 5 °C at 600 rpm for 12 h to obtain the aqueous solution of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and methyl-β-cyclodextrin.

[0083] (3) Place the solution obtained in step (2) in a freeze dryer for drying. After pre-freezing at (-60) °C for 4 h, carry out sublimation drying at a vacuum degree of 0.05 mbar and -10 °C for 15 h, and continue to dry at 15 °C for 6 h to obtain the solid powder of the inclusion complex of the volatile oil of Shufeng Jiedu Capsules and methyl-β-cyclodextrin.

[0084] Example 4

[0085] Scanning Electron Microscope (SEM) analysis:

[0086] Appropriately take the volatile oil of Shufeng Jiedu Capsule (SFJDC-EOs), β-cyclodextrin (β-CD), the physical mixture of SFJDC-EOs and β-CD, and select the sample of Example 1, fix them on the sample stage, and perform vacuum gold spraying treatment to improve the conductivity. Observe the surface morphology of the samples through a scanning electron microscope at an accelerating voltage. The results are shown in Figure 1 . The physical mixture is the mixture of SFJDC-EOs and β-cyclodextrin by a vortex shaker. Since there is no inclusion process, SFJDC-EOs cannot enter the cavity of β-cyclodextrin. Therefore, the physical mixture is compared with the inclusion compound to confirm whether SFJDC-EOs enters the cavity of β-cyclodextrin in the example.

[0087] Figure 1 In A, it is the SEM image of SFJDC-EOs, showing an oily structure; Figure 1 In B, it is the SEM image of β-CD, showing a crystalline block or its fragment structure; Figure 1 In C, it is the physical mixture of SFJDC-EOs and β-CD, showing the structure of β-CD and indicating an attached oil layer; Figure 1 In D, it is the sample of Example 1. The structural characteristics of SFJDC-EOs and β-CD both disappear, and a smooth amorphous fragment structure is formed. It can be seen that the volatile oil of Shufeng Jiedu Capsule in Example 1 and β-cyclodextrin act on each other, indicating the formation of an inclusion compound.

[0088] Example 5

[0089] Fourier Transform Infrared Spectroscopy (FT-IR) analysis:

[0090] Use a Fourier transform infrared spectrometer to analyze SFJDC-EOs, β-CD, the physical mixture of SFJDC-EOs and β-CD, and select Example 1. Appropriately take the above samples and mix them well with potassium bromide (KBr) powder, grind and press them into tablets. Set the wavenumber range to 4000 - 400 cm -1 , the number of scans is 32, and the resolution is 2 cm -1 , and the results are shown in Figure 2 .

[0091] The small absorption peak of SFJDC-EOs at 3072 cm -1 is the stretching vibration of aromatic C-H; at 2923 cm -1The strong and broad absorption peak at this position is the C-H stretching vibration of methyl and methylene groups; 1642 cm -1 The absorption peak at this position is the stretching vibration of olefin (C=C); 1464~1366 cm -1 The multiple peaks in this range are the symmetric and asymmetric bending vibrations of C-H of methyl groups, as well as the scissoring vibration of C-H of methylene groups; 882 cm -1 are the in-plane and out-of-plane bending vibrations of alkane C-H. β-CD shows a relatively broad peak at 3365 cm -1 The peak at this position is the stretching vibration of hydroxyl group (O-H); 2930 cm -1 The absorption peak is the stretching vibration of methylene (C-H); at 1646 cm -1 There is an absorption band at this position for the stretching vibration of the hemiacetal carbonyl (C=O) at the 1-position, and the stretching vibration of C-O is in the range of 1158~1029 cm -1 The infrared spectra of the physical mixture of SFJDC-EOs and β-CD simultaneously show the characteristic absorption peaks of SFJDC-EOs and -β-CD, indicating that there is no interaction between the two molecules through simple physical mixing. The infrared spectrum of the sample of the inclusion complex of SFJDC-EOs and -β-CD (Example 1) is similar to that of β-CD, indicating that the molecules of SFJDC-EOs are included in the hydrophobic cavity of β-CD. It shows that SFJDC-EOs and β-cyclodextrin in Example 1 interact to form an inclusion complex.

[0092] Example 6

[0093] Thermogravimetric Analysis (TGA)

[0094] Appropriately take the volatile oil of Shufeng Jiedu Capsule (SFJDC-EOs), β-cyclodextrin (β-CD), the physical mixture of SFJDC-EOs and β-CD, and select the sample of Example 1. Test conditions: aluminum crucible; reference substance: alumina; carrier gas: nitrogen, flow rate 10 mL / min; heating rate 10℃ / min; detection temperature 30~400℃, and the results are shown in Figure 3 .

[0095] The TGA curve of SFJDC-EOs showed weight loss starting at around 30 °C and complete weight loss when reaching around 170 °C, indicating that SFJDC-EOs is volatile and unstable under high temperature conditions. The TGA curve of β-CD showed two weight loss stages, namely 0 - 100 °C and 300 - 350 °C, which were attributed to the volatilization of adsorbed water and the thermal decomposition of β-CD, respectively. The TGA curve of the physical mixture of SFJDC-EOs and β-CD showed three stages. Weight loss started at 30 °C due to the volatilization of volatile oil, and the volatile oil was completely volatilized at 170 °C. The third stage was 300 - 350 °C, which was attributed to the cleavage of β-CD. The TGA of the inclusion complex of SFJDC-EOs and β-CD (Example 1) was similar to that of β-CD, divided into two stages, 0 - 100 °C and 300 - 350 °C, indicating that SFJDC-EOs was included in the cavity of β-CD, improving the thermal stability of SFJDC-EOs. The weight loss at the 0 - 100 °C stage was smaller than that of β-CD, indicating that adsorbed water could be effectively removed by the freeze-drying method. It shows that SFJDC-EOs in Example 1 interacted with β-cyclodextrin to form an inclusion complex.

[0096] Example 7

[0097] Proton nuclear magnetic resonance 1 H-NMR analysis:

[0098] Appropriate amounts of SFJDC-EOs and the sample of Example 1 were taken respectively, using DMSO-d 6 as the solvent, and then 1 H-NMR analysis was carried out.

[0099] In the 1 H NMR spectrum of SFJDC-EOs, the peaks between δ 5.4 ppm and 4.5 ppm were for the double bond; while the peaks in the range of δ 2.5 ppm to δ 0.63 ppm were related to the methyl, methylene and methine structures respectively. The main peaks with chemical shift values of δ 4.604, 4.540, 1.391, 1.367, 1.218 and 0.686 ppm were selected and compared with the inclusion complex of SFJDC-EOs and β-CD (Example 1), and it was found that they all shifted to higher fields, being 4.556, 4.495, 1.336, 1.198, 1.166 and 0.634 ppm respectively, indicating that after inclusion, the hydrogen shielding effect in the molecules of SFJDC-EOs increased, resulting in the shift of chemical shift values. Figure 4 and Figure 5 It shows that SFJDC-EOs interacted with β-cyclodextrin to form an inclusion complex.

[0100] Example 8

[0101] In vitro anti-H1N1 influenza virus activity study:

[0102] (1) Experimental purpose: By establishing a model of H1N1 influenza virus in Madin-Darby canine kidney (MDCK) cells, to explore the in vitro anti-influenza virus activities of volatile oils from Shufeng Jiedu Capsule (SFJDC-EOs), Example 1 and Example 3.

[0103] (2) Experimental materials and test substances: Madin-Darby canine kidney transformed cells (MDCK) and H1N1 influenza virus, obtained from Wannan Medical College; the test substances were volatile oils from Shufeng Jiedu Capsule (SFJDC-EOs), β-cyclodextrin (β-CD), methyl-β-cyclodextrin (Me-β-CD), and the samples of Example 1 and Example 3.

[0104] (3) Reagents and instruments: Oseltamivir phosphate (Guangzhou Baiyunshan Pharmaceutical Co., Ltd.); DMEM (high glucose) medium, premium fetal bovine serum, and penicillin / streptomycin were all purchased from Boshark Biotechnology; CCK-8 kit (Shanghai Beyotime Biotechnology Co., Ltd.); Total RNA rapid extraction reagent (Nanjing Yifeixue Biotechnology Co., Ltd.); Evo M-MLV reverse transcription kit (Hunan Aikerui Biotechnology Co., Ltd.); SYBR Green Permix qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.); FORMA STERI-CYCLE i160 cell culture incubator (Thermo Fisher Scientific); Real-time fluorescence quantitative PCR instrument (Roche, Switzerland); Microplate reader (Tecan, Switzerland).

[0105] (4) Cytotoxicity study: The CCK-8 method was used to determine the effect of the test substances on the viability of MDCK cells. Take MDCK cells in good growth condition and in the logarithmic growth phase, make a cell suspension with DMEM complete medium, count with a cell counting plate, and dilute to 5×10 4 cells / mL. Inoculate 100 μL per well into a 96-well plate and incubate overnight in a 37°C, 5% CO 2 cell culture incubator. After the cells adhered well, aspirate the supernatant, and slowly add the test substances into the 96-well plate. Set 3 replicates for each concentration. After culturing for 24 h, add 10 μL of CCK-8 solution to each well, incubate in the incubator for another 4 h, then detect the OD value of each well at a wavelength of 450 nm with a microplate reader, and calculate the cell viability according to the following formula. The results are shown in Figure 6 .

[0106]

[0107] Figure 6It can be seen that β-CD has no cytotoxicity in the range of 100μg / mL to 2000μg / mL, and has significant cytotoxic activity when it reaches 4000μg / mL, and Me-β-CD shows toxicity when it reaches 8000μg / mL; low concentrations of SFJDC-EOs (12.5μg / mL to 50μg / mL) have no obvious toxicity, and show toxicity when it reaches 100μg / mL, while SFJDC-EOs in samples of Examples 1 and 3 have no toxicity at this concentration, and when it reaches 200μg / mL, Example 3 shows cytotoxicity, so the use of cyclodextrin inclusion SFJDC-EOs can reduce its toxic effects. Among them, DMSO represents the solvent control group, that is, the culture medium containing 0.5% DMSO. Control represents the blank control group, that is, cells without any additional conditions.

[0108] (5) Virus titer determination: MDCK cells growing in a culture dish were digested with trypsin and counted at 3×10 4 MDCK cells were seeded into 96-well cell plates at 35°C and 5% CO. 2 Incubate for 24 hours. Discard the DMEM complete medium in the 96-well plate and wash the cells once with 100 μL PBS per well. Take 1 tube of frozen H1N1 virus allantoic fluid and dilute it 1:100 with virus maintenance solution. Then make a series of half-logarithmic dilutions to finally obtain an influenza virus dilution of 10 -2 , 10 -2.5 , 10 -3 , 10 -3.5 , 10 -4 , 10 -4.5 , 10 -5 , 10 -5.5 , 10 -6 In a 96-well plate, the total volume of each well is 100 μL, and each dilution is repeated for 6 wells. Incubate at 35°C for 1 hour, and shake the 96-well plate once every 30 minutes. Discard the virus dilution in the 96-well plate, and wash the cells once with 100 μL PBS in each well. Add 100 μL of virus maintenance solution to each well, incubate at 35°C for 72 hours, and observe cell lesions every day. Wells with cell lesions are recorded as positive wells, and wells without cell lesions are recorded as negative wells. Calculate the half cell culture infection dose (TCID) of the virus according to the Reed-Muench formula 50 ), the results are shown in Table 4, showing TCID 50 ≈10 -3 / 100μL.

[0109] Table 4 TCID calculated by Reed-Muench method 50 Virus dilution ratio of titer

[0110] Dilution Number of positive Number of negative Cumulative number of positive Cumulative number of negative Ratio Percentage of positive number <![CDATA[10 -2 > 6 0 18 0 18 / 18 100 <![CDATA[10 -2.5 > 5 1 12 1 12 / 13 92 <![CDATA[10 -3 > 1 5 7 6 7 / 13 54 <![CDATA[10 -3.5 > 3 3 6 9 6 / 15 40 <![CDATA[10 -4 > 3 3 3 12 3 / 15 20 <![CDATA[10 -4.5 > 0 6 0 18 0 / 18 0 <![CDATA[10 -5 > 0 6 0 24 0 / 24 0 <![CDATA[10 -5.5 > 0 6 0 30 0 / 30 0 <![CDATA[10 -6 > 0 6 0 36 0 / 36 0

[0111] (6) Anti-H1N1 influenza virus assay: MDCK cells in good growth state and in the logarithmic growth phase were taken and 3×10 5 Cells / well were inoculated in a 12-well plate, 1 mL of cell culture medium was added to each well, and the plate was incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator for 24 h. After the cells were well attached to the wall, the supernatant was discarded and the cells were cultured at 100 TCID 50 / mL infection, calculate the virus stock solution to be inoculated at 100μL / well, and add virus maintenance solution to each well to make up to 1mL. Adsorb in a 35℃ incubator for 2h. After the virus enters the cells, the virus maintenance solution is discarded, and after washing with PBS, blank auxiliary control β-CD and M-β-CD (concentration of 2000μg / mL) and different concentrations of the test substance SFJDC-EOs and sample solutions containing Examples 1 and 3 are added respectively, and an equal volume of virus maintenance solution is added as the model group. Each group has 3 replicate wells and is cultured in a 35℃ incubator. After 24h, the supernatant is discarded and the cells are collected. RNA was extracted, reverse transcribed and RT-qPCR were used to detect the mRNA expression of H1N1 in MDCK cells. Madin-Darby canine kidney-actin was used as the internal reference gene, and 2 was calculated. -△△Ct , the results are shown in Figure 7 .

[0112] The results showed that the blank excipients β-CD and Me-β-CD had no effect on the viral load; the volatile oil of Shufeng Jiedu Capsule (SFJDC-EOs) showed that it had a significant inhibitory effect on the mRNA expression of H1N1 only when it reached 50μg / mL; while Example 1 at 50μg / mL and Example 3 at 12.5-50μg / mL had antiviral activity, and showed a dose-effect relationship. Since SFJDC-EOs has low polarity and low solubility in water, although the solvent DMSO is used to dissolve it when preparing its stock solution, DMSO has high cytotoxicity and must be diluted with water-soluble culture medium before adding MDCK cells, which may cause SFJDC-EOs to precipitate, resulting in uneven distribution in the well plate, affecting its antiviral activity; and after inclusion with β-CD and Me-β-CD, the solubility of SFJDC-EOs in the culture medium is improved, and the solubility of the volatile oil is 0.00018mol·L -1 The solubility of volatile oil after inclusion by β-CD is 0.00036 mol·L -1 The solubility of the volatile oil after inclusion by Me-β-CD is 0.00035 mol·L -1 , which can then fully contact with infected MDCK cells and exert an antiviral effect. Therefore, cyclodextrin inclusion improves the antiviral activity of SFJDC-EOs.

[0113] In addition, after detection, the encapsulation efficiency of the volatile oil encapsulated by β-CD in Example 1 was 103.00 ± 4.86%; the encapsulation efficiency of the volatile oil encapsulated by Me-β-CD in Example 3 was 63.22 ± 2.35%.

[0114] This project is supported by the "Key Research and Achievement Transformation Project" of Wuhu City, and the project number is 2023yf001.

[0115] The descriptions of the above embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A volatile oil composition of Shufengjiedu Capsule, characterized in that: The Shufengjiedu capsule volatile oil composition comprises Shufengjiedu capsule volatile oil and cyclodextrin; The mass ratio of the cyclodextrin to the volatile oil of Shufengjiedu Capsule is 5:1 to 200:

1.

2. The volatile oil composition of Shufengjiedu Capsules according to claim 1, characterized in that: The cyclodextrins include β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, α-cyclodextrin, and γ-cyclodextrin.

3. A method for preparing the Shufengjiedu Capsule volatile oil composition according to claim 1 or 2, characterized in that: The preparation method adopts a saturated aqueous solution method, a grinding method or a freeze-drying method.

4. The preparation method according to claim 3, characterized in that: The method for preparing the volatile oil composition of Shufeng Jiedu Capsules by saturated aqueous solution method is as follows: (1) adding cyclodextrin to purified water, stirring at a constant temperature until the cyclodextrin is completely dissolved, to obtain a cyclodextrin aqueous solution; (2) adding the volatile oil of Shufeng Jiedu Capsule dropwise to the cyclodextrin aqueous solution, and after the addition is completed, stirring at 20° C. to 40° C. under nitrogen protection and in the dark to obtain an aqueous solution of the volatile oil of Shufeng Jiedu Capsule and cyclodextrin inclusion complex; (3) The solution obtained in step (2) is allowed to stand at low temperature, filtered, and the solid is freeze-dried or vacuum-dried to obtain the volatile oil composition of Shufeng Jiedu Capsules.

5. The preparation method according to claim 4, characterized in that: In step (2), the stirring is carried out at a rate of 200-600 rpm for a period of 0.5 h to 24 h.

6. The preparation method according to claim 3, characterized in that: In step (3), the vacuum drying is carried out at a temperature of 40°C to 60°C.

7. The preparation method according to claim 3, characterized in that: The method for preparing the volatile oil composition of Shufeng Jiedu Capsules by grinding is as follows: S1: Add β-cyclodextrin and purified water into a mortar and grind thoroughly to form a β-cyclodextrin paste; S2: adding the volatile oil of Shufeng Jiedu Capsule drop by drop into the β-cyclodextrin paste, grinding, and obtaining the volatile oil of Shufeng Jiedu Capsule and β-cyclodextrin inclusion complex; S3: freeze-drying or vacuum-drying the inclusion compound obtained in step S2 to obtain the volatile oil composition of Shufengjiedu Capsules.

8. The preparation method according to claim 7, characterized in that: In step S2, grinding is continued for 20 min to 120 min.

9. The preparation method according to claim 3, characterized in that: The method for preparing the volatile oil composition of Shufeng Jiedu Capsules by freeze drying is as follows: A. Add cyclodextrin into purified water and stir at a constant temperature until it is completely dissolved to obtain a cyclodextrin aqueous solution; B. continuously adding the volatile oil of Shufeng Jiedu Capsule drop by drop to the cyclodextrin aqueous solution; or, dissolving the volatile oil of Shufeng Jiedu Capsule in ethanol solution, and then continuously adding the volatile oil of Shufeng Jiedu Capsule drop by drop to the cyclodextrin aqueous solution; C. After completing step B, fill with N2 gas, and stir at 200-800 rpm / min for 0.5h-24h at a constant temperature of 10°C-30°C and in the dark to obtain an aqueous solution of the volatile oil of Shufeng Jiedu Capsule and cyclodextrin inclusion complex; D. The solution obtained in step C is freeze-dried to obtain Shufengjiedu Capsule volatile oil composition powder.

10. An application of the Shufengjiedu Capsule volatile oil composition according to claim 1 or 2, characterized in that: Used to prepare drugs against influenza viruses.

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