Preparation process of traditional Chinese medicine compound nanoemulsion

Abstract

The invention discloses a preparation process of a traditional Chinese medicine compound nanoemulsion. The preparation process is characterized by comprising the following steps: S1, weighing the following components in parts by weight: 10-20 parts of chrysanthemum, 10-20 parts of honeysuckle, 10-20 parts of cortex pseudolaricis, 10-20 parts of radix sophorae flavescentis, 10-15 parts of dandelion, 10-15 parts of herba violae, 10-20 parts of wild chrysanthemum flower and 10-15 parts of radix semiaquilegiae; s2, crushing and extracting, namely extracting by adopting a double extraction method, namely firstly extracting by using supercritical CO2 and an entrainer absolute ethyl alcohol, then extracting decoction dregs after extraction once by using a water decoction method, and finally combining with an extracting solution; s3, concentration: according to the mass percentage, concentrating the extracting solution through a vacuum concentration method until each milliliter of the extracting solution is equivalent to 0.5%-2.0% of the raw medicinal materials; s4, preparing nano-emulsion: adding an oil phase, an emulsifier, a co-emulsifier and a water phase into the concentrated solution, performing high-pressure circulating homogenization to obtain oil-in-water type nano-emulsion, and preparing the nano-emulsion by adopting a high-pressure homogenization method. The nanoemulsion is wide in antibacterial spectrum, few in organic solvent residue and high in stability.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation process of a traditional Chinese medicine compound nanoemulsion. BACKGROUND

[0002] As a new drug delivery system, the nanoemulsion has shown wide application advantages in the field of traditional Chinese medicine preparations in recent years. The nanoemulsion is composed of an aqueous phase, an oil phase, a surfactant and a co-surfactant in a certain proportion, and has the characteristics of small particle size, high stability, good biocompatibility and the like. In the traditional Chinese medicine preparation, the nanoemulsion has the following excellent performances: 1. The solubility of active ingredients of traditional Chinese medicines can be significantly improved, so as to solve the problems of low solubility and low bioavailability of traditional Chinese medicines; 2. The nanoemulsion has good targeting and sustained-release properties, can accurately deliver drugs to lesion sites, reduce side effects of drugs in the body and improve treatment effect; 3. The nanoemulsion can enhance the stability of traditional Chinese medicine preparations and prolong the shelf life of drugs.

[0003] At present, as an important breakthrough of modern preparation technology, the traditional Chinese medicine nanoemulsion has shown potential in improving the performance of traditional Chinese medicines, but still has the following core defects, such as stability, and needs to rely on emulsifiers and stabilizers to optimize the formula, but the control difficulty is relatively large; and the nano technology has high requirements for equipment conditions and process, so that the production cost is significantly increased, at present, it is still in the transition stage from laboratory to large-scale production, which restricts the clinical application and popularization. SUMMARY

[0004] The technical problem to be solved by the application is to provide a preparation process of a traditional Chinese medicine compound nanoemulsion, which has less organic solvent residue, high stability and is convenient for large-scale production and popularization.

[0005] In order to achieve the above application purpose, the following technical scheme is adopted: The application provides a preparation process of a traditional Chinese medicine compound nanoemulsion, which comprises the following steps: S1: selecting the following components by weight parts: chrysanthemum (10-20) parts honeysuckle (10-20) parts garcinia xanthachylum (10-20) parts kurchi (10-20) parts dandelion (10-15) parts purple flower (10-15) parts wild chrysanthemum (10-20) parts malvastrum (10-15) parts S2: crushing, extraction: the above-mentioned traditional Chinese medicine compound is first crushed, then sieved, and extracted by double extraction method, i.e. first extracted by supercritical CO2 with a carrier solvent anhydrous ethanol, and the extraction conditions are: extraction time 30 min-90 min, extraction temperature 35℃-50℃, extraction pressure 30 MPa-60 MPa, and carrier solvent flow rate 0.5 m L·min -1 -1.0 m L·min -1 Then the extracted residue is extracted once by decocting with 4-10 times the amount of water, and finally combined with the above extraction liquid; S3: concentration: the above-mentioned extraction liquid is concentrated by vacuum concentration method to 0.5%-2.0% of the original medicinal material per milliliter; S4: preparation of nanoemulsion: In terms of weight parts, the prescription ratio is concentrated liquid (1-10) parts, oil phase (2-10) parts, emulsifier (5-15) parts, co-emulsifier (10-25) parts, and water phase (10-30) parts; high-pressure circulation homogenization to oil-in-water nanoemulsion, and high-pressure homogenization method is used to prepare nanoemulsion, and the high-pressure homogenization pressure is 250bar-350bar, and the circulation is 18-30 times.

[0006] As a further improvement of the present application: the oil phase is one or a mixture of the two selected from isopropyl myristate and ethyl oleate; The emulsifier is one or a mixture of the two selected from poloxamer and EL-35; The co-emulsifier is 1-2 propylene glycol; and the water phase is distilled water.

[0007] As a further improvement of the present application: the extraction time is 60 min, the extraction temperature is 42℃, the extraction pressure is 45 MPa, and the carrier solvent flow rate is 0.7 m L·min -1 .

[0008] As a further improvement of the present application: it further comprises step S5: determining the components chlorogenic acid, luteoloside, baeckea acid and baeckea acid in the nanoemulsion.

[0009] As a further improvement of the present application: the determination method of chlorogenic acid and luteoloside in the nanoemulsion is as follows: Agilent1290 Infinity II ultra-high liquid chromatograph, ACQUITY UPLC BEH C 18 chromatographic column, diode array detector DAD, acetonitrile A-0.1% phosphoric acid aqueous solution B as mobile phase, gradient elution program; flow rate: 0.2 mL / min; column temperature: 25℃; detection wavelength: 348 nm, sample size: 2 μL; The ACQUITY UPLC BEH C 18 Column specification: 2.1 mm x 100 mm, 1.7 μm.

[0010] As a further improvement of the present application: the content determination method of malotil and malotilic acid in nanoemulsion is as follows: Using Shimadzu 20A high performance liquid chromatograph, Agilent Zorbax SB C 18 The chromatographic column is gradient eluted with acetonitrile A-0.09% formic acid B as the mobile phase; the flow rate is 1.0 mL / min; the column temperature is 25℃; the detection wavelength is 262nm; the injection amount is 10 μL; The Agilent Zorbax SB C 18 The column specification is: 4.6mm x 250mm, 5μm.

[0011] Wuwei Xiaodu Yin is from Medical Classics · Surgical Heart Methods, which is composed of honeysuckle, wild chrysanthemum, dandelion, purple flower groundsel and malotil. It has the effects of detoxifying heat, resolving toxins, and resolving and detumescing. It has been widely used in the treatment of acne, dermatitis, eczema and ulcer for many years. Modern pharmacological studies have shown that Wuwei Xiaodu Yin has good anti-inflammatory and immunoregulatory effects on common pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. Because its pharmacological effect is focused on clearing heat and resolving toxins, its dosage form is often made into a traditional dosage form for oral administration. The traditional Chinese medicine compound nanoemulsion of the present application is a new prescription formed by adding and subtracting the basis of Wuwei Xiaodu Yin: chrysanthemum, honeysuckle, malotil, sophora, dandelion, purple flower groundsel, wild chrysanthemum and malotil: Honeysuckle: sweet and cold, clearing and resolving heat and toxin throughout the body, detoxifying core of the prescription, and also considering penetrating into sore and toxin; Sophora, dandelion, purple flower groundsel and wild chrysanthemum: strengthen the power of clearing heat and resolving toxins, and dry dampness Insecticidal and antipruritic, respectively focusing on resolving, analgesic, clearing skin heat and toxin, and synergistically removing fire toxin stagnation; Purple flower groundsel and malotil: clearing heat and resolving toxins + cooling blood and removing stasis, breaking boils and hardening, and guiding drugs to the disease site.

[0012] Malotil: the core effect is to dispel wind and dampness, kill insects and relieve itching, and soften and resolve nodules. When heat and toxin are accompanied by dampness and downward injection, skin exudation (yellow water flow), and itching, this prescription solves the "wet" and "insect" complex symptoms that "Wuwei Xiaodu Yin" cannot completely cover.

[0013] The above traditional Chinese medicine components play a role through multiple pathways such as destroying microbial cell membrane or cell wall, inhibiting protein synthesis, and interfering with energy metabolism, producing a synergistic effect of multiple targets, and not easily developing drug resistance.

[0014] Experiments have verified that traditional five-flavor disinfectant drinks primarily target Escherichia coli and Staphylococcus aureus, and have some effect on Candida albicans, but have no effect on Trichophyton rubrum. The herbal compound nanoemulsion of this invention, through the synergistic effect between its components, exhibits a significant effect against Trichophyton rubrum, and enhanced effects against Staphylococcus aureus, Escherichia coli, and Candida albicans, with a larger inhibition zone and a lower minimum inhibitory concentration, effectively achieving broad-spectrum antibacterial efficacy.

[0015] The traditional Chinese medicine compound provided by this invention can encapsulate lipophilic components in an oil core and dissolve hydrophilic components in an aqueous phase, thereby achieving the co-transportation of effective ingredients. The use of nanoscale particles enables it to penetrate the stratum corneum of the skin more effectively and deliver the drug to the lesion site. It can slowly release the drug to prolong the duration of action and has strong stability. Combined with the preparation process of this nanoemulsion, it achieves less organic solvent residue and is safer than oral use.

[0016] In addition, for information on chrysanthemums and wild chrysanthemums, please refer to the Records of Medicinal Materials and Processed Slices in Part I of the 2020 Pharmacopoeia. Attached Figure Description

[0017] Figure 1 for Figure 1 Diagram showing the size of a nipple droplet.

[0018] Figure 2 The chromatogram of the nanoemulsion sample is shown; where 1. chlorogenic acid; 2. luteolin.

[0019] Figure 3 Chromatogram of nanoemulsion sample; where 1. pyrenoidosa acetic acid and 2. pyrenoidosa formic acid. Detailed Implementation

[0020] The following specific embodiments further illustrate the preparation process of a traditional Chinese medicine compound nanoemulsion provided by the present invention in more detail: I. Related Experiments The following is the preparation process of the traditional Chinese medicine compound nanoemulsion of the present invention, including the following steps: The first step is to select the prescription: The Chinese herbal compound prescription is chrysanthemum (10-20 parts), honeysuckle (10-20 parts), hibiscus bark (10-20 parts), sophora flavescens (10-20 parts), dandelion (10-15 parts), violet (10-15 parts), wild chrysanthemum (10-20 parts), and safflower seed (10-15 parts). The second step, pulverization and extraction: The above-mentioned compound Chinese medicine was first pulverized and then passed through a sieve of No. 1-4. A double extraction method was used, namely, first extraction with supercritical CO2 and anhydrous ethanol as an entrainer, recorded as extraction method ①. The extraction conditions were: extraction time 60 min, extraction temperature 42℃, extraction pressure 45 MPa, and entrainer flow rate 0.7 mL·min. -1; The extraction residue after extraction was decocted with 4-10 times the amount of water once, recorded as extraction method 2, and finally combined with the above extraction liquid; the specifications of No. 1-4 sieve refer to the Chinese Pharmacopoeia.

[0021] The innovation points of this extraction method are: 1. Because the active substances in the prescription are more, there are volatile oils, flavonoids such as syringin, alkaloids such as matrine, organic acids such as phellopterin and chlorogenic acid, and the polarity difference of the components is large. In order to increase the extraction rate of active substances, supercritical CO2 with entrainer anhydrous ethanol is used for extraction, mainly extracting small polarity and medium polarity substances, and water extraction can further extract active substances with large polarity in the residue. The combination of the two extraction methods significantly increases the extraction rate of chlorogenic acid and flavonoid active substances, as shown in Table 1 below; 2. The use of supercritical CO2 with entrainer anhydrous ethanol for extraction is a green extraction method that can effectively reduce the residual organic solvent in the final product and increase the stability of the system. Through the above design, a system with scientificity, innovation and practicality can be formed.

[0022] Table 1 Difference between products extracted by different methods

[0023] Third step, concentration: the above extraction liquid is concentrated by reduced pressure concentration method to 0.5%-2.0% of the original medicinal material per milliliter. Rotary evaporator is used for reduced pressure concentration, membrane filtration and high pressure production; Fourth step, preparation of nanoemulsion: the above concentrated liquid is added with oil phase, emulsifier, co-emulsifier and water phase, and high pressure circulation homogenization is carried out to obtain oil-in-water nanoemulsion. Myristyl isopropylate and ethyl oleate are used as oil phase, poloxamer and EL-35 are used as emulsifier, 1-2 propylene glycol is used as co-emulsifier, and distilled water is used as water phase. High pressure homogenization method is used to prepare nanoemulsion, and the high pressure homogenization pressure is 250-350 bar and the circulation is 18-30 times. The prescription ratio is concentrated liquid 1-10 parts, oil phase 2-10 parts, emulsifier 5-15 parts, co-emulsifier 10-25 parts and water phase 10-30 parts. The innovation points of this extraction method are: 1. Myristyl isopropylate and ethyl oleate are used as oil phase, and myristyl isopropylate and ethyl oleate have the effect of promoting penetration and absorption; 2. Poloxamer is used as emulsifier, which can prolong the action time of the drug and has a sustained-release effect.

[0024] Fifth step, detection of nanoemulsion droplet size and main active substances One, determination of nanoemulsion droplet size The LS-609 laser particle size analyzer of Zhuhai Eurofina Instrument Co., Ltd. is used to detect the droplet size of nanoemulsion, and the results show that more than 95% of the droplet size is within 1-100 nm, as shown in Figure 1 .

[0025] Two, determination of the content of chlorogenic acid and syringin in nanoemulsion Agilent 1290 Infinity II ultra-high performance liquid chromatograph (Qingdao Huapai Keji Instrument Co., Ltd.), ACQUITY UPLC BEH C 18 chromatographic column (specification: 2.1 mm x 100 mm, 1.7 μm), using a diode array detector (DAD), using acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as the mobile phase, using gradient elution procedure; flow rate: 0.2 mL / min. Column temperature 25℃. Detection wavelength is 348 nm, injection volume 2 μL, see Table 2 and Figure 2 .

[0026] Table 2 Ultra-high performance liquid chromatography elution conditions

[0027] Determination of the content of oroxylin A and chrysosplenetin in three nanoemulsions Shimadzu 20A high performance liquid chromatograph, Agilent Zorbax SB C 18 (4.6mm x 250mm, 5μm) chromatographic column, using acetonitrile (A)-0.09% formic acid (B) as the mobile phase (gradient elution); flow rate 1.0 mL / min; column temperature 25℃; detection wavelength 262 nm; injection volume 10 μL, see Table 3 and Figure 3 .

[0028] Table 3 HPLC gradient elution conditions

[0029] Four, test method for determination of antibacterial ability 1. Filter paper disc diffusion method for determination of antibacterial ring diameter The required instruments are sterilized and autoclaved, and then the operation is carried out in a super-clean platform. Agar medium is prepared and the bacterial liquid is evenly smeared on the medium, which is then placed in a constant temperature incubator at 37°C for 24-36 hours to activate the culture. After the first generation of bacteria is obtained, the bacterial colonies are picked up with a inoculation loop and evenly smeared on the same sterile medium, which is then placed in a constant temperature incubator under the same conditions for another 24 hours to obtain the second generation of bacteria. After the second generation of bacteria is obtained, the bacterial colonies are picked up and placed in sterile broth medium, which is then placed in a constant temperature incubator under the same conditions for 16-24 hours to obtain the third generation of bacterial suspension. The bacterial concentration is calculated by viable count method. Filter paper sheets are selected to be 5 mm in diameter, and each sheet is dripped with 20 μL of sample. The negative control is dripped with 20 μL of sterile distilled water, and then dried by baking or natural drying. A test bacterial suspension with a concentration of 5×105 CFU / mL~ 5×106 CFU / mL is prepared, and a sterile cotton swab is used to dip the suspension and smear it on a nutrient agar medium plate containing Staphylococcus aureus, Escherichia coli and Rhodotorula rubra, and on a Sabouraud medium plate containing Candida albicans. Each plate is evenly smeared three times, and each time the plate is rotated by 60°. In each test, one bacterial plate is placed, and each plate is placed with four test sample sheets and one negative control sample sheet. After being cultured in a constant temperature incubator at 37°C for 16-18 hours, the diameter of the inhibition zone is observed and measured with a vernier caliper.

[0030] 2.96-Well Plate Method for Determining Minimum Inhibitory Concentration Prepare a 96-well plate, sample solution and bacterial suspension, wherein the bacterial suspension contains Staphylococcus aureus, Escherichia coli, Rhodotorula rubra and Candida albicans, and the concentration is 5×105 CFU / mL~ 5×106 CFU / mL. The bacterial suspension is prepared by the method described in the first embodiment of the present application. 5 CFU / mL~ 5×10 6 CFU / mL, which refers to the experimental material of the bacterial suspension. 200 μL of sample solution, i.e. Wuwei Disinfecting Beverage and nanoemulsion of the present application, is added to the first column of the 96-well plate, and then 100 μL of culture medium is added to the remaining wells. Using a pipette, the sample solution is diluted with water by a factor of 1:2 from the first column to the last column. 100 μL of bacterial suspension is added to each well to maintain a consistent bacterial concentration. The 96-well plate is placed in an incubator for 24 hours at a suitable temperature and humidity. Observe the bacterial growth and record the lowest sample concentration that can inhibit bacterial growth, which is the MIC value.

[0031] Table 4 Test results of the minimum inhibitory concentration MIC and inhibition zone diameter of the nanoemulsion of the present application on four kinds of bacteria

[0032] Table 5 Test results of the minimum inhibitory concentration and inhibition zone diameter of A-Wuwei Disinfecting Beverage solution and B-nanoemulsion of the present application, respectively

[0033] Note: Dotted line---no obvious bacteriostatic effect.

[0034] From the above table, it can be seen that compared with Wuwei Xiaodu Yin, the inhibition effect of the traditional Chinese medicine compound nanoemulsion provided by the application on Staphylococcus aureus, Escherichia coli, Candida albicans and Trichophyton rubrum is that the minimum inhibitory concentration is obviously reduced, and the inhibition ring is obviously increased. Due to the unexpected synergistic effect between components, the pharmacological effect is more prominent in efficacy, and the infection problem of skin caused by bacteria and fungi can be treated by external use. Combined with the preparation process of the application, the minimum inhibitory concentration is reduced, the inhibition ring is obviously increased, the residual amount of organic solvent is small, and the use is safer.

[0035] Fifth, the stability test of the traditional Chinese medicine compound nanoemulsion of the application 5.1 Dilution experiment: Three equal amounts of the traditional Chinese medicine compound nanoemulsion sample of the application were taken, and distilled water was added to dilute 5 times, 10 times and 20 times, respectively, to observe whether there was a stratification phenomenon after dilution. The results show that when the prepared traditional Chinese medicine compound nanoemulsion of the application is diluted 5 times, 10 times and 20 times, respectively, the solution does not have stratification and demulsification phenomenon, which indicates that it has good stability.

[0036] 5.2 Centrifugation experiment: Ten 10 mL nanoemulsions were taken into clean and dry centrifuge tubes, and then placed in a centrifuge. The centrifuge parameters were set to 4000 r / min for 40 minutes or 6000 r / min for 30 minutes. Then, whether the nanoemulsion sample in the centrifuge tube had stratification and demulsification phenomenon was observed. When the compound nanoemulsion sample of the application was centrifuged at 4000 r / min for 40 minutes or 6000 r / min for 30 minutes, it still remained clear and transparent without stratification. The results show that the traditional Chinese medicine compound nanoemulsion of the application can still maintain good stability under high-speed centrifugation.

[0037] 5.3 Temperature influence experiment of nanoemulsion: The prepared nanoemulsion solution was divided into 5 parts, each part was sealed well, and was placed in a constant temperature condition of-20℃, 0℃, 10℃, 25℃ and 60℃, respectively. The changes of each part at 1 day, 7 days and 15 days were recorded, and the color, odor and stratification stability were observed. The results show that the color and odor of the traditional Chinese medicine compound nanoemulsion have no change, and no stratification phenomenon occurs, indicating that the traditional Chinese medicine compound nanoemulsion is less affected by temperature and has good stability.

[0038] It should be understood that the examples are only used for explaining the present application and are not intended to limit the scope of protection of the present application. In addition, it should also be understood that after reading the technical content of the present application, those skilled in the art can make various modifications, changes and / or variations to the present application, and all these equivalent forms also fall within the protection scope defined by the claims attached to the present application. It is known by common technical knowledge that the present application can be realized by other embodiments without departing from the spirit or essential characteristics thereof. Therefore, the above disclosed embodiments are only examples and are not the only ones. All changes within the scope of the present application or within the scope equivalent to the present application are included in the present application.

Claims

1. A preparation process of a traditional Chinese medicine compound nanoemulsion, characterized in that, Comprising the following steps: S1: selecting the following components by weight parts: Chrysanthemum (10-20) parts Honeysuckle (10-20) parts Fructus Physalis (10-20) parts Sophora (10-20) parts Dandelion (10-15) parts Purple violet (10-15) parts Wild chrysanthemum (10-20) parts Malva sylvestris (10-15) parts S2: crushing, extraction: the above compound of traditional Chinese medicine by weight parts is first crushed, then sieved, and extracted by double extraction method, that is, first extracted by supercritical CO2 with entrainer anhydrous ethanol, and the extraction conditions are: extraction time 30 min-90 min, extraction temperature 35℃-50℃, extraction pressure 30 MPa-60 MPa and entrainer flow rate 0.5 m L·min-1-1.0 m L·min-1, then the extracted residue is extracted once by 4-10 times the amount of water decoction method, and finally combined with the above extract; S3: concentration: the above extract is concentrated by vacuum concentration method to 0.5%-2.0% of the original medicinal material per milliliter; S4: preparation of nanoemulsion: By weight parts, the prescription ratio is concentrated liquid (1-10) parts, oil phase (2-10) parts, emulsifier (5-15) parts, co-emulsifier (10-25) parts, water phase (10-30) parts; High pressure circulation homogenization to oil-in-water nanoemulsion, nanoemulsion is prepared by high pressure homogenization method, the high pressure homogenization pressure is 250bar-350bar, and the circulation is 18-30 times.

2. The preparation process of the traditional Chinese medicine compound nanoemulsion according to claim 1, characterized in that: The oil phase is one or a mixture of two selected from isopropyl myristate and ethyl oleate; The emulsifier is one or a mixture of two selected from poloxamer and EL-35; The co-emulsifier is 1-2 propylene glycol; the water phase is distilled water.

3. The preparation process of the traditional Chinese medicine compound nanoemulsion according to claim 1, characterized in that: Step S2: extraction time 60 min, extraction temperature 42℃, extraction pressure 45 MPa and entrainer flow rate 0.7 m L·min-1.

4. The preparation process of the traditional Chinese medicine compound nanoemulsion according to claim 1, characterized in that Also including step S5: determining the components of nanoemulsion chlorogenic acid, luteoloside, physalinic acid and physalide acetic acid.

5. The preparation process of the traditional Chinese medicine compound nanoemulsion according to claim 4, characterized in that: The determination method of chlorogenic acid and luteoloside in the nanoemulsion is as follows: Agilent 1290 Infinity II ultra-high liquid chromatograph, ACQUITY UPLC BEH C18 chromatographic column, diode array detector DAD, acetonitrile A-0.1% phosphoric acid aqueous solution B as mobile phase, gradient elution program; flow rate: 0.2 mL / min; column temperature 25℃; detection wavelength 348 nm, injection volume 2 μL; The specification of ACQUITY UPLC BEH C18 chromatographic column is: 2.1 mm×100 mm, 1.7 μm.

6. The preparation process of the traditional Chinese medicine compound nanoemulsion according to claim 4, characterized in that: The content determination method of nanoemulsion physalinic acid and physalide acetic acid is as follows: Shimadzu 20A high performance liquid chromatograph, Agilent Zorbax SB C18 chromatographic column, acetonitrile A-0.09% formic acid B as mobile phase gradient elution; flow rate 1.0 mL / min; column temperature 25℃; detection wavelength 262 nm; injection volume 10 μL; The Agilent Zorbax SB C18 chromatographic column has the following specifications: 4.6 mm x 250 mm, 5 μm. The Agilent Zorbax SB C18 chromatographic column has the following specifications: 4.6 mm x 250 mm,

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