CN110664750B

CN110664750B - Radix bupleuri nano preparation, preparation method, detection method and application

Info

Publication number: CN110664750B
Application number: CN201911009813.6A
Authority: CN
Inventors: 宋信莉; 刘�文; 金阳; 汪云霞; 陈欢欢
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2022-03-15
Anticipated expiration: 2039-10-23
Also published as: CN110664750A

Abstract

The invention relates to a radix bupleuri nano preparation which is prepared from the following components in parts by weight: 1.00-3.01 g of oil phase, 0.50-3.00 g of water phase, 4.00-6.76 g of emulsifier and 1.76-4.51 g of co-emulsifier. The preparation method comprises the following steps: weighing the emulsifier, the co-emulsifier and the oil phase according to the formula ratio, placing the mixture into a beaker, uniformly mixing the mixture on a magnetic stirrer at the rotating speed of 500r/min, weighing the water phase, carrying out ultrasonic treatment to completely dissolve the mixture, slowly adding the mixture into the beaker which is stirring to uniformly mix the oil phase and the water phase to obtain the bupleurum root nanoemulsion, and adding pharmaceutically acceptable auxiliary materials into the nanoemulsion to prepare a pharmaceutically acceptable dosage form. The radix bupleuri nanoemulsion is used as a massage medium in the process of massage, is used for treating infantile fever, has obvious antipyretic effect, does not bring discomfort such as redness, swelling and pain to the skin, and has low toxic and side effects.

Description

Radix bupleuri nano preparation, preparation method, detection method and application

Technical Field

The invention relates to the field of medicine invention, in particular to a radix bupleuri nano preparation, a preparation method, a detection method and application.

Background

Fever is a common acute and severe symptom in pediatrics, and the armpit body temperature is higher than 37.3 ℃, namely fever. The disease names of infantile fever in the traditional Chinese medicine literature are tidal fever, dysphoria with smothery sensation, high fever, accumulated heat, bone steaming heat, alternating cold and heat, night fever and fright. Li wenlu, based on the ancient Chinese medical literature and the diagnosis and treatment theory of infantile fever diseases [ D ]. Liaoning traditional Chinese medicine university, 2017 causes the infantile fever to mainly cause external six excesses and internal injuries, and the external six excesses mainly cause wind pathogen, cold pathogen, summer heat, wind cold, wind heat and seasonal warm heat; internal injury is mainly caused by food retention, fetal heat, maltreatment, retained heat of breast milk mother, residual toxicity after illness, fright and the like. The pathogenesis of infantile fever mainly includes deficiency and excess. Deficiency heat includes "yin deficiency with internal heat, yin deficiency with internal heat", "qi failing to return to original qi, yang floating outside" and "weakness after illness". The excess heat is divided into exterior heat, interior heat and half-exterior and half-interior heat. Exterior heat includes "summer-heat pathogen, fumigating the skin surface", "wind-cold attack, yang-qi stagnation"; the interior heat includes "food stagnation, stagnated heat and external eruption", "sudden fright, transformation of five emotions into fire", "heat generation of five zang organs, and fuming to the outside"; the heat in the interior and exterior of the body is the pathogenesis of the gallbladder. Li Yongchun, Zhou Mian Ying, Lu Yin, six meridians dialectical treatment of infantile fever [ J ] in Chinese medicine J2016, 31(01): 156-. ② sweating due to heat, and heat will not be withdrawn due to sweating. And fever with heat in the palms and soles. Fourthly, fever is accompanied with cool palms and soles and abdominal heat. Fifthly, fever with vomiting. Sixthly, fever with dry stool or constipation. And seventhly, fever with diarrhea. Eighty percent febrile convulsion. Ninthly, fever without sweat, perspiration after taking the medicine and fever after heat is subsided but the medicine effect is over-heated.

The traditional Chinese medicine is simple and easy to implement, exact in curative effect and small in side effect when used for treating infantile fever, and is widely applied to pediatric diseases. Li Shiming, Shihua, Fan Zhu Wen, Liyangxian, traditional Chinese medicine for treating infantile fever test case will experience [ J ] Guangming traditional Chinese medicine, 2009,24(01): 128-doped 129. summarized six clinical traditional Chinese medicine defervescence methods, respectively: the traditional Chinese medicine has the effects of relieving fever by acupuncture, relieving fever by massage, relieving fever by external application, relieving fever by enema, relieving fever by oral administration and relieving fever by oral administration.

Infant tuina is a therapeutic method of regulating qi, blood, yin and yang of the zang-fu organs by acupoint, pressing, tui and Na based on the syndrome differentiation theory of traditional Chinese medicine. Indulgence in dreaminess, Liangwengwang, external treatment of traditional Chinese medicine for treating infantile fever general [ J ], J. of pediatrics, 2016,12(04):74-76. methods for treating infantile fever clinically include Kaitian, tuikanggong, Rou Taiyang, Na Fengchi, Rou Feishu, fen Yin Yang and Qingfei Jing. Infant tuina has no toxic or side effects, little pain and is easily accepted by patients, and is one of the main treatment methods in pediatric diagnosis and treatment. Observing clinical curative effects of Zhudongyang, Chinese medicine tuina for treating infantile fever (32 reports in attached cases) [ J ] medical equipment, 2017,30(14):4-5, bringing 64 patients to be diagnosed into a study, randomly dividing the patients into a control group and an experimental group, and carrying out antibiotic infusion treatment on the control group; the experimental group was subjected to TCM tuina based on the control group. The test group shows 23 cases of effectiveness, 8 cases of effectiveness and 1 case of ineffectiveness, and the total effective rate of treatment is 96.9%; the control group has 18 effective cases, 6 effective cases and 8 ineffective cases, and the total effective rate is 75.0 percent; the difference was statistically significant in the two group comparisons.

It is known from the literature that the fever is treated by traditional Chinese medicine massage based on the infusion of antibiotics, the total effective rate is 96.9%, although the treatment effect is better, the treatment needs to be combined with the antibiotics, and the use of the antibiotics has great side effects on children, such as: can cause the side effects of self immune system damage, resistance reduction and the like; in addition, no medium is used in the massage, so that the skin of the child is easily damaged, for example; it is easy to cause the discomfort of red, swelling and pain.

Patent application No. CN201110085917.2 in the prior art, the name of the invention is: the invention discloses an oil-in-water type bupleurum oil nano-emulsion oral liquid and a preparation method thereof, the invention discloses the oil-in-water type bupleurum oil nano-emulsion oral liquid, the particle size of the oral liquid is 1 nm-100 nm, and the oral liquid is prepared by the following raw materials by weight percentage: 20.00 to 40.00 percent of surfactant, 6.00 to 14.00 percent of cosurfactant, 1.00 to 5.00 percent of oil, 0.01 to 10.00 percent of bupleurum oil and the balance of distilled water, wherein the sum of the mass percentages of the raw materials is 100 percent. The bupleurum oil nanoemulsion oral liquid improves the bioavailability of bupleurum oil, enhances the stability of medicines, delays the internal drug replacement time of the bupleurum oil, can effectively avoid various adverse reactions caused by using an injection of the bupleurum oil, has small using amount of auxiliary materials and low production cost, and has wide market prospect in the field of medicines.

The above patent is mainly an oral preparation, improves the bioavailability of the bupleurum oil, enhances the stability of the medicine, delays the drug substitution time of the bupleurum oil in vivo, and can effectively avoid various adverse reactions caused by using the bupleurum oil injection, but the effect of the invention used as a medium in the massage treatment for fever in children is not obvious.

In order to solve the technical problems, the invention develops a radix bupleuri nano preparation, preferably a radix bupleuri nano emulsion.

The nanoemulsion is a transparent or semitransparent liquid medicine carrying system with emulsion droplet particle size of 10-100 nm, and is composed of an oil phase, a water phase, an emulsifier and an auxiliary emulsifier, and the emulsion droplets are uniform in size and mostly spherical. The nanoemulsion can improve the bioavailability and enhance the solubility of insoluble drugs, has the functions of slow release and targeting, can enhance the transdermal absorption of the drugs, and has high safety, thermodynamic stability and low viscosity. The administration route is various, and oral administration, gastrointestinal administration, transdermal administration, topical administration, etc. can be performed. The nano-emulsion can be water-in-oil (W/O), oil-in-water (O/W) and bicontinuous nano-emulsion.

The bupleurum has the main effects of relieving muscles and reducing fever, harmonizing shaoyang, soothing liver and relieving depression, and lifting yang and sinking, and is very common in clinical treatment of fever. The main components of bupleurum root generally comprise saikoside, volatile oil, fatty oil, polysaccharide and the like, and at present, the general research considers that the antipyretic main components of bupleurum root comprise saikoside, volatile oil and sapogenin, wherein the antipyretic effect of the volatile oil and the saponin of bupleurum root is stronger and the dosage is less. The total saikosaponin contains saikosaponin a and saikosaponin d with strongest pharmacological activity.

The invention combines the pharmacological action of the bupleurum volatile oil and the total saponin with the massage action, and utilizes the characteristic of transdermal administration of the nanoemulsion to prepare the O/W type nanoemulsion by taking the bupleurum volatile oil as an oil phase and dissolving the total saponin of the bupleurum in ultrapure water as a water phase, wherein the O/W type nanoemulsion is used as a massage medium during massage and is used for treating infantile fever. The invention determines the essential oil and total saponins of bupleurum root as main effective components through repeated experiments, screens out five groups of prescriptions through early-stage preliminary experiments, screens out the best prescription through drawing a pseudo-ternary phase diagram, and the average grain diameter of the prepared nano-emulsion reaches 77nm and carries out quality evaluation. Transdermal experiments prove that the nanoemulsion can be absorbed through skin. The radix bupleuri nanoemulsion is used as a massage medium in massage, is used for treating infantile fever, has obvious antipyretic effect, does not bring discomfort of red, swelling and pain to the skin, and has low toxic and side effects.

Disclosure of Invention

The invention aims to provide a bupleurum nano preparation.

The other purpose of the invention is to provide a preparation method of the bupleurum nano preparation.

The other purpose of the invention is to provide a detection method of the bupleurum nano preparation;

another purpose of the present invention is to provide an application of the nanometer preparation of radix bupleuri as a massage medium in infant massage for treating fever in infants.

The bupleurum root nano preparation is prepared from the following components in percentage by weight: 1.00-3.01 g of oil phase, 0.50-6.00 g of water phase, 4.00-6.76 g of emulsifier and 1.76-4.51 g of co-emulsifier.

The oil phase of the invention is: bupleuri radix oil; the water phase is: dissolving bupleuri radix total saponin in ultrapure water to obtain water phase; the emulsifier is as follows: any one of Tween-80 and RH-40, preferably Tween-80, and the coemulsifier is: any one of 1, 3-butanediol, 1, 2-propanediol and n-butanol, preferably n-butanol.

The dissolving of the total saikosaponin in ultrapure water specifically comprises the following steps: each 2g of total saikosaponin is dissolved in 20ml of ultrapure water.

The bupleurum root nano preparation is preferably prepared from the following components in parts by weight: tween-804.00-6.76 g, n-butanol 1.76-4.51 g, bupleurum oil 1.00-3.01 g, and bupleurum total saponin 0.50-6.00 g dissolved in 5-30 ml of ultrapure water.

The bupleurum root nano preparation of the invention is further preferably prepared by the following components according to the mixture ratio: tween-806 g, n-butanol 3g, bupleuri radix oil 2g, and bupleuri radix total saponin 2g are dissolved in 20ml ultrapure water.

The preparation method of the bupleurum nano preparation comprises the following steps: weighing the emulsifier, the co-emulsifier and the oil phase according to the formula ratio, placing the mixture into a beaker, uniformly mixing the mixture on a magnetic stirrer at the rotating speed of 500r/min, weighing the water phase, carrying out ultrasonic treatment to completely dissolve the mixture, slowly adding the mixture into the beaker which is stirring to uniformly mix the oil phase and the water phase to obtain the bupleurum root nanoemulsion, and adding pharmaceutically acceptable auxiliary materials into the nanoemulsion to prepare a pharmaceutically acceptable dosage form.

The preparation method of the bupleurum nano preparation with the optimized formula comprises the following steps: weighing Tween-80, n-butanol and bupleuri radix oil according to formula ratio, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, and adding pharmaceutically acceptable adjuvants to prepare pharmaceutically acceptable dosage forms after the nanoemulsion is formed.

The acceptable auxiliary materials are one or more of dextrin, starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose, hydroxypropyl methyl cellulose, polyvinyl alcohol, carbomer, cellulose derivatives, alginate, gelatin or polyvinylpyrrolidone, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose, stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, superfine silica gel powder, talcum powder or magnesium stearate, sodium dodecyl benzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, sorbitan fatty acid or polysorbate (Tween), aspartame, sucralose or saccharin sodium, lactose, glycerol, water and the like.

The pharmaceutically acceptable dosage forms comprise radix bupleuri nanoemulsion, granules, capsules, paste, pills, patches and the like.

The method for detecting the content of the active ingredients in the bupleurum nano preparation comprises the following specific steps:

the method for detecting the content of the saikosaponin A comprises the following specific steps:

chromatographic conditions and System suitability test

A chromatographic column: ZORBAX SB-C18, 5 μm, 4.6 × 250 mm; using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution under the following conditions: 0min, wherein the concentration of acetonitrile is 10-35%; 10min, the concentration of acetonitrile is 36-55%; 20min, wherein the concentration of acetonitrile is 56-70%; 30min, the concentration of acetonitrile is 30 percent, the flow rate is 1ml/min, the column temperature is 22-28 ℃, the detection wavelength is 210nm, and the sample amount is 10 mul;

preparation of control solutions: accurately weighing saikosaponin A5.00 mg, placing in A5 ml volumetric flask, adding methanol, ultrasonically dissolving, diluting to scale, and shaking to obtain saikosaponin A reference solution with mass concentration of 1.00 mg/ml;

preparation of a test solution: taking about 0.05g of radix bupleuri extract, precisely weighing, placing in a 10ml measuring flask, adding methanol, ultrasonically dissolving, diluting to scale, shaking, and filtering with 0.45 μm microporous membrane;

the method for detecting the content of the total saponins of bupleurum comprises the following steps:

preparation of control solutions

Precisely weighing saikosaponin A9.00 mg, placing in a 25ml volumetric flask, adding methanol to obtain solution containing 0.36mg per 1ml, and shaking to obtain reference stock solution;

preparation of test solution

Taking about 0.05g of the bupleurum extract, precisely weighing, placing in a 10ml volumetric flask, adding methanol to dissolve and fix the volume to a scale, and shaking up to obtain the bupleurum extract;

measurement method

Precisely absorbing 0.2mL of each of the test solution and the reference solution, respectively adding 0.10-0.15 mL of 1-2% p-dimethylaminobenzaldehyde ethanol solution, heating at 70 ℃ for 10 minutes, taking out, cooling, adding 4.0-4.5 mL of phosphoric acid, reacting at 65-75 ℃ for 25-35 minutes, cooling, taking methanol as a blank solution, and respectively measuring the absorbance of the test solution and the reference solution at the wavelength of 545 nm.

Preferably, the method for detecting the content of the active ingredients in the bupleurum root nano preparation comprises the following steps:

chromatographic conditions and System suitability test

A chromatographic column: ZORBAX SB-C18, 5 μm, 4.6 × 250 mm; using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution under the following conditions: 0min, the concentration of acetonitrile is 30 percent; 10min, the concentration of acetonitrile is 50%; 20min, wherein the acetonitrile concentration is 60%; 30min, the concentration of acetonitrile is 30%, the flow rate is 1ml/min, the column temperature is 25 ℃, the detection wavelength is 210nm, and the sample injection amount is 10 mu l;

preparation of control solutions

preparation of test solution

measurement method

Precisely absorbing 0.2mL of each of the test solution and the reference solution, respectively adding 0.1mL of 1% p-dimethylaminobenzaldehyde ethanol solution, warming at 70 ℃ for 10 minutes, taking out, cooling, adding 4.0mL of phosphoric acid, reacting at 70 ℃ for 30 minutes, cooling, taking methanol as a blank solution, and respectively measuring the absorbance of the test solution and the reference solution at the wavelength of 545 nm.

The bupleurum nano preparation is applied to children's massage as a massage medium for treating children's fever.

The invention has the beneficial effects that:

1. compared with the prior art:

1) in the prior art, the clinical curative effect observation of Zhubai, Chinese medicine tuina for treating infantile fever (attached 32 reports) [ J ] medical equipment, 2017,30(14):4-5, and the total effective rate of treatment is 96.9%. The literature records that the traditional Chinese medicine massage treatment is carried out on the basis of antibiotic infusion, the total effective rate is 96.9%, although the traditional Chinese medicine massage treatment has good curative effect, the traditional Chinese medicine massage treatment needs to be combined with antibiotic treatment, and the antibiotic use has great side effect on children, such as: can cause the side effects of the destruction of the immune system and the reduction of resistance.

2) In the prior art, the effect of treating fever by simple massage is recorded to a certain extent, but the effect is slow.

3) For fever treatment by massage, no medium or non-nano emulsion medium is used in the massage process, so that the skin of children is easily damaged, for example; it is easy to cause the discomfort of red, swelling and pain.

4) Patent application No. CN201110085917.2 in the prior art, the name of the invention is: the invention discloses an oil-in-water type bupleurum oil nano-emulsion oral liquid and a preparation method thereof, and the oil-in-water type bupleurum oil nano-emulsion oral liquid does not contain bupleurum total saponin, and the content of saikosaponin A is low, so that the effect of using as a medium in the massage treatment for treating infantile fever is not obvious.

2. The invention combines the pharmacological action of the bupleurum volatile oil and the total saponin with the massage action, and utilizes the characteristic of transdermal administration of the nanoemulsion to prepare the O/W type nanoemulsion by taking the bupleurum volatile oil as an oil phase and dissolving the total saponin of the bupleurum in ultrapure water as a water phase.

3. The invention determines the essential oil and total saponins of bupleurum root as main effective components through repeated experiments, screens out five groups of prescriptions through early-stage preliminary experiments, screens out the best prescription through drawing a pseudo-ternary phase diagram, and the average grain size of the prepared nano-emulsion reaches 77 nm. The nano-emulsion is used as a new formulation, can improve the transdermal speed of the medicine, delay the medicine release and improve the bioavailability by transdermal administration, is very suitable as a massage medium, uses the nano-emulsion of the traditional Chinese medicine with the exterior syndrome relieving function as the massage medium, combines the massage manipulation to treat the infantile fever, and has low toxic and side effects and good curative effect.

4. The quality evaluation is carried out on the bupleurum root nanoemulsion, and the specific quality conditions are as follows:

1) a dilution test shows that the prepared bupleurum nano-emulsion is O/W type; the high-speed centrifugation experiment shows that the phenomena of layering, emulsion breaking and the like do not occur after centrifugation, and the color is not changed.

2) The detection of the particle size of the nano-emulsion shows that the distribution range of the particle size of the bupleurum nano-emulsion is narrow, and the particle size distribution is relatively uniform.

3) The nanoemulsion was diluted 10 and 50 times by dilution stability test, and the color of the nanoemulsion gradually faded with the increase of dilution, but it was still clear and transparent.

4) Temperature influence tests show that the materials are taken out after ten days at the temperature of 4 ℃, 25 ℃, 37 ℃ and 60 ℃, and the content and appearance change is less influenced by the temperature.

5) The nano-emulsion is placed for two months at normal temperature, and is sampled and observed in 0 month, 1 month and 2 months, and the result shows that the content change is small in 2 months and the appearance is not influenced.

6) The average content of the total saikosaponin in the bupleurum nano-milk reaches 17.353 mg/ml.

7) The content of saikosaponin A in the bupleurum nano-milk reaches 3.071 mg/ml.

5. The accumulated penetration amount of the radix bupleuri nano-emulsion has a better linear relation with the penetration time, the accumulated penetration amount Q is used for carrying out linear regression on the time t, the obtained Q-t linear equation is Q0.0295 t-0.2544, the r value is 0.995, the accumulated penetration amount within 48h is 1.1548 (mg/square meter), and the transdermal rate constant J (mg/cm) is²H) is 0.0295.

Description of the drawings:

FIG. 1: taking bupleurum oil as an oil phase, Tween80 as an emulsifier, and n-butanol as an auxiliary emulsifier, and taking a pseudo-ternary phase diagram when the Km value is 3: 1.

FIG. 2: taking bupleurum oil as an oil phase, Tween80 as an emulsifier, and n-butanol as an auxiliary emulsifier, and taking a Km value of 2:1 as a pseudo-ternary phase diagram.

FIG. 3: taking bupleurum oil as an oil phase, Tween80 as an emulsifier, and n-butanol as an auxiliary emulsifier, and taking a pseudo-ternary phase diagram when the Km value is 1: 1.

FIG. 4: oil phase: bupleurum oil and emulsifier: tween80, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km ═ 3: 1.

FIG. 5: oil phase: bupleurum oil and emulsifier: tween80, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km ═ 2: 1.

FIG. 6: oil phase: bupleurum oil and emulsifier: tween80, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km 1: 1.

FIG. 7: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km ═ 3: 1.

FIG. 8: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km ═ 2: 1.

FIG. 9: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 3-butanediol, Km 1: 1.

FIG. 10: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for n-butanol, Km 3: 1.

FIG. 11: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for n-butanol, Km 2: 1.

FIG. 12: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for n-butanol, Km 1: 1.

FIG. 13: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 2-propanediol Km ═ 3: 1.

FIG. 14: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 2-propanediol Km 2: 1.

FIG. 15: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: pseudo-ternary phase diagram for 1, 2-propanediol Km 1: 1.

FIG. 16: linear relationship of total saponins of radix bupleuri

FIG. 17: standard curve and linear range

FIG. 18: method for identifying nano-emulsion structure by dyeing method

FIG. 19: nanoemulsion particle size detection No. 1

FIG. 20: nanoemulsion particle size detection No. 2

FIG. 21: nanoemulsion particle size detection No. 3

FIG. 22: nanoemulsion dilution experiment No. 1

FIG. 23: nanoemulsion dilution experiment No. 2

FIG. 24: nanoemulsion dilution experiment No. 3

FIG. 25: cumulative amount of penetration in transdermal experiments

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

Example 1 formulation: tween-806.02 g, n-butanol 3.00g, bupleuri radix oil 2.02g, and bupleuri radix total saponin 0.50g are dissolved in 5ml ultrapure water.

Example 2 formulation: tween-806.00 g, n-butanol 3.00g, bupleuri radix oil 2.02g, and bupleuri radix total saponin 1.00g are dissolved in 10ml ultrapure water.

Example 3 formulation: tween-806.01 g, n-butanol 3.01g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 1.51g are dissolved in 15.1ml ultrapure water.

Example 4 formulation: tween-806.00 g, n-butanol 3.00g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 2.00g by dissolving in 20ml ultrapure water.

Example 5 formulation: tween-806.01 g, n-butanol 3.01g, bupleuri radix oil 2.02g, and bupleuri radix total saponin 2.50g by dissolving in 25ml ultrapure water.

Example 6 formulation: tween-806.02 g, n-butanol 3.00g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 3.00g are dissolved in 30ml ultrapure water.

Example 7 formulation: tween-806.75 g, n-butanol 2.25g, bupleuri radix oil 1.00g, and bupleuri radix total saponin 2.00g by dissolving in 20ml ultrapure water.

Example 8 formulation: tween-806.00 g, n-butanol 2.00g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 2.00g by dissolving in 20ml ultrapure water.

Example 9 formulation: tween-805.26 g, n-butanol 1.76g, bupleuri radix oil 3.01g, and bupleuri radix total saponin 2.50g by dissolving in 25ml ultrapure water.

Example 10 formulation: tween-806.00 g, n-butanol 3.00g, bupleuri radix oil 1.00g, and bupleuri radix total saponin 1.50g by dissolving in 15ml ultrapure water.

Example 11 formulation: tween-805.34 g, n-butanol 2.68g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 1.50g by dissolving in 15ml ultrapure water.

Example 12 formulation: tween-804.68 g, n-butanol 2.34g, bupleuri radix oil 3.00g, and bupleuri radix total saponin 2.00g by dissolving in 20ml ultrapure water.

Example 13 formulation: tween-804.50 g, n-butanol 4.51g, bupleuri radix oil 1.01g, and bupleuri radix total saponin 3.50g are dissolved in 35ml ultrapure water.

Example 14 formulation: tween-804.00 g, n-butanol 4.00g, bupleuri radix oil 2.00g, and bupleuri radix total saponin 4.50g are dissolved in 45ml ultrapure water.

The formulations of examples 1 to 14 were prepared according to the preparation method of any of examples 15 to 20

Example 15 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix the oil phase and the water phase, and obtaining the bupleuri radix nanoemulsion.

Example 16 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/5 starch or dextrin, mixing uniformly, drying, and granulating to obtain granules.

Example 17 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula ratio, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/5 starch or dextrin, mixing uniformly, drying, pulverizing into fine powder, and encapsulating to obtain capsule.

Example 18 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula ratio, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotation speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the stirring beaker to uniformly mix the oil phase and the water phase to obtain bupleuri radix nanoemulsion, decocting and concentrating to obtain paste.

Example 19 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/6 starch or dextrin, mixing uniformly, drying, pulverizing into fine powder, and making into pill.

Example 20 preparation method: weighing Tween-80, n-butanol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/10 starch or dextrin, mixing uniformly, coating the bupleuri radix nanoemulsion on a coating machine, and then laminating with a medical viscose layer to obtain the patch.

Example 21 formulation: tween-806.75 g, 1, 3-butanediol 2.26g, bupleuri radix oil 1.01g, and bupleuri radix total saponin 3.00g by dissolving in 30ml ultrapure water.

Example 22 formulation: tween-806.00 g, 1, 3-butanediol 3.30g, bupleuri radix oil 1.01g, and bupleuri radix total saponin 3.50g by dissolving in 35ml ultrapure water.

EXAMPLES 21-22 formulations were prepared according to any of the methods of preparation of examples 23-28

Example 23 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotating speed of 500r/min, weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the stirring beaker, and mixing uniformly the oil phase and the water phase to obtain the bupleuri radix nanoemulsion.

Example 24 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing total saikosaponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/5 starch or dextrin, uniformly mixing, drying, and granulating to obtain granules.

Example 25 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing total saikosaponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/5 starch or dextrin, uniformly mixing, drying, pulverizing into fine powder, and encapsulating to obtain capsules.

Example 26 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the stirring beaker to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, and decocting and concentrating into paste to obtain the ointment.

Example 27 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the stirring beaker to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/6 starch or dextrin, uniformly mixing, drying, pulverizing into fine powder, and making into pill.

Example 28 preparation method: weighing Tween-80, 1, 3-butanediol and bupleuri radix oil according to formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing total saikosaponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain a bupleuri radix nanoemulsion, adding 1/10 starch or dextrin, uniformly mixing, coating the bupleuri radix nanoemulsion on a coating machine, and then laminating with a medical viscose layer to obtain the patch.

Example 29 formulation: RH-406.76 g, 1, 3-butanediol 2.25g, bupleurum oil 1.00g, bupleurum total saponin 5.00g are dissolved in 50ml of ultrapure water.

Example 30 formulation: RH-406.00 g, 1, 3-butanediol 2.02g, bupleurum oil 2.01g, bupleurum total saponin 6.00g are dissolved in 60ml of ultrapure water.

Example 31 formulation: RH-406.00 g, 1, 3-butanediol 3.02g, bupleurum oil 1.00g, bupleurum total saponin 5.00g are dissolved in 50ml of ultrapure water.

EXAMPLES 29-31 preparation according to any of the preparations of examples 32-37

Example 32 preparation method: weighing RH-40, 1, 3-butanediol and bupleuri radix oil according to the formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the stirring beaker, and uniformly mixing the oil phase and the water phase to obtain the bupleuri radix nanoemulsion.

Example 33 preparation method: weighing RH-40, 1, 3-butanediol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nanoemulsion, adding 1/5 starch or dextrin, uniformly mixing, drying and granulating to obtain granules.

Example 34 preparation method: weighing RH-40, 1, 3-butanediol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nanoemulsion, adding 1/5 starch or dextrin, uniformly mixing the materials, drying, crushing the mixture into fine powder, and filling the fine powder into capsules to obtain capsules.

Example 35 preparation method: weighing RH-40, 1, 3-butanediol and bupleuri radix oil according to the formula ratio, placing into a beaker, uniformly mixing on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the stirring beaker to uniformly mix the oil phase and the water phase to obtain a bupleuri radix nanoemulsion, and decocting and concentrating into a paste to obtain the ointment.

Example 36 preparation method: weighing RH-40, 1, 3-butanediol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nanoemulsion, adding 1/6 starch or dextrin, uniformly mixing the mixture, drying, crushing the mixture into fine powder, and preparing pills.

Example 37 preparation method: weighing RH-40, 1, 3-butanediol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleurum total saponin, dissolving the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain a bupleurum nanoemulsion, adding 1/10 starch or dextrin into the bupleurum nanoemulsion, uniformly mixing the mixture, coating the bupleurum nanoemulsion on a coating machine, and laminating the mixture with a medical viscose layer to obtain the patch.

Example 38 formulation: RH-406.73 g, n-butanol 2.26g, bupleurum oil 1.02g, bupleurum total saponin 2.50g dissolved in 25ml ultrapure water.

Example 39 formulation: RH-406.00 g, n-butanol 2.00g, bupleurum oil 2.00g, bupleurum total saponin 3.00g dissolved in 30ml ultrapure water.

Example 40 formulation: RH-406.00 g, n-butanol 3.00g, bupleurum oil 1.00g, bupleurum total saponin 2.00g dissolved in 20ml ultrapure water.

Example 41 formulation: RH-405.34 g, n-butanol 2.68g, bupleurum oil 2.03g, bupleurum total saponin 3.00g dissolved in 30ml ultrapure water.

Example 42 formulation: RH-404.51 g, n-butanol 4.51g, bupleurum oil 1.02g, bupleurum total saponin 2.50g dissolved in 25ml ultrapure water.

Example 43 formulation: RH-404.00 g, n-butanol 4.02g, bupleurum oil 2.01g, and bupleurum total saponin 2.50g are dissolved in 25ml of ultrapure water.

EXAMPLES 38-43 preparation according to any one of preparation examples 44-49

Example 44 preparation method: weighing RH-40, n-butanol and bupleuri radix oil according to the formula, placing in a beaker, uniformly mixing on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the beaker under stirring to uniformly mix the oil phase and the water phase, thus obtaining the bupleuri radix nanoemulsion.

Example 45 preparation method: weighing RH-40, n-butanol and bupleurum oil with the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleurum total saponin, dissolving the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nano-emulsion, adding 1/5 starch or dextrin, uniformly mixing, drying and granulating to obtain granules.

Example 46 preparation method: weighing RH-40, n-butanol and bupleurum oil with the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, additionally weighing bupleurum total saponin, dissolving the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nano-emulsion, adding 1/5 starch or dextrin, uniformly mixing the mixture, drying, crushing the mixture into fine powder, and filling the fine powder into capsules to obtain capsules.

Example 47 preparation method: weighing RH-40, n-butanol and bupleuri radix oil according to the formula, placing in a beaker, uniformly mixing on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the stirring beaker to uniformly mix the oil phase and the water phase to obtain bupleuri radix nanoemulsion, decocting and concentrating to obtain paste.

Example 48 preparation method: weighing RH-40, n-butanol and bupleuri radix oil according to the formula, placing in a beaker, uniformly mixing on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, performing ultrasonic treatment to completely dissolve, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, adding 1/6 starch or dextrin, uniformly mixing, drying, pulverizing into fine powder, and making into pill.

Example 49 preparation method: weighing RH-40, n-butanol and bupleurum oil with the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, additionally weighing bupleurum total saponin, dissolving the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water to obtain bupleurum nano-emulsion, adding 1/10 starch or dextrin into the bupleurum nano-emulsion, uniformly mixing the mixture, coating the bupleurum nano-emulsion on a coating machine, and then laminating the mixture with medical viscose glue to obtain the patch.

Example 50 formulation: RH-406.76 g, 1, 2-propylene glycol 2.26g, bupleurum oil 1.00g, bupleurum total saponin 3.00g are dissolved in 30ml of ultrapure water.

EXAMPLE 50 preparation of any one of preparation methods of examples 51 to 56

Example 51 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the mixture into a beaker, uniformly mixing the mixture on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to dissolve the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the stirring beaker, and uniformly mixing the oil phase and the water phase to obtain the bupleurum nanoemulsion.

Example 52 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the mixture into a beaker, uniformly mixing the mixture on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nanoemulsion, adding 1/5 starch or dextrin, uniformly mixing, drying and granulating to obtain granules.

Example 53 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nano-emulsion, adding 1/5 starch or dextrin, uniformly mixing the mixture, drying, crushing the mixture into fine powder, and filling the fine powder into capsules to obtain capsules.

Example 54 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to dissolve the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the stirring beaker to uniformly mix the oil phase and the water phase to obtain a bupleurum nano-emulsion, and decocting and concentrating the bupleurum nano-emulsion into a paste to obtain the ointment.

Example 55 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the materials into a beaker, uniformly mixing the materials on a magnetic stirrer at the rotating speed of 500r/min, weighing bupleurum total saponin to be dissolved in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix oil and water phases to obtain bupleurum nano-emulsion, adding 1/6 starch or dextrin, uniformly mixing the mixture, drying, crushing the mixture into fine powder, and preparing pills to obtain pills.

Example 56 preparation method: weighing RH-40, 1, 2-propylene glycol and bupleurum oil according to the formula ratio, placing the mixture into a beaker, uniformly mixing the mixture on a magnetic stirrer at the rotating speed of 500r/min, further weighing bupleurum total saponin, dissolving the bupleurum total saponin in ultrapure water, carrying out ultrasonic treatment to completely dissolve the bupleurum total saponin, slowly adding the mixture into the beaker which is stirring to uniformly mix two phases of oil and water to obtain a bupleurum nanoemulsion, adding 1/10 starch or dextrin into the bupleurum nanoemulsion, uniformly mixing the mixture, coating the bupleurum nanoemulsion on a coating machine, and then laminating the mixture with a medical adhesive layer to obtain the patch.

The product prepared in the above example was tested according to any of the test methods of examples 57-59:

example 57

chromatographic conditions and System suitability test

A chromatographic column: ZORBAX SB-C18, 5 μm, 4.6 × 250 mm; using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution under the following conditions: 0min, acetonitrile concentration 10%; 10min, the concentration of acetonitrile is 36%; 20min, wherein the acetonitrile concentration is 56%; 30min, the concentration of acetonitrile is 30%, the flow rate is 1ml/min, the column temperature is 22 ℃, the detection wavelength is 210nm, and the sample injection amount is 10 mu l;

preparation of control solutions

preparation of test solution

measurement method

Precisely absorbing 0.2mL of each of the test solution and the reference solution, respectively adding 0.10mL of 1% p-dimethylaminobenzaldehyde ethanol solution, warming at 70 ℃ for 10 minutes, taking out, cooling, adding 4.0mL of phosphoric acid, reacting at 65 ℃ for 25 minutes, cooling, taking methanol as a blank solution, and respectively measuring the absorbance of the test solution and the reference solution at the wavelength of 545 nm.

Example 58

chromatographic conditions and System suitability test

A chromatographic column: ZORBAX SB-C18, 5 μm, 4.6 × 250 mm; using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution under the following conditions: 0min, the concentration of acetonitrile is 35 percent; 10min, the concentration of acetonitrile is 55%; 20min, wherein the acetonitrile concentration is 70%; 30min, the concentration of acetonitrile is 30%, the flow rate is 1ml/min, the column temperature is 28 ℃, the detection wavelength is 210nm, and the sample injection amount is 10 mu l;

preparation of control solutions

preparation of test solution

measurement method

Precisely absorbing 0.2mL of each of the test solution and the reference solution, respectively adding 0.15mL of 2% p-dimethylaminobenzaldehyde ethanol solution, warming at 70 ℃ for 10 minutes, taking out, cooling, adding 4.5mL of phosphoric acid, reacting at 75 ℃ for 35 minutes, cooling, taking methanol as a blank solution, and respectively measuring the absorbance of the test solution and the reference solution at the wavelength of 545 nm.

Example 59

chromatographic conditions and System suitability test

preparation of control solutions

preparation of test solution

measurement method

The radix bupleuri nanoemulsion is mainly applied to infant massage as a massage medium for treating the infantile fever.

In order to further verify the feasibility of the invention, the inventor carries out a series of experiments, and because the products of various formulations are prepared after the radix bupleuri nanoemulsion is prepared, the inventor mainly provides the relevant experimental method and data of the radix bupleuri nanoemulsion:

first, experimental design basis and technical route

1. Product research and development thought and purpose

Fever can occur in the process of various diseases of children, and safe and effective defervescence is needed for treating various diseases of children. For treating infantile fever, infant tuina is extremely effective, and has the characteristics of simplicity, easy learning, low price, quick effect, green therapy and the like, so that infant tuina has the advantages of irreplaceable medicine. Infant tuina meets the requirements of "non-invasive medicine" and "natural therapy", and is increasingly being regarded by researchers. To improve the curative effect of tuina, it is important to select and use the medium reasonably except the skilled manipulation.

The nano-emulsion is used as a new formulation, can improve the transdermal speed of the medicine, delay the medicine release and improve the bioavailability by transdermal administration, and is very suitable to be used as a massage medium. In treating infantile fever, infant tuina is very effective, and tuina media with antipyretic action are used in tuina to enhance the action of bringing down fever. The bupleurum has the main effects of relieving muscles and reducing fever, harmonizing shaoyang, soothing liver and relieving depression, and lifting yang and sinking, and is very common in clinical treatment of fever. The main components of bupleurum root generally contain saikoside, volatile oil, fatty oil, polysaccharide and the like, and the bupleurum root antipyretic main components are generally considered to be saikoside, volatile oil and sapogenin by the current general research. Wherein, the bupleurum volatile oil and the bupleurum total saponin have stronger antipyretic effect and less dosage.

The total saikosaponin and the volatile saikooil can be used for delaying the release of the medicine and improving the bioavailability, and the nanoemulsion prepared from the total saikosaponin and the volatile saikooil can be used as a massage medium and can be used for effectively treating the infantile fever.

2. Formulation of

2.1 screening of formulations

The exterior-releasing Chinese medicinal bupleurum is selected, which has an excellent antipyretic effect, and the antipyretic components of the bupleurum are mainly bupleurum volatile oil and bupleurum total saponin. The saponins are mainly researched by panax notoginseng saponins and ginsenoside, the two saponins have good transdermal effect, and transdermal research on saikosaponin is not reported. In the experiment, the volatile oil of the bupleurum can be used as the oil phase of the nano-emulsion, the total saponins of the bupleurum are water-soluble components, and the total saponins of the bupleurum can be dissolved in water to be used as the water phase, so that the bupleurum is selected reasonably.

2.2 formulation basis

The nanoemulsion consists of an oil phase, a water phase, an emulsifier and an auxiliary emulsifier, and the bupleurum volatile oil and the bupleurum total saponin have antipyretic effects. The oil phase adopts volatile oil of radix bupleuri, the total soap of radix bupleuri is dissolved in water as water phase, the emulsifier is any one of O/W type emulsifier Tween-80 and RH-40, the auxiliary emulsifier is any one of 1, 3-butanediol, 1, 2-propanediol and n-butanol, and the O/W type nanoemulsion is prepared by adopting a water phase inversion method.

3. Formulation screening

The nano-emulsion has the advantages of extremely small particle size of 1-100 nm, and has the advantages of transdermal administration, rapid skin penetration, slow release, delayed drug release and prolonged drug action time, so the nano-emulsion is selected as a massage medium and is very effective in treating infantile fever.

Second, nanoemulsion prescription screening

1. Laboratory apparatus and reagent

1.1 Experimental instruments

Model T09-1S magnetic stirrer (shanghai selec instruments ltd); a beaker; a glass rod; an electronic balance; a rubber head dropper; measuring cylinder

1.2 reagent

Bupleurum oil (Jiangxi Hencui spice source manufacturer, batch number: 20170701); tween-80 (chemical purity, Chongqing Jiangchuan chemical Co., Ltd., batch No. 20170519); RH40 (Beijing Kull chemical technology, batch No. Y636871J); span-80 (laboratory reagents, Guangzhou chemical reagents, lot # 20150101-01); n-butanol (Shanghai Shenbo chemical Co., Ltd., lot number: 150310); 1, 3-butanediol (Dow Akoda chemical Co., Ltd., batch No. 201707121); 1, 2-propanediol (Shinshough chemical Co., Ltd., batch No. 151121); absolute ethanol (Chongqing Chuandong chemical Co., Ltd., batch No. 20150501); glycerol (Shanghai Shenbo chemical Co., Ltd., lot number: 150301); ultrapure water, the remainder was analytical grade.

In the experiment, the volatile oil of the bupleurum is fixed as an oil phase, and the bupleurum nanoemulsion is prepared by screening and optimizing an emulsifier and an auxiliary emulsifier. Widely used Tween80, span80 and RH40 are selected as emulsifiers; the method comprises the steps of taking glycerin with low toxicity, 1, 3-butanediol, 1, 2-propanediol, n-butanol and ethanol as auxiliary emulsifiers, primarily screening a prescription, drawing a pseudo-ternary phase diagram by a titration method, investigating the influence of emulsifiers, auxiliary emulsifiers, oil phases, Km values and the like in each system on a nano-emulsion region, and finally determining a group of optimal prescriptions by comparing the phase behaviors of each system with the area of the formed nano-emulsion region. By observing the appearance characteristics, observing the form of the nanoemulsion by adopting a transmission electron microscope, measuring the particle size and the particle size distribution of the nanoemulsion by utilizing a laser particle size analyzer, and evaluating the stability of the nanoemulsion, the physicochemical properties of the nanoemulsion are investigated.

The preparation method of the nano-emulsion adopts a phase inversion method, namely adding the emulsifier and the co-emulsifier into an oil phase, uniformly stirring, slowly dripping a water phase (ultrapure water) into a mixed system while stirring, wherein the system is a clear oil phase with low viscosity at the beginning, the system becomes turbid after adding water, when the water is added to a critical point, the system suddenly becomes very viscous and transparent, and is difficult to stir, and the system is a liquid crystal phase or a gel phase at the moment; continuously dripping water and continuously stirring, and when the water is added to a certain amount, the system becomes thin suddenly and returns to a clear state; forming the oil-in-water type nano-emulsion.

The operation method comprises the following steps: dissolving bupleuri radix total saponin in water phase, placing emulsifier, co-emulsifier, and bupleuri radix oil in a beaker, stirring, slowly adding the water phase dissolved in bupleuri radix total saponin while stirring, observing and recording.

2. Screening of blank prescription

2.1 screening auxiliary emulsifier by taking span80 as emulsifier

Weighing 6g of emulsifier and 3g of co-emulsifier in a beaker, uniformly stirring, adding 0.5g of bupleurum oil, and uniformly stirring to ensure that the emulsifier is: auxiliary emulsifier: the ratio of the bupleurum oil is 6:3:0.5, about 50ml of distilled water is slowly added while stirring, and the phenomenon is observed and recorded. The results are shown in Table 1.

TABLE 1 Span80 formation of nanoemulsions with different coemulsifiers

And (3) test results: as can be seen from the above table, when span80 is used as an emulsifier, it cannot form a nanoemulsion with co-emulsifiers such as glycerin, 1, 3-butylene glycol, 1, 2-propylene glycol, n-butanol, ethanol, etc.

2.2 screening of coemulsifiers with Tween80 as emulsifier

Weighing 6g of emulsifier and 3g of co-emulsifier in a beaker, uniformly stirring, adding 0.5g of bupleurum oil, and uniformly stirring to ensure that the emulsifier is: auxiliary emulsifier: the ratio of the bupleurum oil is 6:3:0.5, about 50ml of distilled water is slowly added while stirring, and the phenomenon is observed and recorded. The results are shown in Table 2.

TABLE 2 conditions of Tween80 with different co-emulsifiers to form a nanoemulsion

And (3) test results: as can be seen from the above table, when tween80 is used as an emulsifier, the nanoemulsion can be formed by the tween80, glycerol, 1, 3-butanediol, 1, 2-propanediol and n-butanol, and the nanoemulsion cannot be formed by the tween80 and ethanol.

2.3 screening of Co-emulsifier with RH40 as emulsifier

Weighing 6g of emulsifier and 3g of co-emulsifier in a beaker, uniformly stirring, adding 0.5g of bupleurum oil, and uniformly stirring to ensure that the emulsifier is: auxiliary emulsifier: the ratio of the bupleurum oil is 6:3:0.5, about 50ml of distilled water is slowly added while stirring, and the phenomenon is observed and recorded. The results are shown in Table 3.

TABLE 3 case of nanoemulsion formation with RH40 and different coemulsifiers

And (3) test results: as can be seen from the above table, RH40 can form a nanoemulsion with glycerol, 1, 3-butylene glycol, 1, 2-propylene glycol, and n-butanol, but cannot form a nanoemulsion with ethanol.

2.4 further screening of blank prescription

Weighing 6g of emulsifier and 3g of co-emulsifier in a beaker, uniformly stirring, adding 1g of bupleurum oil, and uniformly stirring to ensure that the emulsifier is: auxiliary emulsifier: the ratio of the bupleurum oil is 6:3:1, 50ml of distilled water is slowly added while stirring, and the phenomenon is observed and recorded. The results are shown in tables 4 and 5.

TABLE 4 case of nanoemulsion formed by Tween80 and different co-emulsifiers after increasing the content of bupleurum oil

TABLE 5 nanoemulsion formation with RH-40 and different co-emulsifiers after increasing the Bupleurum oil content

And (3) test results: tween80 can form nano-emulsion with 1, 3-butanediol, 1, 2-propanediol and n-butanol, and can not form nano-emulsion with glycerol and ethanol. RH40 can form nanometer emulsion with glycerol, 1, 3-butanediol, 1, 2-propylene glycol and n-butanol, and can not form nanometer emulsion with ethanol.

Weighing 6g of emulsifier and 3g of co-emulsifier in a beaker, stirring uniformly, adding 1g of bupleurum oil, stirring uniformly, weighing 1g of bupleurum total saponin, dissolving in 50ml of distilled water, slowly adding the distilled water in which the bupleurum total saponin is dissolved into the beaker, and stirring while adding to ensure that the emulsifier: auxiliary emulsifier: bupleurum oil: the ratio of total saikosaponin was 6:3:1:1, and the phenomenon was observed and recorded. The results are shown in tables 6 and 7.

TABLE 6 case of nanoemulsion formed by Tween-80 and different co-emulsifiers after adding total saikosaponin

TABLE 7 nanoemulsion formation with RH40 and different co-emulsifiers after addition of total saikosaponin

And (3) test results: the Tween80 can form nano-emulsion with 1, 3-butanediol, Tween80 and n-butanol, RH40 and 1, 3-butanediol, RH40 and 1, 2-propanediol, RH40 and n-butanol. The rest can not form nano-emulsion.

3. Pseudo ternary phase diagram screening prescription

5 groups of blank prescriptions are screened out through preliminary experiments, and are respectively as follows: the 5 blank prescriptions can form the nanoemulsion by the Tween80, 1, 3-butanediol, Tween80, n-butanol, RH40, 1, 3-butanediol, RH40, 1, 2-propanediol and RH 40. The experiment adopts a water drop method pseudo-ternary phase diagram to determine an optimal blank prescription, and the larger the range of a nano-emulsion area in the phase diagram is, the higher the stability of a nano-emulsion system is. Thus, the optimal formulation components and optimal proportions of the nanoemulsion are determined by comparing the size of the regions forming the nanoemulsion.

3.1 pseudo-ternary phase diagram drawing of emulsifier system with Tween80 as emulsifier and n-butanol as co-emulsifier

A first group: oil phase: bupleurum oil and emulsifier: tween80, co-emulsifier: n-butanol

Bupleurum oil is used as an oil phase, Tween80 is used as an emulsifier and n-butyl alcohol is used as a co-emulsifier, and Km values of 3:1, 2:1 and 1:1 are respectively and uniformly mixed to form the mixed emulsifier. The mixed emulsifier and the oil phase were weighed in the mass ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), respectively, placed on a magnetic stirrer and stirred at a constant temperature, titrated with ultrapure water while stirring, the state change during titration was observed, and the water amount was recorded. A pseudo-ternary phase diagram was drawn using Origin 8.0 software. The results are shown in tables 8, 9 and 10, and fig. 1,2 and 3.

TABLE 8 results of pseudo-ternary phase diagram plotting at 3:1 Km (first group)

And (3) test results: when Km is 3:1, (emulsifier and co-emulsifier): the ratio of the oil phase is 9:1, 8:2 and 7:3, and the nano emulsion can be formed.

TABLE 9 results of pseudo-ternary phase diagram plotting with Km of 2:1 (first group)

And (3) test results: when Km is 2:1, (emulsifier and co-emulsifier): the ratio of the oil phase is 9:1, 8:2 and 7:3, and the nano emulsion can be formed.

TABLE 10 results of pseudo-ternary phase diagram plotting at 1:1 Km (first group)

And (3) test results: when Km is 1:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1 and 8:2, the nano-emulsion can be formed.

3.2 pseudo-ternary phase diagram drawing of emulsifier system with Tween80 as emulsifier and 1, 3-butanediol as co-emulsifier

Second group: oil phase: bupleurum oil and emulsifier: tween80, co-emulsifier: 1, 3-butanediol

The bupleurum oil is used as an oil phase, Tween80 is used as an emulsifier and 1, 3-butanediol is used as a co-emulsifier, and Km values of 3:1, 2:1 and 1:1 are respectively and uniformly mixed to form the mixed emulsifier. The mixed emulsifier and the oil phase were weighed in the mass ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), respectively, placed on a magnetic stirrer and stirred at a constant temperature, titrated with ultrapure water while stirring, the state change during titration was observed, and the water amount was recorded. A pseudo-ternary phase diagram was drawn using Origin 8.0 software. The results are shown in tables 11, 12 and 13. See fig. 4, 5, 6.

TABLE 11 results of pseudo-ternary phase diagram plotting at a Km of 3:1 (second group)

And (3) test results: when Km is 3:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1, the nano-emulsion can be formed. The rest of the components can not form the nano-emulsion.

TABLE 12 results of pseudo-ternary phase diagram plotting at 2:1 Km (second group)

And (3) test results: when Km is 2:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1, the nano-emulsion can be formed. The rest of the components can not form the nano-emulsion.

TABLE 13 results of pseudo-ternary phase diagram plotting 1:1 Km (second group)

And (3) test results: when Km is 1:1, the nanoemulsion cannot be formed.

3.3 pseudo-ternary phase diagram of emulsifier system using Rh40 as emulsifier and 1, 3-butanediol as co-emulsifier

Third group: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: 1, 3-butanediol

Bupleurum oil is used as an oil phase, Rh40 is used as an emulsifier, 1, 3-butanediol is used as a co-emulsifier, and Km values of 3:1, 2:1 and 1:1 are respectively and uniformly mixed to form a mixed emulsifier. The mixed emulsifier and the oil phase were weighed in the mass ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), respectively, placed on a magnetic stirrer and stirred at a constant temperature, titrated with ultrapure water while stirring, the state change during titration was observed, and the water amount was recorded. A pseudo-ternary phase diagram was drawn using Origin 8.0 software. The results are shown in Table 14, Table 15, Table 16, FIG. 7, FIG. 8, and FIG. 9.

TABLE 14 results of pseudo-ternary phase diagram plotting at 3:1 Km (third group)

And (3) test results: when Km is 3:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1 and 8:2, the nano-emulsion can be formed.

TABLE 15 results of pseudo-ternary phase diagram plotting 2:1 Km (third group)

TABLE 16 results of pseudo-ternary phase diagram plotting at 1:1 Km (third group)

And (3) test results: when Km is 1:1, no nanoemulsion can be formed.

3.4 pseudo-ternary phase diagram drawing of emulsifier system with Rh40 as emulsifier and n-butanol as co-emulsifier

And a fourth group: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: n-butanol

Bupleurum oil is used as an oil phase, Rh40 is used as an emulsifier and n-butyl alcohol is used as a co-emulsifier, and Km values of 3:1, 2:1 and 1:1 are respectively and uniformly mixed to form the mixed emulsifier. The mixed emulsifier and the oil phase were weighed in the mass ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), respectively, placed on a magnetic stirrer and stirred at a constant temperature, titrated with ultrapure water while stirring, the state change during titration was observed, and the water amount was recorded. A pseudo-ternary phase diagram was drawn using Origin 8.0 software. The results are shown in tables 17, 18 and 19. See fig. 10, 11, 12.

TABLE 17 results of pseudo-ternary phase diagram plotting with Km of 3:1 (fourth group)

And (3) test results: when Km is 3:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1 and 8:2, the nano-emulsion can be formed. The rest of the components can not form the nano-emulsion.

TABLE 18 results of pseudo-ternary phase diagram plotting at 2:1 Km (fourth group)

And (3) test results: when Km is 2:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1 and 8:2, the nano-emulsion can be formed. The rest of the components can not form the nano-emulsion.

TABLE 19 results of pseudo ternary phase diagram plotting 1:1 Km (fourth group)

And (3) test results: when Km is 1:1, (emulsifier and co-emulsifier): when the proportion of the oil phase is 9:1 and 8:2, the nano-emulsion can be formed. The rest of the components can not form the nano-emulsion.

3.5 pseudo-ternary phase diagram drawing of emulsifier System with Rh40 as emulsifier and 1, 2-propylene glycol as co-emulsifier

And a fifth group: oil phase: bupleurum oil and emulsifier: rh40, co-emulsifier: 1, 2-propanediol

Bupleurum oil is used as an oil phase, Rh40 is used as an emulsifier, 1, 2-propylene glycol is used as a co-emulsifier, and Km values of 3:1, 2:1 and 1:1 are respectively and uniformly mixed to form a mixed emulsifier. The mixed emulsifier and the oil phase were weighed in the mass ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), respectively, placed on a magnetic stirrer and stirred at a constant temperature, titrated with ultrapure water while stirring, the state change during titration was observed, and the water amount was recorded. A pseudo-ternary phase diagram was drawn using Origin 8.0 software. The results are shown in tables 20, 21 and 22. Fig. 13, 14, 15.

TABLE 20 results of pseudo-ternary phase diagram plotting at 3:1 Km (fifth group)

TABLE 21 results of pseudo-ternary phase diagram plotting 2:1 Km (fifth group)

And (3) test results: when Km is 2:1, no nanoemulsion can be formed.

TABLE 22 results of pseudo-ternary phase diagram plotting 1 Km 1:1 (fifth group)

And (3) test results: when Km is 1:1, no nanoemulsion can be formed.

3.6 screening of drug-containing prescription

The experiments show that the optimum blank prescription is obtained when Tween-80 is used as an emulsifier, n-butanol is used as an auxiliary emulsifier, and the Km value is 2: 1. Fixing the result as final blank prescription, adding bupleuri radix total saponin with different weight into 20ml ultrapure water, and screening out the optimal medicine-containing prescription according to the dissolving condition of bupleuri radix total saponin in water and whether nanoemulsion can be formed. The results are shown in Table 23.

Table 23 screening results of drug-containing prescription

And (3) test results: if the total saikosaponin is added too little, the medicine content is insufficient, if too much, the total saikosaponin is difficult to dissolve in water, so the optimal prescription screened finally is tween-806 g, n-butanol 3g, bupleurum oil 2g, total saikosaponin 2g dissolved in ultrapure water, and the others are feasible prescriptions.

3.7 preparation method

Weighing tween-806 g, n-butanol 3g, and bupleuri radix oil 2g, placing in a beaker, mixing uniformly on a magnetic stirrer at a rotation speed of 500r/min, further weighing bupleuri radix total saponin 2g, dissolving in 20ml of ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix the oil phase and the water phase.

Thirdly, measuring the content of the bupleurum total saponin in the bupleurum extract

1. Laboratory apparatus and reagent

1.1 instruments

An ultraviolet-visible spectrophotometer (UV-5900 Shanghai chromatography Instrument Co., Ltd.); model HH-S4 electric heating constant temperature water bath (beijing wei Yongxing instruments ltd); 202-3AB type electrothermal constant temperature drying oven (Tester instruments, Inc. of Tianjin); model SK8200H ultrasonic cleaner (shanghai koku ultrasonic instruments ltd); JA2003 electronic balance (Shunhyu constant science instruments, Inc., Shanghai); microanalysis balance (AE 240); 10ml stoppered test tubes; a beaker; a volumetric flask; a test tube clamp; a pipette; a rubber head dropper.

1.2 reagent

Saikosaponin A (Guizhou Di big biological science and technology, Limited liability company, batch number: 20736-09-8, purity:noless than 98%); radix bupleuri total saponin (Haoyu farmer professional cooperative, batch number: HYHZS 171023); methanol (chromatographically pure, national pharmaceutical group chemical reagents, Inc., batch No. 20170303); p-dimethylaminobenzaldehyde (Mimi Chemicals, Inc., Tianjin, Inc., lot number 20170110); phosphoric acid (Chongqing Chuandong chemical Co., Ltd., batch No.: 20160401); the rest are analytically pure.

2. Establishment of method for measuring content of total saikosaponin

2.1 preparation of control solutions

Precisely weighing saikosaponin A9.00 mg, placing in a 25ml volumetric flask, adding methanol to obtain solution containing 0.36mg per 1ml, and shaking to obtain reference stock solution.

2.2 preparation of test solutions

Taking about 0.05g of the bupleurum extract, precisely weighing, placing in a 10ml volumetric flask, adding methanol to dissolve and fix the volume to a scale, and shaking up to obtain the Chinese medicinal preparation.

2.3 measurement method

2.4 determination of the detection wavelength

Taking 0.2ml of the stock solution of the reference substance, adding 0.1ml of 1% p-dimethylaminobenzaldehyde ethanol solution, shaking up, reacting at 70 ℃ for 10 minutes, taking out, cooling down, adding 4.0ml of phosphoric acid, shaking up, reacting at 70 ℃ for 30 minutes, taking out, cooling down, scanning the wavelength within the range of 200-800 nm, and determining 545nm as the detection wavelength because the maximum absorption peak exists at 545 nm.

2.5 Standard Curve and Linear Range

Taking the reference solution, precisely sucking 0.6ml, 1.2ml, 2.5ml, 3.75ml and 5ml into a 5ml measuring flask, adding methanol to a constant volume to scale, shaking up, precisely sucking 0.2ml to 10ml test tubes with plugs, adding 0.1ml of 1% p-dimethylaminobenzaldehyde ethanol solution, shaking up to 70 ℃ water bath for 10min, taking out, cooling down, adding 4.0ml of phosphoric acid, shaking up, reacting in a 70 ℃ constant temperature water bath for 30min, taking out, cooling down, and measuring the absorbance at 545 nm. Taking the concentration of the reference substance as the abscissa and the absorbance as the ordinate,and drawing a standard curve. R²0.9936. The results are shown in Table 24, FIG. 16.

TABLE 24 Standard Curve and Linear Range

2.6 precision

Accurately sucking saikosaponin a reference substance solution (0.36mg/ml), measuring according to color development condition under '2.3', continuously measuring for six times, wherein RSD is 0.71%, and indicating that the precision of the instrument is good. The results are shown in Table 25.

TABLE 25 precision test results

2.7 stability

Precisely sucking 0.2ml of the same test solution, measuring according to the color development condition under the item of '2.3', measuring once every 10min, and obtaining the result that the test solution is stable within 80min and the RSD is 1.93%. The results are shown in Table 26.

TABLE 26 stability test results

2.8 repeatability

Taking 6 parts of total saikosaponin, precisely weighing, preparing test solution in parallel according to the method under item 2.2, and measuring according to the color development condition under item 2.3, wherein the average value of the results is 0.212, and the RSD is 2.16%. The results are shown in Table 27.

TABLE 27 results of the repeatability tests

2.9 sample application recovery test

Accurately weighing six samples with known content, accurately adding appropriate amount of saikosaponin A reference solution (1mg/ml), preparing test solution in parallel according to the method under item 2.2, and measuring according to the color development condition under item 2.3, wherein the average sample recovery rate of saikosaponin is 100.15%, which indicates that the accuracy meets the requirement. The results are shown in Table 28.

TABLE 28 sample recovery test results

2.10 assay

Taking about 0.03g of bupleurum total saponin, precisely weighing, placing in a 10ml measuring flask, adding methanol, ultrasonically dissolving, and fixing the volume to the scale. The measurement was carried out as described above. The results are shown in Table 29.

TABLE 29 results of content measurement

Fourthly, determining saikosaponin A in the bupleurum extract by HPLC method

1. Laboratory apparatus and reagent

1.1 Experimental instruments

A high performance liquid chromatograph; a chromatographic column; a sample injection needle; 0.45 μm microporous filter membrane; centrifuging the tube; a volumetric flask; syringe with a needle

1.2 reagent

Saikosaponin A; total saponins of radix bupleuri; methanol

1.3 chromatographic conditions and System suitability test

A chromatographic column: ZORBAX SB-C18(5 μm, 4.6 × 250 mm); using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution under the following conditions: 0min, the concentration of acetonitrile is 30 percent; 10min, the concentration of acetonitrile is 50%; 20min, wherein the acetonitrile concentration is 60%; 30min, and the concentration of acetonitrile is 30 percent. The flow rate was 1ml/min, the column temperature was 25 ℃, the detection wavelength was 210nm, and the sample size was 10. mu.l.

1.4 preparation of control solutions: accurately weighing saikosaponin A5.00 mg, placing in A5 ml volumetric flask, adding methanol, ultrasonically dissolving, diluting to scale, and shaking to obtain saikosaponin A reference solution with mass concentration of 1.00 mg/ml.

1.5 preparation of test solution: taking about 0.05g of radix bupleuri extract, precisely weighing, placing in a 10ml measuring flask, adding methanol, ultrasonically dissolving, diluting to scale, shaking, and filtering with 0.45 μm microporous membrane.

1.6 examination of the Linear relationship

Taking saikosaponin A about 5mg, precisely weighing, placing in a 5ml volumetric flask, adding methanol for ultrasonic dissolution, diluting to scale, precisely weighing 1ml reference substance solution in the 5ml volumetric flask, and adding methanol to scale. Respectively injecting samples of 1 muL, 5 muL, 10 muL, 15 muL and 20 muL, determining according to chromatographic conditions under the item of '1.3', and performing linear regression by taking a peak area as a vertical coordinate and taking the content of a reference substance as a horizontal coordinate to obtain a regression equation: y is 371540x-6209.3 and r is 0.9998. The results are shown in Table 30, FIG. 17.

TABLE 30 Linear relationship examination of saikosaponin A

1.7 precision test

Respectively and precisely sucking saikosaponin A (1mg/ml), determining according to chromatographic condition under '1.3', and continuously sampling for six times, wherein the peak area RSD of saikosaponin A is 2.43%, which indicates that the precision of the instrument is good. The results are shown in Table 31.

TABLE 31 precision test results

1.8 stability test

Sampling the same sample solution for 0, 1,2, 3, 4, 5, 6, 7 and 8 hours respectively, and determining for nine times, wherein the peak area RSD of the saikosaponin A in the sample solution is 1.90%, which indicates that the sample solution is stable within 8 hours. The results are shown in Table 32.

TABLE 32 stability test results

1.9 repeatability test

Respectively and precisely weighing six parts of the same total saikosaponin, each part is about 0.05g, and measuring according to the chromatographic condition under the item of 1.3, wherein the result shows that the peak area RSD of saikosaponin A in the test solution is 1.23 percent, which indicates that the method has good repeatability. The results are shown in Table 33.

TABLE 33 repeatability test results

1.10 sample recovery test

Precisely weighing 9 parts of sample powder, each part is about 0.025g, adding a proper amount of reference substance solution into nine samples respectively, preparing test sample solution according to a test sample preparation method, measuring according to chromatographic conditions under the item of 1.3, and obtaining results shown in table 34, wherein the average sample adding recovery rate of the saikosaponin A is 98.26%, and the RSD is 1.83%. The sample adding recovery rate measurement result meets the relevant requirements.

TABLE 34 sample recovery test results

1.11 measurement of content

Three samples, each about 0.05g, were taken and assayed according to the chromatographic conditions under "1.3". The results are shown in Table 35.

TABLE 35 results of content determination test

Fifthly, evaluating the quality of the nanoemulsion

1. Laboratory apparatus and reagent

1.1 Experimental instruments

A micro-nano laser particle size analyzer; a high performance liquid chromatograph; a constant temperature magnetic stirrer; a centrifuge; HWHS-250 type constant temperature and humidity incubator (Rihua instruments Co., Ltd., Tan city) and centrifuge tube

1.2 reagent

Bupleuri radix nanoemulsion and ultrapure water

2. Quality evaluation

2.1 external form

The nanoemulsion is a yellow brown clear transparent liquid

2.2 identification of the type of Structure

2.2.1 dyeing method

Placing two parts of radix bupleuri nano emulsion with the same volume in a beaker, simultaneously and respectively dropwise adding one drop of Sudan red III and one drop of methylene blue dye, standing, and observing the diffusion speed and appearance change of the red dye and the blue dye in the nano emulsion. The Sudan red III dye is oil-soluble dye and is easy to diffuse in an oil phase; methylene blue is a water-soluble dye and easily diffuses in the water phase. If the diffusion speed of red is greater than that of blue, the nano-emulsion is W/O type, and if the diffusion speed of blue is greater than that of red, the nano-emulsion is O/W type. The results show that the two color developing agents have different diffusion conditions after being simultaneously dropped into a sample, and the blue diffusion speed is obviously higher than the red diffusion speed, thereby showing that the prepared bupleurum nano-emulsion is O/W type nano-emulsion. See fig. 18.

2.2.2 dilution method

The emulsion is usually diluted with a liquid having the same properties as the external phase. The O/W type nanoemulsion immediately disperses after being dropped into water, but the W/O type nanoemulsion cannot immediately disperse after being dropped into water, and the result shows that the bupleurum root nanoemulsion immediately disperses after being dropped into ultrapure water, is still clear and transparent after being diluted, and the bupleurum root nanoemulsion prepared on the surface is O/W type.

2.3 high speed centrifugation experiment

Taking a proper amount of the bupleurum nano-emulsion in a proper centrifugal tube, centrifuging for 30 minutes at the rotating speed of 8000r/min, observing whether the phenomena of layering, emulsion breaking and the like occur, and repeating the experiment for three times. The results show that the centrifugal separation and demulsification are not generated after centrifugation, and the color is not changed.

2.4 detection of nanoemulsion particle size

The particle size can be distinguished from ordinary milk and nano-emulsion, wherein nano-emulsion is formed when the particle size is 10-100 nm, and sub-microemulsion is formed when the particle size is 100-1000 nm. PDI measures the homogeneity of the dispersion of the nano-emulsion particles, and a laser particle size analyzer is used for measuring the particle size distribution of the nano-emulsion. The bupleuri radix nanoemulsion has the average particle size of 77.21nm and PDI of 0.407. The results show that the bupleurum root nanoemulsion has narrower particle size distribution range and more uniform particle size distribution. The results are shown in Table 36, FIGS. 19, 20 and 21.

TABLE 36 nanoemulsion particle size measurement results

2.5 dilution stability test

The nanoemulsion was diluted 10 and 50 times, and the color of the nanoemulsion gradually faded with the increase of the dilution factor, but it was still clear and transparent. Measuring the particle size of the product by a laser particle size analyzer, wherein the particle size is reduced after 10 times of dilution, but the fluctuation is small and the product is relatively stable; after being diluted by 50 times, the particle size is greatly increased and is relatively unstable. The results are shown in Table 37, FIGS. 22, 23 and 24.

TABLE 37 nanoemulsion dilution stability test results

2.6 temperature Effect test

Preparing a part of radix bupleuri nanoemulsion, taking the appearance and the content of the prepared radix bupleuri nanoemulsion as blank controls, dividing the same part of nanoemulsion into four parts, respectively placing the four parts at the conditions of 4 ℃, 25 ℃, 37 ℃ and 60 ℃, taking out the nanoemulsion after ten days, and observing the content and the change of the appearance. The results are shown in Table 38.

TABLE 38 temperature Effect test results

2.7 sample Retention Observation experiment

And (3) placing the nano-emulsion at normal temperature for two months, sampling and observing in 0 month, 1 month and 2 months, and determining the content change of the nano-emulsion.

The results are shown in Table 39.

TABLE 39 sample Retention Observation test results

2.8 determination of the content of total saikosaponin in nanoemulsion

Precisely sucking 0.2ml of radix bupleuri nanoemulsion, placing in a 10ml volumetric flask, adding methanol to a constant volume to a scale, ultrasonically dispersing uniformly to prepare a test solution, and performing content determination according to the method under the item 2.3. The results are shown in Table 40.

TABLE 40 determination of Total saikosaponin content

4.9 determination of saikosaponin A content in nanoemulsion

Preparing three parts of radix bupleuri nanoemulsion, placing 1ml of each part of radix bupleuri nanoemulsion into a 10ml volumetric flask, adding methanol to a constant volume to a scale, performing ultrasonic treatment to uniformly disperse the mixture to obtain a sample solution, and performing content measurement according to the high performance liquid chromatography. The results are shown in Table 41.

TABLE 41 measurement results of saikosaponin A content

Sixth, percutaneous absorption experiment

1. Laboratory apparatus and reagent

1.1 Experimental instruments

Model RYJ-12B transdermal drug diffusion tester (Shanghai yellow sea drug testing apparatus Co., Ltd.), and high performance liquid chromatograph.

1.2 reagent

Normal saline and bupleurum nano-emulsion.

2. Treatment of nitrocellulose membranes

The nitrocellulose membrane was placed in a beaker, added with an appropriate amount of isopropyl myristate, soaked overnight, taken out, and wiped dry with filter paper.

3. Measuring the cumulative permeation quantity by a transdermal diffusion instrument

Spreading nitrocellulose membrane between the supply chamber and the receiving chamber, and fixing with matched clamp, transdermal diffusion area is 2.8m²And the receiving liquid is physiological saline water, the temperature in the water tank is set to be 37 ℃, 7ml of receiving liquid and a stirrer are added into the receiving pool, bubbles on the liquid surface are removed, the receiving liquid is fully contacted with the nitrocellulose membrane, 2ml of radix bupleuri nano-emulsion is added into the supply chamber after the temperature is stabilized at 37 ℃, and the upper end of the receiving liquid is sealed by sealing glue. The magnetic stirrer is started and the rotating speed is controlled to be 300 r/min. Taking 1ml of receiving solution at 2h, 4h, 6h, 8h, 10h, 12h, 24h, 28h, 30h, 32h, 36h and 48h respectively. And supplementing new receiving solution, and filtering the obtained receiving solution with 0.45 μm microporous membrane to obtain sample solution, and performing content measurement according to the above established method. The cumulative permeation amount thereof was calculated.

The formula is a calculation formula of the cumulative permeation quantity (Q) per unit area, wherein: a is the effective diffusion area (2.8 square meters in the experiment), V is the volume (7ml) of the receiving chamber, Vo is the sampling volume (1ml), Ci is the concentration of the drug in the receiving liquid measured at the ith sampling point (i is less than or equal to n-1), and Cn is the concentration of the drug in the receiving liquid during the sampling. Linear regression is carried out on the time t by the accumulated penetration Q, and the slope of the obtained Q-t linear equation is the transdermal rate constant J (mg/cm)²H). The results are shown in Table 42, FIG. 25.

TABLE 42 cumulative permeation test results

TABLE 43 in vitro transdermal permeation kinetics parameters of saikosaponin A in Bupleurum nanoemulsion

And (3) test results:

as can be seen from tables 42 and 43, the cumulative permeation amount of the bupleurum root nanoemulsion has a better linear relation with the permeation time, the cumulative permeation amount Q is used for carrying out linear regression on the time t, the obtained Q-t linear equation is Q-0.0295 t-0.2544, the r value is 0.995, the cumulative permeation amount in 48h is 1.1548 (mg/square meter), and the transdermal rate constant J (mg/cm) is²H) is 0.0295.

Seven and radix bupleuri nanoemulsion with antipyretic effect on body temperature of dry yeast pyrogenic rat

1 materials of the experiment

1.1 Experimental instruments

An electronic thermometer; an electronic analytical balance; a microplate reader (MuL7ISKAN SKY); an EP tube; a micro-pipetting gun; an ultra-low temperature refrigerator; high speed centrifuge (Kate TD 5B).

1.2 drugs and reagents

Bupleurum nano-emulsion (self-made); aspirin enteric-coated tablet (Bayer medicine Bao, batch number: Chinese medicine standard J20130078); high yeast Anqi high activity dry yeast (Anqi Yeast Co., Ltd.); physiological saline (Guizhou Konlun pharmaceutical Co., Ltd., batch No. H20033939); distilled water; chloral hydrate (national chemical group, Inc., lot number: 1335-2006); liquid paraffin (Chongqing Chuanjiang chemical Co., Ltd., batch No.: Q/CCH 012-; cAMP kit (Nanjing institute of bioengineering); AVP kit (Nanjing institute of bioengineering).

1.3 Experimental animals

Wistar rat, SPF grade, female rat, 180-200 g

2 method of experiment

2.1 animal groups

Wistar rats were randomly divided into 6 groups: normal control group, model control group, positive control group (aspirin group), high, medium and low dosage group of radix bupleuri nanoemulsion, and comparative example group (patent application No. CN201110085917.2), and weighing after marking. The feed is adaptively fed for 3 days at normal temperature before the experiment, and the anal temperature is measured 3 times every day by a thermometer at intervals of 1 h.

2.2 establishment of fever animal model

And (3) fixing the rat on the same day of molding, coating paraffin on a probe of the electronic thermometer, inserting the probe into the rectum of the rat for 4cm, measuring the body temperature for 1 time per hour, continuously measuring for 3 times, and selecting animals with the body temperature of 37.0-38.5 ℃ and the body temperature change of not more than 0.4 ℃ for experimental molding, wherein 6 animals are selected for each group. Except for the blank control group, rats in other groups are injected with 10mL/kg of 15% dry yeast suspension subcutaneously on the back to prepare a pathologic model of the febrile rat.

2.3 administration of drugs

The body temperature of each group of rats was measured 4h after pyrogenicity and administered in the following manner: normal control group: 10ml/kg physiological saline; model control group: 10ml/kg physiological saline; aspirin group: 0.1 g/kg; comparative example group: 14g/kg, and the four groups are administered by intragastric administration; the high (12.0g/kg) to medium (6.0g/kg) and low (3.0g/kg) dose groups of the bupleurum nanoemulsion are as follows: removing the abdominal hair of the rat, and smearing the high, medium and low dosage bupleuri radix nanoemulsion on the abdominal part of the corresponding group of rats. Body temperature was measured 1h after the first dose, and at the same time, the dose was the same as before.

2.4 Observation of fever rat body temperature Change

Body temperature was measured once each 1h, 2h, 3h, 4h after the first administration. The antipyretic effect of the radix bupleuri nanoemulsion is researched by observing the influence of various groups of medicines on the body temperature of a rat at various time intervals after pyrogenicity.

2.5 Collection of rat serum

After the temperature is measured for the last time, 10% chloral hydrate is injected into the abdominal cavity to anaesthetize the rat, after anaesthetizing, the abdominal aorta is used for taking blood, after standing for 1h, the centrifugation is carried out for 10min at 3000r/min, and the supernatant is collected and stored in a refrigerator at minus 80 ℃ for standby.

2.6 measurement of cAMP and AVP levels in rat serum

ELISA is used for measuring the content of cAMP and AVP in rat serum, and the specific operation steps are carried out according to the operation requirements of the kit.

2.7 statistical methods

The experimental data are expressed as "mean ± standard deviation" and were statistically analyzed using GraphPad Prism 4 statistical software. Statistical analysis was performed using one-way anova, which was considered to be significantly different when P was < 0.05.

3 results

3.1 Observation of fever rats on temperature Change

The body temperature change of rats in each group is shown in table 44, and compared with a normal control group, the temperature rise of the model control group is extremely obvious (P is less than 0.01), which indicates that the molding is successful; compared with a model control group, the antipyretic effect of the aspirin group is most obvious (P is less than 0.01) 1h and 2h after the aspirin group is taken for the first time, and the antipyretic effect is obvious (P is less than 0.05) 3h and 4h after the aspirin group is taken for the first time; compared with a model control group, the bupleurum nano-emulsion high-dose group has extremely obvious antipyretic effect (P is less than 0.01) 1h and 2h after the first administration, and has extremely obvious antipyretic effect (P is less than 0.01) 3h and 4h after the first administration; compared with a model control group, the antipyretic effect of the Bupleurum nanoemulsion is significant (P is less than 0.05) 1h and 2h after the first administration, and the antipyretic effect of the Bupleurum nanoemulsion is significant (P is less than 0.01) 3h and 4h after the first administration; 1h and 2h after the first administration, the low-dose group of the bupleurum nano-emulsion has obvious antipyretic effect (P is less than 0.05), and 3h and 4h after the first administration, the antipyretic effect is obvious (P is less than 0.05); the comparative group has significant antipyretic effect (P is less than 0.05) 1h and 2h after the first administration, and has significant antipyretic effect (P is less than 0.01) 3h and 4h after the first administration. See table 44.

TABLE 44 variation of body temperature (x. + -. s) in rats

P < 0.01 compared to normal control group; compared with the model control group, # P < 0.05, # P < 0.01.

3.2 cAMP and AVP levels in rat serum

The cAMP and AVP contents in the serum of rats in each group are shown in Table 45, and compared with a normal control group, the AVP content in the plasma of rats in a model control group is obviously increased (P is less than 0.05); compared with a model control group, the content of AVP in the plasma of rats in the aspirin group and the radix bupleuri nanoemulsion high-dose group is obviously increased (P is less than 0.01); compared with a model control group, the content of AVP in the plasma of rats in the low-dose bupleurum nano-emulsion and the control group is obviously increased (P is less than 0.05); the result shows that the antipyretic effect of the bupleurum nano-emulsion on the rats with dry yeast fever is probably related to the increase of the AVP content in the plasma of the rats. Comparative analysis there was no significant difference in c AMP levels in the plasma of rats in each group (P > 0.05). The suggestion shows that the antipyretic effect of the effective components in the bupleurum root nanoemulsion on the dry yeast pyrogenic rat is probably not related to the c AMP content in the plasma of the rat or the action mechanism is not clear. The results are shown in Table 45.

TABLE 45 arginine vasopressin index and adenosine cyclophosphate index content of rats after application of radix bupleuri for antipyresis

P < 0.01 compared to normal control group; compared with the model control group, # P < 0.05, # P < 0.01

The traditional Chinese medicine has the advantages of good effect of treating infantile fever, small side effect, simplicity, convenience and feasibility, is extremely effective in treating infantile fever, has the characteristics of simplicity, easiness in learning, low price, quick response, green therapy and the like, and has the irreplaceable advantages of medicines. The antipyretic effect can be enhanced by using massage medium with antipyretic effect in massage. The nano-emulsion is used as a new formulation, can improve the transdermal speed of the medicine, delay the medicine release and improve the bioavailability by transdermal administration, and is very suitable to be used as a massage medium. The nanoemulsion with exterior-releasing traditional Chinese medicines is used as a massage medium, and is combined with a massage manipulation to treat infantile fever, so that the traditional Chinese medicine composition is low in toxic and side effects, good in curative effect, wide in market prospect and worthy of research.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The radix bupleuri nano preparation is characterized by being prepared from the following components in parts by weight: tween-804.00-6.76 g, n-butanol 1.76-4.51 g, bupleurum oil 1.00-3.01 g, and bupleurum total saponin 0.50-6.00 g dissolved in 5-30 ml of ultrapure water.

2. The radix bupleuri nano preparation according to claim 1, which is characterized by being prepared from the following components in parts by weight: tween-806 g, n-butanol 3g, bupleuri radix oil 2g, and bupleuri radix total saponin 2g are dissolved in 20ml ultrapure water.

3. The method for preparing the bupleurum nano preparation according to claim 2, which is characterized in that the method comprises the following steps: weighing Tween-80, n-butanol and bupleuri radix oil according to formula ratio, placing into a beaker, mixing uniformly on a magnetic stirrer at a rotating speed of 500r/min, further weighing bupleuri radix total saponin, dissolving in ultrapure water, ultrasonically dissolving completely, slowly adding into the beaker under stirring to uniformly mix oil and water phases to obtain bupleuri radix nanoemulsion, and adding pharmaceutically acceptable adjuvants to prepare pharmaceutically acceptable dosage forms after the nanoemulsion is formed.

4. A detection method for the radix bupleuri nano preparation as claimed in any one of claims 1-2, wherein the detection method for the content of active ingredients in the radix bupleuri nano preparation comprises the following steps:

chromatographic conditions and System suitability test

preparation of control solutions

preparation of test solution

measurement method

5. The method for detecting the bupleurum root nano preparation according to claim 4, wherein the method for detecting the content of the active ingredients in the bupleurum root nano preparation comprises the following steps:

chromatographic conditions and System suitability test

preparation of control solutions

preparation of test solution

measurement method

6. The radix bupleuri nano-preparation according to any one of claims 1-2, wherein the radix bupleuri nano-preparation is used as a medium for infantile fever massage.