CN112870161B

CN112870161B - Cedarol nanoemulsion and optimized preparation method thereof

Info

Publication number: CN112870161B
Application number: CN202110143945.9A
Authority: CN
Inventors: 杨敏; 汤陆扬; 彭黔荣; 付阳洋; 王昊; 杨培昕
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2023-03-21
Anticipated expiration: 2041-02-02
Also published as: CN112870161A

Abstract

The invention belongs to the technical field of preparation of medicinal preparations, and particularly relates to a cedrol nanoemulsion and an optimized preparation method thereof; the nanoemulsion is prepared by adopting a low-energy emulsification method, on the basis of a pseudo-ternary phase diagram screening formula, the particle size and the polydispersity index are used as evaluation indexes, the nanoemulsion formula is optimized by utilizing SLD (super-low-resolution liquid D), so that an oil phase, a water phase, a surfactant and a cosurfactant are fully blended, the particle size of the obtained nanoemulsion is 16.66nm, the polydispersity index is 0.087, the nanoemulsion has good centrifugal stability and physicochemical properties, and the solubility of cedrol reaches 7.44mg.g < -1 >. Lays a foundation for the application of cedrol in external preparations of head skin, the promotion of hair growth, the in vivo drug delivery of injection preparations for treating cardiovascular and cerebrovascular system diseases, the anti-tumor, the anti-bacterial, the anti-plasmodium activity and the like.

Description

Cedarol nanoemulsion and optimized preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of medicinal preparations, and particularly relates to a cedrol nanoemulsion and an optimized preparation method thereof.

Background

The nanoemulsion is also called microemulsion, is a thermodynamic stable system formed by a water phase, an oil phase, a surfactant and a cosurfactant according to a proportion, is used as an emulsion with the average particle size of 10-100nm, has high transparency, and is often applied to the field of pharmaceutical and chemical engineering.

Cedrol (Cedrol, CE), also known as Cedrol, CAS number: 77-53-2, a high boiling sesquiterpene alcohol with a weak fragrance, a mild cedar fragrance, and is mostly present in volatile oils of plants of Cupressaceae, cunninghamiae Lanceolatae, pinaceae, etc. Cedrol has been approved by the U.S. food and drug administration as a flavoring agent or adjuvant, and has been commonly used as a flavoring ingredient in cosmetics, foods and pharmaceuticals, as well as a number of flavoring agents for disinfectants and sanitary products. Cedrol is reported to have a wide range of pharmacological activities including anxiolytic, sedative, hair growth promoting, anti-inflammatory, analgesic, cardiovascular, anti-tumor, etc. effects. However, their poor water solubility limits their use, and thus, increasing their water solubility is an effective way to increase the range of applications. Furthermore, particle size is often the first parameter to assess its effectiveness for drug delivery. In fact, smaller nanoparticles exhibit greater depth of penetration into the hair follicle. Nanoparticles in the size range of less than 100nm are reported to be critical in initiating drug release effects in the skin. Furthermore, a diameter of about 200nm facilitates drug release on the isthmus of the hair follicle. In general, nanoparticle systems can provide superior skin delivery capabilities compared to non-particulate formulations.

Patent document with application number 201711288702.4 discloses a vitamin E nanoemulsion and a preparation method thereof, wherein isopropyl myristate is used as an oil phase, glycerin is used as a cosurfactant, tween 80 and span 20 are compounded into a main surfactant, and the mass of vitamin E accounts for 3-9% of that of a nanoemulsion matrix. The microemulsion capable of improving the transdermal capacity and the permeability of a product and having a good moisturizing effect is prepared, so that the absorptivity of vitamin E is improved, the prepared nanoemulsion is an O/W type nanoemulsion, and the particle size range of the nanoemulsion is 30-60nm.

Disclosure of Invention

The invention provides a cedrol nanoemulsion and an optimized preparation method thereof for solving the problems.

The method is realized by the following technical scheme:

1. a cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: water phase: oil phase solvent: cedrol =15 to 30:15 to 30: 50-60: 0.03 to 0.04.

Further, the surfactant is one of castor oil polyoxyethylene ether (Cremophor EL) and tween 80; the cosurfactant is one of polyethylene glycol 400 (PEG-400) and glycerol; the oil phase is obtained by mixing a cedrol raw material medicament with an oil phase solvent and then carrying out ultrasonic treatment, wherein the oil phase solvent is one of isopropyl myristate (IPM), peppermint oil and octyl dodecanol, and the dosage of the cedrol raw material medicament is 30-40mg.

Further, the optimal formula of the cedrol nanoemulsion is as follows: 30mg of cedrol, 1.2g of castor oil polyoxyethylene ether, 0.2g of isopropyl myristate, 0.6g of polyethylene glycol and 2.0g of water.

2. The preparation method of the cedrol nanoemulsion comprises the following steps:

a. accurately weighing cedrol bulk drug, adding the cedrol bulk drug into an oil phase solvent, and assisting with ultrasound until the cedrol is completely dissolved to obtain an oil phase;

furthermore, the ultrasonic frequency is 40kHz, the power is 240w, and the ultrasonic time is 2min;

b. mixing the following raw materials in a ratio of 1-2:1-2, adding the surfactant and the cosurfactant into an oil phase, uniformly mixing by using a magnetic stirrer at room temperature, slowly dripping water by using a liquid transfer gun, stirring while adding, and balancing for 30min to obtain the clear and transparent cedrol nanoemulsion which is an oil-in-water (O/W) type emulsion with the particle size range of 16.87-26.30 nm.

3. Pseudo ternary phase diagram screening formula

1. Oil phase screening: cremophor EL was chosen as the surfactant, the immobilizing co-surfactant was PEG-400, the oil phase was IPM, peppermint oil and octyldodecanol, the water phase was distilled water, the surfactant to co-surfactant mass ratio (Km) was mixed in a ratio of 1.

2. And (3) screening cosurfactant: selecting Cremophor EL as a surfactant, IPM as an oil phase, PEG-400 and glycerol as cosurfactants, mixing the surfactants and different cosurfactants according to the mass ratio Km =1, and mixing the mixed surfactants and the IPM phases in the ratio of (1, 8: 8.1, mixing under magnetic stirring at room temperature, slowly dripping distilled water, drawing a pseudo-ternary phase diagram by using origin8.0 software, and screening a proper cosurfactant according to the size of a nanoemulsion region.

Km screening: selecting Cremophor EL as a surfactant, IPM as an oil phase, PEG-400 as a co-surfactant, the surfactant and co-surfactant being mixed in different mass ratios Km =1, 2, 1, 3 and 1.

4. The formula of the cedrol nanoemulsion is optimized by adopting a simplex mesh design method (SLD):

on the basis of the pseudo-ternary phase diagram, the surfactant is determined to be Cremophor EL, the cosurfactant is PEG-400, the oil phase is IPM, and the water phase is distilled water. The mass fraction of the fixed oil phase IPM is 5%, the surfactant, the cosurfactant and the distilled water are respectively used as influencing factors X1, X1 and X3, and the particle size and PDI are used as dependent variables Y1 and Y2. Wherein X1=15-30%, X2=15-30%, X3=50-65% is fixed. The formula of the cedrol nanoemulsion was optimized by the SLD Design method in Design Expert 8.0.6, and the particle size and polydispersity index (PDI) were determined, with the results shown in table 1:

TABLE 1 SLD formulation optimization design

Preferably, the optimal formula of the cedrol nanoemulsion is as follows: 30mg of cedrol, 1.2g of castor oil polyoxyethylene ether, 0.2g of isopropyl myristate, 0.6g of polyethylene glycol and 2.0g of water.

4. The cedrol nanoemulsion can be used for preparing skin external preparations for promoting the growth of head hair and can also be used for preparing injection preparations so as to achieve the effect of in vivo drug delivery.

In conclusion, the beneficial effects of the invention are as follows: the invention adopts a low-energy emulsification method to prepare the nanoemulsion, and optimizes the nanoemulsion formula by using SLD (super-low-resolution D) on the basis of a pseudo-ternary phase diagram screening formula by using the particle size and the polydispersity index as evaluation indexes to ensure that an oil phase, a water phase, a surfactant and a cosurfactant are fully blended, so that the obtained nanoemulsion has the particle size of 16.66nm and the polydispersity index of 0.087, has good centrifugal stability and physicochemical property, has the solubility of cedrol of 7.44mg.g < -1 >, can be used for preparing external preparations for head skin, and lays a foundation for in-vivo drug delivery of injection preparations for treating cardiovascular and cerebrovascular system diseases, anti-tumor, anti-bacterial, anti-plasmodium activities and the like.

After the oil phase, the cosurfactant and the Km value are screened by a pseudo-ternary phase diagram method, on the basis, the influence of the proportion of the surfactant, the cosurfactant and the water phase on key factors such as the particle size of the cedrol nanoemulsion, the polydispersity index and the like is more carefully and comprehensively investigated by an SLD method, so that an optimal formula is screened. The application adopts a low-energy emulsification method to prepare the cedrol nanoemulsion, and is widely applied because small liquid drops are formed by using the stored energy of a system. Emulsification can be performed by these methods by varying parameters (temperature or composition) that affect the hydrophilic-lipophilic balance (HLB) of the system. In addition, the low energy method is more energy-saving, so that large-scale industrial production is easier to implement. High energy emulsification techniques require higher forces to break intermolecular attractive forces, hydrogen bonding and van der waals forces, than do high energy emulsification techniques. External energy in the form of shear, ultrasound and pressure can destroy the droplets in the nanometer size range, but very high heat is generated during these processes, which can easily destroy the drug properties of the drug. The nano-emulsion finally prepared by the method has the average particle size of 16.6nm, the polydispersity index of 0.087 and the Zeta potential of-6.14 mV, which indicates that the prepared nano-emulsion is uniform and stable. Compared with the vitamin E nanoemulsion prepared by the patent document with the application number of 201711288702.4, the nanoemulsion obtained by the invention has smaller particle size and wider applicable range. Research reports that the solubility of the cedrol is 21.88mg.kg < -1 >, 30mg of the cedrol is encapsulated in 4.03g of the cedrol nanoemulsion, the solubility of the cedrol reaches 7.44mg.g < -1 >, and the prepared nanoemulsion is an O/W type nanoemulsion, has better dilution stability and greatly improves the solubility of the cedrol. Compared with free cedrol, the nanoemulsion can achieve the effects of slow release and controlled release, and the release amount of the nanoemulsion is about 15.5 times that of the free cedrol. The nano-emulsion is tested by a centrifugal stability test, and the result shows that the prepared nano-emulsion has good centrifugal stability. The invention realizes the entrapment of the cedrol nanoemulsion for the first time, and the prepared nanoemulsion has good physicochemical properties, thereby providing a new thought and method for the research and development of cedrol preparations and expanding the application range of cedrol. The prepared nanoemulsion is mainly used for scalp external application to promote hair growth. The cedrol has poor solubility, and the nano emulsion is coated on the cedrol to play a role in enhancing the solubility. And the nanoemulsion also has the effects of slow release, controlled release, permeation enhancement and the like. Topical application can avoid first-pass effect and gastrointestinal effect, and reduce systemic toxicity.

Drawings

Fig. 1 is a diagram of the structure of cedrol used in this patent application.

FIG. 2 is a pseudo-ternary phase diagram of different oil phases; wherein, the figure a, the figure b and the figure c are respectively pseudo ternary phase diagrams of oil phases of isopropyl myristate, octyl dodecanol and peppermint oil.

FIG. 3 is a pseudo-ternary phase diagram for different co-surfactants; wherein, the figures d and e are respectively pseudo ternary phase diagrams of which the cosurfactants are polyethylene glycol 400 and glycerol.

FIG. 4 is a pseudo ternary phase diagram for different Km values; wherein, fig. f, g, h and j are pseudo ternary phase diagrams of Km =1, km =2, km = 3.

Fig. 5 is a distribution diagram of cedrol nanoemulsion particle size.

FIG. 6 is a photograph showing the discrimination of cedrol nanoemulsion type, wherein A is Sudan red and B is methylene blue.

Fig. 7 is a TEM morphogram of cedrol nanoemulsion.

FIG. 8 is the in vitro release profile of cedrol nanoemulsion

Fig. 9 is a distribution diagram of the particle size of the nanoemulsion prepared by experiment two in the verification experiment.

Fig. 10 is a distribution diagram of the particle size of the nanoemulsion prepared in experiment three of the verification experiment.

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =30:15:5:50.

the method specifically comprises the following steps: 30mg of cedrol, 1.2g of surfactant, 0.2g of oil phase solvent, 0.6g of cosurfactant and 2.0g of water.

Further, the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

a. accurately weighing 30mg of raw cedrol medicine, adding the cedrol medicine into 0.2g of isopropyl myristate, and adding ultrasound to completely dissolve the cedrol to obtain an oil phase;

b. precisely weighing 1.2g of castor oil polyoxyethylene ether and 0.6g of polyethylene glycol 400, adding into the oil phase, uniformly mixing at room temperature by using a magnetic stirrer, slowly dropwise adding 2.0g of water by using a liquid transfer gun while stirring, and balancing for 30min to obtain the clear and transparent cedrol nanoemulsion.

Transparent and stable nanoemulsion with a particle size of 17.01 polydispersity index (PDI) of 0.09 was obtained.

3. The cedrol nanoemulsion can be used for preparing skin external preparations for promoting the growth of head hair, and can also be used for preparing injection preparations so as to achieve the effect of in vivo drug delivery.

Example 2

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =25:17.5:5:52.5, can form transparent stable nano-emulsion with 30mg of cedrol, and has the particle size of 18.21 and the polydispersity index (PDI) of 0.121.

The method comprises the following specific steps: 30mg of cedrol, 1.0g of surfactant, 0.2g of oil phase solvent, 0.7g of cosurfactant and 2.1g of water.

a. accurately weighing 30mg of cedrol, dissolving the cedrol in 0.2g of isopropyl myristate, and assisting with ultrasound until the cedrol is completely dissolved to obtain an oil phase;

b. precisely weighing 1.0g of castor oil polyoxyethylene ether and 0.7g of polyethylene glycol 400, adding into the oil phase, and uniformly mixing at room temperature; then 2.1g of water is dripped while stirring, and the mixture is balanced for 30min to obtain clear and transparent cedrol nanoemulsion which is oil-in-water (O/W) type emulsion.

Example 3

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =22.5:22.5:5:50.0, can form transparent stable nano emulsion with 30mg of cedrol, and has the particle size of 18.86 and the polydispersity index (PDI) of 0.131.

The method specifically comprises the following steps: 30mg of cedrol, 0.9g of surfactant, 0.2g of oil phase solvent, 0.9g of cosurfactant and 2.0g of water.

a. accurately weighing 30mg of cedrol, dissolving the cedrol in 0.2g of isopropyl myristate, and adding ultrasound until the cedrol is completely dissolved to obtain an oil phase;

b. accurately weighing 0.9g of castor oil polyoxyethylene ether and 0.9g of polyethylene glycol 400, adding into the oil phase, and uniformly mixing at room temperature; then 2.0g of water is dripped while stirring, and the mixture is balanced for 30min to obtain clear and transparent cedrol nanoemulsion which is oil-in-water (O/W) type emulsion.

Example 4

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =22.5:15:5:57.5, can form transparent stable nanoemulsion with 30mg of cedrol, and the particle size is 18.96 polydispersity index (PDI) is 0.124.

The method specifically comprises the following steps: 30mg of cedrol, 0.9g of surfactant, 0.2g of oil phase solvent, 0.6g of cosurfactant and 2.3g of water.

b. accurately weighing 0.9g of castor oil polyoxyethylene ether and 0.6g of polyethylene glycol 400, adding into the oil phase, and uniformly mixing at room temperature; then 2.3g of water is dripped while stirring, and the mixture is balanced for 30min to obtain clear and transparent cedrol nanoemulsion which is oil-in-water (O/W) type emulsion.

Example 5

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =15:22.5:5:57.5, can form transparent stable nanoemulsion with 30mg of cedrol, and the particle size is 25.09 and the polydispersity index (PDI) is 0.141.

The method specifically comprises the following steps: 30mg of cedrol, 0.6g of surfactant, 0.2g of oil phase solvent, 0.9g of cosurfactant and 2.3g of water.

b. accurately weighing 0.6g of castor oil polyoxyethylene ether and 0.9g of polyethylene glycol 400, adding into the oil phase, and uniformly mixing at room temperature; then 2.3g of water is dripped while stirring, and the mixture is balanced for 30min to obtain clear and transparent cedrol nanoemulsion which is oil-in-water (O/W) type emulsion.

Example 7

Further, the surfactant is tween 80, the cosurfactant is glycerol, and the oil phase solvent is peppermint oil.

a. accurately weighing 30mg of raw cedrol medicine, adding the cedrol medicine into 0.2g of peppermint oil, and assisting with ultrasound till the cedrol is completely dissolved to obtain an oil phase;

b. precisely weighing 1.2g of Tween 80 and 0.6g of glycerol, adding into the oil phase, uniformly mixing at room temperature by using a magnetic stirrer, slowly dropwise adding 2.0g of water by using a liquid transfer gun while stirring, and balancing for 30min to obtain the clear and transparent cedrol nanoemulsion.

Example 8

1. The formula of the cedrol nanoemulsion comprises the following components in percentage by weight: surfactant (b): cosurfactant: oil phase solvent: pure water =22.5:22.5:5:50.0.

Further, the surfactant is tween 80, the cosurfactant is glycerol, and the oil phase solvent is octyl dodecanol.

a. accurately weighing 30mg of cedrol, dissolving the cedrol in 0.2g of octyl dodecanol, and assisting with ultrasonic waves until the cedrol is completely dissolved to obtain an oil phase;

b. accurately weighing 0.9g of Tween 80 and 0.9g of glycerol, adding into the oil phase, and mixing at room temperature; then 2.0g of water is dripped while stirring, and the mixture is balanced for 30min to obtain clear and transparent cedrol nanoemulsion which is oil-in-water (O/W) type emulsion.

1. Screening formula by adopting pseudo ternary phase diagram

1. Oil phase screening: cremophor EL was chosen as the surfactant, the immobilizing co-surfactant was PEG-400, the oil phase was IPM, peppermint oil and octyldodecanol, the aqueous phase was distilled water, the surfactant to co-surfactant mass ratio (Km) was mixed in a ratio of 1.

on the basis of the pseudo-ternary phase diagram, the surfactant is determined to be Cremophor EL, the cosurfactant is PEG-400, the oil phase is IPM, and the water phase is distilled water. The mass fraction of the fixed oil phase IPM is 5%, a surfactant, a cosurfactant and distilled water are respectively used as influencing factors X1, X1 and X3, and the particle size and PDI are used as dependent variables Y1 and Y2. Wherein X1=15-30%, X2=15-30%, X3=50-65% is fixed. The SLD Design method in Design Expert 8.0.6 is adopted to optimize the formula of the cedrol nanoemulsion.

The optimal formula of the obtained cedrol nanoemulsion is as follows: 30mg of cedrol, 1.2g of castor oil polyoxyethylene ether, 0.2g of isopropyl myristate, 0.6g of polyethylene glycol and 2.0g of water.

2. Cedrol drug-loading screening experiment

2.1 materials of the experiment

0, 5, 10, 20, 30, 40, 50, 60 and 80mg of cedrol are respectively taken for experiments.

2.2 Experimental methods

Adding cedrol into a glass bottle filled with 0.2g of IPM, adding Cremophor EL and PEG-400 by ultrasonic assistance until the cedrol is completely dissolved, then adding Cremophor EL and PEG-400 by 0.9g respectively, uniformly mixing under the condition of constant-temperature magnetic stirring, slowly dripping distilled water to 2ml by using a liquid-transferring gun after all the components are uniformly mixed, observing the physical appearance of the formed nano-emulsion, and testing the physical stability and the light transmittance, wherein the results are shown in Table 2.

2.3 results of the experiment

TABLE 2

The experimental result shows that when the content of the cedrol is 0-40mg, clear and transparent nano-emulsion can be formed, but when the content is 40mg, the clear nano-emulsion just formed is not clear and transparent, and the content is 30mg which is the best drug loading rate according to the experimental result of light transmittance.

3. Emulsification Performance test of surfactants of different oil phases

3.1 Experimental materials

Sample 1: emulsifying by taking Tween 80 as a surfactant, PEG-400 as a cosurfactant and IPM as an oil phase;

sample 2: emulsifying by using Cremophor EL as a surfactant, PEG-400 as a cosurfactant and IPM as an oil phase;

sample 3: emulsifying by taking Tween 80 as a surfactant, PEG-400 as a cosurfactant and IPM/oleic acid (1;

sample 4: emulsifying by taking Cremophor EL as a surfactant, PEG-400 as a cosurfactant and IPM/oleic acid (1;

sample 5: emulsifying by using Tween 80 as a surfactant, PEG-400 as a cosurfactant and oleic acid as an oil phase;

sample 6: emulsifying by using Cremophor EL as a surfactant, PEG-400 as a cosurfactant and oleic acid as an oil phase;

sample 7: emulsifying by using Tween 80 as surfactant, PEG-400 as cosurfactant and oleum Menthae Dementholatum as oil phase;

sample 8: emulsifying by using Cremophor EL as a surfactant, PEG-400 as a cosurfactant and peppermint oil as an oil phase;

sample 9: emulsifying by using Tween 80 as surfactant, PEG-400 as cosurfactant and olive oil as oil phase;

sample 10: emulsifying by using Cremophor EL as a surfactant, PEG-400 as a cosurfactant and olive oil as an oil phase;

sample No. 11: emulsifying by taking Tween 80 as a surfactant, PEG-400 as a cosurfactant and octyl dodecanol as an oil phase;

sample 12: emulsifying by using Cremophor EL as a surfactant, PEG-400 as a cosurfactant and octyl dodecanol as an oil phase.

Wherein Km of the above samples are all 1, and the mixed surfactant and the oil phase are all mixed at a mass ratio of 9.

3.2 Experimental methods

The appearance of samples 1 to 12 was observed to examine the emulsifying properties of the respective surfactants, and the results are shown in Table 3.

3.3 results of the experiment

TABLE 3

According to experimental results, clear and transparent solutions can be formed by tween 80 and Cremophor EL, IPM, octyldodecanol and peppermint oil, but the clear and transparent solution formed by tween 80 has poor stability, is turbid or flocculated when placed, has good emulsifying properties of the sample 2 and the sample 12, but the sample 2 is optimal by combining a pseudo-ternary phase diagram, namely the Cremophor EL is selected as a surfactant, and the IPM is selected as an oil phase, so that the emulsifying property is optimal.

4. Optimum formulation verification experiment

4.1 Experimental materials

Sample No. 13: a nanoemulsion prepared in the manner of example 1.

4.2 Experimental methods

The particle size, polydispersity index (PDI), pH and Zeta potential values of the nanoemulsion were determined and tested three times, with the results averaged as shown in table 4.

The particle size distribution of sample 13 was measured as shown in FIG. 4.

4.3 results of the experiment

TABLE 4

Item	Particle size	PDI	pH value	Value of Zeta potential
					Sample 13	16.66nm	0.087	6.79	-6.14mV

5. Experiment of centrifugal stability

5.1 Experimental materials

Cedrol nanoemulsion prepared in example 1.

5.2 Experimental methods

And (3) measuring the stability of the nano-emulsion by a low-temperature high-speed centrifugation method, centrifuging for 30min at 12000r.min-1, observing the appearance of the nano-emulsion, measuring the peak areas of the sample before and after centrifugation by a gas chromatography, and calculating a centrifugation stability constant K.

Wherein, the gas chromatography conditions are as follows: agilent GC7890 gas chromatograph, DB-5MS column (30 mm. Times.0.25 μm); temperature rising procedure: setting the initial temperature to 110 ℃, and maintaining for 2min; heating to 180 deg.C at 15 deg.C, min-1, and maintaining for 2min; then the temperature is raised to 240 ℃ at 10 ℃ for min-1, and the temperature is maintained for 2min. The injection port temperature and the gasification temperature are both 250 ℃; the flow splitting ratio is 50; the sample size is 1 mul; the detector is an FID detector; the carrier gas is nitrogen.

Centrifuge stability constant K = A1/A0; a0 is the area of the centrifugal front peak, and A1 is the area of the centrifugal rear peak.

5.3 results of the experiment

The nano-emulsion after centrifugation is still in a clear and transparent state, and aggregation, coagulation, phase separation and emulsion breaking are not generated;

calculated from the peak area, the centrifuge stability constant K =432.4/464.7=93.05% (n = 3), indicating good stability.

6. Physical and chemical properties

6.1 Experimental materials

Cedrol nanoemulsion prepared in example 1.

6.2 Experimental methods

And (3) judging the type of the nanoemulsion: two portions of the nanoemulsion prepared in example 1 were added with equal amounts of sudan red and methylene blue, respectively, and the diffusion rate was observed, as shown in fig. 5.

Microscopic morphology: the microscopic morphology of the nanoemulsion at the dimensions of 20nm and 50nm, respectively, and the TEM morphogram is shown in fig. 6.

6.3 results of the experiment

Appearance: the nanoemulsion prepared according to the formulation of example 1 has a yellowish, clear and transparent appearance.

Type of nanoemulsion: as shown in fig. 5, methylene blue was observed to diffuse faster than sudan red, so the nanoemulsion prepared was an O/W type nanoemulsion.

Microscopic morphology: as shown in figure 6, the cedrol nanoemulsion is spherical and uniform in size, has no adhesion among emulsion drops, and is good in formability.

7. In vitro Release assay

Cedrol nanoemulsion (SLD optimized medium particle size minimum formula) release: taking 2ml of freshly prepared nanoemulsion, putting the freshly prepared nanoemulsion into a dialysis bag (MWCO: 7 kDa), wherein the dialysate is 20% ethanol PBS (PH = 7.4), putting the dialysis bag into a beaker containing 50ml of dialysate, stirring and shaking at 37 ℃, taking 2ml of the dialysate from the beaker for 0.25h, 0.1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h and 45h respectively, and supplementing 2ml of the fresh dialysate into the beaker.

Free CE release: 2ml of freshly prepared CE ethanol solution (30 mg/4 ml) was placed in a dialysis bag (MWCO: 7 kDa) containing 20% ethanol in PBS (pH = 7.4), the dialysis bag was placed in a beaker containing 50ml of dialysate, and 2ml of the dialysate was sampled at 0.25h, 0.1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, and 45h, respectively, with shaking at 37 ℃ and supplemented with 2ml of dialysate. The results are shown in FIG. 7.

As can be seen from FIG. 7, the amount of released free cedrol is very low, 45 hours, and the cumulative amount of released cedrol is less than 10%. The nano-emulsion is used for encapsulating the nano-emulsion, so that the effect of increasing the release can be achieved, the 24-hour accumulation can reach 60%, and the 45-hour accumulation release can reach 80%. Compared with free cedrol, the nano-emulsion can play a role in slow release and controlled release, and the release amount is about 15.5 times of that of the free cedrol, so that the cedrol nano-emulsion has the functions of slow release, controlled release, permeation enhancement and the like. Topical application can avoid first-pass effect and gastrointestinal effect, and reduce systemic toxicity.

8. Verification experiment

8.1 Experimental methods

The experiment of preparing cedrol nanoemulsion using the method of the patent document 201711288702.4 is as follows:

experiment one: 67.8mg of cedrol and 0.1560g of isopropyl myristate were weighed out according to example 2 of the vitamin E patent, and cedrol could not be completely dissolved. The next experiment could not be performed.

Experiment two: cedrol nanoemulsion was prepared according to the formula in vitamin E patent example 2, weighing 23.4mg of cedrol (cedrol was weighed according to the ratio of cedrol to isopropyl myristate in the subject group nanoemulsion formula). The prepared nano-emulsion is milky white, is diluted by 20 times, and has the particle size of 308.95nm and the polydispersity index (PDI) of 0.171. The particle size diagram is shown in fig. 8.

Experiment three: cedrol nanoemulsion was prepared according to the formula in vitamin E patent example 2, weighing 8.9mg of cedrol (cedrol was weighed according to the ratio of cedrol to total nanoemulsion mass in the subject group nanoemulsion formula). The prepared nano-emulsion is milky white, is diluted by 20 times, and has the particle size of 321.95nm and the polydispersity index (PDI) of 0.201. The particle size diagram is shown in fig. 9.

8.2 results of the experiment

As can be seen from the experimental results and fig. 8 and 9, the experimental preparation of cedrol nanoemulsion according to the method of the 201711288702.4 patent document cannot be performed; by combining the experimental proportion of the inventor for adjustment, the prepared milky emulsion has the particle sizes of 308.95nm and 321.95nm, can not obtain clear and transparent nano-emulsion, and does not meet the requirement that the medicine with the diameter of about 200nm is beneficial to the release of the medicine on the isthmus of hair follicles.

Claims

1. A cedrol nanoemulsion is characterized by comprising the following components: 30mg of cedrol, 1.2g of surfactant, 0.2g of oil phase solvent, 0.6g of cosurfactant and 2.0g of water; the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

2. A cedrol nanoemulsion is characterized by comprising the following components: 30mg of cedrol, 1.0g of surfactant, 0.2g of oil phase solvent, 0.7g of cosurfactant and 2.1g of water; the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

3. A cedrol nanoemulsion is characterized by comprising the following components: 30mg of cedrol, 0.9g of surfactant, 0.2g of oil phase solvent, 0.9g of cosurfactant and 2.0g of water; the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

4. A cedrol nanoemulsion is characterized by comprising the following components: 30mg of cedrol, 0.9g of surfactant, 0.2g of oil phase solvent, 0.6g of cosurfactant and 2.3g of water; the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

5. A cedrol nanoemulsion is characterized by comprising the following components: 30mg of cedrol, 0.6g of surfactant, 0.2g of oil phase solvent, 0.9g of cosurfactant and 2.3g of water; the surfactant is castor oil polyoxyethylene ether, the cosurfactant is polyethylene glycol 400, and the oil phase solvent is isopropyl myristate.

6. The method for preparing cedrol nanoemulsion according to any one of claims 1 to 5, comprising the following steps:

a. accurately weighing cedrol bulk drug, dissolving the cedrol bulk drug in an oil phase solvent, and assisting with ultrasound until the cedrol is completely dissolved to obtain an oil phase;

b. precisely weighing a surfactant and a cosurfactant, adding the surfactant and the cosurfactant into an oil phase, and uniformly mixing at room temperature; then adding water dropwise under stirring, and balancing for 30min to obtain clear and transparent cedrol nanoemulsion.

7. The method for preparing cedrol nanoemulsion according to claim 6, wherein the ultrasound has the frequency of 40kHz, the power of 240w and the time of 2min.

8. The method for preparing the cedrol nanoemulsion as claimed in claim 6, wherein the cedrol nanoemulsion is an oil-in-water (O/W) emulsion with the particle size ranging from 16.87 to 26.30nm.

9. Use of a cedrol nanoemulsion according to any one of claims 1-5, wherein the cedrol nanoemulsion is used for the preparation of a skin external preparation for promoting head hair growth.

10. Use of a cedrol nanoemulsion according to any one of claims 1-5, wherein the cedrol nanoemulsion is used for the preparation of an injectable formulation.