CN104644553B - A kind of tanshinone IIA microemulsions, tanshinone IIA microemulsion gel preparation and their preparation method - Google Patents
A kind of tanshinone IIA microemulsions, tanshinone IIA microemulsion gel preparation and their preparation method Download PDFInfo
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Abstract
The present invention provides a kind of tanshinone IIA microemulsions, microemulsion gel preparation and their preparation method.Tanshinone IIA microemulsions are made up of following components：Tanshinone IIA, mass ratio are 7:3 Solutrol HS 15 are with the mixture of Tween 80, absolute ethyl alcohol, by Lavender, Rosemary Oil, grapefruit essential oil and tea tree ethereal oil using mass ratio as 2:2:1:The compound plant essential oil and water of 1 composition.Microemulsion gel preparation is made up of following components：Above-mentioned microemulsion formulation, Acritamer 940 and triethanolamine.The present invention have selected suitable emulsifying agent, assistant for emulsifying agent and oil phase by testing sieve, and the proportioning each component is optimized, and obtain spheroidal distribution, uniform in size, rounding microemulsion formulation.The present invention has further been investigated the addition of Acritamer 940, has been obtained the formula of the optimal microemulsion gel preparation of accumulative transit dose based on the microemulsion formulation.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, and in particular to a kind of tanshinone IIA microemulsions, tanshinone IIA micro emulsion coagulate
Glue preparation and their preparation method.
Background technology
The red sage root belongs to labiate, is the traditional Chinese medicine of China, sees earliest《Sheng Nong's herbal classic》, it is listed in
Product.Tanshinone IIA is the liposoluble constituent extracted from the red sage root, is cherry red acicular crystal, and fusing point is 209-210 DEG C, slightly soluble
Yu Shui, is soluble in the organic solvents such as dimethyl sulfoxide (DMSO), ethanol, acetone, ether and benzene.Tanshinone IIA is in high temperature and illumination condition
Under it is unstable, easily occur degradation reaction, but solution pH substantially on the stability of tanshinone IIA without influence.Tanshinone IIA
Water-soluble poor, the μ g/mL of solubility about 7.0 in water, permeable membrane is poor, and itself is easily crossed and imitated by the effect of P- glycoprotein and liver head
The influence answered.Shi Yuan etc. result of study shows that the A of Danshen injection ketone II is metabolized and drained in vivo comparatively fast, average to be detained in vivo
Time (MRT) is 29.14min, average distribution half-life (t1/2α) it is 11.64min.As can be seen here, the intestines and stomach of tanshinone IIA
Absorption difference, in vivo elimination are fast, bioavilability is low.But tanshinone IIA may participate in internal a variety of biochemical reactions and have extensive
Pharmacological activity, if the preferably effect such as protection cardiovascular and cerebrovascular, antitumor, anti-inflammatory, antibacterial, with higher application value.
Microemulsions gel (microemulsion-based gels, MBGs) is by medicinal substances extract and suitable oil
Phase, emulsifying agent, assistant for emulsifying agent, the microemulsion obtained by water add the transparent, homogeneous formed into gel-type vehicle, stable gel
Contain micro emulsion drop in network structure, network structure.Numerous researchs show that micro emulsion has the solubility of increase medicine, promotes to inhale
The features such as receiving, improve medicine stability, extension drug treating time, maintain constant blood concentration, therefore it is increasingly subject to research
The attention of person, but micro emulsion mobility is strong, and poor as external preparation carrier organism adhesion, the holdup time is short, thus limits it
Practical application.Micro emulsion gel has the two-fold advantage of micro emulsion and gel concurrently as novel Drug Delivery Systems, inherits micro emulsion and improves indissoluble
Property medicine solubility, improve medicine bioavilability, improve medicine stability, extension drug treating time, maintain it is constant
Blood concentration advantage, while micro emulsion can be overcome because of long-term storage, surfactant concentration liter caused by moisture easily evaporation
The strong shortcoming of high, skin irritation；Inheriting gel again can stick with site of action for a long time, there is preferable bio-compatible
Property, blood concentration that is lasting, constant and being easily controlled can be produced, it is to avoid the appearance of peak valley phenomenon, reduce times for spraying, improved
The advantage of Drug safety, validity and stability, not pollution clothes.
Tanshinone IIA has many pharmacological activity, and that clinically applies is relatively broad, now by its pharmacological action and faces
Bed application has carried out simple summary.
Current tanshinone IIA mainly has following aspect in clinical application：1. it is used to treat disease in terms of angiocarpy such as
Chronic heart failure, Senile Cor Pulmonale Treated, unstable angina pectoris etc.；2. hepatic fibrosis；3. anti-malignant tumor；4. acute brain
Bleeding.
At present the dosage form research of relevant tanshinone IIA mainly has injection and an oral two kinds of formulations, including injection, orally
Solid dispersions, injection Submicron Emulsion and micro emulsion, oral micro-capsule, oral administration impulse pellet, injection lipid microspheres, oral solid lipid
Nanoparticle and oral administration nanometer capsule.
The content of the invention
The technical problems to be solved by the invention be how to overcome tanshinone IIA in existing preparation intestines and stomach absorption difference,
The low defect of fast, bioavilability is eliminated in vivo, and tanshinone IIA is prepared into the high formulation of bioavilability；And how gram
The solubility for taking tanshinone IIA is low, is difficult to the high microemulsion formulation of bioavilability and micro emulsion gel system is made by prior art
The defect of agent.
In order to solve the above-mentioned technical problem, the invention provides a kind of tanshinone IIA microemulsions and preparation method thereof.
Tanshinone IIA microemulsions provided by the present invention, are made up of following components：Tanshinone IIA, emulsifying agent, help breast
Agent, oil phase and water.
The emulsifying agent is that mass ratio is 6-8:3 (such as 7:3) Solutrol HS-15 and the mixture of Tween 80.
The assistant for emulsifying agent is absolute ethyl alcohol.
The oil phase is using mass ratio as 2 by Lavender, Rosemary Oil, grapefruit essential oil and tea tree ethereal oil:2:
1:The compound plant essential oil of 1 composition.
The mass ratio of the emulsifying agent and assistant for emulsifying agent is 8/2-6/4, such as 8/2,7/3 or 6/4, concretely 6/4.
The mixture of the emulsifying agent and assistant for emulsifying agent is 9/1-4/1 with the mass ratio of the oil phase, such as 9/1,8/1,7/
1st, 6/1,5/1 or 4/1, concretely 6/1.
In the tanshinone IIA microemulsions, the weight/mass percentage composition of water is >=40%, such as 46%.
In the tanshinone IIA microemulsions, the mass concentration of tanshinone IIA is 0.55mg/mL-1.02mg/mL.
1), 2) or 3) tanshinone IIA microemulsions are prepared according to following：
1) first the emulsifying agent, assistant for emulsifying agent, oil phase and water carried out being mixed to get blank micro emulsion, then by tanshinone IIA
It is added in the blank micro emulsion, after stirring, obtains tanshinone IIA microemulsions；
2) first tanshinone IIA is dissolved with the assistant for emulsifying agent under stirring, the emulsifying agent, oil phase and water is added, obtains
To tanshinone IIA microemulsions；
3) first tanshinone IIA is dissolved with the oil phase, emulsifying agent and assistant for emulsifying agent under stirring, water is added, obtains pellet
Join the A microemulsion formulations of ketone II.
In the above method, the speed of the stirring is 250r/min-500r/min, concretely 250r/min, time
It is 0.5h-1.5h, concretely 0.5h.
It is preferred to use and described 1) prepares the tanshinone IIA microemulsions.
Present invention also offers a kind of tanshinone IIA microemulsion gel preparation and preparation method thereof.
Tanshinone IIA microemulsion gel preparation provided by the present invention is made up of following components：Tanshinone IIA, emulsifying agent, help
Emulsifying agent, oil phase, water, Acritamer 940 and triethanolamine.
The emulsifying agent is that mass ratio is 7:3 Solutrol HS-15 and the mixture of Tween 80.
The assistant for emulsifying agent is absolute ethyl alcohol.
The oil phase is using mass ratio as 2 by Lavender, Rosemary Oil, grapefruit essential oil and tea tree ethereal oil:2:
1:The compound plant essential oil of 1 composition.
The mass ratio of the tanshinone IIA and the emulsifying agent, assistant for emulsifying agent, oil phase and water is 0.055-0.102:
27.8:18.5:7.4:46.3。
The quality of Acritamer 940 is the 0.5%- of the gross mass of tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase and water
0.9%, such as 0.5%.
The tanshinone IIA microemulsion gel preparation is prepared by the method comprising the following steps：Use recipe quantity
The water-swellable Acritamer 940 of half, the Acritamer 940 being swelled；By tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase with
Remaining water mixing, obtains mixed liquor；The Acritamer 940 being swelled is mixed with the mixed liquor, three ethanol are eventually adding
The pH value of amine regulation system obtains tanshinone IIA microemulsion gel preparation between 6.5-7.5.
Application of above-mentioned tanshinone IIA microemulsions and/or the tanshinone IIA microemulsion gel preparation in treatment acne also belongs to
In protection scope of the present invention.
The present invention filters out suitable emulsifying agent, assistant for emulsifying agent and oil phase and its proportioning, has investigated mixing time, stirring speed
Influence of the order of degree and addition medicine to micro emulsion drug carrying amount, the micro- of the tanshinone IIA containing effective dose is made by tanshinone IIA
Emulsion formulation, the microemulsion formulation stability is good.The present invention determines the content of Acritamer 940 by vitro permeation assay, will be described
Microemulsion gel preparation has been made in tanshinone IIA microemulsions, in the formulation of percutaneous dosing, and micro emulsion gel is not only retaining biological
Active aspect has certain advantage, and easy to carry, using simple.Pharmacodynamic research has shown that, described to join the A micro emulsions of ketone II
Preparation and microemulsion gel preparation have bacteriostatic activity, available for the treatment of acne, compare and antibiotics, tanshinone IIA
Microemulsion formulation and microemulsion gel preparation are not likely to produce drug resistance.
The present invention prepares tanshinone IIA micro emulsion gel, and a new administration way is provided for the clinical practice of tanshinone IIA
Footpath, while also finding a kind of new method for acne treatment.
Brief description of the drawings
Fig. 1 is the uv absorption spectra of tanshinone IIA standard items.
Fig. 2 is the canonical plotting of tanshinone IIA.
Fig. 3 is the block diagram of tanshinone IIA solubility in different medium.Wherein, a represents 30wt% ethanol-physiological saline,
B represents 40wt% ethanol-physiological saline, and c represents 50wt% ethanol-physiological saline, and d represents 40wt%PEG-400- physiology salts
Water, e represents 50wt%PEG-400- physiological saline.
Fig. 4 is the block diagram of tanshinone IIA solubility in different oil phases.Wherein, OA represents oleic acid, and EO represents oleic acid second
Ester, IPM represents isopropyl myristate, and LEO represents Lavender, and REO represents Rosemary Oil, and TTEO represents tea tree essence
Oil, GEO represents grapefruit essential oil, and CEO represents compound essential oil.
Fig. 5 is the block diagram of tanshinone IIA solubility in different emulsifiers.
Fig. 6 is the block diagram of tanshinone IIA solubility in different assistant for emulsifying agents.Wherein, E represents ethanol, and O represents 1,2- third
Glycol, G represents glycerine.
Fig. 7 A are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=8/2.
Fig. 7 B are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=7/3.
Fig. 7 C are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=6/4.
Fig. 7 D are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=5/5.
Fig. 8 is influence figure of the mixing time to micro emulsion drug carrying amount.
Fig. 9 is influence figure of the different mixing speeds to micro emulsion drug carrying amount.
Figure 10 is the grain size distribution of tanshinone IIA micro emulsion (left figure) and blank micro emulsion (right figure).
Figure 11 is the electron microscope of tanshinone IIA micro emulsion (left figure) and blank micro emulsion (right figure).
Figure 12 is that tanshinone IIA standard items HPLC (A figures), the transdermal HPLC of blank micro emulsion gel (B figures), tanshinone IIA are micro-
Newborn gel transdermal HPLC (C figures).
Figure 13 is the canonical plotting of tanshinone IIA.
Figure 14 is tanshinone IIA cumulative in vitro infiltration capacity curve.
Figure 15 is blank micro emulsion gel (left figure) and the grain-size graph for carrying medicine micro emulsion gel (right figure).
Figure 16 is the transmission electron microscope figure of blank micro emulsion gel (left figure) and load medicine micro emulsion gel (right figure), wherein,
Left figure and right figure are same multiplication factor × 50000.
Embodiment
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., unless otherwise specified, are commercially obtained.
Instrument and material used in following embodiments：
Cary50 ultraviolet specrophotometers (U.S.'s Varian)；
The high performance liquid chromatographs of Agilent 1260 (U.S.'s Agilent)；
LD5-10 low speed centrifuges (Beijing Medical Centrifugal Machine Factory)；
SHA water-bath constant temperature oscillators (Changzhou Guohua Electric Appliance Co., Ltd.)；
JA4003 precision electronic balances (upper current chart day instrument and meter Co., Ltd)；
BSA224S precision electronic balances (Sai Duolisi scientific instrument Co., Ltd)
78-1 type magnetic force heating stirrer (high honour instrument manufacturing Co., Ltd of Jintan City of Jiangsu Province)；
IKAC-MAGHS4 heating magnetic stirring apparatus (Guangzhou Yi Ke laboratory techniques Co., Ltd)；
PHS-25 type digital display pH meters (Shanghai INESA Scientific Instrument Co., Ltd.)；
DDS-11C conductivity meters (Shanghai INESA Scientific Instrument Co., Ltd.)；
The type transmission electron microscopes of JEM-100CX II (JEOL JEOLs)；
The Malvern Laser Scattering Particle analyzers of Zetasizer 3000 (Malvern company of Britain)；
Tanshinone IIA standard items (Chinese food drug assay research institute, 110766-200619)；
Tanshinone IIA bulk drug (Xi'an Honson Biotechnology Co., Ltd.)；
Solutrol HS-15 (BASF Corp. of Germany)；
Lavender, Rosemary Oil, grapefruit essential oil, tea tree ethereal oil (French U.S. of family is happy)；
N-octyl alcohol (analyzes pure, Tianjin Xing Fu fine chemistry industries research institute)；
Polyethylene glycol 400 (analyzes pure, Tianjin Xing Fu fine chemistry industries research institute)；
Oleic acid (analysis is pure, Tianjin recovery fine chemistry industry research institute)；
Ethyl oleate (analysis is pure, Tianjin recovery fine chemistry industry research institute)；
Isopropyl myristate (analyzes pure, Shanghai Xi Run chemical industry Co., Ltd)；
Tween 80 (chemical pure, Tianjin Xing Fu fine chemistry industries research institute)；Solutrol HS-15 (BASF Corp. of Germany)；
EL-35 (BASF Corp. of Germany)；
Absolute ethyl alcohol (analyzes pure, Tianjin Kermel Chemical Reagent Co., Ltd.),
1,2-PD (analyzes pure, Tianjin Feng Chuan chemical reagent Co., Ltd),
Glycerine (analyzes pure, Tianjin Kermel Chemical Reagent Co., Ltd.)；
Methanol (analysis is pure, and rich chemical reagent Co., Ltd is entered in Tianjin)；
Water is distilled water；
Propionibacterium acnes (Guangdong Province's Culture Collection, ATCC11817)；
Staphylococcus aureus (is provided) by affiliated hospital of University Of Shanxi second；
MH agar mediums (Jinan Baibo Biotechnology Co., Ltd., 14061701)；
Pancreas peptone soybean broth dehydrated medium (the silent winged generation that biochemistry product Beijing Co., Ltd of match,
1024004)；
Blood agar culture-medium (Jinan Baibo Biotechnology Co., Ltd.)；
Injection erythromycin lactobionate dry powder (Dalian Merro Pharmaceutical Co., Ltd.)；
Benzylpenicillin sodium for injection (day is credible herein).
The determination of embodiment 1, the prescription of tanshinone IIA microemulsions and preparation method thereof
1st, the foundation of tanshinone IIA method for measurement of concentration
1) selection of wavelength is determined
Tanshinone IIA standard items 2.72mg is taken, dissolves quantitative with 25mL methanol, using methanol as blank medium, 200~
UV scanning is carried out in 400nm wave-length coverages, the uv absorption spectra (Fig. 1) of tanshinone IIA standard items is obtained.
Tanshinone IIA has absorption maximum at 270nm as shown in Figure 1, therefore selection 270nm is Detection wavelength.
2) HPLC chromatogram condition
Chromatographic column：DiamonsilC18Post (250 × 4.6mm, 5 μm)；Mobile phase：Methanol：Water (85:15), flow velocity：1mL/
min；Column temperature：30℃；Detection wavelength：270nm；Sample size：10μL.
3) standard curve and the range of linearity
Precision measures tanshinone IIA standard items 2.72mg, is placed in 25mL brown volumetric flasks, plus simultaneously constant volume is arrived for methanol dissolving
Scale.Obtain the storing solution that concentration is 108.8 μ g/mL.Measured successively from storing solution 0.1mL, 0.2mL, 0.3mL, 0.5mL,
1.0mL, 2.0mL, 4.0mL, with methanol constant volume to scale, obtain a series of standard liquid of concentration in 10mL brown volumetric flasks.Press
According to above-mentioned HPLC chromatogram condition successively sample introduction.The peak area of the tanshinone IIA standard liquid measured is shown in Table 1 with concentration value.
Using the peak area A that measures as ordinate, concentration C is that abscissa draws the canonical plotting of tanshinone IIA (see figure
2)。
Linear regression is carried out with the peak area A and concentration C that measure, regression equation is obtained for y=51.274x-11.646 (x generations
Table concentration, y represents peak area), R2=0.9991, as a result show tanshinone IIA in the μ g/mL scopes of 1.088 μ g/mL~43.52
Interior linear relationship is good.
Table 1, the peak area of tanshinone IIA standard items and concentration table (n=3)
4) Precision Experiment
Take with certain density tanshinone IIA standard liquid (21.76 μ g/mL), continuously repeat sample introduction 6 times, survey Qi Feng faces
Product, the experimental result measured is shown in Table 2.
Table 2, Precision Experiment result table (n=6)
As shown in Table 2, the precision of instrument is good.
5) stability experiment
Take same standard liquid (wherein, the concentration of tanshinone IIA be 10.88 μ g/mL), at room temperature respectively at 0h, 2h,
Each sample introduction of 4h, 6h, 8h, 10h, 12h is once detected, is measured experimental result and is shown in Table 3.
Table 3, stability experiment result table
As shown in Table 3, tanshinone IIA is stable in the range of 0~12h.
6) rate of recovery is tested
The sample solution of basic, normal, high three concentration is prepared, respectively 1.90 μ g/mL, 2.55 μ g/mL, 5.25 μ g/mL are surveyed
Fixed result brings regression equation into and draws measured concentration, is compared with actual concentrations, the computational methods rate of recovery, is shown in Table 4.
Table 4, rate of recovery experimental result table
As shown in Table 4：The rate of recovery of tanshinone IIA is 98.24%~100.43%.
2nd, the research of tanshinone IIA basic physical and chemical
1) measure of oil/water distribution coefficient
Oil/water distribution coefficient (LogP) is to characterize polar drugs and fat-soluble parameter, and the wherein medicine in P=oil phases is dense
Drug concentration in degree/aqueous phase, is to influence one of key factor of percutaneous drug absorption.Cuticula is similar to adipose membrane, fat-soluble
Big medicine easily passes through cuticula；Medicine is passed through after cuticula, need to be distributed and then be absorbed into living Epidermis's layer, because of active table
Cortex is water-soluble tissue, and fat-soluble too big medicine is difficult to distribution and enters living Epidermis's layer, so the medicine of percutaneous dosing is needed
There is suitable oil/water distribution to inhale coefficient.
From conventional n-octyl alcohol/aqueous systems, the LogP of tanshinone IIA is determined using fask oscillating method.
Using water saturated n-octyl alcohol is distilled as oil phase, distilled water is used as aqueous phase.First take a certain amount of tanshinone IIA
It is added in oil phase and is allowed to be completely dissolved, takes this tanshinone IIA solution 3mL to be placed in 10mL EP pipes, add 3mL water, in
72h is shaken in 37 DEG C of water-baths under conditions of shading, then stands and is allowed to be layered, then draw oil phase and water respectively with syringe
Phase, determines the concentration of the tanshinone IIA in two-phase, and horizontal survey is averaged for three times.
Measurement result is LogP=LogCOil phase/CAqueous phaseThe profit distribution of=2.11, i.e. tanshinone IIA in n-octyl alcohol-water body
Coefficient is 2.11.
2) measure of solubility
Potts etc. proposes that the stable state accumulation infiltration rate of medicine is influenceed by reception liquid, and this is primarily due to when selection
Reception liquid it is very poor to the solubility property of medicine when, diffusion carry out a period of time after i.e. up to saturation or tend to saturation, make transdermal
Process stops or slowed down, so as to not be inconsistent with internal actual conditions, causes experimental bias.Therefore, the selection of external accepting medium
It is the key for describing vitro Drug transdermal penetration.In order to preferably simulate physiological disposition, the reception liquid that experiment in vitro is used should have
There is the ability for receiving transdermal drug, conventional reception liquid has physiological saline, ringer's solution and isotonic phosphate buffer etc., right
In insoluble drug, the non-physiologic composition such as ethanol, polyethylene glycol, propane diols can be added, to increase the solubility of medicine.
The poorly water-soluble of tanshinone IIA, physiological saline of the selection containing finite concentration ethanol, PEG-400 is alternative media.
Appropriate tanshinone IIA is taken in 10mL centrifuge tubes, plus appropriate solvent is configured to supersaturated solution, and centrifuge tube is placed in into (37
± 1) 72h is balanced in DEG C thermostatic water bath vibrator, remain with the presence of solid drugs, taken in the defined time in the process
Sample, after 0.45 μm of miillpore filter insulation filtering, is measured with high performance liquid chromatograph.
Fig. 3 is the block diagram of tanshinone IIA solubility in different medium.Wherein, a represents 30wt% ethanol-physiological saline,
B represents 40wt% ethanol-physiological saline, and c represents 50wt% ethanol-physiological saline, and d represents 40wt%PEG-400- physiology salts
Water, e represents 50wt%PEG-400- physiological saline.
As shown in Figure 3：Solubility of the tanshinone IIA in 50wt% ethanol-physiological saline is maximum, therefore 50% second of selection
Alcohol-physiological saline is accepting medium.
3rd, the screening of micro emulsion composition
1) selection of oil phase
Form the region of micro emulsion to improve the solubility of medicine, increase, be typically chosen to medicine have larger solubility and
Nontoxic nonirritant short chain oil phase.The conventional vegetable oil having after aliphatic acid, crude vegetal, structural modification, in being particularly
Etc. the glyceride type of chain length.Lavender (LEO) has anti-inflammatory, antibacterial, antiviral, promotes cytothesis, accelerates wound to be cured
Close, mitigate the pharmacological activity of the dermatological conditions such as psoriasis, dermatitis and eczema；Rosemary Oil (REO) has antibacterial, anti-inflammatory,
The effects such as improving skin turgor and edema, convergence skin, reduction wrinkle, removal speckle；Grapefruit essential oil GEO) there is antibacterial, depth
Layer purification dark sore and the skin of hyperemia, effect of Rosemary Extract and tissue, but grapefruit essential oil has certain photosensitive toxicity, consumption
It is unsuitable big；Tea tree ethereal oil (TTEO) has antibacterial, convergence pore, cleaning skin, improves effect of wound infection suppuration phenomenon.
The characteristics of this research combines the oil phase commonly used in pharmacy and acne have selected following several oil phases, investigate pellet
Join solubility of the A of ketone II in each oil phase.The oleic acid (OA), ethyl oleate (EO), myristic acid that tanshinone IIA is respectively configured are different
Propyl ester (IPM), Lavender (LEO), Rosemary Oil (REO), grape fruit smart (GEO), tea tree ethereal oil (TTEO) and plant
Compound essential oil (uses lavender: rosemary: grape fruit: tea tree=2: 2: 1: 1 compound essential oil) according to effect of various essential oils
Supersaturated solution, in 37 ± 0.5 DEG C of water-baths concussion 72h, then 4000r/min centrifuges 10min, takes supernatant to cross 0.45 μm
Miillpore filter, with the concentration for determining medicine after methanol dilution to suitable concentration with HPLC, every kind of oil phase is measured 3 times, asks it to put down
Average, is as a result shown in Fig. 4.
As shown in Figure 4, solubility of the tanshinone IIA in OA, EO, IPM is smaller, the solubility in plants essential oil compared with
Greatly, consider the price, effect and toxicity of plants essential oil, tentatively from compound essential oil (Lavender, Rosemary Oil,
Grapefruit essential oil and tea tree ethereal oil are using mass ratio compound essential oil made from 2: 2: 1: 1) it is used as oil phase.
2) selection of emulsifying agent
Emulsifying agent is to form one of base substance of micro emulsion, and its main function is to reduce the interface between water-oil phase
Power formation interfacial film, promotes the formation of micro emulsion.A kind of good emulsifying agent should possess advantages below：1. there is stronger emulsifying power
Power.Emulsifying capacity refers to that emulsifying agent can significantly reduce the surface tension between water-oil phase, and can be formed around emulsion droplet firmly
Emulsifying agent film ability.2. have certain physiological fitness, it is nontoxic, it is nonirritant, can orally, external application or be administered to
Medicine.3. influenceed smaller by various factors, stability is good.The species of conventional emulsifying agent has cationic emulsifier, anion
Type emulsifying agent, amphoteric ion type emulsifying agent, nonionic emulsifier and natural emulsifying agent, conventional emulsifying agent have Span classes,
Tween classes, poloxalkol class, polyoxyethylene aliphatic alcohol ether class, Emulsifier EL-60 compound , Unit are hard
Glycerol class etc..Its selection should first consider the hydrophilic lipophilic balance (HLB) of emulsifying agent, consider further that the peace of emulsifying agent
Quan Xing.It is generally acknowledged that the HLB of emulsifying agent can form w/o type micro emulsion at 4~7, O/W type micro emulsions can be formed at 14~20,7
When~14 according to process conditions can be formed can phase inversion micro emulsion.
The characteristics of comprehensive externally applied drug, acne and the toxicity of emulsifying agent, selection Tween-80, HS-15, EL-35 are alternative breast
Agent, has investigated solubility of the tanshinone IIA in various emulsifying agents, and experimental result is as shown in Figure 5.
As shown in Figure 5：Solubility of the tanshinone IIA in different emulsifiers is HS-15 ＞ Tween-80 ＞ EL-35, because
This, preliminary is emulsifying agent from HS-15.
3) selection of assistant for emulsifying agent
Assistant for emulsifying agent is primarily referred to as to merge and increasing the material of emulsion stability with emulsifying agent.The emulsifying power of assistant for emulsifying agent
Power is general very weak or without emulsifying capacity, but can improve the viscosity of emulsion, or adjusts the HLB value of emulsifying agent, is formed with emulsifying agent
Complex condensed film, enhancing emulsification film strength, prevents emulsion droplet from merging.
The assistant for emulsifying agent of conventional increase aqueous phase viscosity includes：Sodium carboxymethylcellulose, methylcellulose, sodium alginate,
Agar, Arabic gum etc.；The assistant for emulsifying agent of conventional increase oil phase viscosity includes：Unit tristerins, stearic acid, hard
Alcohol, cetanol, beeswax etc.；The coemulsifier of conventional formation complex condensed film includes：It is ethanol, propane diols, n-butanol, sweet
Oil, PEG400 etc..
Fig. 6 is the block diagram of tanshinone IIA solubility in different assistant for emulsifying agents.Wherein, E represents ethanol, and O represents 1,2- third
Glycol, G represents glycerine.
As shown in Figure 6：Solubility of the tanshinone IIA in different assistant for emulsifying agents is that absolute ethyl alcohol ＞ 1,2- propane diols ＞ is sweet
Oil.And ethanol has anti-inflammatory, the effect of pore refining, the part bacterium removed on skin, the treatment to acne can also be sterilized
There is certain booster action, therefore, preliminary is assistant for emulsifying agent from ethanol.
4th, the determination of tanshinone IIA microemulsion formulation
1) emulsifying agent composition is determined
Found in preliminary attempt：Solutrol HS-15, which are used alone, can not make the selected formula of the above that micro emulsion is made,
Tween80 and two kinds of emulsifying agents of Solutrol HS-15 must be used in mixed way could produce O/W microemulsion regions in triangle phasor, and
Under equal conditions Solutrol HS-15:Tween 80=7:Micro emulsion best results when 3.
2) what is be formulated primarily determines that
With emulsifying agent (Solutrol HS-15: Tween 80=7：3) mixture, essential oil, distilled water with absolute ethyl alcohol are
Pseudo-ternary phase diagram is drawn on three summits of phasor.
Respectively by the emulsifying agent (Solutrol HS-15: Tween 80=7：3) with ethanol using mass ratio (Km) value as 9:
1st, 8: 2,7: 3,6: 4,5: 5,4: 6,3: 7,2: 8,1: 9 mixing, is then 9: 1,8: 2,7 in mass ratio by mixture and oil phase:
3rd, 6: 4,5: 5,4: 6,3: 7,2: 8,1: 9 in mixed at room temperature, and aqueous phase is added dropwise to titrate in each mixture of normal direction, reaches and is seen after balance
Clarity and mobility are examined, judges whether to form blank micro emulsion.
Found during in above ratio step-by-step operation：When Km values are less than 5: 5, no matter the ratio of oil phase is how many
Only have the big colloid of viscosity and milky emulsion to occur in whole process, O/W microemulsion regions do not occur, so Km values are less than 5：
5 situation is not considered；Emulsifier content is excessive when Km values are 9: 1, can also exclude.It is 8: 2,7: 3,6 directly to draw Km values:
4th, 5: 5 pseudo-ternary phase diagram (see Fig. 7 A, Fig. 7 B, Fig. 7 C, Fig. 7 D), therefrom finds out O/W microemulsion regions.
Fig. 7 A are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=8/2.
Fig. 7 B are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=7/3.
Fig. 7 C are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=6/4.
Fig. 7 D are the pseudo-ternary phase diagram of the micro emulsion formed under the conditions of Km=5/5.
It was found from Fig. 7 A, Fig. 7 B, Fig. 7 C and Fig. 7 D：When Km=8/2,7/3 region of micro emulsion with respect to Km=6/4,5/5 it is micro-
Newborn region area is big, but the content of emulsifying agent and assistant for emulsifying agent is big.
3) formula range is determined in O/W microemulsion regions
It is preferred that principle：1st, good into transparency after breast, viscosity is smaller；2nd, micro emulsion is stablized at room temperature, and institute's reconnaissance can not be
Micro emulsion area edge, otherwise micro emulsion is unstable；3rd, the micro emulsion being made into can use water infinite dilution.
Primarily determine that the scope of formula is according to above principle：The mixing of 9/1 >=Km >=1/1,9/1 >=emulsifying agent and ethanol
Thing/oil phase >=8/2, aqueous phase >=40%.
4) it is formulated preferred
Consider blended emulsifier, ethanol, oil phase and drugloading rate to carry out preferably.
(1) Km values is preferred
Blended emulsifier has micro cytotoxicity, and consumption should try one's best less, and ethanol long-time use can make dry skin, institute
Median should be selected with Km values, experiment is drawn：Km=8/2, Km=7/3, Km=6/4 are desired proportions；
(2) ratio of the mixture and oil phase of blended emulsifier and ethanol is preferred
Because tanshinone IIA is dissolved in, oil does not dissolve in water, so in the case where other conditions allow, being considered as increasing oil phase
Ratio.The minimum value that mixture/oil phase of emulsifying agent and ethanol is understood from above-mentioned experimental result is 8/2, in identical Km
In the case of value, the usage amount of oil is continuously increased, the mixture/oil phase for making emulsifying agent and ethanol is respectively 9/1,8/1,7/1,6/
1st, 5/1,4/1 six micro emulsion samples are made, can be found by observing transparency and stability：The mixture of emulsifying agent and ethanol/
The transparency and stability of sample are substantially better than other during oil phase=6/1.
(3) ratio of aqueous phase is preferred
1. solubility of the tanshinone IIA in water is smaller；
2. the minimum dosage of water is 40% from the above.
In the case where other three-phase consumptions are fixed and meet above principle, the content of water often increases by 1% and makees a sample
Observe the stability of tanshinone IIA in micro emulsion.
As a result show that the content of water does not produce precipitation after three days for 46% sample, and transparency is better than other and contained
The micro emulsion of water.
In summary the preferred micro emulsion formula of experimental result has：(1) mixing of Km=8/2, blended emulsifier and ethanol
Thing/oil phase=6/1, water is 46%；(2) mixture/oil phase=6/1 of Km=7/3, blended emulsifier and ethanol, water is 46%；
(3) Km=6/4, mixed surfactant/oil phase=6/1, water is 46%.
Then three above blank micro emulsion sample is made, transparency is observed after being long placed in and is compared, is as a result shown：Formula three
Transparency is best.Therefore it is preferred that formula be：Mixture/oil phase=6/1 of Km=6/4, blended emulsifier and ethanol, water is
46%.
5th, the determination of the preparation technology of tanshinone IIA micro emulsion
The order of mixing time, mixing speed and addition medicine has a certain impact to micro emulsion drug carrying amount, with single factor test
Test to investigate influence of the three above condition to micro emulsion drug carrying amount, preparation technology is then optimized by orthonormal design of experiments.Institute
There is experiment to carry out three times, take its average value.
1) single factor experiment result
Ith, influence of the mixing time to micro emulsion drug carrying amount
Mixing time has a certain impact to the drugloading rate of micro emulsion, and the time of stirring and emulsifying is according to oil phase, aqueous phase
The factor such as volume ratio, the species of emulsifying agent and consumption, the viscosity of two-phase is determined.Therefore, weigh each composition in prescription and (smoke clothing
Careless 0.074g, rosemary 0.074g, grape fruit 0.037g, tea tree 0.037g, absolute ethyl alcohol 0.555g, Solutrol HS-15
0.5838g, Tween 80 0.2502g, water 1.389g), low whipping speed is 250r/min, the condition of absolute ethyl alcohol dissolving medicine
Under, influence of the different mixings time to micro emulsion drug carrying amount is investigated, each time sample is 3.Different mixings time are carried to micro emulsion
The influence of dose is as shown in Figure 8.
As shown in Figure 8, drugloading rate increases with the time lengthening of stirring within a certain period of time, to specific time point after again
The increase time will not also increase drugloading rate.
IIth, influence of the mixing speed to micro emulsion drug carrying amount
Suitable mixing speed can make oil phase, aqueous phase be sufficiently mixed uniform and reach optimal drugloading rate.Therefore, weigh
Each composition (Lavender 0.074g, Rosemary Oil 0.074g, grapefruit essential oil 0.037g, tea tree ethereal oil in prescription
0.037g, absolute ethyl alcohol 0.555g, Solutrol HS-15 0.5838g, Tween 80 0.2502g, water 1.389g), in stirring
Under conditions of time is 0.5h, absolute ethyl alcohol dissolving medicine, the influence of different mixing speeds to micro emulsion drug carrying amount is investigated, is each stirred
Speed sample is mixed for 3.
Influence of the different mixing speeds to micro emulsion drug carrying amount is as shown in Figure 9.
As shown in Figure 9, in the range of certain mixing speed, with the increase drugloading rate increase of mixing speed.
IIIth, influence of the dosing method to micro emulsion drug carrying amount
Medicine feed postition has a certain impact to micro emulsion drug carrying amount, studies 3 kinds of medicine feed postitions, i.e.,：A, first prepare
Blank micro emulsion adds medicine；B, medicine is dissolved in absolute ethyl alcohol, the other compositions added in prescription；C, with oil phase, breast
Agent, assistant for emulsifying agent dissolve medicine, the other compositions added in prescription.
Each composition (Lavender 0.074g, Rosemary Oil 0.074g, grapefruit essential oil are weighed according to prescription
0.037g, tea tree ethereal oil 0.037g, absolute ethyl alcohol 0.555g, Solutrol HS-15 0.5838g, Tween 80 0.2502g, water
1.389g), it is under conditions of 0.5h, mixing speed are 250r/min, to investigate different dosing methods and micro emulsion is carried in mixing time
The influence of dose, every kind of dosing method sample is 3.Influence of the different dosing methods to micro emulsion drug carrying amount is as shown in table 5.
The influence table of table 5, order of addition of ingredients to micro emulsion drug carrying amount
As shown in Table 5, the drugloading rate of different order of addition of ingredients micro emulsions is different, wherein the micro emulsion drug carrying amount prepared using C methods
It is maximum.
2) orthogonal test
To obtain the process conditions of the optimal drugloading rate of micro emulsion, on the basis of single factor exploration, to mixing time, stirring speed
3 factors of feed postition of degree and medicine carry out orthogonal test, and the content of tanshinone IIA is index using in micro emulsion, prepared by optimization
The experimental factor water-glass of technique is shown in Table 6, and orthogonal experiments are shown in Table 7, and orthogonal test variance analysis is shown in Table 8.
Table 6, orthogonal test factor level table
Note：The blank micro emulsion after mixing the emulsifying agent, assistant for emulsifying agent, oil phase and water by being made.
Table 7, Orthogonal Experiment and Design scheme and result table
The orthogonal test analysis of variance table of table 8
Note：F0.05 (2,2)=19.00, F0.01 (2,2)=99.00
From variance analysis, each factor is B ＞ C ＞ A, A factors on micro emulsion drug carrying amount influence size order compared with other shadows
Sound is smaller, therefore is that error carries out variance analysis with A, as a result shows that B factors have significant, C factors are without conspicuousness
Meaning.In summary analysis and production cost etc., the optimised process for obtaining the preparation of tanshinone IIA micro emulsion be mixing time be 0.5h,
Mixing speed be 250r/min, with blank micro emulsion dissolve medicine when micro emulsion drugloading rate it is optimal.
Confirmatory experiment：It is that 0.5h, mixing speed are 250r/min, the work of medicine dissolved with blank micro emulsion according to mixing time
Skill prepares three batches of samples, surveys its drugloading rate, experimental result is as follows：
Embodiment 2, tanshinone IIA micro emulsion property research
According to following formulas：Tanshinone IIA 28mg, Lavender 1.230g, Rosemary Oil 1.230g, grape fruit essence
Oily 0.61g, tea tree ethereal oil 0.61g, absolute ethyl alcohol 9.25g, Solutrol HS-15 9.73g, Tween 80 4.17g, water
23.15g.Mixing time be 0.5h, mixing speed be 250r/min, to dissolve medicine with blank micro emulsion micro- to prepare tanshinone IIA
Breast (mass concentration of tanshinone IIA is 0.54mg/mL)
1) tanshinone IIA micro emulsion is homogeneous, transparent pink liquid.
2) judgement of tanshinone IIA microemulsion type
Two parts of the micro emulsion of same volume is taken, two is added simultaneously respectively and drips Sudan red dyes and methylene blue dye solution, it is quiet
Only place, blue diffusion velocity is more than red diffusion velocity.
3) measure of tanshinone IIA micro emulsion pH value
Take tanshinone IIA micro emulsion described in 10mL to be measured with pHS-25pH tanshinone IIA micro emulsion pH value be 6.34.
4) measure of tanshinone IIA micro emulsion particle diameter
Taking 0.5mL blank micro emulsion respectively, (composition is：Lavender 0.074g, rosemary 0.074g, grapefruit essential oil
0.037g, tea tree ethereal oil 0.037g, absolute ethyl alcohol 0.555g, Solutrol HS-15 0.5838g, Tween 80 0.2502g and
Water 1.389g) and the tanshinone IIA micro emulsion (formula of tanshinone IIA micro emulsion be tanshinone IIA 28mg, Lavender
1.230g, Rosemary Oil 1.230g, grapefruit essential oil 0.61g, tea tree ethereal oil 0.61g, absolute ethyl alcohol 9.25g, Solutrol
HS-15 9.73g, Tween 80 4.17g and water 23.15g, mixing time be 0.5h, mixing speed be 250r/min, it is micro- with blank
Breast dissolves medicine to prepare), add 9.5mL water to be diluted respectively, its particle diameter, experimental result Figure 10 are surveyed with Malvern laser particle size
It is shown.The particle diameter for measuring blank micro emulsion be 12.15nm, carry medicine micro emulsion particle diameter be 12.78nm.
5) form of tanshinone IIA micro emulsion
Sample is prepared using background stain：Take 0.5mL steps 4) described in blank micro emulsion and 0.5mL steps 4) described in it is red
20 times with distilled water diluting of the ginseng A micro emulsions of ketone II are testing sample, support the copper mesh of film to be placed on stencil plate by Formvar is loaded with,
A little testing sample is added on film, 30min is dried naturally, then the drop of 2% phosphotungstic acid one is added dropwise, 10min is dried naturally, filter paper is used
Unnecessary liquid is siphoned away, is placed under the type transmission electron microscopes of TEM-100X II and observes the form of micro emulsion.As a result it is as shown in figure 11.
As shown in Figure 11, the tanshinone IIA micro emulsion and blank the micro emulsion rounded distribution under Electronic Speculum, and size is equal
Form is without significant change after even, rounding, load medicine.
The determination of embodiment 3, the prescription of tanshinone IIA microemulsion gel preparation
The content of Acritamer 940 is determined by vitro permeation assay
1) screening of carbomer consumption
Carbomer (Carbopol) also known as carbopol, are a kind of macromolecules being crosslinked by acrylic acid and allyl sucrose
Polymer.Produced, taken in by American and Britain, medium multinational pharmacopeia by Goodrich companies of the U.S. earliest.It is extensive at present
Applied to skin, ophthalmically acceptable, oral cavity, Ultrasonic Diagnosis gel.Carbomer is white, loose, acid, tool hygroscopicity and special smelly
The powder of taste, is dissolved in water, ethanol and glycerine, nontoxic, nonirritant, and gel can be formed by meeting water, and average moisture content is 8%, molecule
Contain 56%~58% carboxylic group in structure, as medium pH<When 4, carboxyl is hardly dissociated；Work as pH<3 or pH>It is sticky when 12
Degree declines, the most sticky stable hydrogel for forming homogeneous transparent in pH6~12, with drug release it is fast, applied without greasy, easy
Cloth, it is easy to clean, to skin and nonirritant mucous membrane the characteristics of.
The formula of tanshinone IIA micro emulsion：The mass parts of tanshinone IIA 0.08, plants essential oil (lavender：Rosemary：Grape
Shaddock：Tea tree=2：2：1：1) 7.4 mass parts；The mass parts of assistant for emulsifying agent absolute ethyl alcohol 18.5, emulsifying agent (SolutrolHS-15：
Tween80=7:3) 27.8 mass parts, the mass parts of water 46.3.Formula based on above-mentioned tanshinone IIA micro emulsion investigates carbomer base
Matter concentration, early stage preliminary experiment is found：When carbomer substrate concentration is ＞ 1%, carbomer is difficult to be swelled uniform and solidifying during preparation
Gluing denseness is higher, not easy to apply, and as concentration ＜ 0.4%, gel viscosity is too small, is difficult to retain in skin surface, thus
The substrate concentration of selection 0.5%~0.9% is investigated by the way that tablets in vitro experiment is further.
2) foundation of analysis method
" tanshinone IIA method for measurement of concentration ", under this chromatographic condition, Acritamer 940 and triethanolamine are seen in embodiment 1
Tanshinone IIA detection assay is not disturbed (see Figure 12).
Figure 12 is tanshinone IIA standard solution HPLC (A figures), the transdermal HPLC of blank micro emulsion gel (B figures), Tanshinone II
The transdermal HPLC of A micro emulsion gels (C figures).
Wherein, the composition of the tanshinone IIA micro emulsion gel is：Tanshinone IIA 28mg, Lavender 1.230g, fan
Repeatedly essential oil 1.230g, grapefruit essential oil 0.61g, tea tree ethereal oil 0.61g, absolute ethyl alcohol 9.25g, SolutrolHS-15
9.73g, Tween 80 4.17g and water 23.15g, Acritamer 940 0.25g, triethanolamine 0.5g.
The composition of the blank micro emulsion gel is：Lavender 1.230g, Rosemary Oil 1.230g, grapefruit essential oil
0.61g, tea tree ethereal oil 0.61g, absolute ethyl alcohol 9.25g, Solutrol HS-15 9.73g, Tween 80 4.17g and water
23.15g, Acritamer 940 0.25g, triethanolamine 0.5g.
The transdermal HPLC of tanshinone IIA micro emulsion gel, the transdermal HPLC of blank micro emulsion gel are respectively to fix bag filter
Between the supply pool and acceptance pool of the vertical diffusion cells of Franz, 50% ethanol normal saline solution is filled in acceptance pool, is included
One stirrer, rotating speed is to be separately added into tanshinone IIA micro emulsion gel 0.250g, blank micro emulsion in 200r/min, supply pool to coagulate
Whole device is placed in the drug transdermal diffusion test instrument of (37 ± 0.5) DEG C by glue 0.25g, by acceptance pool when 1
Liquid is all poured out, and carries out HPLC detections.
The tanshinone IIA standard solution is the methanol solution for the tanshinone IIA standard items that concentration is 23.06 μ g/mL.
The concentration of medicine is low during tablets in vitro, it is therefore desirable to which the ginseng A of ketone II ranges of linearity for carrying out low concentration are investigated.
Compound concentration is 0.057 μ g/mL, 0.115 μ g/mL, 0.230 μ g/mL, 1.148 μ g/mL, 2.297 μ g/mL pellet
Join the A standard solutions of ketone II (solvent of standard solution is methanol), according to above-mentioned chromatographic condition successively sample introduction.The red sage root measured
The peak area of the A standard solutions of ketone II is shown in Table 9 with concentration value.
Using the peak area that measures as ordinate, concentration is abscissa, draws the canonical plotting of tanshinone IIA (see figure
13).As a result show that tanshinone IIA linear relationship in the range of the μ g/mL of 0.057 μ g/mL~2.297 is good
Linear regression is carried out with the peak area and concentration that measure, regression equation is obtained for y=53.84x-0.0138, R2=
0.9992。
The peak area of the tanshinone IIA reference substance of table 9 and concentration table (n=3)
3) tablets in vitro experimental study
To select suitable substrate concentration, we use Franz diffusion cell methods, using bag filter as the barrier film of infiltration,
50% ethanol normal saline solution is receives medium, under the conditions of (37 ± 0.5) DEG C, 200r/min, determines tanshinone IIA micro-
6 hours cumulative release amounts of curdling glue.
By with the water-swellable Acritamer 940 of recipe quantity half, the Acritamer 940 being swelled；By tanshinone IIA, emulsification
Agent, assistant for emulsifying agent, oil phase are mixed with remaining water, obtain mixed liquor；The Acritamer 940 being swelled and the mixed liquor are mixed
Close, be eventually adding the pH value of triethanolamine regulation system between 6.5-7.5, obtain tanshinone IIA microemulsion gel preparation, respectively
It is 0.5%, 0.7%, 0.9% to prepare carbomer content, and each sample is 3.Bag filter is fixed on the vertical diffusion cells of Franz
Supply pool and acceptance pool between, filled in acceptance pool 50% ethanol normal saline solution, include a stirrer, rotating speed is
200r/min, add in supply pool tanshinone IIA micro emulsion gel 0.250g, micro emulsion 0.25mL whole device is placed in (37 ±
DEG C 0.5) in drug transdermal diffusion test instrument, the liquid in acceptance pool is all fallen when 0.5,1,2,3,4,5,6h
Go out, while adding 50% ethanol solution of the same volume with equitemperature, with 0.22 μm of filtering with microporous membrane, inject efficient liquid phase
Chromatograph is determined.Bring the concentration that standard curve calculates medicine into, then calculate the unit Percutaneous permeability Q of medicine respectivelyn, stable state
Infiltration rate constant Js.
Wherein, Cn is the drug monitoring concentration at t time points, CiFor the drug monitoring concentration of t time point former points, V0To connect
By the cumulative volume (6.5mL) of medium in pond, V is sample volume (6.5mL), and A is effective diffusion area (2.54cm2).During with Q pairs
Between t make linear regression, the slope of gained straight line is steady-state permeation speed constant Js (μ gcm-2·h-1)。
Figure 14 is tanshinone IIA cumulative in vitro infiltration capacity curve.
Each system body outer osmotic absorbs as shown in table 10.
Each system body outer osmotic of table 10 absorbs table (n=3)
From table 10 and Figure 14, within the identical time, when the consumption of carbomer is 0.5%, the accumulation of medicine is passed through
Amount is most, therefore is 0.5% from the consumption of carbomer；The Percutaneous permeability of tanshinone IIA micro emulsion is big within the identical time
In the Percutaneous permeability of micro emulsion gel.
The preliminary examinations of embodiment 4, tanshinone IIA micro emulsion gel property
The formula of tanshinone IIA micro emulsion gel be tanshinone IIA 25mg, Lavender 1.230g, rosemary 1.230g,
Grapefruit essential oil 0.61g, tea tree 0.61g, absolute ethyl alcohol 9.25g, Solutrol HS-15 9.73g, Tween 80 4.17g, water
23.15g, Acritamer 940 0.25g and triethanolamine 0.5g.
1) appearance character of tanshinone IIA micro emulsion gel
This product is pale red clear gum material.
2) measure of pH value
The above-mentioned tanshinone IIA micro emulsion gels of 1.0g are taken, 10mL distilled water is added, dissolving mixes, determined with pH meter, measure pH
It is worth for 7.08.
3) measure of viscosity
Suitable rotor and rotating speed are selected, the viscosity for measuring tanshinone IIA micro emulsion gel is 104.2Pas.
4) centrefuge experiment
Take tanshinone IIA micro emulsion gel to be put into centrifuge tube, 30min is centrifuged with 4000r/min, gel is without lamination.
5) measure of particle diameter
Take tanshinone IIA micro emulsion gel (tanshinone IIA 25mg, Lavender 1.230g, rosemary 1.230g, grape
Shaddock oil 0.61g, tea tree 0.61g, absolute ethyl alcohol 9.25g, Solutrol HS-15 9.73g, Tween 80 4.17g, water
23.15g, Acritamer 940 0.25g and triethanolamine 0.5g), 20 multiples are diluted with water, with Nanophox nano-particle size analysis instrument
Determine, measure and carry the particle diameter of medicine micro emulsion gel for 29.94nm.
Take blank micro emulsion gel (Lavender 1.230g, rosemary 1.230g, grapefruit essential oil 0.61g, tea tree
0.61g, absolute ethyl alcohol 9.25g, Solutrol HS-15 9.73g, Tween 80 4.17g, water 23.15g, Acritamer 940
0.25g and triethanolamine 0.5g), 20 multiples are diluted with water, is determined with Nanophox nano-particle size analysis instrument, measures blank micro emulsion
The particle diameter of gel is 20.63nm.
6) the microscopic pattern observation of tanshinone IIA micro emulsion gel
Sample is prepared using background stain：Above-mentioned blank micro emulsion gel and above-mentioned tanshinone IIA micro emulsion gel is taken to use respectively
20 times of distilled water diluting is testing sample, supports the copper mesh of film to be placed on stencil plate by Formvar is loaded with, plus a little on film
Testing sample, dries 30min naturally, then the drop of 2% phosphotungstic acid one is added dropwise, and 10min is dried naturally, unnecessary liquid is siphoned away with filter paper
Body, is placed under the type transmission electron microscopes of TEM-100X II and observes the form of micro emulsion gel.
Figure 16 is blank micro emulsion gel (left figure) and the transmission electron microscope figure for carrying medicine micro emulsion gel (right figure).Wherein,
Left figure and right figure are same multiplication factor × 50000.
As seen from Figure 16, tanshinone IIA micro emulsion gel emulsion droplet rounded distribution under Electronic Speculum, and uniform in size,
Form is without significant change, particle diameter increase after rounding, load medicine.
The Pharmacodynamic research of embodiment 5, micro emulsion gel
1) strain is recovered
Take a certain amount of propionibacterium acnes strain on blood agar culture-medium with disposable sterilized oese, detest at 37 DEG C
48h is cultivated under the conditions of oxygen；A certain amount of staphylococcus aureus strain is taken with disposable sterilized oese on agar medium,
24h is cultivated under 37 DEG C of aerobic conditions.
2) preparation of drug sensitive plate
Using doubling dilution by Benzylpenicillin sodium salt, erythromycin lactobionate, blank micro emulsion and micro emulsion gel, carry medicine micro emulsion and micro-
Curdling glue is configured to finite concentration pastille meat soup.
48 sterile orifice plates are taken, meat soup 0.5mL is added per hole, antibacterials stoste 0.5mL is added in the 1st hole and is mixed,
Then draw 0.5mL and 0.5mL drawn into the 2nd hole, after mixing into the 3rd hole, so continuous doubling dilution, make Benzylpenicillin sodium salt,
Erythromycin lactobionate is that final concentration is 1024,512,256,128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/mL
The pastille meat soup of (totally 14 concentration gradients), last 2 hole is not added with decoction for blank control；Blank micro emulsion and micro emulsion gel, load medicine
Micro emulsion and micro emulsion gel be final concentration be 512,256,128,64,32,16,8,4,2,1,0.5,0.25,0.125g/mL (totally 13
Individual concentration gradient) pastille meat soup, last 3 hole is not added with decoction for blank control.
3) preparation of bacteria suspension
24h golden yellow will be cultivated under propionibacterium acnes and 37 DEG C of aerobic conditions that 48h is cultivated under 37 DEG C of anaerobic conditions
Staphylococcus is corrected to 0.5 Maxwell than turbid standard (about 1.5 × 10 with the physiological saline of sterilizing8Cfu/mL), meat soup is then used again
Culture medium is diluted to 1.0 × 106cfu/mL。
4) it is inoculated with
The bacterium solution prepared is added in drug sensitive plate, meat soup 0.5mL is added per hole, makes the final concentration of of bacterium in every hole
1.0×105Cfu/mL, Benzylpenicillin sodium salt, erythromycin lactobionate final concentration of 512,256,128,64,32,16,8,4,2,1,0.5,
0.25th, 0.125,0.0625 μ g/mL, blank micro emulsion and blank micro emulsion gel, carry medicine micro emulsion and carry medicine micro emulsion gel final concentration (with
Tanshinone IIA meter) it is 256,128,64,32,16,8,4,2,1,0.5,0.25,0.125,0.0625 μ g/mL.
5) culture of bacterial strain
Propionibacterium acnes drug sensitive plate is placed in anaerobism bag, anaerobic gas generation bag, anaerobism bar is put into, sack is close
Envelope, is placed under 37 DEG C of anaerobic conditions and cultivates 48h；Staphylococcus aureus drug sensitive plate is placed under 37 DEG C of aerobic conditions and cultivates 24h.
It the results are shown in Table shown in 11.
Minimal inhibitory concentration (MIC) of the tanshinone IIA of table 11 to propionibacterium acnes and staphylococcus aureus
As seen from the above table, for staphylococcus aureus, the MIC of Benzylpenicillin sodium salt is 32 μ g/mL, the MIC of Erythromycin Lactobionate
It is 256 μ g/mL for the MIC of 256 μ g/mL, tanshinone IIA micro emulsion and micro emulsion gel；For propionibacterium acnes, Benzylpenicillin sodium salt
MIC is that the MIC that 0.0625 μ g/mL, the MIC of Erythromycin Lactobionate are 0.5 μ g/mL, tanshinone IIA micro emulsion and micro emulsion gel is 64 μ
g/mL。
Compared to antibiotics such as Benzylpenicillin sodium salt and Erythromycin Lactobionates, although tanshinone IIA micro emulsion and micro emulsion gel
Bacteriostatic activity it is lower slightly, but tanshinone IIA micro emulsion and micro emulsion gel will not produce drug resistance compared with antibiotics.
Claims (6)
1. a kind of tanshinone IIA microemulsions, are made up of following components：Tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase and
Water；
The emulsifying agent is that mass ratio is 6-8:3 Solutrol HS-15 and the mixture of Tween 80；
The assistant for emulsifying agent is absolute ethyl alcohol；
The oil phase is using mass ratio as 2 by Lavender, Rosemary Oil, grapefruit essential oil and tea tree ethereal oil:2:1:1 group
Into compound plant essential oil；
The mass ratio of the emulsifying agent and assistant for emulsifying agent is 8/2-6/4；
The mixture of the emulsifying agent and assistant for emulsifying agent is 9/1-4/1 with the mass ratio of the oil phase；
In the tanshinone IIA microemulsions, the weight/mass percentage composition of water is >=40%；
In the tanshinone IIA microemulsions, the mass concentration of tanshinone IIA is 0.55mg/mL-1.02mg/mL.
2. a kind of method of the tanshinone IIA microemulsions prepared described in claim 1, including it is following 1), 2) or 3)：
1) emulsifying agent, assistant for emulsifying agent, oil phase and water are carried out being mixed to get blank micro emulsion, tanshinone IIA is added to institute
State in blank micro emulsion, after stirring, obtain tanshinone IIA microemulsions；
2) tanshinone IIA is dissolved with the assistant for emulsifying agent under stirring, the emulsifying agent, oil phase and water is added, obtains tanshinone
II A microemulsion formulations；
3) tanshinone IIA is dissolved with the oil phase, emulsifying agent and assistant for emulsifying agent under stirring, water is added, obtains tanshinone IIA micro-
Emulsion formulation.
3. method according to claim 2, it is characterised in that：The speed of the stirring is 250r/min-750r/min,
Time is 0.5h-1.5h.
4. a kind of tanshinone IIA microemulsion gel preparation, including following components：Tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase,
Water, Acritamer 940 and triethanolamine；
The emulsifying agent is that mass ratio is 7:3 Solutrol HS-15 and the mixture of Tween 80；
The assistant for emulsifying agent is absolute ethyl alcohol；
The oil phase is using mass ratio as 2 by Lavender, Rosemary Oil, grapefruit essential oil and tea tree ethereal oil:2:1:1 group
Into compound plant essential oil；
The mass ratio of the tanshinone IIA and the emulsifying agent, assistant for emulsifying agent, oil phase and water is 0.055-0.102:27.8:
18.5:7.4:46.3；
The quality of Acritamer 940 is the 0.5%-0.9% of the gross mass of tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase and water.
5. a kind of method of the tanshinone IIA microemulsion gel preparation prepared described in claim 4, including it is following：With recipe quantity one
Half water-swellable Acritamer 940, the Acritamer 940 being swelled；By tanshinone IIA, emulsifying agent, assistant for emulsifying agent, oil phase with it is remaining
Under water mixing, obtain mixed liquor；The Acritamer 940 being swelled is mixed with the mixed liquor, triethanolamine is eventually adding
The pH value of regulation system obtains tanshinone IIA microemulsion gel preparation between 6.5-7.5.
6. the tanshinone IIA microemulsion gel preparation described in tanshinone IIA microemulsions or claim 4 described in claim 1
Application in Retinoids, Retin-A, Renova, Accutane is prepared.
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| CN111012687A (en) * | 2019-12-31 | 2020-04-17 | 中山大学新华学院 | Compound fisetin microemulsion gel and preparation method and application thereof |
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| CN113952292B (en) * | 2021-10-28 | 2023-11-03 | 河南中医药大学 | Rosemary essential oil nanoemulsion gel, preparation method and application thereof |
| CN115154408A (en) * | 2022-05-12 | 2022-10-11 | 中国人民解放军空军特色医学中心 | Novel nano drug-loading system of tanshinone extract microemulsion hydrogel as well as preparation method and application of novel nano drug-loading system |
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