CN114796115A - Dendrobium officinale polysaccharide external preparation as well as preparation method and application thereof - Google Patents

Dendrobium officinale polysaccharide external preparation as well as preparation method and application thereof Download PDF

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CN114796115A
CN114796115A CN202210271181.6A CN202210271181A CN114796115A CN 114796115 A CN114796115 A CN 114796115A CN 202210271181 A CN202210271181 A CN 202210271181A CN 114796115 A CN114796115 A CN 114796115A
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emulsion
emulsifier
preparation
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water
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CN114796115B (en
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蒋献
聂宇
郭玲宏
宋旭
向阳
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West China Hospital of Sichuan University
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Abstract

The invention provides a dendrobium officinale polysaccharide external preparation, a preparation method and application thereof, the preparation is a w/o/w type emulsion prepared by taking dendrobium officinale polysaccharide as an active ingredient and adding pharmaceutically acceptable auxiliary materials, the preparation method is simple, the preparation stability is good, and concentric vesicles formed by alternating multilayer water and oil with a similar onion structure can realize the slow-release long-acting function of active substances; meanwhile, the stability, the drug loading rate and the encapsulation rate of the medicine can be improved, the transdermal absorption of the dendrobium officinale polysaccharide can be effectively increased, and the dendrobium officinale polysaccharide is used as an external preparation for treating skin photoaging and has excellent clinical application prospect.

Description

Dendrobium officinale polysaccharide external preparation as well as preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a dendrobium officinale polysaccharide external preparation as well as a preparation method and application thereof.
Background
Skin photoaging is the damage caused by prolonged exposure of the skin to sunlight. It is manifested by rough, thickened and dry exposed skin, loose skin, increased wrinkle, excessive pigmentation or telangiectasia of local part, and various benign or malignant tumors (such as solar keratosis, squamous cell carcinoma, malignant melanoma, etc.).
Studies have shown that the underlying mechanism of skin photoaging is related to the production of ROS and subsequent oxidative stress. ROS are produced by mitochondria, ultraviolet radiation and other external factors. ROS can activate various intracellular kinases, including MAPK, AKT and ERK, in a direct or indirect manner, resulting in the production of activin 1(AP-1) and nuclear factor- κ B (NF-kB). Activated AP-1 and NF-kB regulate transcription of Matrix Metalloproteinases (MMPs). Four members of MMPs, including MMP1, MMP2, MMP3, and MMP9, can completely degrade collagen. Notably, MMPs are members of the elastase family, having the ability to degrade elastin and lower elastin levels. Simultaneously, activated AP-1 and NF-kB may also increase expression of MMP Tissue Inhibitor (TIMP). Nevertheless, MMP activation is still more dominant than TIMP activity, leading to degradation of collagen, elastin, fibrils and some other dermal extracellular matrices. In addition, NF-kB is a major activator of inflammatory cell infiltration by inducing the production of the proinflammatory cytokines TNF- α, Interleukin (IL) -1, IL-6 and VEGF, which stimulates further MMP production. ROS-induced AP-1 also reduced collagen biosynthesis. The decrease in type I and type III collagen results from the down-regulation of transforming growth factor-b receptors by AP-1 and impairment of subsequent cascades and pathways. Eventually, collagen and elastin levels in the dermal layer decrease, thus accelerating skin aging.
Dendrobium officinale is a perennial epiphytic herb of the genus Dendrobium of the family Orchidaceae and has the reputation of "Zhonghuaxiancao" and the like. In recent years, research results show that dendrobium officinale contains substances such as polysaccharide, flavonoid, phenols, vitamins and amino acids, wherein Dendrobium Officinale Polysaccharide (DOP) is the main active component. The Dendrobium officinale polysaccharide has various effects of moisturizing, resisting oxidation, inhibiting inflammatory reaction and the like. Specifically, the dendrobium officinale polysaccharide can effectively reduce oxidative stress load through ways of inhibiting the generation of ROS, enhancing an antioxidant enzyme system, inhibiting inflammatory reaction and the like. In the process of researching that the compound polysaccharide inhibits ultraviolet-induced photoaging of skin cells, Yangkai industry and the like find that the dendrobium officinale polysaccharide has a certain photoaging resistance effect, and the effect is superior to brown alga polysaccharide, ganoderma lucidum polysaccharide or bamboo fungus polysaccharide. However, this research only has been limited to the effect of polysaccharide solutions on cells, which is not practical.
Because the transdermal absorption of dendrobium officinale polysaccharide is relatively low, a proper carrier needs to be found for transdermal administration. An emulsion is a liquid preparation consisting of an aqueous phase, an oil phase, an emulsifier and a drug, and is a relatively mature drug delivery system that can stably deliver active ingredients contained therein to the skin so as not to be changed. In addition, some components in the emulsion have the function of promoting penetration, and the type and the dosage of the oil phase can change the affinity of the medicine and the skin, thereby being beneficial to the medicine entering the stratum corneum. Therefore, the research on the external dendrobium officinale polysaccharide emulsion which has high stability, can realize long-acting slow release and high-efficiency transdermal absorption has very important value in the field of medicines for treating skin photoaging. However, for the preparation of the emulsion, selection of different raw material medicines, auxiliary materials and proportions can greatly affect the stability and the actual drug effect of the emulsion, and at present, no emulsion with good stability, good transdermal administration effect and capability of effectively treating skin photoaging is reported, wherein dendrobium officinale polysaccharide is used as an active ingredient.
Disclosure of Invention
The invention aims to provide a dendrobium officinale polysaccharide external preparation as well as a preparation method and application thereof.
The invention provides a preparation for treating skin photoaging, which is prepared by taking dendrobium officinale polysaccharide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Further, the above preparation is an external preparation.
Further, the external preparation is a solution, powder, tincture, spirit, lotion, oil, emulsion, ointment, plaster, cream, cataplasm, plastics, pellicle, cataplasma, gel, liniment, aerosol, patch or patch, preferably an emulsion.
Further, the emulsion is a multilayer emulsion, preferably a w/o/w type multilayer emulsion.
Furthermore, the w/o/w type multilayer emulsion comprises an internal water phase, an oil phase, an external water phase and an emulsifier, wherein the weight ratio of the internal water phase to the oil phase to the external water phase to the emulsifier is as follows: 1: (1-4): (2-5): (1 to 3), preferably 1: (3-4): (4-5): (1.6-2.3).
Furthermore, the inner water phase is formed by dissolving dendrobium officinale polysaccharide and a water phase thickening agent A in water, and the outer water phase is formed by dissolving a water phase thickening agent B in water; the mass ratio of the dendrobium candidum polysaccharide to the water phase thickening agent is (0.1-5) to (0.1-3); preferably (0.5-5) 0.25; the mass of the aqueous phase thickener is the total mass of the aqueous phase thickener a and the aqueous phase thickener B.
Further, the aqueous phase thickener a and the aqueous phase thickener B are each independently selected from at least one of hyaluronic acid, xanthan gum, carbomer, gelatin, or acacia.
Further, the oil phase is one or more of capric triglyceride, polydimethylsiloxane, polyoxyethylene castor oil, soybean oil and ceramide or a mixture of the same and an oil phase thickener, and the oil phase thickener is hydrogenated oil or cetyl alcohol, preferably cetyl alcohol; the oil phase thickener accounts for 3-8 wt% of the oil phase.
Further, the emulsifier includes colostrum emulsifier and multiple milk emulsifier; the colostrum emulsifier is a compound emulsifier of linear alkyl quaternary ammonium salt and fatty alcohol, and the multiple emulsion emulsifier is a compound emulsifier of polysorbate-20 and sodium lauroyl lactylate.
Furthermore, the linear alkyl quaternary ammonium salt is behenyl trimethyl ammonium methyl sulfate, and the fatty alcohol is cetearyl alcohol; the mass ratio of the linear alkyl quaternary ammonium salt to the fatty alcohol is 1 (1-6), and preferably 1:3.
Furthermore, the mass ratio of the polysorbate-20 to the sodium lauroyl lactylate is (8-15): 1, preferably 9: 1.
Furthermore, the w/o/w type multilayer emulsion also contains a humectant and a preservative.
Further, the humectant is at least one of sodium hyaluronate, glycerin, allantoin, propylene glycol, polyethylene glycol and sorbitol.
Further, the preservative is at least one of ethylhexyl glycerol, phenoxyethanol and sorbitol.
Furthermore, the mass fraction of the dendrobium officinale polysaccharide is 0.1-5%, and the pharmaceutically acceptable auxiliary materials or auxiliary components comprise: 0.1-3% of water phase thickening agent, 1-5% of oil phase thickening agent, 5-15% of primary emulsion emulsifier and 5-15% of multiple emulsion emulsifier; preferably, the beverage also comprises a humectant accounting for 2-10% of the total weight of the beverage and/or a preservative accounting for 0.01-3% of the total weight of the beverage.
Preferably, the mass fraction of the water phase thickening agent is 3%, the mass fraction of the oil phase thickening agent is 2%, the mass fraction of the colostrum emulsifying agent is 5.6%, and the mass fraction of the multiple emulsion emulsifying agent is 11.8%.
The invention also provides a method for preparing the preparation, which comprises the following steps: the method comprises the following steps:
(1) weighing a colostrum emulsifier according to a certain amount, adding the colostrum emulsifier into an oil phase, heating to 60-80 ℃ for dissolving, and preserving heat to obtain a standby liquid;
(2) dissolving dendrobium officinale polysaccharide and a water phase thickener in water to prepare an inner water phase, heating to the same temperature as the standby liquid phase in the step (1), adding the standby liquid obtained in the step (1), and stirring and dispersing uniformly at the temperature of 60-80 ℃ to prepare primary emulsion;
(3) dissolving the water phase thickener in water to obtain an external water phase, adding the multiple emulsion emulsifier into the external water phase for dissolving, adding into the primary emulsion obtained in the step (2), and stirring and dispersing uniformly to obtain the final product.
Further, the temperature in step (1) and/or step (2) is 80 ℃.
Further, the stirring and dispersing speed of the step (2) is 12000-15000 rpm, and the stirring time is 5-20 min; preferably, the stirring speed is 14500rpm, and the time is 10-15 min.
Further, the stirring and dispersing speed in the step (3) is 10000-12000 rpm, and the stirring time is 5-20 min; preferably, the stirring speed is 11500rpm, and the time is 10-15 min.
The invention also provides application of the preparation in preparing a medicament for treating skin photoaging.
Further, the above-mentioned drugs for treating skin photoaging are drugs which are administered topically through the skin.
Experimental results show that the concentric vesicles formed by the multilayer water-oil alternation and the multilayer emulsion with the onion-like structure can be observed under a microscope, and the slow-release long-acting function of active substances can be realized; the multi-layer emulsion of the dendrobium officinale polysaccharide can effectively increase the transdermal absorption of the dendrobium officinale polysaccharide, simultaneously improves the stability of the medicine, has excellent stability, and is beneficial to clinical application and popularization.
The w/o/w type multilayer emulsion of the invention refers to a water-in-oil-in-water type multiple emulsion.
"colostrum emulsifier" refers to an emulsifier used in the process of preparing colostrum, i.e. an emulsifier dissolved in the oil phase or the internal water phase; "double emulsion emulsifier" means an emulsifier used in the preparation of w/o/w double emulsions from colostrum, i.e. an emulsifier dissolved in the external aqueous phase.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the appearance of a Dendrobium Officinale Polysaccharide (DOP) multilayer emulsion prepared by the invention (examples 1-3 are shown from left to right in sequence).
FIG. 2 shows the appearance of the emulsion prepared in the comparative example (comparative examples 2 to 7 in the order from left to right).
FIG. 3 is the microstructure observation of the Dendrobium Officinale Polysaccharide (DOP) multilayer emulsion prepared by the invention (examples 1-3 are shown from left to right in sequence).
Fig. 4 is a microstructure observation of the single milk prepared in comparative example 1.
FIG. 5 is a microstructure observation of the emulsions prepared in comparative examples 2 to 7.
Fig. 6 is a microstructure observation of the emulsion prepared in comparative example 8.
FIG. 7 shows the change of the character of the Dendrobium Officinale Polysaccharide (DOP) multilayer emulsion prepared by the invention at 70 ℃ (examples 1-3 are shown from left to right in sequence).
FIG. 8 shows the results of measuring the temperature response to heat of the Dendrobium Officinale Polysaccharide (DOP) multilayer emulsion prepared in examples 1-3 of the present invention.
FIG. 9 is a DOP concentration-ultraviolet absorbance standard curve.
FIG. 10 shows the results of transdermal delivery experiments of Dendrobium Officinale Polysaccharide (DOP) multi-layer emulsion prepared in examples 1-3 of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Example 1 preparation of Dendrobium officinale polysaccharide emulsion of the invention
The preparation steps are as follows:
(1) preparation of oil phase: weighing a colostrum emulsifier (a composite emulsifier formed by 0.07g of behenyl trimethyl ammonium methyl sulfate and 0.21g of cetearyl alcohol), adding an oil phase (1.8 mL of caprylic/capric triglyceride, 0.1mL of polydimethylsiloxane oil and 0.1g of oil phase thickener cetyl alcohol) into the mixture, heating the mixture in a water bath, performing ultrasonic treatment to dissolve the mixture, and keeping the temperature at 80 ℃ for later use;
(2) preparation of the internal aqueous phase: mixing 0.025g Dendrobium Officinale Polysaccharide (DOP) with 0.5mL of 0.5 wt% aqueous phase thickener xanthan gum aqueous solution.
(3) Preparation of external water phase: mixing multiple emulsion emulsifier (composite emulsifier prepared from 0.53g polysorbate-20 and 0.06g sodium lauroyl lactylate) with 2.5mL of 0.5 wt% aqueous phase thickener xanthan gum aqueous solution.
(4) Preparing colostrum: slowly adding the oil phase into an internal water phase (the mass ratio of the internal water phase to the oil phase is 1:4) heated to the same temperature, and uniformly stirring by using a dispersion machine in a water bath at the temperature of 80 ℃ to obtain primary emulsion, wherein the stirring speed is 14500rpm, and the stirring time is 10-15 min;
(5) slowly adding the external water phase (the mass ratio of the external water phase to the oil phase is 5:4) into the primary emulsion, and stirring by a dispersion machine to obtain the multilayer emulsion; stirring at 11500rpm for 10-15min to obtain the emulsion with the mass fraction of the dendrobium officinale polysaccharide being 0.5%.
Example 2 preparation of Dendrobium officinale polysaccharide emulsion of the invention
The preparation steps are as follows:
(1) preparation of oil phase: weighing a colostrum emulsifier (a composite emulsifier formed by 0.07g of behenyl trimethyl ammonium methyl sulfate and 0.21g of cetearyl alcohol), adding an oil phase (1.8 mL of caprylic/capric triglyceride, 0.1mL of polydimethylsiloxane oil and 0.1g of oil phase thickener cetyl alcohol) into the mixture, heating the mixture in a water bath, performing ultrasonic treatment to dissolve the mixture, and keeping the temperature at 80 ℃ for later use;
(2) preparation of the internal aqueous phase: mixing 0.05g Dendrobium Officinale Polysaccharide (DOP) with 0.5mL of 0.5% aqueous phase thickener (xanthan gum).
(3) Preparation of external water phase: mixing compound emulsion emulsifier (composite emulsifier prepared from 0.53g polysorbate-20 and 0.06g sodium lauroyl lactylate) with 2.5mL 0.5% water phase thickener (xanthan gum).
(4) Preparing colostrum: slowly adding the oil phase into an internal water phase (the mass ratio of the internal water phase to the oil phase is 1:4) heated to the same temperature, and uniformly stirring by using a dispersion machine in a water bath at the temperature of 80 ℃ to obtain primary emulsion, wherein the stirring speed is 14500rpm, and the stirring time is 10-15 min;
(5) taking the external water phase (the mass ratio of the external water phase to the oil phase is 5:4), slowly adding the primary emulsion, and stirring by a dispersion machine to obtain the multilayer emulsion; stirring at 11500rpm for 10-15min to obtain the emulsion with the mass fraction of the dendrobium officinale polysaccharide being 1%.
Example 3 preparation of Dendrobium officinale polysaccharide emulsion of the invention
The preparation steps are as follows:
(1) preparation of oil phase: weighing a primary emulsion emulsifier (a composite emulsifier formed by 0.07g of behenyl trimethyl ammonium methyl sulfate and 0.21g of cetostearyl alcohol), adding an oil phase (1.8 mL of caprylic/capric triglyceride, 0.1mL of polydimethylsiloxane oil and 0.1g of oil phase thickener cetyl alcohol) into the primary emulsion emulsifier, heating the mixture in a water bath, performing ultrasonic treatment to dissolve the mixture, and keeping the temperature at 80 ℃ for later use;
(2) preparation of an inner water phase: mixing 0.25g Dendrobium Officinale Polysaccharide (DOP) with 0.5mL of 0.5% water phase thickener (xanthan gum).
(3) Preparation of external water phase: mixing compound emulsion emulsifier (composite emulsifier prepared from 0.53g polysorbate-20 and 0.06g sodium lauroyl lactylate) with 2.5mL 0.5% water phase thickener (xanthan gum).
(4) Preparing colostrum: slowly adding the oil phase into an internal water phase (the mass ratio of the internal water phase to the oil phase is 1:4) heated to the same temperature, and uniformly stirring by using a dispersion machine in a water bath at the temperature of 80 ℃ to obtain primary emulsion, wherein the stirring speed is 14500rpm, and the stirring time is 10-15 min;
(5) taking the external water phase (the mass ratio of the external water phase to the oil phase is 5:4), slowly adding the primary emulsion, and stirring by a dispersion machine to obtain the multilayer emulsion; stirring at 11500rpm for 10-15min to obtain the emulsion with the mass fraction of the dendrobium officinale polysaccharide being 5%.
COMPARATIVE EXAMPLE 1 preparation of O/W type Single emulsion
[ FORMULATION ]
The mass ratio of oil to water is 1:3.5
Oil phase: caprylic capric triglyceride, adding oil phase thickener: 5 wt% cetyl alcohol (mass fraction of oil phase)
Water phase: water, 0.5 wt% of dendrobium officinale polysaccharide, and a water phase thickener: 0.5 wt% xanthan gum (mass fraction of inner water phase)
Emulsifier: 4% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3)
[ PREPARATION METHOD ] adding emulsifier into oil phase, dispersing uniformly, heating water phase and oil phase to 75 deg.C respectively in water bath to melt, mixing the two phases under stirring in a disperser, and stirring to condense.
[ results ] milky semisolid, thicker, less stable, and flocculated. The emulsion droplets are not uniform in size, the larger size is about 45 μm, the smaller size is about 5 μm, and the surface of some droplets is broken.
Comparative example 2 preparation of W/O/W type multiple emulsion
[ FORMULATION ]
The mass ratio of oil phase to water phase is W1 (internal water phase), O: W (external water phase) is 1:1
Oil phase: caprylic capric triglyceride, oil phase thickener: 5 wt% cetyl alcohol (mass fraction of oil phase)
Internal water phase: water, 0.5 wt% of Dendrobium officinale polysaccharide (mass fraction of total emulsion, i.e. final concentration), 0.5 wt% of xanthan gum (mass fraction of inner water phase)
External water phase: water, 0.5 wt% xanthan gum (mass fraction of the outer aqueous phase)
Colostrum emulsifier: 4 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) (mass fraction of colostrum)
[ PREPARATION METHOD ] mixing cetyl alcohol, BTMS and cetearyl alcohol and caprylic/capric triglyceride (as oil phase), heating in water bath to melt to 75 deg.C, slowly adding the oil phase into the inner water phase heated to the same temperature, stirring with a disperser at room temperature to obtain colostrum, slowly adding the outer water phase at 75 deg.C into colostrum, and stirring with the disperser until condensation.
Comparative example 3 preparation of W/O/W type multiple emulsion
Oil phase thickener: 10 wt% of cetyl alcohol (mass fraction of the oil phase); the rest of the formulation and the preparation method are the same as in comparative example 2.
Comparative example 4 preparation of W/O/W type multiple emulsion
Oil phase thickener: 15 wt% cetyl alcohol (mass fraction of oil phase); the rest of the formulation and the preparation method are the same as in comparative example 2.
Comparative example 5 preparation of W/O/W type multiple emulsion
The formulation was the same as comparative example 2, but 4 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) was used as the multiple emulsion emulsifier.
[ PREPARATION METHOD ]: mixing cetyl alcohol and caprylic capric triglyceride (as oil phase), heating in water bath to melt to 75 deg.C, slowly adding the oil phase into the inner water phase heated to the same temperature, stirring uniformly at room temperature with a dispersion machine to obtain colostrum, dissolving BTMS and cetearyl alcohol in the outer water phase, slowly adding colostrum into the outer water phase at 75 deg.C, and stirring with the dispersion machine until condensation.
Comparative example 6 preparation of W/O/W type multiple emulsion
Oil phase thickener: 10 wt% of cetyl alcohol (mass fraction of the oil phase); the remaining formulation and preparation were the same as in comparative example 5.
Comparative example 7 preparation of W/O/W type multiple emulsion
Oil phase thickener: 15 wt% cetyl alcohol (mass fraction of oil phase); the remaining formulation and preparation were the same as in comparative example 5.
Comparative example 8 preparation of W/O/W type multiple emulsion
The mass ratio of the oil phase to the water phase is W1 (inner water phase), O: W (outer water phase) is 1:3.5:2.5
Oil phase: caprylic capric triglyceride, oil phase thickener: 10 wt% cetyl alcohol (mass fraction of oil phase)
Internal water phase: water, 0.5 wt% of dendrobium officinale polysaccharide (mass fraction of the total emulsion, i.e. final concentration), 0.5 wt% of xanthan gum (mass fraction of the inner water phase)
External water phase: water, 0.5 wt% xanthan gum (mass fraction of the outer aqueous phase)
Colostrum emulsifier: 2 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) (mass fraction of colostrum)
[ PREPARATION METHOD ] mixing cetyl alcohol, BTMS, cetearyl alcohol and caprylic/capric triglyceride (as oil phase), heating in water bath to melt to 75 deg.C, slowly adding the oil phase into the inner water phase heated to the same temperature, stirring uniformly at room temperature with a disperser to obtain colostrum, slowly adding colostrum into the outer water phase at 75 deg.C, and stirring with the disperser until it is condensed.
Comparative example 9 preparation of W/O/W type multiple emulsion
The mass ratio of oil phase to water phase is W1 (internal water phase), O: W (external water phase) is 1:2
Oil phase: caprylic capric triglyceride, oil phase thickener: 12 wt% cetyl alcohol ((mass fraction of oil phase)
Internal water phase: water, 0.5 wt% of Dendrobium officinale polysaccharide (mass fraction of total emulsion, i.e. final concentration), 0.5 wt% of xanthan gum (mass fraction of inner water phase)
External water phase: water, 0.5 wt% xanthan gum (mass fraction of the outer aqueous phase)
Colostrum emulsifier: 2 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) (mass fraction of colostrum)
[ PREPARATION METHOD ] mixing cetyl alcohol, BTMS, cetearyl alcohol and caprylic/capric triglyceride (as oil phase), heating in water bath to melt to 80 deg.C, slowly adding the oil phase into the inner water phase heated to the same temperature, stirring with a disperser under water bath to obtain colostrum (14500rpm, 10-15min) (without taking out, placing in water bath all the time), adding colostrum into the outer water phase at 80 deg.C, and stirring with the disperser (11500rpm, 10-15min) until condensation.
Comparative example 10 preparation of W/O/W type Complex emulsion
Colostrum emulsifier: 4 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) (mass fraction of colostrum); the remaining formulation and preparation were the same as in comparative example 9.
Comparative example 11 preparation of W/O/W type multiple emulsion
Colostrum emulsifier: 4 wt% (behenyltrimethylammonium methylsulfate (BTMS): cetearyl alcohol 1: 3) (mass fraction of colostrum); the mass fraction of the xanthan gum in the internal and external water phases is 0.5 wt%; the remaining formulation and preparation were the same as in comparative example 9.
Comparative example 12 preparation of W/O/W type Complex emulsion
The mass fraction of the xanthan gum in the internal and external water phases is 0.5 wt%; the remaining formulation and preparation were the same as in comparative example 9.
The beneficial effects of the dendrobium officinale polysaccharide emulsion are proved by the following experimental examples.
Experimental example 1 DOP multilayer emulsion Observation and Observation under the lens
1. Experimental methods
Taking a picture of the emulsion prepared in part of the examples and the comparative examples, and observing the characteristics of the emulsion, such as the property, the texture, the fluidity, the fineness and the like; and diluting a small amount of emulsion with ultrapure water, and observing the microstructure of the emulsion under a microscope.
2. Results of the experiment
2.1, as shown in figure 1, in the preparation process of the multilayer emulsion prepared by the embodiment of the invention, colostrums are uniform in character, and the further prepared compound emulsion is in a cream shape, smooth and fine, good in continuity, free of obvious particles, free of layering and free of oil stains. The emulsion of example 1 is milky white, and the multi-layer emulsions of examples 2 and 3 respectively have ginger yellow and dark brown colors along with the increase of the content of dendrobium officinale.
The single-layer emulsion prepared in comparative example 1 was milky semisolid, thick, and poor in stability, and flocculated.
The colostrum of comparative examples 2, 3, 4, 6 shows phase separation, one phase is liquid and the other phase is milky cotton-like substance, but the liquid phase disappears after stopping stirring, and an emulsion with uniform dispersion can be formed after adding the external water phase. The colostrum of comparative examples 5, 7 is in an oil-condensed state, the oil and water are separated by heating, and an emulsion with uniform dispersion can be formed after the addition of the external water phase. However, the emulsions of comparative examples 2 to 7 are still inferior to examples 1 to 3 in shape, although they are fine, and generally have low viscosity and contain a large number of bubbles (fig. 2, comparative examples 2 to 7 are shown in the order from left to right). The emulsion of comparative example 8 is inferior in fineness.
The emulsion prepared in the comparative examples 9-12 is uniform in colostrum property and is in a milk white solid state, and the fact that the colostrum prepared under the heat preservation condition in the water bath is the key for uniform dispersion of the colostrum is verified. However, the compound emulsions finally obtained in comparative examples 9 to 12 were thin, non-uniformly dispersed, and floccules were present.
2.2, the emulsion droplets of the emulsions prepared in examples 1 to 3 are observed under a microscope to be multi-layer concentric vesicles with onion-like structures, and the alternating multi-layer composite structures are confirmed and the structures are uniform and stable (fig. 3).
The size of the emulsion droplets observed in comparative example 1 was not uniform, and the size was as large as about 45 μm and as small as about 5 μm, and part of the droplet surface was broken (FIG. 4).
As can be seen from FIG. 5, the particle size of the emulsion droplets of comparative examples 2 to 7 is related to the amount of cetyl alcohol, and the larger the amount of cetyl alcohol, the smaller the particle size of the emulsion droplets; furthermore, BTMS and cetearyl alcohol are used as colostrum emulsifiers, and the emulsion droplets are dispersed more uniformly. However, in general, the emulsions of comparative examples 2 to 7 had inferior dispersion effect of emulsion droplets, larger particle diameter, no significant multi-layer structure, and unstable structure, compared to the emulsions of examples 2 to 4.
As can be seen from fig. 6, the emulsion prepared in comparative example 8 has a small number, a large particle diameter, and a poor dispersion effect although there are multiple layers of emulsion droplets.
Therefore, the emulsion with the specific composition prepared by the exploration of a large amount of emulsion auxiliary materials of the inventor has the advantages of optimal properties, smoothness, fineness, good continuity, no obvious floccules, precipitates, particles, oil stains, layering and the like; and has obvious characteristic multi-layer vesicle structure, uniformity and stability, and the particle size is suitable for skin absorption.
Experimental example 2 stability examination of DOP multilayer emulsion
1. Experimental methods
The emulsions prepared in examples 2 to 4 were placed at 4 ℃, 25 ℃ and 70 ℃ for 6 hours, respectively, and the changes in the appearance properties and the changes in the multilayer structure were observed under a microscope.
2. Results of the experiment
The multilayer emulsion is placed in the environment of 4 ℃ and 25 ℃ for 6 hours, the appearance form is not obviously changed, and the layering phenomenon is avoided; after the multi-layer emulsion is placed in an environment of 70 ℃ for 6 hours, the layering phenomenon of the low, medium and high dosage multi-layer emulsion of Dendrobium Officinale Polysaccharide (DOP) occurs, floccule deposits, and liquid precipitates are not dissolved in the original emulsion (figure 7). The phenomenon shows that the high temperature can destroy the composite water-oil structure of the w/o/w of the multilayer emulsion. The DOP multilayer emulsion with three dose gradients is placed in the environment of 4 ℃ and 25 ℃, the microstructure is stable, the DOP multilayer emulsion is not obviously changed, and clear concentric round vesicles can be seen. Under the high temperature of 70 ℃, the microstructure of DOP multilayer emulsion is destroyed, wherein complete concentric circle-shaped structure can not be seen under a DOP low-dosage and medium-dosage multilayer emulsion microscope, a large amount of broken emulsion drop fragments can be seen under the DOP high-dosage multilayer emulsion microscope, and the normal concentric circle-shaped structure is kept only in a small amount (figure 8).
The above results show that the emulsion of the present invention has good stability at low to room temperature and is easy to manufacture and transport.
Experimental example 3 measurement of drug-loading and encapsulation efficiency of multilayer emulsion of the present invention
1. Experimental methods
Precisely weighing 10mg of DOP in a 10ml volumetric flask, dissolving the DOP in 2ml of aqueous solution, diluting the DOP to form a standard solution with the volume of 1mg/ml by using water, diluting the DOP to 5 groups of 0.1mg/ml, 0.05mg/ml, 0.02mg/ml, 0.01mg/ml and 0.005mg/ml, measuring an absorbance value at a wavelength of 268nm by using an ultraviolet visible spectrophotometer, and obtaining a standard curve of the DOP by using the mass concentration as a horizontal coordinate and the absorbance as a vertical coordinate. The encapsulation efficiency of the emulsion was determined by centrifugation. Taking a proper amount of the multilayer emulsion prepared in the examples 2-4, respectively, centrifuging the multilayer emulsion in a centrifuge tube at 12000rpm for 20 minutes, absorbing lower-layer liquid, moderately diluting the lower-layer liquid, measuring an absorbance value at a wavelength of 268nm, calculating according to a standard curve to obtain the content of the drug, and calculating according to the following formula to obtain the drug loading rate and the encapsulation rate: the drug loading is the amount of drug contained in the emulsion/total mass of the emulsion x 100%; encapsulation efficiency (amount of drug encapsulated in emulsion/total amount of drug encapsulated and unencapsulated in emulsion) x 100%; the amount of drug contained in the emulsion was m measurement value × dilution factor.
2. Results of the experiment
Drawing a standard curve according to the ultraviolet absorption values corresponding to DOP solutions with different concentrations to obtain a curve regression equation of y ═ 0.0283x +0.0011, R 2 0.9989 (fig. 9). According to calculation, the DOP low-dose multilayer emulsion has the drug loading rate of 0.46 percent and the encapsulation rate of 93.58 percent; the drug loading rate of the dose multilayer emulsion in DOP is 0.92 percent, and the encapsulation rate is 92.03 percent; the DOP high-dose multilayer emulsion has the drug loading of 4.69 percent and the encapsulation efficiency of 93.91 percent.
The results show that the drug loading of the emulsion can reach more than 4 percent, the encapsulation efficiency is more than 90 percent, and the emulsion has good application prospect.
Experimental example 4 transdermal Release assay of the multilayer emulsion of the present invention
1. Experimental methods
Transdermal permeation tests were performed on DOP solutions and the multi-layer emulsions of examples 2-4 using a Franz diffusion cell apparatus. The reserve rat skin was held between the supply and receiving chambers of the diffusion cell with the stratum corneum facing the supply chamber. The effective diffusion area of the diffusion cell is 2.27cm 2 The magnetic stirring speed is 300 r/min. Accurately adding DOP emulsion and DOP multilayer emulsion 0.3g into the administration pool, uniformly distributing onto the surface of mouse skin, placing the diffusion pool into 32 deg.C water bath, adding 14mL PBS buffer preheated to 32 deg.C into the receiving poolAnd (4) sampling the receiving solution for 0.5, 1, 2, 4 and 6 hours respectively, taking out all the receiving solution and rapidly supplementing the receiving solution with the same amount of preheating. The absorbance was then measured at a wavelength of 268nm and drug content was obtained from the standard curve.
2. Results of the experiment
The experimental results show that the transdermal absorption of the DOP multilayer emulsion prepared by the invention for six hours is far higher than that of the DOP solution with high dose (equal mass fraction) (figure 10).
The results show that the DOP emulsion prepared by the invention obviously increases the transdermal absorption degree of DOP, has excellent transdermal administration effect and is beneficial to clinical application.
In conclusion, the invention provides a multi-layer emulsion of dendrobium officinale polysaccharide and a preparation method thereof, the preparation method is simple and convenient to operate, the prepared emulsion has concentric vesicles formed by alternating multilayer water and oil with onion-like structures, and can be observed under a microscope, and the slow-release long-acting function of active substances can be realized; the multi-layer emulsion of the dendrobium officinale polysaccharide can improve the stability of the medicine, has excellent stability, drug loading rate and encapsulation rate, can effectively increase the transdermal absorption of the dendrobium officinale polysaccharide, is an external preparation for treating skin photoaging, and has excellent clinical application prospect.

Claims (10)

1. A preparation for treating skin photoaging is characterized in that the preparation is a w/o/w type multilayer emulsion prepared by taking dendrobium officinale polysaccharide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
2. The formulation according to claim 1, wherein the w/o/w type multi-layer emulsion comprises an internal aqueous phase, an oil phase, an external aqueous phase and an emulsifier, wherein the weight ratio of the internal aqueous phase, the oil phase, the external aqueous phase and the emulsifier is: 1: (1-4): (2-5): (1 to 3), preferably 1: (3-4): (4-5): (1.6-2.3).
3. The preparation of claim 2, wherein the inner aqueous phase is formed by dissolving dendrobium officinale polysaccharide and/or aqueous phase thickener A in water, and the outer aqueous phase is formed by dissolving aqueous phase thickener B in water; the water phase thickening agent A and the water phase thickening agent B are respectively and independently selected from at least one of hyaluronic acid, xanthan gum, carbomer, gelatin or acacia;
the mass ratio of the dendrobium candidum polysaccharide to the water phase thickening agent is (0.1-5): 0.1-3, preferably (0.5-5): 0.25; the mass of the aqueous phase thickener is the total mass of the aqueous phase thickener a and the aqueous phase thickener B.
4. The formulation of claim 2, wherein the oil phase is one or more of capric triglyceride, polydimethylsiloxane, polyoxyethylated castor oil, soybean oil, ceramide, or a mixture thereof with an oil phase thickener which is a hydrogenated oil or cetyl alcohol, preferably cetyl alcohol; the oil phase thickener accounts for 3-8 wt% of the oil phase.
5. The formulation of claim 2, wherein the emulsifier comprises a colostrum emulsifier and a multiple milk emulsifier; the colostrum emulsifier is a compound emulsifier of linear alkyl quaternary ammonium salt and fatty alcohol, and the multiple emulsion emulsifier is a compound emulsifier of polysorbate-20 and sodium lauroyl lactylate.
6. The formulation of claim 5, wherein the linear alkyl quaternary ammonium salt is behenyltrimethylammonium methylsulfate, the fatty alcohol is cetearyl alcohol; the mass ratio of the linear alkyl quaternary ammonium salt to the fatty alcohol is 1 (1-6);
and/or the mass ratio of the polysorbate-20 to the sodium lauroyl lactylate is (8-15): 1.
7. the formulation of claim 2, wherein the w/o/w type multilayer emulsion further comprises a humectant and/or a preservative; preferably, the humectant is at least one of sodium hyaluronate, glycerin, allantoin, propylene glycol, polyethylene glycol, and sorbitol; the preservative is at least one of ethylhexyl glycerol, phenoxyethanol or sorbitol.
8. The preparation according to any one of claims 1 to 7, wherein the mass fraction of the dendrobium officinale polysaccharide is 0.1 to 5%, and the pharmaceutically acceptable auxiliary materials or auxiliary components comprise 0.1 to 3% of a water phase thickener, 1 to 5% of an oil phase thickener, 5 to 15% of a colostrum emulsifier and 5 to 15% of a multiple emulsion emulsifier; preferably, the beverage also comprises a humectant accounting for 2-10% of the total weight of the beverage and/or a preservative accounting for 0.01-3% of the total weight of the beverage.
9. A method of preparing a formulation according to any one of claims 1 to 14, comprising the steps of:
(1) weighing a colostrum emulsifier according to a certain amount, adding the colostrum emulsifier into an oil phase, heating to 60-80 ℃ for dissolving, and preserving heat to obtain a standby liquid;
(2) dissolving dendrobium officinale polysaccharide and a water phase thickener in water to prepare an inner water phase, heating to the same temperature as the standby liquid phase in the step (1), adding the standby liquid obtained in the step (1), and stirring and dispersing uniformly at the temperature of 60-80 ℃ to prepare primary emulsion;
(3) dissolving the water phase thickener in water to obtain an external water phase, adding the multiple emulsion emulsifier into the external water phase for dissolving, adding into the primary emulsion obtained in the step (2), and stirring and dispersing uniformly to obtain the final product.
10. Use of a formulation according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment of photoaging of skin; preferably, the medicament for treating skin photoaging is a medicament for topical administration through the skin.
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CN105434350A (en) * 2015-12-24 2016-03-30 中国药科大学 Diclofenac sodium frozen compound emulsion transdermal delivery preparation and preparation method thereof
CN107157872A (en) * 2017-05-08 2017-09-15 宁波易中禾生物技术有限公司 A kind of oxidation resistant dendrobium candidum facial treatment milk of moisturizing and preparation method thereof

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