KR20150104727A - Preparation method of w/o/w multiple emulsions - Google Patents

Abstract

The present invention relates to a production method of a water-in-oil-in-water (W/O/W) type multiple emulsion which can be used as a delivery system of collected materials for promoting bioavailability and maintains stability during long-term storage.

Description

PREPARATION METHOD OF W / O / W MULTIPLE EMULSIONS [0002]

The present invention relates to a method for producing multiple emulsions of water-in-oil-in-water (W / O / W) type. More specifically, the present invention relates to a method for producing an emulsion having improved stability as a multiple emulsion for capturing phytochemicals.

Emulsions are dispersed in the form of particles in a liquid by a surfactant in one of two liquids which are not mixed with each other, and are widely used in various industries such as pharmaceuticals, cosmetics, foods, agrochemicals and paints.

The two unmixed liquids of the emulsion are generally classified into a water phase component and an oil phase component. When the oil is dispersed in the water phase in a water phase, it is called an oil-in-water (O / W) type emulsion, / O) type emulsion.

In an O / W type emulsion, an oil phase is called a disperson phase or an inner phase, and a water phase is called a continuous phase or an outer phase. The characteristics of the O / W type or W / O type emulsion depend on the kind of the surfactant, the composition ratio of the two liquid phases, and the mechanical conditions at the time of production. Of these, the selection of a surfactant is particularly important. If a hydrophilic surfactant is used, the surfactant has a property of dissolving in the water phase due to affinity with the water phase, and thus an O / W type emulsion existing as an oil phase-dispersed particle is formed. Conversely, a W / O type emulsion is produced.

Multiple emulsions are systems in which the W / O type emulsion and the O / W type emulsion are simultaneously present in one emulsion. Specifically, a water-in-oil type heavy oil (O / W / O) type emulsion in which the W / O type emulsion is redispersed in the oil phase and a W / O type emulsion in which the W / / W) type emulsion is referred to as multiple emulsion.

O / W type, O / W / O / W type, W / O / W / O type and O / W / O / O type according to the composition and manufacturing method of the emulsifier. Type W emulsion, which has high applicability in terms of stability and usability, has been extensively studied.

The W / O / W type multiple emulsion has a semipermeable liquid film property in which the oil phase separates the inner water phase from the outer water phase. This feature makes multiple emulsions available as a controllable emission delivery system. Therefore, such W / O / W type multiple emulsions can be applied to medicine, cosmetics and solvent extraction.

Recently, as an in vivo delivery system, the development of multi-emulsion technology for capturing phytochemicals is actively under way. These phytochemicals are synthetic words of phyto which means plants and chemicals which means chemicals, and are collectively referred to as plant-derived compounds, and they are attracting attention recently because they have useful physiological activities such as antioxidation and cholesterol lowering. In particular, luteolin is a flavonoid present in fruits and vegetables. It is an antioxidant and a very excellent free radical scavenger. It is attracting attention as a substance that affects various biological reactions such as antioxidation, anti-inflammation, neuroprotection, Has attracted great interest as a polyphenol antioxidant having a 4-trihydroxystilbene structure. Quercetin, one of the phytochemicals, is also a type of flavonoid, and has attracted attention as a 5,7,3 ', 4'-tetrahydroxy derivative of flavonol having a strong antioxidant activity.

However, a W / O / W emulsion containing a capture material such as a phytochemical can easily become unstable over time due to its low emulsion stability and physical or chemical interaction between the capture material and the emulsion. This destabilization occurs primarily in the form of an external aqueous phase and an internal aqueous phase. This ultimately leads to the separation of the phases, so that the effective components captured in the multiple emulsions can not reach the target point smoothly.

In order to solve such a problem, Korean Patent Laid-Open Publication No. 2002-0027788 discloses a method for producing a W / O / W type multiple emulsion using emulsion polymerization and hybridization of a microfluidic device, but in this hybrid method, There is a limit in improving the stability of the battery. Korean Unexamined Patent Publication No. 2002-0055908 discloses a W / O / W type multiple emulsion having improved cohesion of an aqueous phase using a hydrogen bonding inhibitor. However, when the internal water phase is excessively hydrophobic due to the use of a hydrogen bonding inhibitor, There is a risk of fusion with the oil phase.

As described above, there is a growing need to develop a W / O / W type multiple emulsion which can be used as a delivery system for collecting materials for enhancing bioavailability and maintains stability even after storage for a long time.

Thus, the present invention is internal aqueous phase (W ₁₎ oil by dispersing the internal aqueous phase (W ₁₎ to the phase, the oil and oil phase (O) a surfactant is added to add a trapping substance and the median to (W ₁ / O) type (W ₁ / O / W ₂ ) type emulsion by dispersing the emulsion of the water-in-oil type (W ₁ / O) type in the external water phase (W ₂ ) A method for producing multiple emulsions is provided.

When preparing multiple emulsions according to the production method of the present invention, it is possible to produce a W / O / W type multiple emulsion which contains a capturing substance and is excellent in stability even for long-term storage.

1 is a graph showing the particle size and stability of an emulsion according to the type of surfactant.
2 is a graph showing the particle size and stability of the emulsion according to the surfactant concentration difference.
3 is a graph showing the particle size and stability of the emulsion according to high pressure treatment.
4 is a graph showing the particle size and stability of the emulsion according to the weight ratio difference between the water-in-water type emulsion and the external water type.
5 is a graph showing the particle size and stability of the emulsion according to the type of trapping material.
FIG. 6 is a graph showing the change in particle size during storage of the emulsion according to the difference in viscosity of the oil.
7 is a graph showing the stability change during storage of the emulsion according to the viscosity difference of the oil.
8 is a graph showing the turbidity change during storage of the emulsion according to the viscosity difference of the oil.
9 is a photograph showing the storage characteristics of the emulsion with time.
10 is a photograph showing the storage characteristics of the emulsion according to the cold-thawing repetition.

Hereinafter, the present invention will be described in detail. However, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.

In one embodiment, as a method for producing multiple emulsions, the present invention provides a method for preparing a multiple emulsion comprising: a) adding a capture material to an internal aqueous phase (W ₁ ); b) dispersing the internal water phase (W ₁ ) in an oil phase (O) to which an oil and a surfactant are added to prepare a water-in-oil type (W ₁ / O) type emulsion; And c) dispersing the emulsion of the water-in-oil type (W ₁ / O) in the external water phase (W ₂ ) to prepare an emulsion of water-in-oil emulsion type (W ₁ / O / W ₂ ).

In one embodiment, the internal aqueous phase (W ₁ ) in step a) may be composed of purified water, ethanol, or a mixture of purified water and ethanol, and in particular, to improve the storability of the particulate matter added to the internal aqueous phase (W ₁ ) Is preferably 100% ethanol.

In one embodiment, the capture material added to the internal water phase (W ₁ ) in step a) is phytochemical. More specifically, phytochemicals that can be used as capture materials include EGCG, selenium, curcumin, saponin, lycopene, anthocyanin, luteolin, resveratrol and quercetin. Preferably, the capture material added to the inner aqueous phase (W ₁ ) may be selected from the group consisting of luteolin, resveratrol and quercetin. More preferably, the capture material added to the internal water phase (W ₁ ) in step a) is luteolin.

In one embodiment, in step b), various natural oils or synthetic oils, including mineral oils, vegetable oils, animal oils, essential oils, synthetic oils, etc., may be used as the oil. The oil used in the production of multiple emulsions should be appropriately selected according to the purpose of the collecting material delivery system, and vegetable oil having little biohazardiness is suitable as the oil of the present invention. More specifically, vegetable oils such as soybean oil, canola oil, olive oil, orange oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, peanut oil and poppy oil are suitable as oils. In particular, soybean oil, canola oil, olive oil, orange oil, or a mixture thereof is preferably used as the oil of step b). The most suitable oil for step b) is soybean oil. This is because, when soybean oil is used as an oil, it is possible to produce a multi-emulsion having a small particle size and excellent stability, and is an oil suitable for the emulsion capture material transfer function.

In one embodiment, in step b), a nonionic surfactant may be used as the surfactant. This is because the hydrophilic group of the nonionic surfactant does not have a charge and thus the stimulus in vivo is the least. More specifically, as non-ionic surfactants, it is preferable to use, as the nonionic surfactant, a phage-60 glyceryl ester having an HLB value of 13 to 20, a saxatolate, a phage-40 glyceryl esterate, Tween 60, Tween 80, Or Ceteth-20, or a hydrophilic non-ionic surfactant such as Ceteth-20, or a phage-3 glyceryl having an HLB value of 1.5 to 6 may be used as a source of thiosate, sorbitan laurate, sorbitan stearate, Oleophilic surfactants such as stearate, stearate-2, ole-2, and PGPR may be used singly or in combination. Tween 80 is preferably used as the surfactant in step b). This is because, when Tween 80 having an HLB value of 15 is used in the preparation of an emulsion, the effect of reducing the particle size of multiple emulsions is excellent and a stable emulsion can be produced.

In one embodiment, in step b), the oil is soybean oil and the surfactant is Tween 80. This is because the stability of multiple emulsions formed by the combination of soybean oil and Tween 80 is excellent, which is advantageous for long-term storage of the emulsion.

In one embodiment, in step b), the surfactant in oil phase (O) may be from 10 to 40% by weight. In particular, it is preferable that the amount of the surfactant in the oil phase (O) is 15 to 30% by weight. This is because, when the surfactant in the oil phase (O) is less than 15 wt% or exceeds 30 wt%, the particle size of the emulsion is relatively large and the stability can be reduced.

In one embodiment, in step b), the weight ratio of oil to surfactant in oil phase (O) is preferably 85:15 to 70:30. If the weight ratio of oil to surfactant is out of this range, the stability of the emulsion may be reduced. In view of the stability of the emulsion, the weight ratio of the oil to the surfactant in the oil phase (O) is preferably 80:20.

In one embodiment, in step b), the oily phase (O) is treated with an internal aqueous phase (W ₁₎ by any standard method such as homogenizer method, high pressure homogenizer method, high speed stirring method, ultrasonic emulsification method, ), And heat can be applied as needed.

In one embodiment, the internal aqueous phase (W ₂ ) in step c) can be purified water, ethanol, or a mixture of purified water and ethanol.

In one embodiment, the emulsion can be treated at high pressure using a microfludizer to produce an emulsion with nanoscale particle size. In particular, such high pressure treatment can be carried out in step c). At this time, the pressure applied to the emulsion is preferably 15,000 to 30,000 psi, more preferably 20,000 to 25,000 psi. There is little change in the particle size and stability of the emulsion when a pressure of less than 15,000 psi is applied, and when the pressure exceeds 30,000 psi, the stability of the emulsion may deteriorate as the phases forming multiple emulsions are combined. Therefore, in view of the stability of the emulsion, it is preferable to apply a pressure of 20,000 to 25,000 psi.

In one embodiment, in step c), the weight ratio of water-in-oil (W ₁ / O) emulsion to external water-phase (W ₂ ) may be 0.5 to 5:10. Preferably, the weight ratio of the water-in-oil (W ₁ / O) emulsion to the external water-phase (W ₂ ) is 0.5 to 3:10, more preferably 1:10. External water phase (W _2), based on the weight of the water-in-oil (W ₁ / O) type emulsion, if it exceeds 30% by weight of the oil-in-water heavy water _{(W 1 / O / W 2} ) type water-in-oil (W present in the emulsion (W ₁ / O) type emulsion is too high, the stability of the emulsion may be deteriorated. When the emulsion of the water (W ₁ / O) type is less than 5% by weight based on the weight of the external water phase (W ₂ ) ₁ / O) type emulsion is too low, it is difficult to carry out the function of delivering the absorbing substance smoothly. Considering the stability of the emulsion and the function of transferring the collected blister, it is preferable that the weight ratio of the water-in-oil (W ₁ / O) emulsion to the external water-phase (W ₂ ) is 1:10.

In one embodiment, in step c), the water-in-oil (W ₁ / O) type emulsion may be prepared by any of the standard methods such as homogenizer method, high pressure homogenizer method, high speed stirring method, ultrasonic emulsification method, And can be dispersed in the outer water phase (W ₂ ), and heat can be applied if necessary.

In one embodiment, the water-in-oil-in-water (W ₁ / O / W ₂ ) type emulsion of the present invention may further contain one or more additives as needed. Such additives include antioxidants, antibacterial agents, pH adjusting agents, isotonic agents, and soothing agents. Specific examples of the antioxidant include sodium metabisulfite, sodium sulfite, sodium bisulfite, potassium metabisulfite, potassium sulfite, sodium thiosulfate and the like. Specific examples of the antibacterial agent include sodium caprylate, methyl benzoate, sodium metabisulfite, sodium edetate and the like. Specific examples of the pH adjusting agent include hydrochloric acid, acetic acid, lactic acid, malic acid, citric acid, sodium hydroxide and the like. Specific examples of the isotonizing agent include sugars such as glycerol, glucose, fructose and maltose, and sugar alcohols such as sorbitol and xylitol. Among them, the lipophilic substance may be used in advance in an oil-forming oil or the like, and the hydrophilic substance may be used in advance in the inner water or purified water constituting the outer water phase.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

Example

Analytical methods and samples

The particle size of the prepared emulsion was measured using Dynamic Light Scattering (Nanotrac 250, Microtrac).

To confirm the stability of the emulsion prepared, zeta-potential (zeta plus seta potential abolyzer, Brookhaven instruments corporation) was measured.

The turbidity was measured by measuring the absorbance at a wavelength of 660 nm using ELISA by diluting the prepared emulsion 1000 times.

Absorption of luteolin by cell model was measured by HPLC analysis.

In the preparation of the emulsion, olive oil (soybean oil), soybean oil (snow white) and canola oil (purified water) were used as the oil and Tween 80 (Sigma) and PGPR (Palsgaard) were used as surfactants. As a capture material, luteolin (Santa Cruz Biotechnology) was used.

Depending on surfactant type emulsion  characteristic

After adding luteolin to the inner aqueous phase consisting of 100% ethanol, the inner aqueous phase was added to an oil phase in which soybean oil and Tween 80 were mixed at a weight ratio of 8: 2, and the mixture was homogenized at a speed of 10,000 rpm And stirred for 1 minute to form a water-in-oil emulsion. Then, the water-in-oil type emulsion was added to the external water phase composed of 100% purified water so that the weight ratio of the water-in-water emulsion to the external water was 1:10, and the mixture was stirred at a speed of 12,000 rpm for 1 minute using a homogenizer, To prepare a water-in-oil emulsion. Another oil-in-water emulsion was prepared in the same manner as above using PGPR instead of Tween 80.

Two oil - in - water emulsions were prepared for olive oil and canola oil in the same manner as for soybean oil. The particle size and stability of the six emulsions thus prepared were measured.

As shown in FIG. 1, it was confirmed that the emulsion prepared using Tween 80 as a surfactant regardless of the oil type was a relatively stable emulsion having a smaller particle size than the emulsion prepared using PGPR.

Depending on the surfactant content emulsion  characteristic

The emulsion was prepared in the same manner as in the emulsion characteristics test according to the type of surfactant using soybean oil as the oil and Tween 80 as the surfactant except that the weight ratio of the soybean oil to the Tween 80 was varied, .

As shown in FIG. 2, there was almost no change in the particle size of the emulsion until 8 wt% of Tween 80 was added based on the weight of soybean oil. When 10 wt% or more of the surfactant was added, the particle size of the emulsion was constant As shown in Fig. The stability of the emulsion was not changed until 10 wt% of Tween 80 was added based on the weight of soybean oil, but stability was improved when 15 wt% to 30 wt% of Tween 80 was added. In particular, when 20 wt% of Tween 80 was added based on the weight of the soybean oil, it was confirmed that the stability of the emulsion was greatly improved.

High pressure treatment Emulsion  characteristic

The emulsion was prepared by using soybean oil as the oil and Tween 80 as the surfactant in the same manner as in the emulsion characteristics test according to the type of the surfactant. After dispersing the water-in-oil emulsion in the external oil phase, the microfluidizer Lt; RTI ID = 0.0 > psi. ≪ / RTI >

As shown in FIG. 3, the emulsion prepared at a pressure of 15,000 psi or less had no change in particle size, but it was confirmed that the emulsion prepared by applying a pressure of 20,000 to 30,000 psi had a reduced particle size. In the case of the emulsion stability, it was confirmed that the stability of the emulsion prepared by applying the pressure of 20,000 to 25,000 psi was excellent.

Water type emulsion  Depending on the difference in weight ratio emulsion  characteristic

The emulsion was prepared by using soybean oil as the oil and Tween 80 as the surfactant in the same manner as in the emulsion characteristics test according to the type of the surfactant except that the weight ratio of the water- 1: 10, 3: 10, 5: 10.

As shown in FIG. 4, it was confirmed that as the ratio of the water-in-oil emulsion decreased, the particle size of the emulsion decreased and the stability of the emulsion increased.

Capture material  Depending on the type emulsion  characteristic

Emulsions were prepared by using soybean oil as the oil and Tween 80 as the surfactant in the same manner as in the emulsion characteristics test according to the type of the surfactant. However, in order to confirm the characteristics of the emulsion according to the type of the collected material, , Resveratrol and quercetin were used to prepare three emulsions.

As shown in FIG. 5, it was confirmed that the particle size of emulsion prepared using luteolin as a trapping material was relatively small and the stability was also excellent.

Depending on the difference in viscosity of the oil emulsion  Storage characteristics

The emulsion was prepared by using Tween 80 as the surfactant in the same manner as in the emulsion characteristics test according to the kind of the surfactant except that soybean oil (79.1 cP) and orange oil (0.79 cP, An emulsion of branches was prepared. The emulsion was stored at room temperature (20 ° C) and refrigerated temperature (4 ° C) for 14 days, and frozen (-20 ° C) - thawed (30 ° C) was repeated 6 times.

As shown in FIG. 6, the emulsion prepared using orange oil tended to gradually increase in particle size over time at room temperature and in a refrigerator, and the particle size rapidly increased from the storage day 5 . The oil with soybean oil also tended to gradually increase in size over time at room temperature and in cold storage, but the increase was slower than that of the orange oil emulsion. Even after 14 days, the particle size was relatively small Respectively. In the cold-thaw repeated experiment, the particle size of the emulsion using orange oil rapidly increased from the 4th day, but the particle size of the emulsion using soybean oil did not change greatly even after 6 repetitions.

Further, as shown in Fig. 7, the storage stability of the emulsion was superior to that of the emulsion prepared using soybean oil having a low viscosity compared with the emulsion prepared using orange oil, both at room temperature storage, refrigeration storage and cold- appear.

Further, as shown in Fig. 8, it can be seen that emulsions prepared using soybean oil having a low viscosity are better retained in storage during storage at room temperature, refrigerated storage, and cold-thaw repeatedly compared to emulsions prepared using orange oil Respectively. With respect to turbidity change during storage, similar results were obtained when the emulsion was visually observed, as shown in Figs. 9 and 10.

Cell penetration experiment

Emulsion was prepared by using luteolin as a trapping agent and Tween 80 as a surfactant in the same manner as in the emulsion characteristic test according to the type of surfactant, except that soybean oil and orange oil were used as the oil, Emulsion. Luteolin adsorption by cell model (Caco-2, co-culture, triple co-culture) was measured with the thus prepared emulsion. Specifically, Caco-2 cells were seeded at a density of 2 × 10 ⁴ cells / well and the change of TEER (transepithelial electrical resistance, Ω · cm ² ) was observed while the medium was changed for 20 days. cultured cells were seeded by mixing Caco-2 cells and HT-29 cells at a density of 2 × 10 ⁴ cells / well at a final concentration of 9: 1, and then the electrical resistance was measured while changing the medium for 20 days. Cultured cells were seeded with Caco-2 cells and HT-29 cells at a density of 2 × 10 ⁴ cells / well at 3.3 × 10 ⁴ cells / well and then measured for electrical resistance for 6 days. On the 20th day, the emulsion samples (final concentration 20 μM) were treated in the upper layer, incubated for 6 hours, and then the upper and lower buffers were recovered and lyophilized to be used as a sample for HPLD analysis.

Table 1. Luteolin absorption by cell model of emulsion prepared with soybean oil

Table 2. Luteolin absorption by cell model of emulsion prepared with orange oil

"C: H = 9: 1" in Tables 1 and 2 means co-culture cells in which Caco-2 cells and HT-29 cells were mixed at a ratio of 9: 1. As shown in Tables 1 and 2, luteolin was not found on the basolateral side of the Caco-2, co-culture, and triple co-culture cells in the case of the emulsion prepared using orange oil, In the case of the prepared emulsion, a small amount of luteolin was found in the lower part of Caco-2 cells.

Claims (10)

Adding a capturing material to the internal water phase (W ₁ );
(W ₁ / O) type emulsion by dispersing the internal water phase (W ₁ ) in an oil phase (O) to which an oil and a surfactant are added; And
(W ₁ / O / W ₂ ) type emulsion by dispersing the emulsion of the water-in-oil type (W ₁ / O) type in the external water phase (W ₂ ). The method according to claim 1,
Characterized in that the entrapping material is selected from the group consisting of luteolin, resveratrol and quercetin. 3. The method of claim 2,
RTI ID = 0.0 > 1, < / RTI > wherein the entrapping material is luteolin. 4. The method according to any one of claims 1 to 3,
Wherein the oil is soybean oil. 4. The method according to any one of claims 1 to 3,
Wherein the surfactant is a nonionic surfactant. 6. The method of claim 5,
≪ / RTI > wherein the nonionic surfactant is < RTI ID = 0.0 > Tween 80. < / RTI > 4. The method according to any one of claims 1 to 3,
Wherein the oil is soybean oil and the surfactant is Tween 80. 4. The method according to any one of claims 1 to 3,
Characterized in that the concentration of the surfactant added to the oil phase (O) is from 15% (w / v) to 30% (w / v). 4. The method according to any one of claims 1 to 3,
Characterized in that a pressure of 20,000 psi to 25,000 psi is applied in the step of producing an emulsion of water-in-oil-in-water (W ₁ / O / W ₂ ) type emulsion. 4. The method according to any one of claims 1 to 3,
Wherein the weight ratio of the water-in-oil (W ₁ / O) emulsion to the external water-phase (W ₂ ) is 1:10.

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