KR102296341B1 - liposome nanosphere containing lipophilic antiaging ingredints and preparation method of the same - Google Patents

Abstract

The present invention relates to liposome nanoparticles capable of loading a fat-soluble component and a method for preparing the same, and more particularly, includes a certain amount of phospholipids and cholesterol in the lipids forming the liposome nanoparticles, and increases the solubility of poorly soluble substances in aqueous phase It relates to liposome nanoparticles having excellent biocompatibility, which can be fused with skin cells to promote absorption into the skin and improve delivery of cosmetic ingredients by increasing the encapsulation efficiency, and a method for preparing the same.

Description

Liposome nanoparticles loaded with a fat-soluble anti-aging ingredient and a method for preparing the same

The present invention relates to liposomal nanoparticles and a method for preparing the same, and more particularly, includes a certain amount of phospholipids in lipids forming liposome nanoparticles, and increases the encapsulation efficiency of poorly soluble components in an aqueous phase by introducing cholesterol, and By increasing the solubility of poorly soluble substances and increasing the encapsulation efficiency, liposomal nanoparticles with excellent biocompatibility that can be fused with skin cells to promote absorption into the skin and improve delivery of cosmetic ingredients, and a method for preparing the same will be.

Skin aging occurs through two major processes. Endogenous aging refers to the changes that occur with time to anyone without special environmental factors, and changes that appear on the face, neck, and hands due to prolonged exposure to environmental factors such as UV irradiation. This is called photo aging or exogenous aging.

The main cause of endogenous aging is the accumulation of damage to the components of our body by reactive reactive oxygen radicals created in the metabolic process of our body. Once free radicals are created and cannot be effectively removed by various protective devices in the body, a series of inflammatory reactions occur, resulting in skin damage.

The main mechanism related to skin wrinkle formation is that the skin is continuously exposed to oxidative stress from harmful environments such as air pollution, UV exposure, stress or disease, and radicals in the body increase, and collagen, the connective tissue of the dermis, ), elastin, hyaluronic acid, etc., are explained to be able to cause subsidence (wrinkle) in a certain area of the skin. In addition, radicals are involved in the formation of melanin and cause spots, freckles and wrinkles.

In addition, the greatest influence on the skin wrinkle formation is the expression amount and activity of collagenase, which is a collagen degrading enzyme that reduces the amount of collagen produced and the amount of collagen. In particular, the collagenase is excessively expressed during photoaging by ultraviolet rays rather than natural aging.

Collagen is a high molecular substance present in the dermal layer of the skin and is known as one of the main components that give elasticity to the skin. Therefore, as a method of improving skin wrinkles, efforts are being made to find a substance that can promote collagen biosynthesis and simultaneously inhibit collagenase activity. Since collagenase is a major factor in the wrinkle-generating process, studies to develop natural extracts that inhibit the activity of collagenase as effective wrinkle-improving substances have been actively conducted.

As a result of this, in Patent Document 1, anti-wrinkle improvement comprising an anti-sense oligonucleotide for mRNA of collagenase gene and an anti-sense oligonucleotide for mRNA of c-Jun gene, which has an excellent wrinkle-improving effect by inhibiting collagen degradation It discloses the contents of the active substance.

In addition, in order to improve the skin wrinkles, ascorbic acid, α-tocopherol, retinol and its derivatives, super oxide dismutase (SOD), etc. are used in cosmetics or pharmaceuticals as a material for free radical scavenging function or skin wrinkle improvement. Although formulated and used to prevent wrinkles and other skin diseases, they have a problem in that it is difficult to expect practical effects due to poor long-term stability. For this reason, developing a safer and more effective material for improving skin wrinkles has emerged as an important task not only in the pharmaceutical and food fields but also in the cosmetic industry, and many studies are being conducted.

Here, the retinol is known to play an important role in maintaining the original function of the epidermal cells of the skin as a representative compound of fat-soluble vitamin A. Retinol quickly penetrates into the skin and activates genes involved in cell growth and differentiation from the cell nuclear DNA of skin cells. In addition, by activating mRNA involved in protein (collagen, etc.) synthesis, collagen biosynthesis is promoted, thereby increasing the connective tissue of the skin. This is known to have the effect of improving skin elasticity.

However, while retinol has excellent effects, it is very unstable to light, temperature, and oxygen, so it is easily oxidized and discolored. Various studies are being conducted for effective retinol with long-term stability against factors.

In addition, natural substances derived from silkworm larvae, pupae, and cocoons are also known to be effective in improving wrinkles, such as ascorbic acid, α-tocopherol, retinol, and the like. For example, Patent Document 2 discloses a cosmetic composition comprising a powder, extract or hydrolyzate of a golden silkworm-derived material as an active material, and the silkworm-derived material has excellent wrinkle improvement, whitening and moisturizing effects, and other It has excellent bonding strength with cosmetic materials, so it is confirmed that there is an effect of easy formulation of lotions, essences, lotions, creams, packs, gels, ointments, patches, mask sheets, sprays, and detergents.

Similar liposomal nanoparticles use U.S. Patent No. 5,059,591 amphotericin B and cholesterol-polyethylene glycol (PEG) to reduce the toxicity of amphotericin B. In the above patent, amphotericin B was combined with cholesterol-PEG in the form of a salt.

U.S. Patent No. 5,965,156 relates to a method for preparing a liposome encapsulating amphotericin B. The above patent encapsulated amphotericin B in liposomes and ionically combined amphotericin B and anionic phosphatidylglycerol in an acidic organic solvent to increase the encapsulation rate and reduce toxicity in the body when formed into a formulation.

Therefore, there is a need for a new method that improves aqueous solubility of amphotericin B, reduces drug toxicity, increases circulation time in the body, and enables mass production.

Republic of Korea Patent Publication 10-2006-0065813 Republic of Korea Patent Publication 10-2013-0108805

Accordingly, the present inventors have been conducting research to develop nanoparticles with excellent long-term stability in which silkworm oil, which is well known as an effective wrinkle improvement ingredient, for example, is encapsulated, the wrinkle improvement effective substance according to the present invention It was confirmed that the encapsulated nanoparticles can be useful as a wrinkle-improving cosmetic composition having excellent long-term stability and excellent long-term stability by solving the problem of low solubility in water of the wrinkle-improving material by including a skin permeation promoter, and the present invention was completed.

The present invention features liposome nanoparticles in which fat-soluble components such as silkworm oil and retinol are encapsulated in particles formed of phospholipids and cholesterol.

In other words, as shown in Fig. 1, cholesterol with strong hydrophobicity surrounds the fat-soluble component, and phospholipids with good affinity for cholesterol act as a surfactant, thereby producing stable liposome nanoparticles. The hydrophilic phosphate groups of the phospholipids are arranged on the surface of the nanoparticles, so they are well dispersed and stabilized in an aqueous solution.

Also, the present invention

(A-1) preparing a lipid solution by dissolving in an organic solvent with respect to 100 parts by weight of a lipid prepared by mixing phosphatidylcholine, ionic phospholipids and sterols in a weight ratio of 40 to 70: 5 to 20: 10 to 40;

(A-2) preparing a drug solution by dissolving an anti-aging component in a linear or branched alcohol having 1 to 6 carbon atoms;

(A-3) mixing the lipid solution of (A-1) and the drug solution of (A-2) in a volume ratio of 1:1 to 1:9 to prepare a lipid-drug mixture solution;

(A-4) dispersing the mixed solution of (A-3) in a volume ratio of 2:1 to 1:10 in an aqueous phase to form lipid nanoparticles;

(A-5) distilling the lipid nanoparticle solution of (4) under reduced pressure at 20-50 ° C. to remove the organic solvent, and filtering to prepare liposome nanoparticles encapsulated with cosmetic ingredients of uniform size;

The present invention will be described in more detail as follows.

In the present invention, cholesterol with strong hydrophobicity surrounds the fat-soluble cosmetic ingredient among the lipids forming the liposome nanoparticles, and the phospholipids having good affinity with cholesterol act as a surfactant, thereby preparing stable liposome nanoparticles. The hydrophilic phosphate groups of the phospholipids are arranged on the surface of the nanoparticles, so they are well dispersed and stabilized in an aqueous solution.

In particular, for the preparation of the liposome nanoparticles encapsulated with the cosmetic component of the present invention, it is characterized by using an ionic phospholipid as a forming lipid of the nanoparticles. The ionic phospholipid is preferably phosphatidylic acid having a hydrophobic group and an alkyl chain having 16 to 18 carbon atoms. More preferably, dimyristylglycerophosphate (DMPA), dipalmitoylglycerophosphate (DPPA), dimyristylglycerophosphate (DMPG), disteroylglycerophosphate (DSPA), disteroylglycerophosphate Polyglycerol (DSPG), dipalmitoylglycerophosphoraglycerol (DPPG), dimyristylglycerophosphoserine (DMPS), dipalmitoylglycerophosphoserine (DPPS) and disteroylglycerophosphoserine (DSPS) , including one or two or more selected from among. When the carbon number of the anionic phospholipid is less than 14, the phase transition temperature falls below body temperature, and the stability of the lipid nanoparticles in the body decreases. It is undesirable because the particle size increases.

The ionic lipid is preferably contained in an amount of 5 to 20% by weight based on the total lipid composition forming the lipid nanoparticles.

In addition, it is preferable to use hydrogenated phosphatidylcholine or phosphatidylcholine as the lipid for forming the lipid nanoparticles in the present invention, and soybean phosphatidylcholine, egg yolk phosphatidylcholine or bovine phospholipid may be used, More preferably, the number of carbon atoms of the hydrophobic group is 16 to 18 It has a phosphorus alkyl chain, and for example, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine, etc. may be used. Even if the carbon number is less than 16, it forms a strong bond with silkworm oil, but the phase transition temperature falls below body temperature, which reduces the stability of the lipid nanoparticles in the body. It is not preferable because the encapsulation efficiency of the decreases and the size of the lipid nanoparticles increases. The phosphatidylcholine is preferably contained in an amount of 40 to 70% by weight based on the total lipid composition forming the lipid nanoparticles.

In addition, sterols are used as lipids used to form lipid nanoparticles. Preferred sterols include cholesterol, cholesterol hexasuccinate, and 3β-[N-(N',N'-dimethylaminoethane)carba. Moyl] cholesterol, ergosterol, stigmasterol or lanosterol and the like. The sterols are preferably contained in an amount of 10 to 40% by weight based on the total lipid composition forming the lipid nanoparticles.

The average particle diameter of the liposome nanoparticles in which the cosmetic ingredient of the present invention is encapsulated with cholesterol and phospholipids, hydrogenated phosphatidylcholine or phosphatidylcholine, is stabilized as a surfactant in the aqueous phase is 20 to 100 nm, preferably 20 to 30 nm. When the average particle diameter of the lipid nanoparticles exceeds the above range, the particles are too large to be absorbed into the skin, and the stability in aqueous solution is rapidly reduced. If it is less than the above range, the loading amount (drug payload) of the cosmetic ingredient in the nanoparticles is insufficient.

Hereinafter, a method of encapsulating a cosmetic ingredient and producing a surface liposome nanoparticles will be described step-by-step.

In step (A-1), phosphatidylcholine, ionic phospholipids and sterols are mixed in a weight ratio of 40 to 70: 5 to 20: 10 to 40 to prepare lipids. In this case, when the phosphatidylcholine used is less than the above range, the formation of lipid nanoparticles is not easy, and when it exceeds, the stability of the lipid nanoparticles is reduced. When the content of anionic phospholipids is outside the lower limit, there are problems such as an increase in the size of the lipid nanoparticles in the lipid nanoparticles, aggregation between the lipid nanoparticles, and the like. There is a problem of increasing the particle size. In addition, when the content of cholesterol is out of the lower limit, there is a problem in that the drug encapsulation efficiency is reduced, and when it is out of the upper limit, there is a problem in that the stability of the lipid nanoparticles is reduced. In addition, as the weight ratio of sterols increases, the drug encapsulation rate increases, but above the above range, the particle size increases, so that it is preferably prepared within the above range.

In step (A-2), a drug solution is prepared by dissolving the drug in a linear or branched alcohol having 1 to 6 carbon atoms. Examples of the linear or branched alcohol having 1 to 6 carbon atoms include methanol, ethanol, propanol, butanol, isobutanol, and isopropanol. It is preferable to dissolve the drug in the alcohol at 0.1 to 1 mg/ml. When the concentration of amphotericin B is out of the lower limit, the drug encapsulation concentration is lowered, and when it is out of the upper limit, there is a problem in that the drug is not completely dissolved in alcohol. In addition, when another organic solvent such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) is used for the drug, the solvent is not easily removed and biocompatibility is reduced.

In step (A-3), the lipid mixed solution of (A-1) and the drug solution of (A-2) are mixed in a volume ratio of 1:1 to 1:9 to prepare a lipid-drug mixed solution. When the ratio of the (A-1) solution exceeds 50 (v/v)%, there is a problem in that the concentration of the drug is reduced, and when it is less than 10 (v/v)%, the amount of phospholipids is excessively reduced to form a formulation there are difficulties in

In step (A-4), the mixed solution of (A-3) is preferably dispersed in a volume ratio of 2:1 to 1:10 in the aqueous phase, more preferably 1:1 to 1:3 by dispersing the lipid nanoparticle. to form particles. As the aqueous phase, a sugar solution such as distilled water, a phosphate buffer solution, a saline solution, a sucrose solution, a maltose solution, and a mannitol solution, a buffer solution, or an isotonic solution may be used. When the aqueous phase is less than the volume ratio range, the dispersed lipid nanoparticles agglomerate and the particle size of the final lipid nanoparticles increases. there is a case to do In addition, for the formation of lipid nanoparticles, it is preferable to disperse the lipid-drug mixture solution of step (A-3) in water using a syringe at a rate of 1 to 5 ml/min in tip sonication. When the dispersion rate exceeds the above range, the size of the particles increases, and even if it is less than the above range, it is difficult to form particles of a certain size or less, so a dispersion rate within the above range is preferable.

In step (A-5), the lipid nanoparticle solution of (A-4) is distilled under reduced pressure at 20 to 50° C. to remove the organic solvent, and filtered to filter the lipid nanoparticle having a PEG functional group encapsulated with a drug of a uniform size. to manufacture When the temperature at the time of the vacuum distillation is less than 20 ° C, the removal time increases and the complete removal of the organic solvent is difficult, and when it exceeds 50 ° C, the destruction of the formed lipid nanoparticles and the drug may be denatured. It is desirable to keep In addition, in order to completely remove the organic solvent, it is preferable to remove 0.5 to 5 times the volume of the organic solvent together with the organic solvent during the vacuum distillation.

As described above, in the present invention, a stable liposome nanoparticle is prepared by enclosing the fat-soluble component with cholesterol with strong hydrophobicity, and phospholipids having good affinity for cholesterol serve as a surfactant. It is a technology that has the characteristics of being well dispersed and stabilizing in an aqueous solution by arranging the hydrophilic phosphate groups of phospholipids on the surface of the nanoparticles. Liposomal nanoparticles with excellent biocompatibility that can be encapsulated in excellent liposome nanoparticles to increase solubility of poorly soluble substances in aqueous phase and increase encapsulation efficiency, thereby promoting absorption into the skin and improving delivery of cosmetic ingredients, and their By developing a manufacturing method, the present invention was completed.

1 shows a Cryo-TEM photograph and a conceptual diagram calculated in Example 7.
2 shows the results for the skin permeability calculated in Examples 1 and 7.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited by these examples.

Example 1-6

Hydrogenated distearoylphosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) 60 mg of any one selected from phosphatidylcholine (PC) and 20 mg of cholesterol (CHOL) in 2 ml of chloroform was dissolved in to prepare a lipid mixture solution.

Silkworm oil or retinol was dissolved in methanol at a concentration of 0.5 mg/ml, and 2 ml of the lipid mixture solution and 8 ml of the silkworm oil solution were mixed to prepare 10 ml of a silkworm oil-lipid mixture solution.

After dispersing 10 ml of the silkworm oil or retinol-lipid mixed solution in 20 ml of distilled water at a rate of 2 ml/min while tip sonicating using a syringe to form lipid nanoparticles, using a reduced pressure distiller at 35° C. Remove 10 ml of organic solvent and 10 ml of distilled water until the total amount of the solution is 10 ml, and pass through a 0.2 μm semipermeable membrane using an extruder to equalize particle size distribution.

The particle size and surface charge of the lipid nanoparticles prepared by the above process were measured with a particle analyzer (electrophoretic light scattering spectrophotometer, ELS-8000, Osuka Electronics, Japan), and are shown in Table 1 below.

In addition, the encapsulation efficiency was measured at a maximum absorption wavelength of 260 nm using HPLC, and was calculated by Equation 1 below. The encapsulation efficiency is shown together in Table 2 below.

In Formula 1, where C _f is the drug concentration after dissolving the liposome with chloroform : methanol (8 : 2) (volume ratio), and C _i is the concentration of the initially added drug.

ingredient Phospholipids particle size
(nm) surface charge
(mV) Encapsulation efficiency
(%) Example 1 silkworm oil HSPC 55 4.5 75 Example 2 silkworm oil DPPC 62 11.2 78 Example 3 silkworm oil DSPC 88 8.3 66 Example 4 retinol HSPC 65 3.8 71 Example 5 retinol DPPC 77 8.7 76 Example 6 retinol DSPC 84 6.9 69

On the other hand, except that melatonin (Comparative Example 1) and beta-histin (Comparative Example 2), which are comparative drugs, were used instead of the silkworm oil and retinol as active ingredients, it was prepared and tested in the same manner as in Example 1, and as a result of the experiment, the encapsulation efficiency It was confirmed that this Example 1-6 did not reach (Comparative Example 1: 32%, Comparative Example 2: 37%).

manufacturing example 7-10: anionic containing phospholipids of lipid nanoparticles Produce

In the preparation of lipid nanoparticles in the same manner as in Preparation Example 1, the anionic phospholipid dipalmitoylglyceride is more preferably 50 nm or less in size and to increase the encapsulation rate of Sample 3 to 90% or more. Anionic lipid nanoparticles were prepared by adding lophosphate (DPPA) and disteroylglycerophosphoglycerol (DSPG), respectively.

Changes in the particle size of lipid nanoparticles, zeta potential, and encapsulation efficiency according to the content of DPPA and DSPG were measured, and are shown in Table 2 below, respectively.

ingredient Phospholipids particle size
(nm) surface charge
(mV) Encapsulation efficiency
(%) Example 7 silkworm oil HSPC/DPPA 35 - 42 91 Example 8 silkworm oil HSPC/DPPG 42 - 55 94 Example 9 retinol HSPC/DPPA 43 - 38 91 Example 10 retinol HSPC/DPPG 53 - 61 95

test example 1: Nanoparticle Stability Experiment

Changes in particle size due to precipitation and agglomeration with time of the liposome nanoparticles prepared in Examples 7 and 9 according to the present invention in order to measure the particle size for long-term stability evaluation

In order to evaluate, the prepared nanoparticles were placed at room temperature (25 ℃) and 40 ℃, respectively, and the particle size according to time was measured with a particle analyzer (Electrophoretic light scattering spectrophotometer, ELS-8000, Osuka Electronics, Japan), and the result is shown below. Table 3 shows.

Room temperature (25°C) ingredient 1 month 2 months 3 months 4 months 5 months 6 months Example 1 silkworm oil 57 61 65 71 sedimentation Example 4 retinol 63 68 82 88 Precipitation/discoloration Example 7 silkworm oil 35 36 41 38 43 46 Example 9 retinol 43 48 45 49 51 55 Room temperature (40°C) ingredient 1 month 2 months 3 months 4 months 5 months 6 months Example 1 silkworm oil 55 64 69 sedimentation Example 4 retinol 64 77 Precipitation/discoloration Example 7 silkworm oil 39 36 44 45 48 51 Example 9 retinol 46 49 55 53 58 59

As shown in Table 3, in the case of Examples 1 and 4, which do not contain an antioxidant, the particle size was changed and discolored by external conditions, indicating that the long-term stability was not excellent, but anionic liposomes were In the case of including Examples 7 and 9, the particle size change due to external conditions hardly occurred and discoloration did not occur, indicating that the long-term stability was very good. Therefore, it can be seen that the nanoparticles encapsulated with an effective wrinkle improvement material according to the present invention preferably include anionic phospholipids.

test example 2: percutaneous penetration exam

A French cell was used for the transdermal permeation experiment, and 1% SDS PBS was used as a permeate for the receptor cell. The Strat-Mⓡ Membrane (Size 25mm) is fixed to the skin penetrating device with the stratum corneum facing up.

As announced in the guidelines for in vitro skin absorption test, the temperature of the permeate in the device was kept constant at 32±1℃ and the RPM at 600.

Put 1 ml of each of Examples 1 and 7, which are substances for the purpose of permeation, into the prepared skin penetrating device donor compartment, so that the Strat-Mⓡ Membrane can be thoroughly wetted.

After 2h, 6h, 12h, and 24h after permeation, the content of Alpha-Linolenic Acid in the permeate was analyzed through GC analysis after sampling using a 1ml syringe.

As a result, in the case of Example 7, the permeation amount was higher than in Example 1, which is evaluated as a result due to the particle size, surface charge and stability of the particles.

Claims (9)

Anionic lipid nanoparticles for improving skin wrinkles, comprising phospholipids and sterols lipids, and containing at least one skin anti-aging component selected from silkworm oil and retinol,
The phospholipids include (i) phospholipids and (ii) anionic phospholipids for forming lipid nanoparticles, and the (i) phospholipids for forming lipid nanoparticles have a hydrophobic group having an alkyl chain having 16 to 18 carbon atoms. a phospholipid, and (ii) the anionic phospholipid is a phosphatidylic acid having an alkyl chain having 14 to 18 carbon atoms as a hydrophobic group,
The anionic lipid nanoparticles do not contain polyethylene glycol (PEG) and have an average particle diameter of 20 nm to 53 nm. delete According to claim 1, wherein the phosphatidylic acid having an alkyl chain having 14 to 18 carbon atoms in the hydrophobic group is dimyristyl glycerophosphate (DMPA), dipalmitoyl glycerophosphate (DPPA), dimyristyl glycerophosphate (DMPG), Disteroylglycerophosphate (DSPA), disteroylglycerophosphoglycerol (DSPG), dipalmitoylglycerophosphoraglycerol (DPPG), dimyristylglycerophosphoserine (DMPS), dipalmitoylglycero At least one selected from phosphoserine (DPPS) and disteroylglycerophosphoserine (DSPS),
The phospholipid having an alkyl chain having 16 to 18 carbon atoms is anionic lipid nanoparticles, characterized in that at least one selected from hydrogenated phosphatidylcholine, phosphatidylcholine, soybean phosphatidylcholine, egg yolk phosphatidylcholine and bovine phospholipid. The anionic lipid nanoparticles according to claim 1, wherein the anionic phospholipids are contained in an amount of 5 to 20% by weight based on the total lipid composition forming the lipid nanoparticles. delete (1) phosphatidylcholine, anionic phospholipids and sterols 40 to 70: 5 to 20: preparing a mixed solution of 100 parts by weight of a lipid prepared by mixing in a weight ratio of 10 to 40;
(2) preparing a drug solution by dissolving one or more skin anti-aging ingredients selected from silkworm oil and retinol in linear or branched alcohol having 1 to 6 carbon atoms;
(3) mixing the lipid mixture solution of (1) and the drug solution of (2) in a volume ratio of 1:1 to 1:9 to prepare a lipid-drug mixture solution;
(4) dispersing the mixed solution of (3) in a volume ratio of 2:1 to 1:10 in an aqueous phase to form lipid nanoparticles; and
(5) removing the organic solvent by distilling the lipid nanoparticle solution of (4) under reduced pressure at 20-50 ° C., and filtering to prepare anionic lipid nanoparticles encapsulated with a drug of a uniform size;
Method for producing anionic lipid nanoparticles encapsulated with a drug, comprising:
Here, the anionic lipid nanoparticles do not contain polyethylene glycol (PEG) and have an average particle diameter of 20 nm to 53 nm. The preparation of anionic lipid nanoparticles according to claim 6, wherein in step (2), the skin anti-aging component is dissolved in a linear or branched alcohol having 1 to 6 carbon atoms at a concentration of 0.1 to 1 mg/ml. Way. The anionic according to claim 6, wherein the mixed solution of step (3) is dispersed using a syringe at a rate of 1 to 5 ml/min using tip sonication in step (4). A method for producing lipid nanoparticles. [Claim 7] The method of claim 6, wherein in step (5), 0.5 to 5 times the volume of the organic solvent is removed with water together with the organic solvent.

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