WO2016143939A1 - Production method for complex of steviol glycoside and sparingly soluble material having improved solubility, and complex of steviol glycoside and sparingly soluble material having improved solubility produced thereby - Google Patents

Abstract

The present invention relates to a production method for a complex of a steviol glycoside and a sparingly soluble material having improved solubility, and to a complex of a steviol glycoside and a sparingly soluble material having improved solubility produced thereby. More specifically, the present invention relates to a method for improving the solubility of a sparingly soluble material by producing a complex of a steviol glycoside and the sparingly soluble material, by mixing the steviol glycoside and the sparingly soluble material and dissolving in a solvent, and relates to the complex of the steviol glycoside and the sparingly soluble material produced thereby.

Description

Method for preparing a composite of a poorly soluble material with improved solubility and steviol glycosides and a composite of a poorly soluble material with improved solubility and steviol glycosides

The present invention relates to a method for preparing a complex of poorly soluble material and steviol glycoside with improved solubility, and a complex of poorly soluble material and steviol glycoside with improved solubility. Specifically, the present invention is a method of improving the solubility of the poorly soluble material and steviol glycosides prepared by mixing the poorly soluble material and steviol glycosides to prepare a complex of poorly soluble material and steviol glycosides It is about a complex.

In general, solubilization of the drug is closely related to its effect since the drug may exhibit physiological action in a dissolved state. Drugs that are well soluble in water are easily absorbed for their manufacturing process and action, but solubilization is essential for poorly soluble drugs. Poorly soluble drugs exist in large aggregates without solubilization, and do not dissolve as a single molecule, so they are not absorbed at all, so the effect cannot be expected.

On the other hand, steviol glycosides (steviol glycosides) is a sweetener that is classified as a natural additive, and is produced by extracting from the leaves of Chrysanthemum Stevia. The sweetness is about 200 to 300 times the sugar. Stevia is a South American paraguay plant, and locals have long used dry leaves as a sweetener. It is currently used in low-calorie foods, carbonated drinks, confectionery, pickled foods. As a result of safety evaluation by JECFA (Joint Food Additives Expert Committee), the daily intake allowance (ADI) is set to 0-4 mg / kg body weight / day (steviol). Types of steviol glycosides include stevioside, stevioside, rebaudioside, dulcoside, rubusoside, steviolside, and the like.

In particular, Rubusoside (13-O-β-glucosyl-19-O-β-d-glucosyl-steviol) is a rare ingredient which is obtained from a plant called Rubus suavissimus in addition to stevia and is also included as a component of Chinese sweet tea. It is a natural sweetener. Rubusoside is a sugar-free, calorie-free, anti-cavity sweetener with a taste of about 115 times that of sugar, and is a substitute for sugar and is used for people with diseases such as obesity, diabetes, cardiovascular disease and kidneys. It can have a good auxiliary effect. In addition, the role of anti-diabetic, anti-hypertensive, anti-tumor (anti-mutant), anti-viral, intestinal flora, renal function, etc. has been reported. It is known that it can be applied as a cosmetic material because of its excellent moisturizing effect.

The present inventors completed the present invention by finding that the solubility of the poorly soluble material was remarkably improved when using the complex of the poorly soluble material and the steviol glycoside while conducting a study on the method for improving the solubility of the poorly soluble material.

According to one aspect of the invention,

A first step of mixing and stirring a steviol glycoside and a poorly soluble material in ethanol;

A second step of centrifuging the mixture;

A third step of separating the supernatant; And

A fourth step of removing ethanol contained in the separated supernatant;

Disclosed is a method for preparing a complex of poorly soluble material and steviol glycosides with improved solubility.

According to one aspect of the invention, it is possible that the steviol glycoside comprises an enzyme product that reacts with the enzyme. According to an aspect of the present invention, the method further comprises the step of preparing a steviol glycoside enzyme reaction solution by mixing the steviol glycoside and the enzyme before the first step, wherein the prepared steviol glycoside enzyme reaction It may be to use a liquid. That is, in the method for improving the solubility of the poorly soluble material according to the present invention, the steviol glycoside may be purified or an enzyme reaction product.

According to one aspect of the invention, the enzyme may be lactase.

According to an aspect of the present invention, the steviol glycoside per 100 parts by weight of the poorly soluble material may be mixed in a ratio of 800 to 1200 parts by weight.

In the method for producing a complex of a poorly soluble material and steviol glycosides according to the present invention, the steviol glycosides are stevioside, rebaudioside A, rebaudioside B, rebaudioside B Rebaudioside C, rebaudioside D, rebaudioside E, rubusoside, dulcoside a, steviolside or mixtures thereof Can be.

In the method for producing a complex of a poorly soluble material and a steviol glycoside according to the present invention, the poorly soluble material may be a substance or food additive which is an active ingredient of any one of medicines, animal medicines, quasi-drugs, cosmetics and pesticides have.

In the method for producing a complex of a poorly soluble material and a steviol glycoside according to the present invention, the poorly soluble material is oleanolic acid (oleanolic acid, OA), idebenone (ID), phytosphingosine (PS), ceramide Ceramide IIIB (CR), epigallocatechin gallate, or astragalin.

In the method for producing a composite of a poorly soluble material and a steviol glycoside according to the present invention, when mixing rubusoside (Ru) as a steviol glycoside and oleanoic acid (OA) as a poorly soluble material, It may be established.

[Relationship 1]

Y = 0.90 × X-0.38

In the above formula, Y represents the concentration of oleanoic acid (OA) dissolved in a solvent (mg.ml ⁻¹ ), and X represents the concentration of added Ru (mg.ml ⁻¹ ).

In the method for producing a complex of a poorly soluble material and a steviol glycoside according to the present invention, the following relational formula 2 is established when rubusoside (Ru) is mixed as steviol glycoside and idebenone (ID) as a poorly soluble material. It may be.

[Relationship 2]

Y = 2.94 × X-4.58

In the above formula, Y represents the concentration of Idebenone (ID) dissolved in a solvent (mg.ml ^-1 ), and X represents the concentration of Ru (mg.ml ^-1 ).

In the method for improving the solubility of the poorly soluble material according to the present invention, when mixing rubisoside (Ru) as a steviol glycoside and phytosphingosine (PS) as a poorly soluble material, the following relation 3 may be established.

[Relationship 3]

Y = 0.94 × X-0.90

In the above formula, Y represents the concentration of phytosphingosine (PS) dissolved in a solvent (mg.ml ⁻¹ ), and X represents the concentration of Ru (mg.ml ⁻¹ ).

In the method for producing a complex of a poorly soluble material and a steviol glycoside according to the present invention, when mixing rubusoside (Ru) as a steviol glycoside and ceramide IIIB (CR) as a poorly soluble material, the following relation 4 is established. It may be.

[Relationship 4]

Y = 0.15 × X-0.29

In the above formula, Y represents the concentration of CR dissolved in the solvent (mg.ml ⁻¹ ), and X represents the concentration of Ru (mg.ml ⁻¹ ).

According to one aspect, it provides a complex of poorly soluble material and steviol glycosides improved in the solubility produced by the production method of the present invention.

According to one aspect, a fifth step of dissolving the product obtained in the fourth step in a solvent, stirred and centrifuged; And a sixth step of separating the supernatant centrifuged; providing a method for preparing a complex solution of a poorly soluble material and a steviol glycoside.

Figure 1 shows the results of TLC analysis for the case of dissolving the oleanoic acid (OA) and steviol glycoside complex according to Example 1 in a solvent.

Figure 2 shows the results of the oleanoic acid (OA) solubility increase in the stevioside and lactase enzyme reaction solution according to Example 1.

Figure 3 shows a graph of the increase in oleanoic acid (OA) solubility according to the concentration of stevioside and lactase enzyme reaction solution according to Example 1.

4 shows the results of TLC analysis for the idebenone (ID) steviol glycoside complex according to Example 2. FIG.

Figure 5 shows the results of the solubility increase of Idebenone (ID) in the stevioside and lactase enzyme reaction solution according to Example 2.

Figure 6 shows a graph of increasing solubility of idebenone (ID) according to the concentration of stevioside and lactase enzyme reaction solution according to Example 2.

Figure 7 shows the results of TLC analysis for phytosphingosine (PS) and steviol glycoside complex according to Example 3.

Figure 8 shows the results of phytosphingosine (PS) solubility increase in the stevioside and lactase enzyme reaction solution according to Example 3.

Figure 9 shows a graph of the increase in phytosphingosine (PS) solubility according to the concentration of stevioside and lactase enzyme reaction solution according to Example 3.

Figure 10 shows the results of TLC analysis for the ceramide and steviol glycoside complex according to Example 4.

11 shows the results of increasing ceramide water solubility in the stevioside and lactase enzyme reaction solution according to Example 4.

12 is a graph showing the increase in ceramide solubility according to the concentration of stevioside and lactase enzyme reaction solution according to Example 4.

Figure 13 is a graph showing the effect of rubusoside and oleanoic acid complex on the proteolytic enzyme (3CL ^pro ).

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific events, including the terms included herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those of ordinary skill in the art, they can be used in a variety of ways, and therefore should not be construed as limiting the invention to particular methods, protocols, and reagents.

As used herein, the singular encompasses the plural objects unless the context clearly dictates otherwise. As used herein, "or" means "and / or" unless stated otherwise. Moreover, the terms “comprising” as well as other forms, such as “having”, “consisting of” and “consisting of” are not limiting.

The numerical range includes the numerical values defined in the range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limitations as if the higher numerical limit were clearly written. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical range, as the narrower numerical limitations are clearly written. The headings provided herein are not to be construed as limiting the following embodiments, as a reference to the specification in various aspects or as a whole.

The present invention is to provide a method for improving the solubility of poorly soluble material by forming a complex with a poorly soluble material steviol glycosides.

According to one embodiment,

A first step of mixing and stirring a steviol glycoside and a poorly soluble material in ethanol;

A second step of centrifuging the mixture;

A third step of separating the supernatant; And

A fourth step of removing the ethanol contained in the separated supernatant is provided, a method for producing a complex of poorly soluble material and steviol glycosides with improved solubility.

As used herein, the "complex of poorly soluble material and steviol glycoside" is a material prepared by mixing and stirring a poorly soluble material and a steviol glycoside in an ethanol solvent, centrifuging, and removing ethanol. Although a clear covalent bond is not formed between steviol glycosides, it means a state in which a certain structure is physically formed by intermolecular bonds.

In the present invention, "dissolution" includes not only a completely dissolved state in a solvent containing water, but also a solubilized state by micelles or the like, or a liquid uniformly dispersed in an aqueous solvent and visually transparent, and generally used to measure solubility of each substance. Means the condition measured by the test method used.

In the method for preparing a complex of a poorly soluble material and a steviol glycoside according to the present invention, a complex of a poorly soluble material and a steviol glycoside is obtained by dissolving in ethanol and then removing the ethanol in the supernatant again. Although ethanol was used as the solvent in the first step, the present invention is not limited thereto, and the method for removing ethanol from the supernatant after complex formation is not particularly limited.

As used herein, the term “poorly soluble material” means “slightly insoluble”, “hardly soluble”, “extremely insoluble” and “nearly insoluble,” and includes pharmaceuticals, veterinary medicines, quasi-drugs, cosmetics, It may include substances used in food or pesticides. The medicine, animal medicine, quasi-drug, cosmetics, food or pesticides are not particularly limited.

For example, the poorly soluble material is more specifically oleanolic acid (oleanolic acid), idebenone (idebenone), phytosphingosine, ceramide (ceramide), epigallocatechin gallate, astragalin (astragalin), paclitaxel, paclitaxel derivatives, taxotere, adriamycin, teniposide, etoposide, daunomycin, methotrexate, mitomycin Mitomycin C, carmustine, busulfan, dactinomycin, lomustine, megestrol acetate, melphalan, mitoxanthrone (mitoxantrone), indomethacin (indomethacin), etodolac, ibuprofen, camptothecin, topotecan, aspirin, pyroxicam, cimetidine , s Estrogen, prednisolone, cortisone, hydrocortisone, diflorasone, phenesterine, daunorubicin, mitotane, bisadine (visadine), halonitrosouureas, anthrocyclines, ellipticine, diazepam, omeprazole, methoxyfluorane, isofluorane ), Enfluorane, halotane, benzocaine, benzocaine, dantrolene, barbiturates, cyclosporin A, azathioprine, ampho Amphotericin B, nystatine, itraconazole, biphenyl dimethyl dicarboxylate (BDD), piposulfan, danazole, hemoglobin ), Curcuminoid It may be a compound, resveratrol (resveratrol), silico-glycidyl alginic acid (glycyrrhizinic acid) or a derivative thereof, and the like. According to the present invention, as the poorly soluble material, oleanolic acid (oleanolic acid), idebenone (idebenone), phytosphingosine (certosphingosine), ceramide IIIB (ceramide IIIB), epigallocatechin gallate (epigallocatechin gallate) and astragalin ( astragalin) was used.

The oleanoic acid (Oleanolic acid, 3β-hydroxy-olea-12-en-28-oic acid; OA) is a substance having a structure of the following formula (1), triterpernoid saponin is present as a free acid or non-glycoside. Used in wrinkle improvement, anti-inflammatory or sebum care, but there is a phenomenon that precipitates in the water system and the melting point in the oil is very high, there is a problem that the product development is not easy.

Formula 1

Idebenone (Idebenone, Hydroxydecyl Ubiquinone, Id) is a substance having a structure of the following formula (2), a short chain benzoquinone structurally similar to coenzyme Q10, but because the antioxidant effect is very high compared to Q10, currently in the cosmetic market It is a newly noticed ingredient. Idebenone is a strong antioxidant, an electron transporter, and in addition to the effect of inhibiting skin aging is known to be effective in the protection of nerves and mitochondria, relieve cardiac hypertrophy, but it is difficult to stabilize due to precipitation phenomenon in liquid (aqueous) products.

Formula 2

The phytosphingosine (Phytosphingosine, 2-amino-4-octadecene-1, 3-diol) is an 18-carbon amino alcohol having a structure of Formula 3 below, which has an unsaturated hydrocarbon chain to form sphigolipids, which are important phospholipids. It is also involved in intercellular signaling and is used in atopy creams and regeneration creams because it has a high skin barrier repair effect, such as ceramide, but liquid products have not been developed yet because it is not a highly studied ingredient.

Formula 3

The ceramide IIIB (Cereminde IIIB or Ceremide C-6) is a type of ceramide, a waxy lipid having the structure of Formula 4 below, and ceramide is generally insoluble in water, but recently, many cosmetic products have been developed due to the development of cosmetic prescription technology. Developed. However, the actual distribution of ceramide IIIB in the skin is the highest, but the liquid ceramide is currently applied to cosmetics ceramide II is required to liquefy the ceramide III.

Formula 4

The epigallocatechin gallate (EGCG, gallate-3-epigallocatechin) or catechin is a kind of polyphenol which is an extract of green tea leaf having the following formula (5) and is known to have a strong antioxidant activity. .

Formula 5

The astragalin (kaempferol-3-O-glucopyranoside.astragalin) has the following formula 6, and contains a flavonoids-based compound, is known to be effective in reducing anti-inflammatory, anti-allergic, dermatitis, etc. .

Formula 6

As used herein, the term “steviol glycoside” refers to a substance that has a sweet taste as a compound present in the leaves of stevia. Steviol glycosides in stevia leaves are, for example, stevioside, rebaudioside, rebaudioside A, rebaudioside C, dulcoside A, as well as rebaudioside B, D, E, F, rubuososide, svethiol monoside, steviol bioside or mixtures thereof.

The chemical structure of the steviol glycosides is as follows:

Formula 7

According to one embodiment, further comprising the step of preparing a steviol glycoside enzyme reaction solution by mixing the steviol glycoside and the enzyme before the first step, wherein the prepared steviol glycoside enzyme reaction solution Provided is a method for producing a poorly soluble material and steviol glycoside complex having improved solubility. That is, in the present invention, it is possible to use the steviol glycoside itself or to use both the steviol glycoside enzyme reaction solution. According to one embodiment, the enzyme is possible lactase.

According to one embodiment, the steviol glycoside per 100 parts by weight of the poorly soluble material may be mixed in a ratio of 800 to 1200 parts by weight. When the mixing ratio of the steviol glycoside is higher than 8: 1 (w / w) or lower than 12: 1 (w / w), the formation efficiency of the complex of the steviol glycoside and the poorly soluble material is lowered and the egg caused by the tebiol glycoside is reduced. The degree of solubility improvement of the soluble material may be small or reduced.

In addition, the first step of mixing and stirring the steviol glycosides and the poorly soluble material in ethanol may be performed for 10 to 20 minutes. When the mixing time is 10 minutes or less, a sufficient reaction for complex formation does not occur, and when the mixing time is 20 minutes or more, the reaction efficiency may decrease due to unnecessary mixing process. The poorly soluble material may be at least 15%, at least 50%, at least 60%, at least 80% soluble, or substantially fully soluble.

The present invention also provides a complex of a poorly soluble material and steviol glycosides having improved solubility produced by the production method of the present invention.

Hereinafter, various examples are presented to help understand the invention. The following examples are merely provided to more easily understand the invention, but the protection scope of the invention is not limited to the following examples.

<Example>

Experimental material

Oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid, OA) and Idebenone (ID) were purchased from Tokyo chemical industry company (Japan), and steviol glycoside mixtures were purchased from domestic companies. Stevioside and Levadioside were purified by MPLC (Grace Discovery Science, China). Rubusoside was prepared using the method developed by the present inventors.

Example 1 Experiment to Confirm Increased OA Solubility Using Steviol Glycosides

1-1. Solubility Preliminary Test

10 mg of oleanoic acid (OA) was mixed with 100 mg of rubusoside (Ru), stevioside (Ste), rebadioside (Reb), and a mixture thereof (M), respectively. Each mixture was added to 1 mL anhydrous ethanol and vigorously stirred for 15 minutes, then centrifuged for 10 minutes at 12,000 rpm. The supernatant was transferred to another eppendorf tube. Ethanol was evaporated from the separated supernatant to obtain a poorly soluble material and a steviol glycoside complex.

The oleanoic acid (OA) -rubisoside complex, the oleanoic acid (OA) -stevioside complex, and the oleanoic acid (OA) -levaodioside complex which are obtained poorly soluble material oleanoic acid (OA) and a steviol glycoside complex And each of the oleanoic acid (OA) -M complex was dissolved in 1 mL of water, centrifuged at 12,000 rpm for 10 minutes, and then the solubility was measured by checking the amount of each component present in the supernatant. Using 12 mg.ml ^-1 - developing solvent include acetonitrile / water 85:15 (v / v) 0.25 mg.ml dissolved in was used as the standard solution are N, N- dimethylformamide (DMF) ^-1 It was. Oleanoic acid (OA) was identified at 254 nm UV on TLC plates. The results are shown in FIG.

In FIG. 1, each lane is as follows.

Lane 1: 10 mg.ml ^-1 OA in DMF

Lane 2: 10% (w / v) aqueous solution of rubus side

Lane 3: Oleanoic acid (OA) in 10% (w / v) aqueous solution of rubusside

Lane 4: 10% (w / v) stevioside aqueous solution

Lane 5: Oleanoic acid (OA) in 10% (w / v) stevioside aqueous solution

Lane 6: 10% (w / v) aqueous solution of levodioside

Lane 7: Oleanoic acid (OA) in 110% (w / v) aqueous solution of levadioside

Lane 8: Aqueous 10% steviol glycoside mixture

Lane 9: Oleanoic acid (OA) in 10% (w / v) steviol glycoside mixture solution

The solubility of the rubisoside, stevioside, levaodioside and mixtures thereof and the poorly soluble oleanoic acid (OA) complex in water is shown in Table 1 below. Solubility of oleanoic acid (OA) was the highest in the case of the complex with rubusoside.

Table 1 water Ru Ste Reb M OA - 8.69 ± 0.27 0.14 0.22 ± 0.02 3.04 ± 0.34

1-2. Solubility Test of Oleanoic Acid (OA) with Various Concentrations of Rubussoside for Complex Formation

10 mg oleanoic acid (OA) was added to 1.0% (w / v), 2.0% (w / v), 4.0% (w / v), 6.0% (w / v), 8% (w / v) and After mixing 10.0% (w / v) of rubussoside and ethanol, the solubility was confirmed in the same manner as in Example 1-1. The results are shown in Table 2 below.

TABLE 2 Addition of Ru (%) OA (mg.ml ^-1 ) Amount Amount of dissolved OA 1.0 10.0 0.40 ± 0.17 2.0 1.59 ± 0.47 4.0 3.21 ± 0.15 6.0 5.0 ± 0.14 8.0 6.63 ± 0.46 10.0 8.69 ± 0.45

Referring to Table 2, it was confirmed that the solubility of oleanoic acid (OA) increased with increasing concentration of rubusside (Ru) added for complex formation. Based on the results in Table 2, a linear relationship between the solubility of OA and the concentration of rubussoside (Ru) was derived as follows.

[Relationship 1]

Y = 0.90 x X-0.38

Wherein Y represents the concentration of dissolved OA (mg.ml ⁻¹ ) and X represents the concentration of added Ru (mg.ml ⁻¹ ).

1-3. Increased solubility of oleanoic acid (OA) using enzyme reaction solution

Stevioside and lactase were mixed together to prepare the enzyme reaction solution by enzymatic reaction of stevioside with lactase, and the result of increasing the solubility of oleanoic acid (OA) in water without purifying rubusoside was confirmed. 2 and 3 are shown.

Each lane in FIG. 2 is as follows.

Lane 1: 0.25 mg.ml ^-1 OA in DMSO

Lane 2: 1 mg.ml ^-1 OA in DMSO

Lane 3: 2 mg.ml ^-1 OA in DMSO

Lane 4: 4 mg.ml ^-1 OA in DMSO

Lane 5: 8 mg.ml ^-1 OA in DMSO

Lane 6: 12 mg.ml ^-1 OA in DMSO

Lane 7: 24 mg.ml ^-1 OA in DMSO

Lane 8: 10 mg.ml ^-1 OA in aqueous solution of steviol glycoside enzyme reaction solution

Lane 9: 20 mg.ml ^-1 OA in aqueous solution of steviol glycoside enzyme reaction solution

Lane 10: 40 mg.ml ^-1 OA in an aqueous solution of steviol glycoside enzyme reaction solution

Lane 11: 60 mg.ml ^-1 OA in aqueous solution of steviol glycoside enzyme reaction solution

Referring to FIGS. 2 and 3, even when the lactase enzyme reaction solution of steviol glycosides is mixed, a poorly soluble oleanoic acid (OA) and a steviol glycoside complex are formed, and as a result, solubility in water is increased. Confirmed. In particular, the solubility of oleanoic acid (OA) in water was the highest when 40 mg of stevioside lactase enzyme reaction solution was mixed.

Example 2. Idebenone (ID) solubility increase experiment

2-1. Solubility Preliminary Test

Except for the use of idebenone (ID) as a poorly soluble material was carried out to confirm the solubility increase of idebenone (ID) in the same manner as in Example 1-1, the results are shown in Figure

In FIG. 4, each lane is as follows.

Lane 1: 10 mg.ml ^-1 idebenone (ID) in DMSO

Lane 2: 10% (w / v) aqueous solution of rubusside

Lane 3: Idebenone (ID) in 10 mg.ml ^-1 aqueous solution of rubusside

Lane 4: 10% (w / v) stevioside aqueous solution

Lane 5: Idebenone (ID) in 10% (w / v) stevioside aqueous solution

Lane 6: 10% (w / v) aqueous solution of leviodioside

Lane 7: Idebenone (ID) in 10% (w / v) aqueous solution of levadioside

Lane 8: Aqueous 10% steviol glycoside mixture

Lane 9: Idebenone (ID) in 10% (w / v) steviol glycoside mixture solution

Referring to FIG. 4, an idebenone (ID) rubusoside complex, an idebenone (ID) stevioside complex, an idebenone (ID) leviodioside complex and an idebenone (ID) steviol glycoside mixture complex are formed. It was confirmed that the solubility of Benon (ID) was increased.

The solubility of water of Idebenone (ID) in the case of forming an insoluble idebenone (ID) complex with the rubusoside, stevioside, leviodioside and mixtures thereof is shown in Table 3 below.

TABLE 3 water Ru Ste Reb M Ide Benon (ID) - 28.79 ± 0.15 1.26 ± 0.07 4.92 ± 0.28 11.8 ± 0.23

Referring to Table 3, the solubility in water of the rubusside idebenone (ID) complex was 28.79 mg.ml ^-1, and the 10.8 mg.ml of the 10% (w / v) steviol glycoside mixture isidebenone (ID) complex ^-1, 10% (w / v) when the lever-side audio Ide Vernon (ID) complex 4.92 mg.ml ^-1, and 10% (w / v) in water 0.26 for stevioside Ide Vernon (ID) conjugate When solubility of mg.ml ^-1 was present, it was confirmed that the solubility of Idebenone (ID) was the highest when Idebenone (ID) forms a complex with rubussoside.

2-2. Experimental study on the increase of solubility of Rubussoside Idebenone (ID) according to the concentration of Rubussoside added for complex formation

10 mg idebenone (ID) for 1.0% (w / v), 2.0% (w / v), 4.0% (w / v), 6.0% (w / v), 8% (w / v) and 10.0 After preparing a composite by mixing with rubussoside of% (w / v), the degree of solubilization was confirmed in the same manner as in Example 2-1, and the results are shown in Table 4 below.

Table 4 Addition of Ru (%) ID (mg.ml ^-1 ) Amount Amount of dissolved OA 1.0 30.0 0.33 ± 0.07 2.0 0.86 ± 0.17 4.0 8.25 ± 0.14 6.0 9.24 ± 0.46 8.0 15.07 ± 0.45 10.0 28.79 ± 0.27

Referring to Table 4, it was confirmed that the water solubility of the Rubussoside Idebenone (ID) complex increases with increasing concentration of Rubussoside (Ru) added during complex formation. Based on the results of Table 4, a linear relationship between the solubility of idebenone (ID) and the added rubisoside (Ru) was derived as follows.

[Relationship 2]

Y = 2.94 x X-4.58

In the above formula, Y represents the concentration of dissolved Idebenone (ID) (mg.ml ⁻¹ ), and X represents the concentration of added Ru (mg.ml ⁻¹ ).

2-3. Idebenone Solubility Increase Experiment Using Enzyme Reaction Solution

Stevioside and lactase were mixed together to prepare the enzyme reaction solution by enzymatic reaction of stevioside with lactase, and the result of increasing the solubility of Idebenone (ID) in water was confirmed through SDS page without purifying rubusoside. The results are shown in FIGS. 5 and 6.

In FIG. 5, each lane is as follows.

Lane 1: DMSO with 1.0 mg.ml ^-1 ID

Lane 2: DMSO with 1.5 mg.ml ^-1 ID

Lane 3: DMSO with 2.0 mg.ml ^-1 ID

Lane 4: DMSO with 3.0 mg.ml ^-1 ID

Lane 5: DMSO with 4.0 mg.ml ^-1 ID

Lane 6: DMSO with 5.0 mg.ml ^-1 ID

Lane 7: DMSO with 6.0 mg.ml ^-1 ID

Lane 8: 10 mg.ml ^-1 ID in aqueous solution of stevioside enzyme reaction solution

Lane 9: 20 mg.ml ^-1 ID contained in aqueous solution of stevioside enzyme reaction solution

Lane 10: 40 mg.ml ^-1 ID in the aqueous solution of stevioside enzyme reaction solution

Lane 11: 60 mg.ml ^-1 ID in aqueous solution of stevioside enzyme reaction solution

Referring to FIGS. 5 and 6, even when the lactase enzyme reaction solution of steviol glycosides is mixed, an insoluble material idebenone (ID) and a steviol glycoside complex are formed, and as a result, the water of the idebenone (ID) It was confirmed that the solubility was increased. In particular, when 20 mg of stevioside lactase enzyme reaction solution was mixed, the solubility of oleanoic acid (OA) in water was the highest.

Example 3 Experiment to Confirm Increased Solubility of Phytosphingosine (PS) Using Steviol Glycosides

3-1. Solubility Preliminary Test

Except that phytosphingosine (PS) was used as a poorly soluble material, the solubility increase test of phytosphingosine (PS) was performed in the same manner as in Example 1-1, and the results are shown in FIG. 7.

In FIG. 7, each lane is as follows.

Lane 1: 10 mg.ml ^-1 PS in DMSO

Lane 2: 10% (w / v) aqueous solution of rubusside

Lane 3: PS in 10% (w / v) aqueous solution of rubusside

Lane 4: 10% (w / v) stevioside aqueous solution

Lane 5: PS in 10% (w / v) stevioside aqueous solution

Lane 6: 10% (w / v) aqueous solution of leviodioside

Lane 7: PS in 10% (w / v) aqueous solution of levodioside

Lane 8: Aqueous 10% steviol glycoside mixture

Lane 9: PS contained in an aqueous 10% (w / v) steviol glycoside mixture

Referring to FIG. 7, the solubility of phytosphingosine (PS) is increased in lanes 3, 5, 7 and 9, which form a complex by mixing rubisoside, stevioside, levaodioside, and a mixture thereof with phytosphingosine (PS). Confirmed.

The solubility of phytosphingosine (PS) in DMSO in each case where rubitoside, stevioside, leviodioside and mixtures thereof and phytosphingosine (PS) were mixed to form a complex is shown in Table 5 below.

Table 5 water Ru Ste Reb M PS - 8.86 ± 0.35 0.83 ± 0.07 1.98 ± 0.15 4.83 ± 0.21

3-2. Experimental Study of Solubility of Phytosphingosine (PS) with Concentration of Rubussoside Added for Complex Formation

10 mg phytosphingosine each at 1.0% (w / v), 2.0% (w / v), 4.0% (w / v), 6.0% (w / v), 8% (w / v) and 10.0% (w / v After preparing a complex by mixing with rubisoside and ethanol in the ratio of), the solubility of phytosphingosine (PS) was confirmed in the same manner as in Example 3-1. The results are shown in Table 6 below.

Table 6 Addition of Ru (%) PS (mg.ml ^-1 ) Amount Amount of dissolved OA 1.0 10.0 0.21 ± 0.03 2.0 1.25 ± 0.05 4.0 2.65 ± 0.17 6.0 4.14 ± 0.07 8.0 6.65 ± 0.23 10.0 8.86 ± 0.20

Referring to Table 6, it was confirmed that the solubility of phytosphingosine (PS) increased with increasing concentration of rubussoside added to form a complex with phytosphingosine (PS). Based on the results of Table 6, a linear relationship between the solubility of phytosphingosine (PS) and the concentration of added rubussoside was derived as follows.

[Relationship 3]

Y = 0.94 x X-0.90

In the above formula, Y represents the concentration of phytosphingosine (PS) (mg.ml ⁻¹ ), and X represents the concentration of Ru (mg.ml ⁻¹ ).

3-3. Experimental Study on Increased Solubility of Phytosphingosine (PS) in Stevioside Enzyme Solution

Stevioside and lactase were mixed together to prepare an enzyme reaction solution by enzymatic reaction of stevioside with lactase, and the result of increasing the solubility of phytosphingosine (PS) in water without purifying rubusoside was confirmed. And FIG. 9.

Each lane in FIG. 8 is as follows.

Lane 1: 2 mg.ml ^-1 PS in DMSO

Lane 2: 4 mg.ml ^-1 PS in DMSO

Lane 3: 6 mg.ml ^-1 PS in DMSO

Lane 4: 8 mg.ml ^-1 PS in DMSO

Lane 5: 10 mg.ml ^-1 PS in DMSO

Lane 6: 30 mg.ml ^-1 PS in aqueous solution of stevioside enzyme reaction

Lane 7: 40 mg.ml ^-1 PS in aqueous solution of stevioside enzyme reaction

Lane 8: 50 mg.ml ^-1 PS in aqueous solution of stevioside enzyme reaction

Referring to FIGS. 8 and 9, even when mixing the lactase enzyme reaction solution of steviol glycosides, a soluble phytosphingosine (PS) and a steviol glycoside complex are formed, and as a result, the solubility of phytosphingosine (PS) in water It was confirmed to increase.

Example 4 Experiment of Confirming Increased CR Solubility Using Steviol Glycosides

4-1. Solubility Preliminary Test

Except for using ceramide IIIB (CR) as a poorly soluble material, the solubility increase of the ceramide IIIB (CR) was confirmed in the same manner as in Example 1-1, and the results are shown in FIG.

In FIG. 10, each lane is as follows.

Lane 1: 10 mg.ml ^-1 CR in DMSO

Lane 2: 10% (w / v) aqueous solution of rubusside

Lane 3: CR in 10% (w / v) aqueous solution of rubus side

Lane 4: 10% (w / v) stevioside aqueous solution

Lane 5: CR in 10% (w / v) stevioside aqueous solution

Lane 6: 10% (w / v) aqueous solution of leviodioside

Lane 7: CR in aqueous solution of 10% (w / v) levoaudioside

Lane 8: Aqueous 10% steviol glycoside mixture

Lane 9: CR in aqueous 10% (w / v) steviol glycoside mixture

Referring to Figure 10, ceramide IIIB (CR) in lanes 3, 5, 7 and 9, which is a mixture of rubusside, stevioside, levaodioside and mixtures thereof and ceramide IIIB (CR) in ethanol to form a complex It was confirmed to be dissolved.

4-2. Increase in ceramide IIIB (CR) solubility according to the concentration of rubussoside added for complex formation

10 mg CR was 1.0% (w / v), 2.0% (w / v), 4.0% (w / v), 6.0% (w / v), 8% (w / v) and 10.0% (w / v) After mixing the leubusoside and ethanol of v) to form a complex, the degree of solubility was confirmed in the same manner as in Example 4-1. The results are shown in Table 7 below.

TABLE 7 Addition of Ru (%) CR (mg.ml ^-1 ) Amount Amount of dissolved OA 1.0 10.0 0.48 ± 0.02 2.0 0.59 ± 0.02 4.0 0.85 ± 0.02 6.0 1.17 ± 0.02 8.0 1.53 ± 0.05 10.0 1.81 ± 0.02

Referring to Table 7, it was confirmed that the solubility of ceramide IIIB (CR) increased with increasing concentration of rubusside (Ru) added for complex formation. Based on the results in Table 7, a linear relationship between the solubility of CR and Ru was derived as follows.

[Relationship 4]

Y = 0.15 x X-0.29

In the above formula, Y represents the concentration of dissolved ceramide IIIB (CR) (mg.ml ⁻¹ ), and X represents the concentration of Ru added (mg.ml ⁻¹ ).

4-3. Increased CR Solubility in Stevioside Enzyme Reaction

Stevioside and lactase were mixed together to prepare the enzyme reaction solution by enzymatic reaction of stevioside with lactase, and the result of increasing the solubility of ceramide IIIB (CR) in water without purifying rubusoside was confirmed. 11 and FIG. 12.

In FIG. 11, each lane is as follows.

Lane 1: 1 mg.ml ^-1 CR in DMSO

Lane 2: 1.5 mg.ml ^-1 CR in DMSO

Lane 3: 2 mg.ml ^-1 CR in DMSO

Lane 4: 3 mg.ml ^-1 CR in DMSO

Lane 5: 4 mg.ml ^-1 CR in DMSO

Lane 6: 5 mg.ml ^-1 CR in DMSO

Lane 7: 60 mg.ml ^-1 CR in aqueous solution of stevioside enzyme

Lane 8: 80 mg.ml ^-1 CR in aqueous solution of stevioside enzyme

Lane 9: 100 mg.ml ^-1 CR in aqueous solution of stevioside enzyme

Referring to FIGS. 11 and 12, the ceramide IIIB (CR) steviol glycoside complex was formed even when the lactase enzyme reaction solution of the steviol glycoside was mixed, and thus the solubility was increased. In particular, the best solubility was shown in 80 mg of stevioside lactase enzyme reaction solution.

Experimental Example 1: Material characteristics study on 3CL ^pro inhibition of SARS

東)성에서 발생, 홍콩을 거쳐 벨기에를 제외한 유럽 각국과 미국·캐나다 등 북아메리카, 그리고 한국·일본을 제외한 아시아 각국 등 세계 32개국에서 83,000여 명이 감염되었으며, 이 중 10%가 사망하였다. Severe Acute Respiratory Syndrome (SARS) is an infectious disease caused by SARS-Coonavirus (SARS-CoV) invading the human respiratory system. One or more of the symptoms of pneumonia on the radiography will appear. November 2002 South China Guangdong

In the eastern provinces, over 83,000 people have been infected in 32 countries, including Hong Kong, Europe, the United States and Canada, and North America, including the United States and Canada, and Asia, except Korea and Japan.

The World Health Organization (WHO) has identified the causative agent of SARS as a variant coronavirus, which causes mild or severe upper respiratory diseases in humans and respiratory, gastrointestinal tract, liver and neurological diseases in animals. Corona virus is a virus that contains a strand of RNA as a genetic material. The first and second serotypes that cause infection in humans are mainly known, and other new types are emerging, and thus prevention and treatment of the occurrence of new strains are needed. The path of transmission of the virus has not yet been fully understood, but the path of infection known to date is known to be the droplets that spout when a patient sneezes or coughs, and is known to be transmitted when entering another's respiratory tract. The drop propagation distance is usually reported to be 1m, and claims have been made that can be transmitted through the air, but it has not been confirmed.

Two to seven days after incubation with SARS-Corona virus, fever, helplessness, headache, and muscle pain develop throughout the body. Cough and respiratory distress develop, followed by diarrhea in 25% of patients, and in severe cases, symptoms persist for more than two weeks, significantly worsening respiratory function and progressing to acute respiratory distress syndrome and multi-organ failure.

1-1. Transformation and preparation experiment

The study was carried out by producing SARS-Coronavirus 3CL protease as a vector capable of efficiently expressing the recombinant SARS-Coronavirus 3CL protease in yeast. Gene acquisition and recombinant expression vector construction were performed by genetic information of SARS-CoV 3CL protease [Activity Inhibitor and Screening Method of SARS-Corona Virus 3CL Protease Caused by Severe Acute Respiratory Syndrome, 2014, Domestic Registration Patent 10-1418898] synthesizes the SARS-Corona virus 3CL protease gene and introduces it into the multiple cloning position of the pPICZαA (Invitrogen, USA) vector to express the active protein and immediately after the α-factor signal peptide. A recombinant vector pPICZαA-3CLpro comprising a corona virus 3CL protease gene was constructed.

Transformation of yeast was carried out in a prepared competent cell, yeast, Pichia pastoris GS115 (Invitrogen, USA), 240 μl 50% polyethylene glycol 3350, 36 μl 1M LiCl, 25 μl single strand salmon sperm DNA (2 mg / ml) and recombinant pPICZαA-3Clpro DNA (5 μg) were added and mixed vigorously for 1 minute. In order to insert the DNA was left for 25 minutes at 30 ℃ and heat shock was applied for 25 minutes at 42 ℃. After that, only cells were recovered by centrifugation, and again, 1 ml of YPD medium [1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose] was added. Incubated at 30 ° C. for 1 hour. The culture was incubated for 2 days at 30 ° C. in YPD solid medium to which zeocin (100 μg / ml) was added. Production of the recombinant SARS-Coronavirus 3CL protease was performed by culturing the transformed strain with BMGY [1% (w / v) yeast extract, 2% (w / v) peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base. with ammonium sulfate without amino acids, 4 x 10-5% Biotin, 1% (w / v) glycerol] incubated at 28 ℃ for 18 hours. The cells recovered by centrifugation of the culture were obtained by BMMY [1% (w / v) yeast extract, 2% (w / v) peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base with ammonium sulfate without amino. acids, 4 x 10-5% Biotin, 0.5% (w / v) methanol] was suspended in OD 600 to 1.0, and then incubated at 28 ℃ for 4 days. For enzyme expression, 1.0% methanol was added to the culture solution every 24 hours, and SARS-Corona virus 3CL protease activity was confirmed as the supernatant of the obtained culture solution.

To investigate the activity of the recombinant SARS-Corona virus 3CL protease, it is present in the polyprotein (pp1ab) synthesized by the SARS-CoVreplicase gene as a substrate used for SARS-Corona virus 3CL protease analysis. Synthetic peptides designed to mimic the cleavage site (DABCYL-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS, Bachem, Switzerland) were used. The substrate is bound to the fluorescent donor EDANS and the fluorescent destructor DABCYL [Matayoshi, E.D., Wang, G.T., Kraff, G.A.and Erickson, J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science. 247: 954-958 (1990). Fluorescence is seen only when the substrate is cleaved and the EDANS group is separated from the DABCYL group [Luker, K.E., Francis, S.E., Gluzman, I.Y. and Goldberg. Kinetic analysis of plasmepsin I and II aspartic protease of the Plasmodium falciparum digestive vacuole. Mol. Biochem.Parasitol. 79: 71-78 (1996).

1-2. SARS-Corona Virus 3CL Protease Preparation and Inhibition Experiments

To confirm the activity of the recombinant SARS-Corona virus 3CL protease, the substrate was dissolved in 20 mM Tris buffer (pH 7.5) at a final concentration of 20 μM. After 3 μg of recombinant SARS-Corona virus 3CL protease was mixed with 45 inhibitory activity candidates (final concentration 100 μM) and reacted for 25 minutes at 25 ° C., the reaction product was fluorescence microplate reader SpectraMax Gemini XPS (Molecular Devices, USA The fluorescence intensity (exitation 355 nm, emission 538 nm) was measured using an instrument. Inhibition of the inhibitory candidates was measured using the property of increasing fluorescence intensity as the substrate was degraded by SARS-Corona virus 3CL protease. In order to compare the inhibition of recombinant SARS-Corona virus 3CL protease activity, a reaction solution containing only recombinant SARS-Corona virus 3CL protease and substrate was added as a control without addition of inhibitory active substance, and excellent inhibition of activity among candidates for inhibition of species activity was used. Inhibitory activity of the recombinant SARS-Corona virus 3CL protease when no inhibitory active substance was added was determined as 0% and the activity indicated by the inhibitory effect after the addition of the compound was expressed as%. .

The Km value of the enzyme for the fluorescent substrate was 15.16 ± 1.26 μM. Rubusoside (Ru), oleanoic acid (OA), and oleanoic acid rubusoside complex (OA-Ru) mixed with rubusoside were used as samples, and the enzyme digest (100 µl) was 3 µg enzyme, 16 μM FRET substrate, 200 μM each material, 20 mM Tris buffer (pH 7.5) were mixed and the reaction proceeded at 25 ° C. for 20 minutes. Inhibition degree was calculated using the following formula, and the results are shown in Figure 13 and Table 8.

% inhibition = 100-remaining activity (%), Remaining activity (%) = [(S-So) / (C-Co)] × 100

Wherein C represents fluorescence after 18 minutes of incubation of the controls (enzyme, buffer, and substrate), Co represents 0 minutes of fluorescence, and S is 20 minutes of the experimental samples (enzyme, test sample, buffer and substrate). Fluorescence after incubation is shown, and So represents fluorescence of the test sample at 0 minutes.

Table 8 Compound IC ₅₀ (μM) Rubusoside 10.73 x 10 ³ Oleanoic acid in DMSO 96.59 Oleanoic acid mixed with rubisoside 17.38

Referring to FIG. 13, in the case of preparing a complex by mixing oleanoic acid (OA) with rubusoside, the concentration of 3CL protease was significantly lower than that of simple oleanoic acid (OA) in DMSO. It was confirmed that the activity is inhibited.

Referring to Table 8, when the composite is prepared by mixing oleanoic acid (OA) with rubusoside, it is confirmed that the inhibition ability is improved by about 5.6 times or more as compared with the case of dissolving simple oleanoic acid (OA) in DMSO. It became.

1-3: Preparation of Human Tyrosinase and Enzyme Inhibitory Experiments of OA, ID, PS and CR

As a method for synthesizing and expressing human tyrosinase (HTY) gene, the inventors used a conventionally known method (Gene bank M62238.1) [Thi, THN, YH Moon, YB Ryu, YM Kim, SH Nam, MS Kim, A. Kimura, and D. Kim (2013) The influence of flavonoid compounds on the in vitro inhibition study of a human fibroblast collagenase catalytic domain expressed in E. coli. Enzyme Microb Tech. 52: 26-31], IC ₅₀ values confirmed the inhibition properties for tyrosinase. The enzyme activity inhibitory ability of OA, ID, PS and CR against HTY is shown in Table 9 below.

Table 9 Compound IC ₅₀ (μM) in DMSO or DMF IC ₅₀ (μM) in rubisoside Oleanoic acid 41.1 39.6 Idebenon 74.8 - Phytosphingosine 23.2 - Ceramide IIIB Complete no solubility (can not detected) -

Referring to Table 9, when oleanoic acid (OA) is mixed with rubusoside to form a complex, the solubility of oleanoic acid (OA) is increased, so that only oleanoic acid (OA) is dissolved in DMSO. It was confirmed to have an excellent inhibitory effect on enzyme activity.

Experimental Example 2 Confirmation of Increased EGCG Solubility Using Steviol Glycosides

2-1. Solubility test

Except for using EGCG as a poorly soluble material, the same experiment as in Example 1-1 was carried out to confirm the increase in solubility of EGCG, and rubusoside, stevioside, leviodioside and steviol glycosides with which EGCG forms a complex The solubility of EGCG in the case is shown in Table 10 below.

Table 10 Steviol Glycosides Treatment with water EGCG solubility (mg / ml) SM (10%) 355 Stevioside (10%) 312 Rebaudioside (10%) 241 Rubusoside 1% 396 water 20

As can be seen from Table 10, the soluble EGCG complexes with rubisoside, stevioside, levaodioside and steviol glycosides and EGCG complexes with rubisoside, stevioside, levaodioside and steviol glycosides It was confirmed that the increase of about 10 to about 20 times or more compared to the case that does not form.

2-2. Inhibition of Human Small Intestine Maltase Activity

In order to confirm the steviol glycosides and EGCG complexes inhibiting human small intestine maltase activity, 0.04 U HMA (human small intestine maltase) / mL, 5 mM maltose, and 100 μM test samples were prepared using 50 mM potassium phosphate buffer (pH 6.5). The reaction was carried out by mixing at.

The amount of glucose decomposed from maltose by human small intestine maltase was confirmed using Glucose-E kit (Korea), and the results are shown in Table 11 below.

Table 11 EGCG prepared Human small intestine maltase inhibition level [% inhibition upon treatment with 100 μM] EGCG dissolved in steviol glycoside-free DMSO 75.6 EGCG dissolved in steviol glycoside-free water 58.2 EGCG-SM 10% 79.9 EGCG-stevioside 10% 61.2 EGCG- Rebaudioside 10% 76.7 EGCG-Roubusoside 1% 76.7 EGCG-Roubusoside 10% 92.15

As can be seen from Table 11, in the case of steviol glycoside mixed with and dissolved in the steviol glycosides EGCG complex, the effect of inhibiting the activity of human small intestine maltase is increased by about 1.5 times compared to the case where EGCG is dissolved in water without mixing with steviol glycosides It was confirmed. In addition, in the case of the composite mixture of rubussoside in a 10% weight ratio, it showed the best inhibitory effect of small intestine maltase activity.

Experimental Example 3: Confirmed increase in astragalin solubility

3-1. Solubility test

The solubility of astragalin was measured using the same method as Experimental Example 2, and the results are shown in Table 12 below.

Table 12 Rubusoside added amount (%) Astragalin Astragalin amount added (mg.ml ^-1 ) Detected in Ru solution (mg. Ml ^-1 ) 0.5 50 8.9 ± 0.8 1.0 12.7 ± 1.2 2.0 13.3 ± 1.3 3.0 20.9 ± 0.7 4.0 35.3 ± 2.2 5.0 37.0 ± 0.4 7.5 37.4 ± 2.2 10 39.0 ± 3.4

As can be seen from Table 12, it was confirmed that the solubility of astragali increased as the content of rubussoside added for complex formation increased.

3-2. Inhibition of SARS 3CL ^pro activity

By using the same method as in Experimental Example 1 20μM astragaline was mixed with rubussoside and dissolved in DMSO to measure the activity inhibition of SARS 3CL ^pro and the results are shown in Table 13 below.

Table 13 sample IC ₅₀ (μM) Rubusoside 10.73 x 10 ³ Astragalin dissolved in DMSO 115.40 ± 3.64 Astragalin dissolved after rubusoside treatment 69.16 ± 2.37

Referring to Table 13, it was confirmed that when the astragalin is dissolved by mixing with rubusoside, it is superior to the 3CL protease activity inhibiting ability compared to the case of dissolving in DMSO alone without mixing with rubussoside.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will understand that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Explanation of the sign

OA: Oleanolic acid

ID: Idebenone

PS: Phytosphingosine

CR: Ceramide IIIB

Ru: Rubusoside

Ste: Stevioside

Reb: Rebaudioside

M: Steviol glycoside mix

Claims (13)

A first step of mixing and stirring a steviol glycoside and a poorly soluble material in ethanol; A second step of centrifuging the mixture; A third step of separating the supernatant; And A fourth step of removing ethanol contained in the separated supernatant; Method for producing a complex of poorly soluble material and steviol glycosides with improved solubility. The method of claim 1, A method for preparing a complex of a poorly soluble material and steviol glycoside with improved solubility, further comprising preparing a steviol glycoside enzyme reaction solution by mixing a steviol glycoside and an enzyme before the first step. The method of claim 2, The first step is to use the steviol glycoside enzyme reaction solution, a method of producing a complex of poorly soluble material and steviol glycosides with improved solubility. The method of claim 2, The enzyme is a lactase, a method of producing a complex of poorly soluble material and steviol glycosides with improved solubility. The method of claim 1, The steviol glycosides per 100 parts by weight of the poorly soluble material is mixed in a ratio of 800 to 1200 parts by weight of the poorly soluble material and steviol glycosides composite manufacturing method. The method of claim 1, The steviol glycosides are stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, and rebaudioside D. A complex of poorly soluble materials with improved solubility and steviol glycosides, including rebaudioside E, rubusoside, dulcoside a, steviolside or mixtures thereof Manufacturing method. The method of claim 1, The poorly soluble material is composed of oleanolic acid (oleanolic acid), idebenone (idebenone), phytosphingosine, ceramide IIIB (ceramide IIIB), epigallocatechin gallate and astragalin Will be selected from, solubility improved poorly soluble material and steviol glycoside complex manufacturing method. The method of claim 1, In the case of mixing rubusoside (Ru) as steviol glycoside and oleanoic acid (OA) as poorly soluble material, the following formula 1 holds, making a complex of poorly soluble material having improved solubility and steviol glycoside Way. [Relationship 1] Y = 0.90 × X-0.38 In the above formula, Y represents the concentration of OA (mg.ml ^-1 ), and X represents the concentration of Ru (mg.ml ^-1 ). The method of claim 1, In the case of mixing Rubusoside (Ru) as a steviol glycoside and Idebenone (ID) as a poorly soluble material, the following formula 2 holds, solubility-improved poorly soluble material and steviol glycoside complex manufacturing method . [Relationship 2] Y = 2.94 × X-4.58 In the above formula, Y represents the concentration of Idebenone (ID) (mg.ml ^-1 ), and X represents the concentration of Rubussoside (Ru) (mg.ml ^-1 ). The method of claim 1, A method for producing a composite of a poorly soluble material having improved solubility and a steviol glycoside, wherein the following relation 3 is established when rubusoside (Ru) as a steviol glycoside is mixed with phytosphingosine (PS) as a poorly soluble material. [Relationship 3] Y = 0.94 × X-0.90 In the above formula, Y represents the concentration of phytosphingosine (PS) (mg.ml ⁻¹ ), and X represents the concentration of rubisoside (Ru) (mg.ml ⁻¹ ). The method of claim 1, In the case of mixing rubussoside (Ru) as a steviol glycoside and ceramide IIIB (CR) as a poorly soluble material, the following formula 4 holds, and the method for preparing a complex of a poorly soluble material having improved solubility and steviol glycosides is established. . [Relationship 4] Y = 0.15 × X-0.29 In the above formula, Y represents the concentration of ceramide IIIB (CR) (mg.ml ⁻¹ ), and X represents the concentration of rubussoside (Ru) (mg.ml ⁻¹ ). 12. A complex of a poorly soluble material and steviol glycosides improved in solubility prepared by the method of any one of claims 1 to 11. The method of claim 1, A fifth step of dissolving the complex product of the poorly soluble material and the steviol glycoside obtained in the fourth step in the aqueous solvent, stirring, and centrifuging; And The sixth step of separating the centrifuged supernatant; It further comprises a method of producing a complex solution of a poorly soluble material and steviol glycosides.

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