KR100770204B1 - Use of Exopolysaccharide Produced from Hahella Chejuensis Mutant m10356 - Google Patents

Abstract

The present invention relates to the use of the extracellular polysaccharide produced by the Hahel jeju enses mutant m10356, more specifically, whitening or UV protection containing the extracellular polysaccharide produced by the hahel jeju mutant strain m10356 as an active ingredient The present invention relates to a cosmetic composition for use, an ACE inhibitor containing the extracellular polysaccharide as an active ingredient, and a method for producing beads for drug delivery using the extracellular polysaccharide.

The extracellular polysaccharides produced by the haella jeju nsis mutant m10356 according to the present invention have excellent whitening and UV blocking effects and can be used as functional cosmetic raw materials. It can be applied to the treatment and prevention of geriatric diseases (myocardial infarction, heart failure, etc.) caused by blood vessel deformity, and the drug carrier beads prepared using the extracellular polysaccharides have a sustained and excellent drug release effect at neutral pH. Or for drug delivery to the large intestine.

Extracellular Polysaccharides, Tyrosinase, Inhibition, ACE, Ultraviolet Absorption, Drug Delivery

Description

Use of Exopolysaccharide Produced from Hahella Chejuensis Mutant m10356}

Figure 1 shows the tyrosinase activity inhibitory effect of the extracellular polysaccharide according to the present invention.

Figure 2 shows the ultraviolet absorption spectrum of the extracellular polysaccharide according to the present invention.

Figure 3 shows the ACE activity inhibitory effect of the extracellular polysaccharide according to the present invention.

Figure 4 schematically shows a method for producing extracellular polysaccharide beads according to the present invention.

Figure 5 shows the form of the extracellular polysaccharide beads according to the present invention.

Figure 6 shows the drug release effect of the extracellular polysaccharide beads according to the present invention.

Field of invention

The present invention relates to the use of exopolysaccharides (EPS) produced by the Hahel jejuensis mutant m10356 (KCTC 10469 BP), more specifically, the extracellular polysaccharides produced by the hahel jeju mutant strain m10356 It relates to a cosmetic composition for whitening or sunscreen containing as an active ingredient, an ACE inhibitor containing the extracellular polysaccharide as an active ingredient, and a method for producing beads for drug delivery using the extracellular polysaccharide.

Background of the Invention

Polysaccharides secreted extracellularly by microorganisms protect microorganisms from other organisms, participate in the defense against antibodies, neutralize toxic substances in the environment, form complexes with metal ions, and evaporate intracellular moisture in dry environments. It is involved in preventing. In addition, it has been shown to perform several functions related to the survival of microorganisms (Sutherland, IW, Biotechnology , 3: 533, 1983).

Extracellular polysaccharides of microorganisms have various functions such as gel formation ability, emulsification stability, surface tension control ability, water absorption ability, adhesion ability, lubrication ability, and film formation ability depending on conditions. (Fu, JF and YH Teeng, Appl . Environ. , 56: 919, 1990). In addition, the extracellular polysaccharides have a heavy metal adsorption capacity (Norberg, AG and H. Persson, Biotech. Biochem . , 115: 239, 1984) and antitumor activity (Oda, M. et al. , Agro . Biol . Chem . , 47: 1623, 1983).

In particular, extracellular polysaccharides of microorganisms have high potential for industrial use, as described above, high productivity through improvement of production culture conditions, mass production through continuous culture using short-term fermenters, and ease of produced extracellular polysaccharides. As it has advantages such as separation and recovery, research on this is being actively conducted.

Melanin, a substance related to the skin whitening effect, absorbs ultraviolet rays to protect the skin, but when exposed for a long time, skin photoaging is promoted and pigmentation occurs. At this time, studies on materials that inhibit the activity of tyrosinase involved are actively underway. Currently, whitening products and sunscreens are mostly made of chemicals. However, the chemicals used therein may be deteriorated in biocompatibility, and may cause deterioration of skin function, acceleration of skin aging, etc., and absorb moisture in the air, resulting in skin diseases caused by adsorption of bacteria and fungi. In addition, hormonal components such as estrogen and steroids have excellent temporary effects such as skin softening effect, but may cause toxic symptoms due to commercial use.

On the other hand, angiotensin-II (angiotensin-II) is a substance produced by the conversion of angiotensin-I by an angiotensin convertin enzyme (ACE), causing a change in the locally generated blood vessel diameter and raising blood pressure, such as myocardial infarction, heart failure, etc. May cause disease. Since the disease is already complicated, the treatment method is complicated and not easy, and research for the development of ACE inhibitors and angiotensin-II antagonists, which are easy to use in the treatment, is being conducted.

On the other hand, injections used as the most common drug delivery agents have the disadvantage that they are difficult to take and painful, and should be administered periodically. In addition, the side effect is higher than oral drug delivery agent is also difficult to expect the long-term effective drug delivery efficiency.

Most of the mucous membranes in the intestine are easy to deliver drugs, rapid absorption and low molecular weight substances, as well as relatively high molecular weight substances, such as drugs with peptide structure, so that the main administration of bioactive substances Its value is very high as a route In order to use such gastrointestinal mucosa, it is urgent to develop a drug carrier that exhibits a property of inhibiting drug degeneration and release from low pH conditions in the stomach.

On the other hand, the present inventors have developed a patent for the production of extracellular polysaccharides from the haella jejuensis mutant strain m10356, which is a non-pigmented mutant strain (1010-0495272).

In the present invention, as a result of intensive efforts to determine the use of the extracellular polysaccharide prepared by the Haella jejuensis mutant m10356 (KCTC 10469 BP), the extracellular polysaccharide is melanin-producing tyrosinase inhibitory ability, ultraviolet absorption capacity, ACE (angiotensin) converting enzyme) confirmed that it has the ability to inhibit and bead formation that can be used as a drug delivery agent was completed the present invention.

An object of the present invention to provide a cosmetic composition for whitening or sun protection containing the extracellular polysaccharide as an active ingredient.

It is also an object of the present invention to provide an ACE inhibitor containing the extracellular polysaccharide as an active ingredient.

It is also an object of the present invention to provide a method for producing beads for drug delivery using the extracellular polysaccharide and a drug delivery device accordingly.

In order to achieve the above object, the present invention provides a cosmetic composition for whitening or sun protection containing an extracellular polysaccharide (exopolysaccharide) showing the following characteristics as an active ingredient:

(a) glucose, galactose and galactosamine, per week, in a molar ratio of 1: 1: 1.1;

(b) the copolysaccharide heterologous acid is galactronic acid; And

(c) molecular weight is 1.39 × 10 ⁶ Da.

In the present invention, the extracellular polysaccharide may be characterized in that it was prepared by the Hahel chejuensis mutant m10356 (KCTC 10469 BP).

The present invention also provides an ACE (angiotensin converting enzyme) inhibitor containing an exopolysaccharide as an active ingredient exhibiting the following characteristics:

(a) glucose, galactose and galactosamine, per week, in a molar ratio of 1: 1: 1.1;

(b) the copolysaccharide heterologous acid is galactronic acid; And

(c) molecular weight is 1.39 × 10 ⁶ Da.

In the present invention, the extracellular polysaccharide may be characterized in that it was prepared by the Hahel chejuensis mutant m10356 (KCTC 10469 BP).

The present invention also provides a method for preparing a drug carrier, comprising the following steps:

(a) admixing exopolysaccharides, oils and the desired drug with the following characteristics (i) to (iii), followed by stirring to form an emulsion;

   (i) glucose, galactose and galactosamine, per week, in a molar ratio of 1: 1: 1.1;

    (ii) the heterosaccharide, a subsidiary sugar, is galactronic acid; And

(iii) the molecular weight is 1.39 × 10 ⁶ Da.

(b) reacting the emulsion by adding acetic acid with stirring; And

(c) centrifuging the reactant of step (b) to recover the beads.

In the present invention, the extracellular polysaccharide may be characterized in that it is prepared by the Hahel chejuensis mutant strain m10356 (KCTC 10469BP), the amount of the extracellular polysaccharide is added to about 10 parts by weight of oil It may be characterized by a weight part. In addition, the target drug is mesalazine, sulfasalazine, ibuprofen, prednisolone, dexamethasone, budesonide, prednisolone, beclomethasone, fluticasone, thioxocortal, hydrocortisone, cyclosporin, methotrexate, domperidone, 5-fluorouracil , Bisacodeyl, dietary fiber, lactic acid bacteria, insulin, vasopressin, growth hormone, growth hormone stimulating factor, colony stimulating factor, calcitonin, immunoglobulin, diltiazem, verapamil, nifedipine, captopril, theophylline, naproxen and mixtures thereof It may be characterized in that it is selected from the group.

The present invention also provides a drug carrier which is prepared by the above method, cured at pH 6 or lower, dissolved in the range of pH 6-8, and in the form of a spherical bead having a diameter of about 150 μm.

Hereinafter, the present invention will be described in detail.

First, according to the method described in the inventors Patent No. 10-0495272 of the present invention, an extracellular polysaccharide (exopolysaccharide; EPS) was obtained, and the effect on the tyrosinase activity and the ultraviolet absorbing ability, as a result, the extracellular polysaccharide was determined in the concentration It was confirmed that it exhibits a tyrosinase activity inhibitory effect in proportion and absorbs ultraviolet rays (particularly UV-B and UV-C). It was also confirmed that the extracellular polysaccharides showed an inhibitory effect on ACE activity.

In addition, extracellular polysaccharide beads were prepared according to the method shown in FIG. 4, and the prepared beads were confirmed to have a high drug release effect at neutral pH.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Production of Extracellular Polysaccharides

According to the method described in Korean Patent Registration No. 10-0495272, the mutant strain m10356 (KCTC 10469 BP) of Hahel Jeju Ensis 95CJ10356 was prepared using M-m10356 containing sucrose, organic nitrogen, inorganic phosphoric acid, MgSO ₄ , NaCl, and trace elements. The medium was inoculated, cultured at 25 ° C. under a pH of 7, and extracellular polysaccharides were obtained from the cultured mutants.

Example 2: Inhibitory Effect of Tyrosinase Activity by Extracellular Polysaccharides

0.9 ml of the aqueous solution of extracellular polysaccharides of various concentrations (0.1-1.5% (w / v)) was added to 1 ml of Potassium phosphate buffer, and 1 ml of 0.03% L-tyrosine was added, followed by tyrosinase ( tyrosinase) 0.1 ml (1250 unit / ml) was added and incubated at 37 ° C. for 10 minutes. The reaction mixture was poured into iced water to terminate the reaction, and the absorbance of melanin was measured at 475 nm. No extracellular polysaccharide (0%) was used as a control.

Since L-tyrosine is converted to melanin by tyrosinase, by measuring the amount of melanin produced by the above reaction using absorbance, it is known whether the tyrosinase is inhibited by the extracellular polysaccharide and the extent thereof. Can be.

As a result, as shown in Figure 1, the addition of the extracellular polysaccharide was inhibited the activity of tyrosinase, it was confirmed that the degree of inhibition is proportional to the concentration of the added extracellular polysaccharide. When 1.2% or more of the extracellular polysaccharide was added, it showed high inhibitory effect on tyrosinase activity of 70% or more.

Example 3 UV Absorption Effect by Extracellular Polysaccharides

Absorbance of 0.1% (w / v) extracellular polysaccharide was measured at a wavelength ranging from 200 to 600 nm using a spectrometer (UV-160, Shimadzu, Japan).

As a result, as shown in Figure 2, the extracellular polysaccharides exhibit a characteristic absorption peak at 200 ~ 290nm (UV-C) and 290 ~ 320nm (UV-B), 320 ~ 400nm (UV-A) Showed weak absorption.

Example 4 Inhibition Effect of ACE Activity by Extracellular Polysaccharides

Extracellular polysaccharide was dissolved in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl to prepare a 5 mM extracellular polysaccharide solution. The lung acetone powder was added to the 5 mM extracellular polysaccharide solution so as to be saturated and dissolved, and centrifuged at 15,000xg for 40 minutes (Mega 17R, Hanil science industrial, Korea) while maintaining 4 ° C. ACE (angiotensin converting enzyme) was used as a coenzyme solution. HHL (hippuryl-l-histidyl-l-leucine) was used as the ACE specific substrate.

To 100 μl of 5 mM HHL, add 100 μl of 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl and various concentrations (0.5, 1.0 and 1.5%) of the extracellular polysaccharide solution, and then pre-incubate each at 37 ° C. for 20 minutes. After preincubation), 100 μl of 8mU ACE coenzyme solution was added and left at 37 ° C. for 30 minutes. Then, 0.25 ml of 1N HCl was added to terminate the reaction. 1.5 ml EtOAc was added and stirred for 15 seconds and centrifuged at 600 × g to give a supernatant. 1 ml of the obtained supernatant was dried at 120 ° C. for 30 minutes, and then dissolved in 3 ml of tertiary distilled water to measure absorbance of HA (hyppuric acid) at 228 nm.

HA (hyppuric acid) is an ACE reaction product, by measuring the amount of HA produced by the reaction using the absorbance, it is possible to determine the presence and extent of ACE inhibition by extracellular polysaccharides.

As a result, as shown in Figure 3, the addition of the extracellular polysaccharide was inhibited the activity of the ACE, it was confirmed that the degree of inhibition is proportional to the concentration of the added extracellular polysaccharide. Addition of 1.5% extracellular polysaccharides showed an inhibitory effect on ACE activity of about 60%.

Example 5 Preparation of Extracellular Polysaccharide Beads

30 ml of soybean oil was added to two tubes, 3 ml of 1.0% extracellular polysaccharide was added to one side, and 3 ml of 1.5% extracellular polysaccharide was added to the other side. Each of the two tubes containing soybean oil and extracellular polysaccharides was added to 0.3 ml of ovalbumin to form a W / O emulsion with vigorous stirring. 15 ml of acetic acid was added while the stirring was continued while the W / O emulsion was reacted for 2 hours. Thereafter, beads are recovered by centrifugation, washed with diethyl ether, dried at 50 ° C. for 2 hours, and lyophilized to finally form extracellular polysaccharide beads (drug carriers). Obtained. Ovalbumin (OVA) was used to observe the drug release effect.

Figure 4 schematically shows a method for producing extracellular polysaccharide beads according to the present invention. Figure 5 is a photograph showing the shape of the extracellular polysaccharide beads prepared by the above method, it was confirmed that the extracellular polysaccharide beads of the present invention having a spherical particle size of about 150μm.

Example 6 Drug Release Effect of Extracellular Polysaccharide Beads

Two tubes containing 50 ml of PBS buffer (pH 7.4) were prepared, and two types of extrapolysaccharide beads (prepared with 1.0% and 1.5% extracellular polysaccharides) prepared in Example 5 were prepared, respectively. Put in. In addition, the mixture according to the above procedure was prepared using HCl-KCl buffer (pH 1.4) instead of PBS buffer.

While incubating the reaction at 37 ° C, a portion of the buffer was extracted at regular intervals and absorbance was measured at 590 nm. Using the measured absorbance, the concentration of ovalbumin released from the extracellular polysaccharide beads was measured according to a previously prepared calibration curve.

As a result, as shown in FIG. 6, in the case of beads prepared using 1.0% extracellular polysaccharide, release of ovalmin was not observed until 12 hours of culture under the condition of pH 1.4, and about 20 hours after 48 hours of culture. It showed a release effect of%, and under the condition of pH 7.4, it showed a release effect of about 55% at 4 hours of cultivation, and then gradually increased to about 98.2% at 48 hours of cultivation. .

In addition, in the case of beads prepared using 1.5% extracellular polysaccharide, drug release proceeded more slowly than beads prepared using 1.0% extracellular polysaccharide, which showed about 50% release effect after 8 hours of culture. Afterwards, it gradually increased and showed a release effect of about 94.54% after 48 hours of culture.

For both types of beads, release at pH 7.4 was about 70 times higher than release at pH 1.4 at 12 hours of incubation.

In addition, through the above experimental results, the extracellular polysaccharide beads of the present invention exhibits a hardening property at low pH, while the pH-dependent drug release tends to dissolve at neutral pH, which is an enteric condition. It was confirmed that the formulation is very suitable for drug release using gastrointestinal mucosa.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing a cosmetic composition and ACE inhibitor for whitening or sun protection containing the extracellular polysaccharides produced by the haella jeju ntsis strain m10356 as an active ingredient. The present invention also has an effect of providing a method for producing a drug carrier beads using the extracellular polysaccharide and a drug carrier accordingly.

The extracellular polysaccharides produced by the haella jeju nsis mutant m10356 according to the present invention have excellent whitening and UV (especially UV-B and UV-C) blocking effects and can be used as functional cosmetic raw materials, and have the effect of inhibiting ACE. It inhibits the production of angiotensin, which causes changes in vascular caliber, and can be applied to the treatment and prevention of adult diseases (myocardial infarction, heart failure, etc.) caused by vascular deformity, and drug delivery system beads prepared using the extracellular polysaccharides have side effects. Not only that, but also a sustained and excellent drug release effect at neutral pH is easy for drug delivery to the small or large intestine.

Claims (9)

Cosmetic composition for sun protection containing an extracellular polysaccharide (exopolysaccharide) having the following characteristics as an active ingredient: (a) glucose, galactose and galactosamine, per week, in a molar ratio of 1: 1: 1.1; (b) the copolysaccharide heterologous acid is galactronic acid; And (c) The molecular weight of the extracellular polysaccharide is 1.39 × 10 ⁶ Da. The cosmetic composition according to claim 1, wherein the extracellular polysaccharide is manufactured by Hahella chejuensis mutant m10356 (KCTC 10469BP). delete delete delete delete delete delete delete

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