Pharmaceutical composition for preventing or treating diabetes comprising nanovesicles derived from jerusalem artichoke as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient. The nanovesicles derived from Jerusalem artichoke exhibit a blood sugar reduction effect and a collagen synthesis inhibition effect, which are confirmed to be superior to those of Jerusalem artichoke extract, and thus the composition can be effectively utilized as a composition for preventing, treating or improving diabetes.

Description

Pharmaceutical composition for the prevention or treatment of diabetes containing Jerusalem artichoke-derived nanovesicles as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0002] Diabetes has established itself as a major global health issue, and its prevalence is rapidly increasing, particularly in South Korea. According to the Diabetes Fact Sheet in Korea recently released by the Korean Diabetes Association, there were approximately 6 million diabetic patients aged 30 or older in Korea as of 2020. This figure surpasses the 5.91 million patients predicted by the 2012 Diabetes Fact Sheet for 2050, 30 years ahead of schedule. According to statistics from the Korea Disease Control and Prevention Agency, medical expenses for diabetes increased by over 60% in five years, rising from approximately 1.8 trillion won in 2015 to approximately 2.9 trillion won in 2020. Diabetes poses the greatest disease burden on Koreans, maintaining its undisputed number one position in disease burden over the past decade. Consequently, interest in the prevention and management of diabetes is growing daily, with dietary therapy playing a crucial role in this effort.

[0003] Recently, Jerusalem artichokes have been gaining attention as a beneficial food for diabetic patients. They are known to be a natural food that does not cause a rapid rise in blood sugar levels and can have a significant impact on blood sugar control. The reason diabetic patients have become interested in Jerusalem artichokes is due to inulin, a major component found in the plant. Inulin is a group of naturally occurring polysaccharides produced by numerous plants; unlike ordinary plants that store carbohydrates as starch, Jerusalem artichokes are known to store carbohydrates as a fructose polymer called inulin.

[0004] Inulin is a water-soluble dietary fiber that facilitates bowel movements and aids in the excretion of bile acids, thereby helping to reduce cholesterol levels in the blood. Additionally, inulin is not broken down by enzymes in the body, such as ptyalin or amylase, which break down starch; thus, it provides a feeling of fullness and delays the rise in blood sugar levels.

[0005] Recently, secretion in the form of plant-derived nanovesicles has been reported. These plant-derived nanovesicles are secreted at a size of approximately 50–300 nm, and their physiological activity has been reported in various plant cells. Furthermore, their potential for clinical application is increasing as natural liposomes capable of delivering drugs. However, research on the anti-diabetic effects of Jerusalem artichoke-derived nanovesicles is currently insufficient.

[0006] The object of the present invention is to provide a composition for the prevention, treatment, or improvement of diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0007] Another objective of the present invention is to provide a composition for the prevention, treatment, or improvement of diabetic nephropathy comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for preventing or improving diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0010] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetic nephropathy comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0011] In addition, the present invention provides a health functional food composition for preventing or improving diabetic nephropathy, comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0012] According to the present invention, nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) exhibit blood glucose reduction and collagen synthesis inhibition effects, and by confirming that these effects are superior to those of Jerusalem artichoke extract, they can be usefully utilized as a composition for the prevention, treatment, or improvement of diabetes.

[0013] Figure 1 is a schematic diagram showing a method for isolating nanovesicles from Jerusalem artichokes.

[0014] Figure 2 shows the results of analyzing the size of nanovesicles derived from Jerusalem artichoke.

[0015] Figure 3 shows the results of analyzing the protein content of nanovesicles derived from Jerusalem artichoke.

[0016] Figure 4 shows the results of analyzing the blood glucose-lowering effect of Jerusalem artichoke-derived nanovesicles. **p<0.01(vs CON), #p<0.05(vs HFD).

[0017] Figure 5 shows the results of analyzing the collagen synthesis inhibitory effect of nanovesicles derived from Jerusalem artichoke.

[0018] The present invention will be described in more detail below.

[0019]

[0020] The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0021] The average diameter of the above nanovesicles may be 100 to 140 nm.

[0022] In addition, the above nanovesicles may exhibit a blood sugar-reducing or collagen synthesis-inhibiting effect.

[0023] In addition, the above nanovesicles can exhibit an inhibitory effect on Collagen type 4 protein expression.

[0024] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0025] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0026] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0027] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0028] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0029] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0030] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0031] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0032]

[0033] In addition, the present invention provides a health functional food composition for preventing or improving diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0034] The present invention can be generally used as a commonly used food.

[0035] The food composition of the present invention may be used as a health functional food. The term “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0036] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0037] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0038] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0039] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0040] In the present invention, the term “prevention” refers to any act of suppressing or delaying diabetes by administering a composition according to the present invention.

[0041] In the present invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of diabetes through the administration of a composition according to the present invention.

[0042] In this invention, the term “improvement” refers to any act of improving a poor condition of diabetes through the administration of a composition according to this invention.

[0043]

[0044] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetic nephropathy comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0045]

[0046] In addition, the present invention provides a health functional food composition for preventing or improving diabetic nephropathy, comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

[0047] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0048]

[0049] [Example 1] Separation of Jerusalem artichoke-derived nanovesicles

[0050] Jerusalem artichokes (Helianthus tuberosus) were purchased at the Andong Nonghyup Farmers Market. As shown in Fig. 1, nanovesicles were isolated from the Jerusalem artichokes. Specifically, the Jerusalem artichokes were washed with clean water to remove impurities and peeled. Subsequently, 300g of Jerusalem artichokes and 1500ml of iced 1X PBS (phosphate buffer saline) were placed in a blender and ground for 1 minute. 50ml of the ground Jerusalem artichokes were placed into 50ml conical tubes to ensure a uniform weight. Then, the mixture was centrifuged once at 500g for 10 minutes using a centrifuge to obtain the supernatant. The supernatant was transferred to a new 50ml conical tube and centrifuged once at 2000g for 20 minutes to obtain the supernatant. The above supernatant was transferred to a new 50 ml conical tube and centrifuged twice at 10,000 g for 30 minutes to obtain the supernatant. Afterwards, the supernatant was placed in an 8 ml ultra-high speed centrifuge tube and centrifuged at 100,000 g for 2 hours. The supernatant was discarded, and the remaining Jerusalem artichoke pellets were dissolved by adding 1 ml of 1×PBS.

[0051]

[0052] [Example 2] Analysis of Protein Content in Nanovesicles

[0053] To determine the protein content of the Jerusalem artichoke-derived nanovesicles, the nanovesicles were crushed by adding a 1×RIPA solution, and then the protein was quantified by performing a BCA (bicinchoninic acid, Thermo Fisher Scientific) assay.

[0054]

[0055] [Example 3] Preparation of Jerusalem Artichoke Extract

[0056] The Jerusalem artichoke extract used as a control for the Jerusalem artichoke-derived nanovesicles in this invention was obtained from the Central Bank of Natural Products. 40.18 g of the obtained freeze-dried Jerusalem artichoke extract was dissolved in 16 ml of DMSO (Dimethyl sulfoxide) and used.

[0057]

[0058] [Example 4] Preparation of an animal model

[0059] The 5-week-old male C57BL / 6 mice used as the animal model were purchased from Coagen (Busan, South Korea). The animal housing environment was maintained under constant conditions with constant temperature, humidity, and a photoperiod of 12 hours (08:00–20:00), and the experimental animals were housed individually in polycarbonate housing boxes. This animal experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (Approval No.: 2024-4-1111-04-01). The experimental animals were acclimatized to the animal housing environment for one week, and the experimental groups were established as follows. The high-fat diet group used a diet containing 60% fat, and all experimental groups were administered nanovesicles orally once a day (at a dose of 1 mg / kg per dose) for 7 days.

[0060] 1) Experimental Group 1 (CON): Normal diet (n=10)

[0061] 2) Experimental Group 2 (HFD): High-fat diet (n=10)

[0062] 3) Experimental Group 3 (Jerusalem Artichoke NVs): High-fat diet + administration of Jerusalem artichoke-derived nanovesicles (n=10)

[0063]

[0064] [Example 5] Analysis of blood glucose reduction effect

[0065] To confirm the blood glucose-lowering effect of the nanovesicles derived from Jerusalem artichoke, fasting blood glucose was measured on the 7th day after the first oral administration of the nanovesicles to the experimental group in Example 3 above. Blood glucose was measured once a week using a blood glucose meter (Accu-Check; Roche, Basel, Switzerland) by collecting blood from the tail vein after fasting for at least 12 hours.

[0066]

[0067] [Example 6] Preparation of a cell model

[0068] HK-2 cells (human proximal tubular epithelial cells) used as a cell model were purchased from the Korea Cell Line Bank. HK-2 cells were cultured in a humid incubator at 37°C and 5% CO2 conditions using RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0069] 3×10⁶ of the cultured cells above were placed in a 6-well plate. 5 A diabetes cell model was constructed by seeding cells / well and treating them with high glucose (30 mM D-glucose). The experimental groups were set up as follows. Specifically, normal glucose and high glucose were treated at the same concentration, and nanovesicles and Jerusalem artichoke extract were treated for 3 days after treatment with high glucose.

[0070] 1) Experimental Group 1 (CON): No glucose treatment

[0071] 2) Experimental Group 2 (HG): Treated with normal glucose (5.5 mM D-glucose)

[0072] 3) Experimental Group 3: Treatment with high glucose (30mM D-glucose) + Jerusalem artichoke-derived nanovesicles (10μg / mL)

[0073] 4) Experimental Group 4: Treatment with high glucose (30mM D-glucose) + Jerusalem artichoke extract (10μg / mL)

[0074]

[0075] [Example 7] Analysis of Collagen Synthesis Inhibitory Effect

[0076] Western blot analysis was performed to confirm the collagen synthesis inhibitory effect of Jerusalem artichoke-derived nanovesicles. Protein was extracted from the experimental group of Example 6 and quantified using a BCA assay (performed in the same manner as in Example 2). Subsequently, an equal amount of the quantified protein was separated by SDS-PAGE, transferred to a PVDF membrane, and non-specific binding was blocked with 5% BSA. Then, the samples were reacted with a primary antibody and an HRP-conjugated collagen type 4 secondary antibody, respectively, and protein expression was detected using an ECL solution.

[0077]

[0078] [Experimental Example 1] Nanovesicle Size Analysis

[0079] To confirm the size of the separated nanovesicles, Nanoparticle Tracking Analysis (NTA) was performed. As a result, as shown in Figure 2, the average diameter of the nanovesicles was found to be approximately 126 nm, and it was confirmed that the size was within the range of 60 to 250 nm.

[0080]

[0081] [Experimental Example 2] Analysis of Protein Content in Nanovesicles

[0082] According to Example 2 above, the protein content of the nanovesicles was analyzed, and as shown in Figure 3, it was confirmed that the nanovesicles contain about 4.98 mg / ml of protein.

[0083]

[0084] [Experimental Example 3] Analysis of Blood Sugar Reduction Effect

[0085] According to Example 5 above, the blood glucose-lowering effect of the Jerusalem artichoke-derived nanovesicles was analyzed, and as shown in Figure 4, it was confirmed that blood glucose was significantly reduced in experimental group 3 (Jerusalem artichoke NVs) compared to experimental group 2 (HFD).

[0086]

[0087] [Experimental Example 4] Analysis of Collagen Synthesis Inhibitory Effect

[0088] According to Example 7 above, the collagen synthesis inhibitory effect of the Jerusalem artichoke-derived nanovesicles was analyzed. As shown in Figure 5, the expression of Collagen type 4 protein decreased in experimental groups 3 and 4 compared to experimental group 2 (HG), and the decrease in protein expression was particularly more significant in experimental group 3 compared to experimental group 4. From the above results, it was confirmed that the collagen synthesis inhibitory effect of the Jerusalem artichoke-derived nanovesicles is superior to that of the Jerusalem artichoke extract.

[0089]

[0090] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of diabetes comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

2. A pharmaceutical composition according to claim 1, characterized in that the average diameter of the nanovesicles is 100 to 140 nm.

3. A pharmaceutical composition according to claim 1, characterized in that the nanovesicles exhibit a blood glucose-reducing or collagen synthesis-inhibiting effect.

4. A health functional food composition for preventing or improving diabetes containing nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

5. A pharmaceutical composition for the prevention or treatment of diabetic nephropathy comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

6. A health functional food composition for the prevention or improvement of diabetic nephropathy comprising nanovesicles derived from Jerusalem artichoke (Helianthus tuberosus) as an active ingredient.

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