WO2022071692A1 - Composition comprising hippophae rhamnoides leaf extract as active ingredient for alleviating, preventing, or treating diabetes mellitus complication - Google Patents

Abstract

The present invention relates to a composition for alleviating, preventing, or treating diabetes mellitus complications. More specifically, the composition contains a Hippophae rhamnoides leaf extract as an active ingredient and as such, can alleviate, prevent, or treat diseases caused by diabetes mellitus complications and can be used as a food composition and furthermore as a health functional food or a pharmaceutical composition, a feed composition, or a pharmaceutical composition for animals.

Description

Composition for improvement, prevention or treatment of diabetic complications containing vitamin tree leaf extract as an active ingredient

The present invention relates to a composition capable of improving, preventing or treating diabetic complications by containing a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

Diabetes mellitus (DM) is a progressive disease, often associated with obesity, characterized by insulin deficiency, insulin resistance, or both. Elevated fasting and postprandial blood glucose exposes patients to acute and chronic complications (micro- and macrovascular) leading to blindness, renal failure, heart disease, stroke and amputation.

Improved blood sugar control has been proven to lower the risk of these diabetic complications. Because of the progressive nature of these diseases, evolving therapeutic strategies are needed to maintain glycemic control. There are two types of diabetes. Type 1 diabetes is juvenile diabetes or insulin dependent diabetes mellitus (IDDM), and type 2 diabetes is adult type diabetes or non-insulin dependent diabetes mellitus (NIDDM). The type 1 diabetes patient is absolutely deficient in insulin due to immunological destruction of pancreatic β-cells that synthesize and secrete insulin. The type 2 diabetes mellitus is more complex in etiology and is characterized by relative insulin deficiency, reduced insulin action and insulin resistance.

On the other hand, diabetic complications include diabetic neuropathy, diabetic nephropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, diabetic vascular complications, or diabetic foot ulcer, the main cause of which is aldose in the polyol pathway. Excessive sorbitol accumulation, including due to aldose reductase activity, is known. Therefore, it is known that inhibition of aldose reductase plays an important role in the treatment and prevention of diabetic complications. Aldose reductase inhibitors such as zopolrestat, ponalrestat, sorbinil, tolrestat, fidarestat, ranireatat, and epalrestat, which have been developed so far, have been reported to prevent and delay diabetic complications in several animal experiments.

However, in clinical trials, zopolrestat and ponalrestat had low efficacy and were discontinued during development due to side effects such as sorbinil hypersensitivity reaction and tolrestat liver dysfunction. Clinical trials of ranireatat and fidarestat are currently underway in Japan and the United States. Epalrestat has not been approved by the US Food and Drug Administration (FDA), but was approved and marketed in Japan only in 1992.

Advanced glycation encproducts (Advanced glycation encproducts) are (Nonenzymatic glycation reaction), a Maillard reaction in which an amino acid group such as a lysine residue of a protein and a reducing sugar occur without the action of an enzyme. It is a substance formed as a result of this reaction. The non-enzymatic glycosylation of proteins is the free amino group lysine of the protein, but arginine and the carbonyl group of the reducing sugar react to form a Shciff base, which is the initial glycation reaction product, and the compound formed at this time It is a reaction that continuously forms a brown compound through a series of complex reactions such as condensation, retranslocation, oxidation, cleavage, and cyclization to form an irreversible final glycation product of the overall concept.

Since the final glycation product is an irreversible reaction product, once it is created, it is not decomposed even when blood sugar is restored to normal, but accumulates in the tissue during the protein's survival period and abnormally changes the structure and function of the tissue. Collagen, which has a relatively long half-life, easily undergoes a glycation reaction and forms cross-linking between the already formed final glycation product and collagen, resulting in abnormal physicochemical changes in the structure of the body and other protein-connected tissues. In addition, diabetic complications such as diabetic retinopathy, diabetic neuropathy, diabetic cataract, and diabetic nephropathy and diabetes , chronic kidney disease, heart disease, vascular disease, and various diseases such as aging.

Therefore, studies to treat diseases caused by the final glycation product by inhibiting the final glycation product are being attempted, and conventionally developed representative inhibitors of the final glycation product include aminoguanidine and ALT-711. Synthetic drug, aminoguanidine, is a nucleophilic hydrazine and binds to the product of the condensation reaction to inhibit the production of final glycation products, thereby preventing the development of complications. Aminoguanidine is the most promising drug for the prevention and treatment of diabetic complications and has progressed up to phase 3 clinical trials, but there is a problem that toxicity is induced when administered for a long period of time, so the development of safer drugs and preventive agents is required. In addition, ALT-711 was developed as a drug to break down the cross-links of the already formed final glycation products, and as a nucleophilic substance, it attacks the dicarbonyl group, which is the electrophilicity of the cross-links of the already formed final glycation products. There is a report that administration of ALT-711 to streptozotocin-induced diabetic rats alleviates complications caused by diabetes.

However, since most of the conventionally developed inhibitors of final glycation products are synthetic compounds, safety issues arise in long-term administration, such as causing side effects in the body.

On the other hand, the vitamin tree (Hippophae rhamnoides L.) is a shrub belonging to the Bodhi family and contains more than 100 kinds of ingredients. there is. It grows well in the cold in winter and high in the summer, and has strong adaptability to grow well in barren areas. Various research fields, such as antioxidant activity, cancer prevention, and immune system fields, have already been made using the vitamin tree. In addition, various foods are made and used by using the vitamin tree, but the study has not been actively conducted in Korea.

Korean Patent No. 1224196 discloses a 'method for producing omega-3 containing vitamin tree extract and herbal medicine', and Korean Patent No. 1229748 discloses 'cosmetic composition containing fermented vitamin tree extract', A composition for improving, preventing or treating diabetic complications containing the vitamin tree leaf extract as an active ingredient has not been disclosed.

It is an object of the present invention to provide a pharmaceutical composition capable of preventing or treating diabetic complications by containing a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

In addition, another object of the present invention is to provide a food composition that can prevent or improve diabetic complications by containing the leaf extract of a vitamin tree ( Hippophae rhamnoides ) as an active ingredient.

In addition, another object of the present invention is to provide a feed composition that can prevent or improve diabetic complications by containing a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for animals capable of preventing or treating diabetic complications by containing a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

The pharmaceutical composition capable of preventing or treating diabetic complications of the present invention for achieving the above object may contain a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

The diabetic complications may be at least one selected from the group consisting of diabetic neuropathy, diabetic nephropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, and diabetic foot ulcer.

The diabetic complications can be prevented or treated by inhibiting the production of advanced glycation end products (AGEs) and promoting degradation.

The vitamin tree leaf extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The mixed solvent may be an aqueous solution of 20 to 80% by volume of methanol, ethanol, butanol or propanol.

The mixed solvent may be an aqueous solution of 20 to 80% ethanol.

In addition, the food composition capable of preventing or improving diabetic complications of the present invention for achieving the above other object may contain a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

The food composition may be a health functional food for preventing or improving diabetic complications.

In addition, the feed composition capable of preventing or improving diabetic complications of the present invention for achieving the above another object may contain a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

In addition, the pharmaceutical composition for animals capable of preventing or treating diabetic complications of the present invention for achieving the above another object may contain a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

In addition, the vitamin tree ( Hippophae rhamnoides ) leaf extract of the present invention for achieving the above another object may have the activity of inhibiting the production of advanced glycation end products (AGEs) and promoting degradation.

The composition containing the vitamin tree ( Hippophae rhamnoides ) leaf extract of the present invention as an active ingredient reduces the diabetic complications caused by the accumulation of AGEs through the effect of inhibiting the production of final glycation products (AGEs) and the cross-linking effect between the final glycation products and collagen. can be improved, prevented or treated.

In addition, since the composition of the present invention is non-toxic, it can be used as a pharmaceutical composition, a feed composition, a pharmaceutical composition for animals, a food composition, and even a health functional food.

1 is a graph showing the degree of inhibition of final glycation products (AGEs) production of vitamin tree extracts prepared according to Example 1 and Comparative Example 1 of the present invention.

Figure 2 is a graph showing the degree of inhibition of cross-linking of the final glycation products (AGEs) of the vitamin tree extract prepared according to Example 1 and Comparative Example 1 of the present invention.

3 is a graph showing the degree of cross-linking cleavage of the final glycation products (AGEs) of the vitamin tree extract prepared according to Example 1 and Comparative Example 1 of the present invention.

4 is a graph showing the degree of inhibition of final glycation products (AGEs) production of the leaf extract of vitamin tree prepared according to Examples 1 to 4 of the present invention.

5 is a graph showing the degree of inhibition of cross-linking of final glycation products (AGEs) of vitamin tree leaf extracts prepared according to Examples 1 to 4 of the present invention.

6 is a graph showing the degree of cross-linking cleavage of the final glycation products (AGEs) of the vitamin tree leaf extract prepared according to Examples 1 to 4 of the present invention.

7 is a graph measuring the cytotoxicity of a vitamin tree leaf extract prepared according to Example 1 of the present invention.

8 is a graph measuring changes in fasting blood glucose in the normal group, control group 1, control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)) am.

9 is a photograph after PAS staining of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

10 is a photograph confirming the AGEs IHC of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

11 is a photograph confirming the RAGE IHC of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

Figure 12a is a Western blot measuring GLO1 in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention; 12B is a Western blot for measuring AGEs in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

13 is a graph showing GLO1 activity in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

14 is a photograph confirming GLO1 and AGEs in the kidney tissue of db/db mice in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

15 is a graph of MGO-derived AGEs measured by an ELISA method in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

Figure 16a is a Western blot measuring NOX4 in the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention; Figure 16b is a Western blot measuring NOX2 in the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention.

17 is a graph showing the content of 8-OHdG in the urine of a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

18 is a photograph confirming Nrf2, NOX4 and 8-OHdG in the kidney tissue of a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

19 is a diagram illustrating the measurement of the content of isorhamnetin and rutin according to the solvent of a vitamin tree leaf extract and a vitamin tree fruit extract prepared according to Example 1 of the present invention.

The present invention relates to a composition capable of improving, preventing or treating diabetic complications by containing a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

Specifically, the composition of the present invention contains a vitamin tree leaf extract as an active ingredient, thereby improving, preventing or treating diabetic complications by inhibiting the production of advanced glycation end products (AGEs) and promoting decomposition.

The diabetic complications of the present invention may include one or more selected from the group consisting of diabetic neuropathy, diabetic nephropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, and diabetic foot ulcer.

Hereinafter, the present invention will be described in detail.

The composition for improving, preventing or treating diabetic complications of the present invention contains vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.

The vitamin tree leaf extract is prepared by mixing the vitamin tree leaf and the extraction solvent in a weight ratio of 1: 5 to 25, preferably 1: 8 to 15, and extracting at 30 to 60 ° C. When the weight ratio of the vitamin tree leaf and the extraction solvent is out of the above range, the active ingredient of the vitamin tree leaf may be extracted in a small amount in the extract. In addition, if the extraction temperature is less than the lower limit, the active ingredient of the vitamin tree leaf may not be extracted, and if it exceeds the upper limit, the active ingredient of the vitamin tree leaf is extracted in a small amount, and in particular, if it is 80 ° C or higher, the extract may be altered. there is.

The extraction solvent may include water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The water does not need to be particularly limited when suitable for food production, but for example, groundwater, purified water, distilled water, deionized water, etc. may be used.

The alcohol having 1 to 4 carbon atoms does not need to be particularly limited, but for example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol or normal-butanol may be used, and preferably ethanol. .

The mixed solvent does not need to be particularly limited, but preferably 20 to 80% by volume of an aqueous solution of methanol, ethanol, butanol or propanol, and more preferably 20 to 80% by volume of an aqueous ethanol solution.

The preparation of the water extract does not need to be particularly limited, but it can be prepared by extracting the leaves of the vitamin tree with water at 10 to 100° C. for 2 to 60 hours.

The preparation of the alcohol extract or the extract of a mixed solvent of water and alcohol does not need to be particularly limited, but for example, it is prepared by extracting vitamin tree leaves with 20 to 80% by weight of an ethanol aqueous solution at 20 to 100° C. for 2 to 48 hours. do.

The extract of the vitamin tree leaf with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof includes a fraction obtained by refractionation of an extract using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof with an organic solvent. The organic solvent may be one or more organic solvents selected from alcohols having 1 to 4 carbon atoms, hexane, acetone, ethyl acetate, chloroform and diethyl ether, and preferably hexane or ethyl acetate.

The term 'extract' as used in the present invention is an extract obtained by extracting the components contained in the leaves of a vitamin tree using the solvent, a fraction fractionated therefrom, a concentrate obtained by further concentrating these extracts or fractions, and a purified or separated purified product. It is used in the sense of including a dried product of the extract, fraction, concentrate or purified product or a powder obtained by pulverizing it.

For the production of the purified product, various additionally performed, such as passing through an ultrafiltration membrane having a molecular weight cut-off value, or separation by various chromatography (those prepared for separation according to size, charge, hydrophobicity or affinity) A purification method may be added.

The present invention relates to a food composition for preventing or improving diabetic complications comprising a vitamin tree leaf extract as an active ingredient.

The 'food composition' includes, as an active ingredient, in addition to the vitamin tree leaf extract, food ingredients that can be used as foods described in the standards and standards ('Food Code') of foods commonly used in food manufacturing, and food additives described in the Food Additives Code. include

Although not particularly limited, examples thereof include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sugar, lactose and the like; oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin and the like; Sugar alcohols such as xylitol, sorbitol, erythritol and the like can be used. As the flavoring agent, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

When preparing a food composition using the vitamin tree leaf extract as an active ingredient, the vitamin tree leaf extract does not need to be particularly limited as long as the content can prevent or treat diabetic complications, for example, 0.1 to 99% by weight, 0.5 to 95% by weight %, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, 5 to 50% by weight may be included.

Although the vitamin tree leaf extract, which is an active ingredient in the food composition, varies depending on the condition, weight, presence or extent and duration of the disease, it may be appropriately selected by those skilled in the art. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. And, the number of administration does not need to be particularly limited, but can be adjusted by those skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The food composition is not particularly limited, but may be, for example, a powder, granules, tablets, capsules, pills, extracts, jelly formulations, tea bag formulations or beverage formulations.

In addition, the vitamin tree leaf extract can be added to general food to give the function of preventing or improving diabetic complications, and the food that can be added is not particularly limited, but for example, food according to Article 7 of the Food Sanitation Act. Confectionery, bread or rice cakes, cocoa processed products or chocolates, edible meat or egg products, fish meat products, tofu or jelly products, noodles, teas, coffee, beverages, special purpose foods, sauces exemplified in the standards and specifications ('Food Code') , seasoned foods, dressings, kimchi, salted fish, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and processed eggs exemplified in the processing standards and ingredient specifications of livestock products according to Article 4 of the Livestock Products Sanitation Control Act ('Livestock Products Code').

On the other hand, the food composition containing the vitamin tree leaf extract as an active ingredient can be used alone as "health functional food for preventing or improving diabetes complications".

The 'health functional food' refers to food manufactured (including processing) according to legal standards using raw materials or ingredients useful for the human body (Article 3, Item 1 of the Health Functional Foods Act). The term 'health functional food' may differ in terms or scope of each country, but 'Dietary Supplement' in the US, 'Food Supplement in Europe', 'Health functional food' or 'Health functional food' in Japan It may correspond to 'Food for Special Health Use (FoSHU)' and 'health food' in China.

The above food composition or health functional food may additionally contain food additives, and the suitability as a food additive is determined by the standards and standards for the item in accordance with the general rules and general test methods of the 'Food Additives Codex', unless otherwise specified. follow

In addition, in the health functional food, as a raw material announced as a 'functional raw material' or an individually recognized raw material used in "health functional food for the prevention or improvement of diabetes complications" together with the vitamin tree leaf extract, indigestible maltodextrin, Banaba alcohol Manufacture health functional food for preventing or improving diabetic complications by combining health functional food ingredients related to blood sugar control such as extract, pinitol, rhododendron extract, guava leaf extract, evening primrose seed alcohol extract, pine needle distillation concentrate, soybean fermented extract, albumin, etc. can do.

The present invention relates to a feed composition for preventing or improving diabetic complications comprising a vitamin tree leaf extract as an active ingredient.

In the 'feed composition', in addition to the vitamin tree leaf extract as an active ingredient, food ingredients that can be used as foods described in food standards and specifications ('Food Code') and food additives described in the Food Additives Code can be used, and used as food Even if it is not a possible food raw material or food additive, raw materials that fall within the range of single feed in Attached Table 1 of 'Standards and Specifications for Feed, etc.' and those that fall within the range of Supplementary Feed in Attached Table 2 may be used.

The 'feed composition' may be an extractant among auxiliary feeds according to the 'standards and specifications of feed, etc.', and may be a compound feed including the auxiliary feed.

When preparing a feed composition using the vitamin tree leaf extract as an active ingredient, the vitamin tree leaf extract does not need to be particularly limited as long as the content shows prevention or improvement of diabetic complications, for example, 0.1 to 99% by weight, 0.5 to 95% by weight %, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, 5 to 50% by weight may be included.

The vitamin tree leaf extract, which is an active ingredient in the feed composition, varies depending on the condition, body weight, presence or absence or extent and duration of the ingested animal, but may be appropriately selected by those skilled in the art. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. And, the number of administration does not need to be particularly limited, but can be adjusted by those skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The present invention relates to a pharmaceutical composition for preventing or treating diabetic complications comprising a vitamin tree leaf extract as an active ingredient.

In addition, the present invention relates to a pharmaceutical composition for animals for preventing or treating diabetic complications comprising a vitamin tree leaf extract as an active ingredient.

The present invention also provides a method for treating diabetic complications by administering the composition to a human or non-human animal.

In addition, the present invention provides a novel use of the vitamin tree leaf extract for the prevention or treatment of diabetic complications, or for the manufacture of medicaments for animals.

The 'pharmaceutical composition', 'medicine', 'pharmaceutical composition for animals' or 'medicine for animals' includes, as an active ingredient, suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, in addition to the vitamin tree leaf extract. may include

The 'carrier' is a compound that facilitates the addition of the compound into a cell or tissue. The 'diluent' is a compound that is diluted in water to not only stabilize the biologically active form of the compound of interest, but also to dissolve the compound.

The carrier, excipient and diluent are not particularly limited, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals or veterinary medicine may vary depending on the age, sex, and weight of the patient or animal to be treated, and above all, the condition of the subject to be treated, a specific category of the disease to be treated, or It will depend on the type, route of administration, and the nature of the therapeutic agent used.

The pharmaceutical composition, medicament, pharmaceutical composition for animals or medicament for animals is appropriately selected according to the absorption rate of the active ingredient in the body, the rate of excretion, the age and weight of the patient or the animal to be treated, sex and condition, the severity of the disease to be treated, etc. , It is generally preferred to administer 0.1 to 1,000 mg/kg, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg per day. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicament, pharmaceutical composition for animals or medicament for animals may be administered individually as a prophylactic or therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical composition, medicine, pharmaceutical composition for animals or medicine for animals may be formulated into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. there is. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, and such solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sugar or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

The method for treating diabetic complications is to administer the composition to a human or non-human animal, particularly a mammal, for example, administering the composition to a subject to be treated with diabetic complications.

Whether the subject to be treated with the diabetic complications may be a case of having diabetic complications.

The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals or medicament for animals.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

[Comparison of vitamin tree leaf extract and fruit extract]

Example 1. Vitamin tree leaf extract

Vitamin leaf (Hoengseong, Gangwon-do) and 70 wt% ethanol aqueous solution were mixed in a weight ratio of 1:10 and extracted under reflux at 50 °C for 3 hours, followed by Whatman NO. The process of filtration with one filter paper was repeated twice. The filtrate was concentrated under reduced pressure at 40 ° C. at a rotation speed of 60 rpm using a rotary vacuum evaporator (rotary vacuum evaporator, eyela, Japan), and then freeze-dried to obtain a vitamin tree leaf extract.

Comparative Example 1. Vitamin Tree Fruit Extract

The same procedure as in Example 1 was performed, except that the vitamin tree fruit extract was obtained using the vitamin tree fruit instead of the vitamin tree leaf.

<Test Example I>

Test Example 1. Efficacy of inhibiting the formation of final glycation products (AGEs)

1 is a graph showing the degree of inhibition of final glycation products (AGEs) production of vitamin tree extracts prepared according to Example 1 and Comparative Example 1 of the present invention.

For the test method, 10 mg/mL of BSA was dissolved in 700 µL of 50 mM phosphate buffer, 0.2 M fructose and 100 µL of glucose were added each, and 0.02% sodium azide was added to prevent contamination of microorganisms during the reaction period. To the mixture, 200 µL of each of the extracts of Example 1 and vitamin 1 or AG, an in vitro standard final glycation product formation inhibitor, was added to make a final 1 mL, and then reacted at 37°C for 14 days. After 14 days of reaction, it was measured by exitation 350 nm and emission 450 nm fluorescence analysis (Molecular Devices, Sunnyvale, CA, USA).

As shown in FIG. 1 , when the AGEs generating ability was expressed as 100% (AGEs, control), 1 mM of AG, a positive control, exhibited 54% AGEs production inhibitory efficacy. On the other hand, the vitamin tree fruit extract of Comparative Example 1 showed AGEs production inhibitory effects of 31% and 75% at 50 μg/mL and 200 μg/mL, respectively, and the vitamin tree leaf extract of Example 1 was 50 μg/mL, It was confirmed that the efficacy of 77% and 88% at 200 μg/mL, respectively.

That is, it was confirmed that the vitamin tree leaf extract of Example 1 was superior to the vitamin tree fruit extract of Comparative Example 1 in inhibiting the formation of final glycation products (AGEs) at both low and high concentrations.

Test Example 2. Analysis of the efficacy of cross-linking of final glycated products

Figure 2 is a graph showing the degree of inhibition of cross-linking of the final glycation products (AGEs) of the vitamin tree extract prepared according to Example 1 and Comparative Example 1 of the present invention.

The test method is a kit-NH ₂ Unit (Dojin do Molecular Technologies, Inc., Tokyo, Japan) tagged with horseradish peroxidase (HRP) as Glycoaldehyde-AGE-BSA (Biovision, CA, USA) for the evaluation of the final glycation product cross-linking inhibition efficacy. ) was used. To evaluate the efficacy of final glycation product cross-linking inhibition, 10 μg/mL AGE-BSA, AG, Example 1, or vitamin 1 extracts were dispensed in a collagen-coated 96-well microtiter plate and incubated at 37° C. for 18 hours. After washing 3 times in 0.05% PBST (prepared to be 0.05% Tween 20 in PBS), and developing color using TMB as a substrate, absorbance was measured at 450 nm. The cross-linking inhibition % of AGE-BSA was calculated according to the following [Equation 1].

[Equation 1]

AGE-BSA(%)=(absorbance of wells with drug added/absorbance of wells without drug) X 100

In order to investigate whether the irreversible cross-linking between the final glycosylated product and collagen was inhibited, the inhibitory efficacy of the final glycosylated product was evaluated in vitro using glycoaldehyde, an intermediate product of AGEs.

As shown in FIG. 2 , it was confirmed that 1 mM of AG, known as a final glycosylation cross-linking inhibitor, had a cross-linking inhibitory effect of 67%. On the other hand, the vitamin tree fruit extract of Comparative Example 1 showed 19% and 19% AGEs cross-linking inhibitory effects at 50 μg/mL and 200 μg/mL, respectively, and the vitamin tree leaf extract of Example 1 was 50 μg/mL , it was confirmed that at 200 μg/mL, AGEs cross-linking inhibitory effects of 52% and 74.5% were shown, respectively.

That is, it was confirmed that the vitamin tree leaf extract of Example 1 was superior to the vitamin tree fruit extract of Comparative Example 1 in inhibiting cross-linking of final glycated products at both low and high concentrations.

Test Example 3. Analysis of Efficacy of Cross-Linking Cleavage of Final Glycosylation Products

3 is a graph showing the degree of cross-linking cleavage of the final glycation products (AGEs) of the vitamin tree extract prepared according to Example 1 and Comparative Example 1 of the present invention.

In the test method, 10 μg/mL AGE-BSA was dispensed in a collagen-coated 96-well microtiter plate in the same manner as in Test Example 2 to evaluate the efficacy of final glycation cross-linkage cleavage, followed by incubation at 37° C. for 4 hours. and collagen were cross-linked. After washing 3 times in 0.05% PBST, 1 mg/mL of ALT-711, a drug known as a cross-linking inhibitor, or vitamin tree extract was dispensed into each well and incubated at 37 °C for 18 hours. Then, each well was washed 3 times with 0.05% PBST, and after color development using TMB as a substrate, absorbance was measured at 450 nm and calculated according to the following [Equation 2].

[Equation 2]

AGE-BSA(%)=(absorbance of wells with drug added/absorbance of wells without drug) X 100

As shown in FIG. 3 , as a result of examining the efficacy of cleaving the irreversible cross-linkage (glycoaldehyde) already formed between the final glycosylated product and collagen, ALT-711, known as a final glycosylated cross-linkage cleaving drug, was 1 It was confirmed that the cleavage efficiency of 78% was shown at mg/mL. On the other hand, the vitamin tree fruit extract of Comparative Example 1 showed 14% and 64% AGEs cross-linking cleavage efficacy at 50 μg/mL and 200 μg/mL, respectively, and the vitamin tree leaf extract of Example 1 was 50 μg/mL , it was confirmed that AGEs cross-linking cleavage efficacy of 88% and 98% at 200 μg/mL, respectively.

That is, it was confirmed that the vitamin tree leaf extract of Example 1 was superior to the vitamin tree fruit extract of Comparative Example 1 in both the low and high concentrations of the final glycation product cross-linking cleavage effect.

[Comparison by extraction solvent]

Example 1. 70 wt% ethanol aqueous solution

It is a vitamin tree leaf extract prepared according to Example 1.

Example 2. 30 wt% ethanol aqueous solution

It was carried out in the same manner as in Example 1, but using a 30% by weight aqueous ethanol solution instead of a 70% by weight aqueous ethanol solution to obtain a vitamin tree leaf extract.

Example 3. 50 wt% ethanol aqueous solution

It was carried out in the same manner as in Example 1, but using a 50 wt% ethanol aqueous solution instead of a 70 wt% ethanol aqueous solution to obtain a vitamin tree leaf extract.

Example 4. Distilled Water

It was carried out in the same manner as in Example 1, but using distilled water instead of 70 wt% ethanol aqueous solution to obtain a vitamin tree leaf extract.

<Test Example II>

Test Example 4. Efficacy of inhibiting the formation of final glycation products (AGEs)

4 is a graph showing the degree of inhibition of final glycation products (AGEs) production of the leaf extract of vitamin tree prepared according to Examples 1 to 4 of the present invention.

The test method was performed in the same manner as in Test Example 1.

As shown in Figure 4, it was confirmed that the vitamin tree leaf extract prepared according to Examples 1 to 3 was superior to the vitamin tree leaf extract prepared according to Example 4 to inhibit the formation of final glycation products (AGEs). It was confirmed that the ability to inhibit the formation of final glycation products (AGEs) was similar between the leaf extracts of the vitamin tree of Examples 1 to 3.

That is, it was confirmed that the ethanol aqueous solution extract was superior to the water extract in inhibiting the formation of final glycation products (AGEs).

Test Example 5. Efficacy analysis of cross-linking of final glycated products

5 is a graph showing the degree of inhibition of cross-linking of final glycation products (AGEs) of vitamin tree leaf extracts prepared according to Examples 1 to 4 of the present invention.

The test method was performed in the same manner as in Test Example 2.

As shown in FIG. 5 , it was confirmed that the vitamin tree leaf extract prepared according to Examples 1 and 3 was superior to the vitamin tree leaf extract prepared according to Examples 2 and 4 to inhibit cross-linking of final glycated products.

That is, it was confirmed that the extract extracted with 50 wt% ethanol aqueous solution and 70 wt% ethanol aqueous solution was superior to the 30 wt% ethanol aqueous solution and the extract extracted with water to inhibit cross-linking of final glycated products.

Test Example 6. Analysis of Efficacy of Cross-Linking Cleavage of Final Glycosylation Products

6 is a graph showing the degree of cross-linking cleavage of the final glycation products (AGEs) of the vitamin tree leaf extract prepared according to Examples 1 to 4 of the present invention.

The test method was performed in the same manner as in Test Example 3.

As shown in FIG. 6 , it was confirmed that the vitamin tree leaf extract prepared according to Examples 1 and 3 was superior to the vitamin tree leaf extract prepared according to Examples 2 and 4 for cross-linking cleavage of final glycated products.

That is, it was confirmed that the extracts extracted with 50 wt% ethanol aqueous solution and 70 wt% ethanol aqueous solution had superior cross-linkage cleavage efficiency of the final saccharified product compared to the 30 wt% ethanol aqueous solution and the extract extracted with water.

Test Example 7. Efficacy of inhibiting cytotoxicity by MGO

7 is a graph measuring the cytotoxicity of a vitamin tree leaf extract prepared according to Example 1 of the present invention.

cell culture

SV40MES13 cells originating from the mouse renal glomerular were purchased from ATCC (Manassas, VA) and used in the experiment. Cells were inoculated in a medium of 3:1 ratio DMEM/F12 containing 5% FBS, 1% P&S, and 14 mM HEPES, and cultured at 37 ° C., 5% CO ₂ condition.

Cytotoxicity measurement

After dispensing 5×10 ⁴ cells/well in a 96 well plate, incubate for 18 hours in an incubator at 37 ° C and CO ₂ , cells to become 1 mM MGO and 10, 50, 100, and 200 μg/mL of vitamin tree leaf extract, respectively. was treated and incubated for 18 hours. After that, 3-(4,5-dimethysssl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT; Sigma) solution was dissolved in PBS at 0.1 mg/mL and treated, and then added to an incubator at 37 ℃, CO ₂ 3 The medium was carefully removed so that the formazan produced by reducing the MTT by culturing for more time did not fall off the medium. After aliquoting 200 μL of DMSO and mixing for 10 minutes, absorbance was measured at 540 nm.

An MGO-induced toxicity model was established in kidney cells, and the renal cytotoxicity inhibitory effect of vitamin tree leaf extract was confirmed with MTT. The cell viability was measured by treating the kidney cell line with vitamin tree extract by concentration.

As shown in Figure 7, it was confirmed that apoptosis occurs by MGO by confirming the 68% survival rate in the MGO-treated group (C). On the other hand, in the cell lines treated with vitamin tree leaf extract at 10, 50, and 100 μg/ml, the survival rates of 73.1%, 79.4%, and 80.2%, respectively, were confirmed, confirming that there was a cytoprotective effect.

<Test Example Ⅲ>

animal testing

Experimental animals were type 2 diabetes animal model BKS.Cg-Dock7m +/+ Leprdb/+/J(db/m) mouse (male, 5 weeks old), BKS.Cg-Dock7m +/+ Leprdb/db/J( db/db) mice (male, 5 weeks old) were imported from Jackson Laboratories (Bar Harbor, Maine, USA) and used. In the environment of the animal breeding room, constant temperature (25±2 ℃) and constant humidity (50±5%) were maintained, and feed and drinking water were fed and reared in a constant environment that alternates day and night at 12 hour intervals. After acclimatization to the breeding environment for 1 week, 50 mg/mL of AG was orally administered to db/m (normal group), db/db (control group 1), and db/db of experimental animals by the egg mass method so that blood sugar and body weight were similar ( Control 2), oral administration of 100 mg/mL of the extract of Example 1 to db/db (Example 1(100)), and oral administration of 200 mg/mL of the extract of Example 1 to db/db (Example 1 ( 200)), each of 8 animals was classified into 5 groups. All mice were fed a normal diet. The experiment was conducted for 13 weeks, and after fasting for 12 hours in 2 weeks, blood was collected from the tail vein and measured using a blood glucose meter. This study on animal experiments was conducted after the deliberation and approval of the Animal Experimental Ethics Committee of the Korea Food Research Institute (IACUC: KFRI-M-20004). Aminoguanidine hydrochloride (AG) used in the experiment was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies for tissue staining were AGEs (ab23722), and RAGE (ab3611) was Abcam (Cambridge, MA, USA). ) was purchased and used.

After breeding, the animals were anesthetized by inhalation using isoflurane, and whole blood was collected from the inferior vena cava. After collecting the kidney tissue of the experimental animal, the tissue was sheared and fixed in 10% formalin for histological staining analysis.

-5 groups of mice-

Normal group: administration of physiological saline to db/m mice

Control 1: Administration of physiological saline to db/db mice

Control 2: AG 50 mg/mL administered to db/db mice

Example 1 (100): Administration of 100 mg/mL of the extract of Example 1 to db/db mice

Example 1 (200): Administration of 200 mg/mL of the extract of Example 1 to db/db mice

Test Example 8. Measurement of changes in fasting blood sugar

8 is a graph measuring changes in fasting blood glucose in the normal group, control group 1, control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)) am.

As shown in FIG. 8 , the normal group had a fasting blood sugar of 59 mg/dl at the start of the experiment and 73.25 mg/dl at the end of the experiment, so there was no significant change, but the diabetes model, Control 1, was at 128 mg/dl at the start of the experiment. At the end of the experiment, it was confirmed that it appeared at 467 mg/dl.

In addition, the control group 2 and the Example 1 (100) group did not differ significantly from the control group 1 at 430 mg/dl and 418 mg/dl, respectively, at the end of the experiment, but the Example 1 (200) group showed 277.75 mg/dl It was confirmed that blood sugar was significantly reduced.

Test Example 9. PAS (Periodic Acid Schiff) staining

After the fixed tissue was subjected to tissue treatment and embedded in paraffin, the paraffin block was cut into 3 micro-thick sections to make a section, followed by deparaffinization and hydrolysis, followed by washing with distilled water. After 5 minutes of reaction with Periodic Acid Solution, washing, and reaction with Schiff's solution for 15 minutes. After washing, it was stained with Mayer hematoxylin for 1 minute.

The pathological findings of diabetic nephropathy are generally found in the glomeruli, tubulointerstitium, and blood vessels, and show a typical or atypical form. In particular, mesangial expansion and proliferation can be identified as a classification for glomerular lesions, which are relatively early lesions that can be observed under a microscope.

Mesangium is composed of mesangium cells and matrix and plays a central role in structurally supporting the glomeruli. Mesangial cells play an important role in determining ultrafiltration by regulating glomerular blood flow by closely interacting with podocytes and vascular endothelial cells while controlling the components and regulation of mesangium matrix. In the early stage of diabetic kidney injury, although blood pressure is normal, intraglomerular hypertension occurs, which secretes various substances due to the stretch of mesangium cells, which causes mesangial enlargement and proliferation as well as mesangial expansion.

9 is a photograph after PAS staining of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

As shown in FIG. 9 , it was confirmed that the mesangium cells, which appeared dark purple in the glomerulus in the normal group, were light purple in the control group 1 and expanded in size at the same time. It was confirmed that the expanded mesangium cells were reduced in the expansion and proliferation of mesangium cells similar to the normal group when AG was administered. In addition, it was confirmed that Example 1 (100) group and Example 1 (200) group reduced the size of mesangium cells compared to Control 1.

Test Example 10. AGEs immunostaining (Immunohistochemistry: IHC)

After the fixed tissue was subjected to tissue treatment and embedded in paraffin, the paraffin block was cut to a thickness of 4 micrometers to make a section, followed by deparaffinization and hydrolysis, followed by washing with distilled water. After antigen restoration, in order to eliminate the activity of endogenous peroxidase, it was treated with 3% hydrogen peroxidase solution at room temperature for 10 minutes. After reacting with primary antibodies AGEs (abcam, 1:100) and RAGE (abcam, 1:100), they were reacted with Envision+ Rabbit for 30 minutes and counterstained with Mayer hematoxylin.

In diabetic nephropathy, mesangial cell damage is caused by an increase in advanced glycation end-products (AGEs), activation of protein kinase C, and reactive oxygen species (ROS) upon high glucose exposure. Among them, AGEs IHC were checked to confirm the accumulation of AGEs.

10 is a photograph confirming the AGEs IHC of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

As shown in FIG. 10 , it was confirmed that a large amount of AGEs were accumulated inside and outside the glomerulus in dark brown color in Control 1 . This means that AGEs produced in large amounts by diabetes accumulated in the kidneys. In the control group 2, it was confirmed that the accumulation of AGEs was significantly reduced, in the Example 1 (100) group, it was confirmed that the staining of AGEs was reduced compared to the control 1, and in the Example 1 (200) group, the AGEs were similar to the control 2 It was confirmed that it was significantly reduced.

Test Example 11. RAGE IHC

Receptors of advanced glycation end-products (RAGE) are representative AGEs receptors. By combining RAGE and AGEs, intracellular signaling systems such as PKC and mitogen-activated protein kinase are activated, ROS increases, and transcription of NF-κB Activation of the factor occurs. Histological lesions were confirmed in the development and progression of diabetic nephropathy by confirming RAGE IHC along with proliferation of mesangial cells due to diabetes.

11 is a photograph confirming the RAGE IHC of the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention (Example 1 (100) and Example 1 (200)).

As shown in FIG. 11 , it was confirmed that RAGE was overexpressed in the glomerular cortex in brown color in Control 1. This means that AGEs influence diabetic nephropathy by binding to RAGE. Control group 2 confirmed that RAGE expression was reduced similarly to the normal group, and it was confirmed that less RAGE was stained similar to the normal group in Example 1 (100) and Example 1 (200).

Test Example 12. Glyoxalase 1 (GLO1) activity measurement

In order to analyze the Glyoxalase 1 (GLO1) activity of mouse kidney tissue, it was measured using the QuantiChrom ^™ Glyoxalase I Assay Kit (BioAssay Systems, CA, USA). 40 μL of each sample was mixed with 160 μL of an assay reagent and reacted at room temperature for 20 minutes, and then the mixture in which 70 μL of 4 M HClO ₄ was added to each reaction was reacted on ice for 15 minutes. Thereafter, the reaction mass was centrifuged at 12,000 rpm for 10 minutes, 200 μL of the supernatant was added to a 96-well plate, and GLO1 activity was measured at 240 nm wavelength using a spectrophotometer.

Figure 12a is a Western blot measuring GLO1 in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention; 12B is a Western blot for measuring AGEs in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIGS. 12A and 12B , it was confirmed that the expression level of control 1 (diabetic nephropathy mouse caused by induction of type 2 diabetes) of GLO1 was decreased compared to the normal group, and the expression was decreased due to the administration of Example 1 200. was confirmed to increase. In addition, in the results of AGEs, it was confirmed that the expression level of Control 1 was significantly increased compared to the normal group, and it was confirmed that the expression was decreased due to the administration of Example 1 200.

Through these results, it was confirmed that the extract of Example 1 regulates the expression of GLO1 and the expression of AGEs.

13 is a graph showing GLO1 activity in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIG. 13 , the GLO1 activity was significantly decreased in Control 1, and in contrast, it was confirmed that the GLO1 activity was significantly increased due to the administration of Example 1 200.

Through this, it was confirmed that the activity of GLO1 was restored due to the administration of the extract of Example 1.

14 is a photograph confirming GLO1 and AGEs in the kidney tissue of db/db mice in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIG. 14 , compared to the normal group, the GLO1 expression level of the control group 1 was significantly decreased in the overall tissue compartment, and in contrast, the GLO1 expression level of the 200 group of Example 1 increased. In addition, in the results of AGEs, the expression level was increased in the tissues of the control group 1 compared to the normal group, and the expression level was decreased due to the administration of Example 1 200, so that the expression of GLO1 and AGEs was regulated by the administration of Example 1 200. confirmed that.

Test Example 13. MGO (methylglyoxal)-derived AGEs analysis

MGO-derived AGEs were analyzed using OxiSelect™ methylglyoxal Competitive ELISA kit (Cell Biolabs, Inc., San Diego, CA, USA). Experiments were performed and analyzed according to the provided manual.

15 is a graph of MGO-derived AGEs measured by an ELISA method in a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIG. 15 , it was confirmed that MGO was significantly increased in the diabetic nephropathy mouse group (control group 1) caused by the induction of type 2 diabetes, and in contrast, MGO was significantly increased due to the administration of Example 1 200 It was confirmed that there was a negative decrease.

This means that the extract of Example 1 promotes the decomposition of MGO, so it is suitable for the prevention, improvement or treatment of diabetic nephropathy.

Test Example 14. Measurement of NOX2, NOX4, 8-OHdG and Nrf2

The NOX2 and NOX4 proteins were extracted from kidney tissue and protein expression levels were measured by Western blot using NOX2 and NOX4 antibodies, and 8-OHdG was 8-Hydroxy-2'-deoxyguanosine ELISA kit (Abcam, Cambridge). , MA, USA) was performed and analyzed according to the provided manual. In particular, Nrf2 and NOX4 expression levels were measured in kidney tissue by Immunohistochemistry method.

Figure 16a is a Western blot measuring NOX4 in the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention; Figure 16b is a Western blot measuring NOX2 in the normal group, the control group 1, the control group 2, and the group administered with the extract prepared according to Example 1 of the present invention.

As shown in FIGS. 16A and 16B , in the results of NOX4, a significant increase in expression was observed in Control 1 compared to the normal group, and it was confirmed that the expression level of NOX4 was decreased in the 200 group of Example 1 compared to that of Control 1. On the other hand, in the results of NOX2, there was no change between the normal group and the control group 1, and the expression level of Example 1 200 group showed a tendency to decrease, but it was confirmed that it was insignificant.

17 is a graph showing the content of 8-OHdG in the urine of a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIG. 17 , the normal group exhibited a level of 3.1 ng/mL, and the control group 1 had a level of 5.7 ng/mL, which increased by about 1.9 times. On the other hand, in the group administered with Example 1 200, it was measured to be 3.5 ng/mL, which was confirmed to represent a significant decrease compared to Control 1, and it was confirmed to show similar results to Control 2 (positive control, AG).

18 is a photograph confirming Nrf2, NOX4 and 8-OHdG in the kidney tissue of a normal group, a control group 1, a control group 2, and a group administered with an extract prepared according to Example 1 of the present invention.

As shown in FIG. 18 , it was confirmed that the expression level of Nrf2 in the kidney tissue of the control group 1 was significantly reduced compared to the normal group, and on the contrary, the expression level of the 200 group of Example 1 increased. In contrast, the expression of NOX4 was increased in the kidney tissue of the control 1 compared to the normal group, and it was confirmed that the expression level was decreased in the 200 group of Example 1. In addition, in the expression result of 8-OGdG, similarly, it was confirmed that the expression level was significantly increased in the control group 1 compared to the normal group, and in contrast, it was confirmed that the expression level was decreased in the 200 group of Example 1 .

It is determined that the extract of Example 1 regulates oxidative stress in kidney tissue.

Through the above results, it promotes the decomposition of MGO, a precursor of AGEs, suppresses renal cytotoxicity by MGO, and reduces GLO-1, NOX4, and 8-OHdG through the transcription factor Nrf2 and RAGE sub-pathways involved in antioxidants By controlling it, it shows a significant increase in the activity of the antioxidant effect, and it can be used for prevention, improvement or treatment of diabetic nephropathy by containing it as an active ingredient.

Test Example 15. Component Analysis of Vitamin Tree Extract

Table 1 and FIG. 19 show isorhamnetin and rutin contents measured according to the solvent of the vitamin tree leaf extract and the vitamin tree fruit extract prepared according to Example 1 of the present invention.

Preparation of standard solutions and samples

To set the analysis conditions, appropriate amounts of isorhamnetin and rutin were weighed and transferred to an eppendorf tube, and then an appropriate amount of acetone was added to have a concentration of 1 mg/mL. Thereafter, standard solutions were prepared at 15.625, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00, 2000.00, 4000.00, and 8000.00 ng/mL using LCMS grade water.

In the case of the extract sample, 4.0 mg to 8.0 mg of the vitamin tree extract was transferred to a 2.0 ML eppendorf tube, and an appropriate amount of 50% ethanol was added so as to have a concentration of 4 mg/mL. Then, sonication was performed for 2 hours to convert isorhamnetin and rutin in the extract to a solution phase.

HPLC-MS/MS analysis

Waters' Xevo TQ MS was used to analyze the two components in the vitamin tree extract. For separation, ACQUITY BEH C18 column from Waters was used, and 0.1% formic acid (FA) water and 0.1% FA methanol were used as mobile phases. In addition, MS conditions for analyzing two components were set using Waters' Intellistart program. For isorhamnetin, cone voltage, collision energy, and mass-to-charge ratio of precursor ion and product ion were set to 38.0 V, 34.0 V, m / z 317.2, m / z 302.1, and for rutin, 14.0 V, 28.0 V, m / z 611.4, m / z 303.2.

ng/mg material	Example 1	Comparative Example 1	Comparative Example 2	Comparative Example 3
isorhamnetin	254.3 ± 26.0	133.3 ± 6.9	156.4 ± 20.8	58.5 ± 2.3
rutin	862.1 ± 27.2	446.6 ± 6.6	506.2 ± 12.9	329.9 ± 3.9

Comparative Example 2 was carried out in the same manner as in Example 1, except that the vitamin tree leaf was extracted with hot water at 110 ° C. with distilled water, and Comparative Example 3 was carried out in the same manner as in Example 1, except that the vitamin tree fruit was extracted with distilled water at 110 ° C. It is a hot water extract. As shown in Table 1 and Figure 19 above, it was confirmed that isorhamnetin and rutin were elution at 14.3 minutes and 11.1 minutes. Also, through quantitative analysis, the content of both isorhamnetin and rutin was higher in the leaf than in the fruit, and in all cases, it was confirmed that alcohol extraction was effective in increasing the content of the two components in the extract.

Hereinafter, a formulation example of a composition comprising the extract of the present invention will be described, but the present invention is not intended to limit the present invention, but merely to describe it in detail.

Formulation Example 1: Preparation of powder

20 mg of extract powder of Example 1

Lactose 100 mg

talc 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

Formulation Example 2: Preparation of tablets

10 mg of extract powder of Example 1

100 mg cornstarch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

Formulation Example 3: Preparation of capsules

10 mg of extract powder of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4: Preparation of granules

1,000 mg of extract powder of Example 1

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

potassium phosphate monobasic 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is mixed with ingredients suitable for health functional food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the ingredients are mixed according to a conventional health functional food manufacturing method. Next, the granules can be prepared and used in the preparation of a health functional food composition according to a conventional method.

Formulation Example 5: Preparation of beverage formulations

1,000 mg of extract powder of Example 1

1,000 mg citric acid

100 g of oligosaccharides

2 g of plum concentrate

1 g taurine

Add purified water to total 900 mL

After mixing the above ingredients according to a conventional beverage manufacturing method, after stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then refrigerated. used in the manufacture of functional beverage compositions.

Formulation Example 6: Preparation of feed composition

Example 1 extract powder 0.1 kg, corn 25.5 kg, wheat 15.04 kg, wheat flour 8.15 kg, rice bran 7.4 kg, soybean meal 18 kg, okgurten 1 kg, chicken by-product 14 kg, animal fat 9 kg, processed salt 0.3 kg, ginseng phosphate An animal (dog, pet dog) feed composition was prepared by mixing 0.3 kg of calcium, 1 kg of limestone, 0.01 kg of choline chloride, 0.05 kg of vitamins, 0.05 kg of minerals and 0.1 kg of digestive enzymes.

The vitamin tree ( Hippophae rhamnoides ) leaf extract of the present invention can be used as an active ingredient to be used as a pharmaceutical composition, a feed composition, a pharmaceutical composition for animals, a food composition, and even a health functional food.

Claims (12)

1. Vitamin tree ( Hippophae rhamnoides ) Food composition for preventing or improving diabetic complications containing leaf extract as an active ingredient.
2. The diabetes according to claim 1, wherein the diabetic complications are at least one selected from the group consisting of diabetic neuropathy, diabetic nephropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, and diabetic foot ulcer. A food composition for preventing or improving complications.
3. The food composition for preventing or improving diabetic complications according to claim 1, wherein the diabetic complications are prevented or improved by inhibiting the production of advanced glycation end products (AGEs) and promoting decomposition.
4. The food composition for preventing or improving diabetic complications according to claim 1, wherein the vitamin tree leaf extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
5. The food composition for preventing or improving diabetic complications according to claim 4, wherein the mixed solvent is an aqueous solution of 20 to 80% by volume of methanol, ethanol, butanol or propanol.
6. The food composition for preventing or improving diabetic complications according to claim 4, wherein the mixed solvent is an aqueous solution of 20 to 80% ethanol.
7. The food composition according to claim 1, wherein the food composition is a health functional food for preventing or improving diabetic complications.
8. A pharmaceutical composition for the prevention or treatment of diabetic complications comprising a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.
9. The diabetes according to claim 8, wherein the diabetic complications are at least one selected from the group consisting of diabetic neuropathy, diabetic nephropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, and diabetic foot ulcer. A pharmaceutical composition for the prevention or treatment of complications.
10. Vitamin tree ( Hippophae rhamnoides ) Feed composition for preventing or improving diabetic complications containing leaf extract as an active ingredient.
11. A pharmaceutical composition for animals for the prevention or treatment of diabetic complications comprising a vitamin tree ( Hippophae rhamnoides ) leaf extract as an active ingredient.
12. Vitamin tree ( Hippophae rhamnoides ) leaf extract having activity to inhibit the production of advanced glycation end products (AGEs) and promote degradation.

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