KR20220064448A - A composition for improving insulin sensitivity comprising mixture of herbal extracts of broussonetia kazinoki and juglans mandshurica - Google Patents

Abstract

The present invention relates to a composition for alleviating insulin sensitivity containing a complex extract of Broussonetia kazinoki and Juglans mandshurica. The composition according to the present invention alleviating insulin sensitivity by alleviating impaired glucose tolerance and reducing ITIH1 protein expression, thereby being able to be usefully used as a therapeutic agent for diabetes, diabetic complications, impaired glucose tolerance, and insulin resistance syndrome.

Description

Composition for improving insulin sensitivity comprising a complex extract of mulberry and mulberry trees {A COMPOSITION FOR IMPROVING INSULIN SENSITIVITY COMPRISING MIXTURE OF HERBAL EXTRACTS OF BROUSSONETIA KAZINOKI AND JUGLANS MANDSHURICA}

The present invention relates to a composition for improving insulin sensitivity comprising a complex herbal extract of mulberry and mulberry, and more particularly, by improving impaired glucose tolerance and reducing the expression of ITIH1 (Inter-alpha Trypsin Inhibitor Heavy chain 1) protein. It relates to a composition characterized in that it improves insulin sensitivity.

In normal cases, the body maintains homeostasis by regulating the blood glucose concentration, but in metabolic diseases and diseases accompanied by insulin resistance, blood sugar is excessively increased.

Insulin resistance refers to a case in which the sensitivity to insulin is reduced compared to the normal standard, and an increase in insulin in muscle and adipose tissue is not detected or the action of insulin does not occur effectively even if it is detected. In this case, the blood sugar cannot be lowered, and the balance of cell metabolism and metabolism cannot be effectively maintained.

To improve insulin sensitivity, increase the secretion of insulin itself by beta (ß) cells of the pancreas (JUN-ICHI MIYAZAKI et al, Establishment of a Pancreatic β Cell Line That Retains Glucose-Inducible Insulin Secretion: Special Reference to Expression of Glucose Transporter Isoforms, Endocrinology, Volume 127, Pages 126-132, 1990), a method for amplifying the signal that the insulin receptor binds to insulin and transmits in the metabolic pathway (Manabu Yamamoto et al, Caveolin Is an Activator of Insulin) Receptor Signaling, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 273, No. 41, Issue of October 9, pp. 26962-26968, 1998) is mainly considered, but based on the biological mechanism causing hyperglycemia, the protein itself involved in this Research on a method for controlling hyperglycemia by regulating the activity is insufficient.

ITIH1 (Inter-alpha Trypsin Inhibitor Heavy chain 1) is a protein that is produced in hepatocytes and secreted into the blood, and its concentration and secretion in cells increases by glucose stimulation (Science Translational Medicine, Vol. 11, Issue 513, eaan4735, 2019). Specifically, the increase in ITIH1 is achieved by a decrease in G13, a type of G protein, and an increase in the expression level of OGT (O-GlcNAc transferase) enzyme in hyperglycemia. In a hyperglycemic environment, OGT causes O-GlcNAcylation modification on the serine residue of ITIH1 to increase the stability of ITIH1.

On the other hand, the mulberry ( Broussonetia kazinoki ) is a deciduous broad-leaved tree of the Morus family of the order Nettle, and it is a male and female tree, and the leaves appear in spring and flowers bloom at the same time. The bark is a material for making paper in Korea, China, and Japan, and the fruit is called 'jeosil' or 'gusuja' and is used medicinally, and the young leaves are also eaten. Paper mulberry is known to have effects such as improvement of inflammatory acne skin, inhibition of angiogenesis, and strengthening of immune function (Korean Patent Publication No. 10-1827771, Korean Patent Publication No. 10-1693725, Korean Patent Publication No. 10- 1039110).

In addition, Juglans mandshurica is classified in the family Juglansaceae or Walnutaceae and exists in the form of trees or shrubs, and is native to America, Eurasia or South Asia. It inhabits mainly in central and northeastern Korea or northeastern China, and is used as a medicinal plant and herbal medicine, and its fruits are used medicinally or edible.

However, until now, the effect of improving insulin sensitivity of extracts of mulberry and mulberry trees was not known. In this regard, the inventors of the present invention have completed the present invention by discovering that the complex extract of mulberry and mulberry can improve insulin sensitivity by improving impaired glucose tolerance and suppressing the expression of ITIH1 protein.

1) Korean Patent Publication No. 10-1827771 (2018.02.09) 2) Korean Patent Publication No. 10-1693725 (2017.01.09) 3) Korean Patent Publication No. 10-1039110 (June 15, 2011)

Science Translational Medicine, Vol. 11, Issue 513, eaan4735 (2019.10.09) JUN-ICHI MIYAZAKI et al, Establishment of a Pancreatic β Cell Line That Retains Glucose-Inducible Insulin Secretion: Special Reference to Expression of Glucose Transporter Isoforms, Endocrinology, Volume 127, Pages 126-132 (1990.07.01) Manabu Yamamoto et al, Caveolin Is an Activator of Insulin Receptor Signaling, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 273, No. 41, Issue of October 9, pp. 26962-26968 (October 9, 1998)

An object of the present invention is to provide a composition comprising a complex extract of mulberry and mulberry, which can improve insulin sensitivity by improving impaired glucose tolerance and reducing the expression of ITIH1 protein.

In order to achieve the above object, the present invention provides a composition for improving insulin sensitivity comprising a complex extract of mulberry and mulberry tree.

The composition may be a pharmaceutical composition, a food composition, or a feed composition for animals.

In one embodiment, the composition has the effect of increasing insulin sensitivity and decreasing insulin resistance.

In one embodiment, the composition may be for preventing, treating or ameliorating one or more diseases selected from the group consisting of diabetes, diabetic complications, impaired glucose tolerance, and insulin resistance syndrome.

In one embodiment, the diabetic complications may be one or more diseases selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy.

In one embodiment, the insulin resistance syndrome may be one or more diseases selected from the group consisting of insulin resistance-induced obesity, hypertension, arteriosclerosis, hyperlipidemia, hyperinsulinemia, nonalcoholic fatty liver, and type 2 diabetes.

In one embodiment, the mulberry extract may be a mulberry root extract, a leaf extract, a fruit extract, an eggplant extract or a stem extract, preferably a mulberry leaf extract.

In one embodiment, the Suspension tree extract may be a shamrock root extract, a leaf extract, a fruit extract, or a stem extract, and preferably may be a shamrock fruit extract.

In one embodiment, the complex extract of mulberry and mulberry may be to reduce the expression of ITIH1 (Inter-alpha Trypsin Inhibitor Heavy chain 1) mRNA or protein.

In one embodiment, the extract may be extracted with a solvent selected from the group consisting of water, C1 to C6 alcohol, acetic acid, and a mixed solvent thereof. Preferably, it may be a 0.01% to 90% ethanol extract, more preferably, a 60% to 80% ethanol extract, and most preferably a 70% ethanol extract.

The mulberry and mulberry extracts according to the present invention have the effect of improving insulin sensitivity by improving the future of glucose tolerance and reducing the expression of ITIH1. Therefore, the composition of the present invention can be usefully used as a therapeutic agent for diabetes, diabetic complications, impaired glucose tolerance, and insulin resistance syndrome.

1 shows the results of glucose tolerance test (GTT) in db/db diabetic mice.
2 shows the inhibitory effect of ITIH1 protein expression in liver tissue.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art to which the present invention pertains can easily carry out. However, the present application may be embodied in various forms and is not limited to the embodiments and examples described herein.

Throughout this specification, when a part "includes" a component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a pharmaceutical composition for improving insulin sensitivity, comprising a complex herbal extract of mulberry ( Broussonetia kazinoki ) and mulberry ( Juglans mandshurica ).

The complex extract of the present invention relates to a pharmaceutical composition for improving insulin sensitivity, characterized in that it has the effect of increasing insulin sensitivity and decreasing insulin resistance.

The insulin sensitivity improvement effect means an increase in insulin sensitivity and a decrease in insulin resistance. Insulin sensitivity refers to the degree to which a person responds to insulin, and a decrease in insulin sensitivity, that is, insulin resistance, refers to a decrease in the response to insulin compared to a normal standard, so that the function of insulin to lower blood sugar is reduced, so that cells cannot effectively burn glucose do.

Specifically, the complex extract of the present invention relates to a pharmaceutical composition, characterized in that it is for preventing or treating one or more diseases selected from the group consisting of diabetes, diabetic complications, impaired glucose tolerance, and insulin resistance syndrome.

The diabetic complications may be one or more diseases selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy.

The insulin resistance syndrome may be one or more diseases selected from the group consisting of obesity, hypertension, arteriosclerosis, hyperlipidemia, hyperinsulinemia, nonalcoholic fatty liver, and type 2 diabetes due to insulin resistance.

The extract of the mulberry tree and the mulberry tree of the present invention may be a whole herb extract, a root extract, a leaf extract, a flower extract, a fruit extract, or a stem extract. The whole plant refers to the whole plant with flowers, roots, leaves, and stems.

Specifically, in the present invention, the mulberry extract may be a mulberry leaf extract, and the mulberry extract may be a mulberry fruit extract.

The composition of the present invention has the effect of inhibiting the expression of ITIH1 (Inter-alpha Trypsin Inhibitor Heavy chain 1). ITIH1 is a protein produced by hepatocytes and secreted into the blood. The intracellular concentration and secretion of ITIH1 increases by glucose stimulation, and it binds to hyaluronic acid and decreases insulin sensitivity in muscle and fat cells.

As the extraction method of the complex extract of mulberry and mulberry trees of the present invention, conventional methods in the art such as stirring extraction, filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction can be used.

The pharmaceutical composition of the present invention may be administered parenterally or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, etc. Its scope is diverse. In addition, the therapeutically effective amount of the composition may vary depending on the method of administration, the target site, and the condition of the patient.

The present invention relates to a food composition for improving insulin sensitivity, comprising a complex extract of mulberry ( Broussonetia kazinoki ) and mulberry ( Juglans mandshurica ).

The food composition of the present invention may be a health functional food, and the "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body, and "functionality" means the structure and function of the human body It refers to ingestion for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological effects.

Food compositions can be manufactured and processed into health functional foods in the form of tea bags, leached teas, health drinks, etc., and can also be processed into other breads, confectionery, ice cream, noodles, and the like. In addition, the above 　 food 　 composition may be a health supplement 　 or a 　 food additive, and whether or not it is suitable as a "food additive" is evaluated according to the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the relevant standards and standards.

The present invention relates to a feed composition for animals for improving insulin sensitivity, comprising a complex extract of mulberry ( Broussonetia kazinoki ) and mulberry ( Juglans mandshurica ).

The present invention will be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Production Example]

Preparation of herbal extracts

Washed and dried mulberry ( Broussonetia kazinoki ) leaves and sputum ( Juglans mandshurica ) fruits were used round or pulverized according to the herbal medicine. The mulberry leaf and mulberry fruit were extracted with 10 times 70% ethanol (v/v) aqueous solution to each herbal medicine at room temperature for 72 hours. After filtering the extract, it was concentrated under reduced pressure at 50 to 65° C. to obtain a single herbal extract in a powder state, and the following experiment was performed based on the obtained extract.

[Example 1]

db/db mouse preparation

The db/db mouse (diabetic mouse) has an autosomal recessive mutation on chromosome 4 in the C57BL/KsJ mouse strain made by Bar Harbor, Marine. In addition, mice with mutations in the diabetic gene encoding the leptin receptor exhibit 1) insulin hyperplasia, 2) obesity during the first month of life, 3) hyperglycemia, 4) hyperinsulinemia and insulin resistance. Thereafter, phenotypes of hypoinsulinemia and hyperglycemia appear at around 3 to 4 months of age.

The db/db mouse used in this test is an animal model for non-insulin dependent diabetes mellitus (NIDDM) and is widely used in diabetes-related drug efficacy tests. The mouse was selected. 7-week-old male db/db diabetic mice (Japan SLC, Japan) were used in the experiment after an acclimatization period of 12 days.

(1) Species and strains: C57BLKS/J Iar -+Leprdb/+Leprdb and C57BLK/6

Producer: Japan SLC. (japan)

Source: JoongAng Experimental Animals Co., Ltd. (7, Baumeuro-gil, Seocho-gu, Seoul)

(2) Quarantine and acclimatization period

28 days after acquisition (check the health status by observing general symptoms during the acclimatization period, and use healthy animals for testing)

(3) Number of animals used: 55 males

(4) identification

Animals were identified using unbuoyancy during the acclimatization period (blue), dosing and observation period (red). An individual identification card distinguished by color was attached to the breeding box, and an animal room usage record was attached to the entrance to the breeding room.

(5) breeding environment

This test was conducted in accordance with the guidelines of the National Institutes of Health (NIH publication No.85-23, revised 1985) at a temperature of 23±3 ℃, a relative humidity of 55±15%, a ventilation rate of 10 to 20 times/hr, and an illumination time of 12 hours ( 8 am to 8 pm lights off) and illuminance was set to 150 to 300 Lux. During the breeding period, environmental conditions such as temperature/humidity, ventilation frequency, and illuminance of the animal room were measured regularly.

(6) Feed, water and litter

Feed was supplied with general feed for laboratory animals and freely consumed, and purified water was freely ingested using a polycarbonate drinking bottle. For the rug, a wooden rug was used.

(7) breeding box and breeding density

During the period of acclimatization, administration and observation, they were bred in a polycarbonate cage for rodents (W 170 x L 235 x H 125 mm) at a maximum of 5 animals / cage.

(8) breeding management

Breeding boxes, rugs and water bottles were exchanged at least once a week.

(9) group separation

Diabetes was confirmed by measuring body weight and blood sugar during the acclimatization period, and then randomly distributed so that the average blood sugar and body weight of each group were distributed as evenly as possible.

[Example 2]

Composition of test group, preparation of administration test substance, and administration

(1) Composition of the test group

The composition of the test group and the setting of the dose were shown in Table 1 below as the group, sex, number of animals, administered material, dose, and dose were set.

army gender Number of animals (number of animals) animal number substance to be administered
(mixing ratio, v/v) Dosage (mg/kg) Dosage volume (mL/kg) G1* M 5 1-5 Distilled water - 5 G2 M 5 6-10 Distilled water - 5 G3 M 5 11-15 metformin 100 5 G4 M 5 16-20 mulberry leaves 30 5 G5 M 5 21-25 Paper mulberry leaves: mulberry fruit (1:1) 30 5

*, Group G1 is a normal animal (C57BLK/6), and G2~G5 is a db/db mouse.

(2) Preparation of administered test substance

According to the test group composition, the test substances mulberry leaf and mulberry fruit were prepared at 6 mg/mL for the G4 group. Group G5 was prepared by mixing the same volume (v/v) of mulberry leaves and mulberry fruit prepared in advance at 6 mg/mL. G3 group metformin (Metformin) is prepared by dissolving 20 mg/mL. When administered, it was prepared and used daily.

(3) administration

Oral administration was selected as the route of administration. The frequency and duration of administration were administered once/day, for 5 weeks. The dose amount was calculated and administered at 5 mL/kg/day based on the body weight measured on the latest weight measurement day. As for the administration method, animals were fixed by the method of fixing the skin of the cervical region and administered directly into the stomach using a sonde for oral administration.

[Example 3]

GTT experiment in db/db diabetic mice

GTT (glucose tolerance test) was performed to confirm the glucose processing ability (glucose tolerance) of pancreatic beta cells. Blood glucose was measured using a blood glucose meter (ACCU-CHEK® Performa, Roche Diagnostics, Mannheim, Germany). After intraperitoneal administration of glucose at a dose of 2 g/kg to the animal on the test day, blood glucose was measured using a blood glucose meter at a predetermined time. Blood glucose was measured at 0 (before glucose administration) and at 15, 30, 60, 90, 120, 180 and 240 minutes after glucose administration. Five weeks after administration of the test substance, the animals were fasted for at least 16 hours. Blood glucose measurement was measured by diluting the blood (3 uL) drawn with PBS (6 uL) three-fold in consideration of the upper limit of the measuring device. The result value was measured by calculating the area-under the curve (AUC).

As shown in FIG. 1 , it was confirmed that the GTT AUC level was increased in the db/db mice compared to the normal group. As a positive control compared to db/db mice, it was confirmed that the AUC values of the metformin-treated group and the mulberry leaf-treated group were hardly reduced. However, it was confirmed that the AUC level was significantly reduced in the mulberry leaf and mulberry fruit (1:1) treatment group when compared with the metformin treatment group.

[Example 4]

Efficacy of reducing ITIH1 expression in db/db diabetic mouse liver tissue

On the day of autopsy, the animals were anesthetized by inhalation with ether, and when anesthesia was confirmed, the animals were opened and blood was collected from the posterior vena cava using a syringe. The collected blood was injected into a vacuum tube containing a clot activator, left at room temperature for about 15-20 minutes to coagulate, and then centrifuged at 3,000 rpm for 10 minutes. After blood collection, the abdominal aorta and posterior vena cava were cut and exsanguinated/killed, and the liver tissue was excised and weighed, and stored at -80°C for protein extraction.

After homogenization of the obtained liver tissue, the expression of ITIH1 protein was confirmed through western blot. Specifically, cell lysis buffer (RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), 120 mM, Protease Inhibitor Cocktail (AEBSF, Aprotinin, Cell lysates were obtained by lysing cells with Bestatin, E64, Leupeptin, and Pepstatin A), Phosphatase Inhibitor Cocktail (sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate), and the cell lysate was mixed with sample buffer (60 mM). Tris-HCl (pH 6.8), 25% glycerol, 0.1% bromophenol blue, 2% SDS, 2% mercapto ethanol) was mixed and incubated for 10 minutes in a heating block at 100°C. The supernatant was collected by centrifugation at 13,000 rpm and 4° C. for 5 minutes, and each sample was loaded and electrophoresed at 120 V by making an SDS PAGE gel (12%). After transferring the entire protein separated in the SDS PAGE gel to the NC membrane, ITIH1 (matured form) and β-actin antibodies were used to confirm the expression of ITIH1 protein and quantify the protein band.

As shown in FIG. 2 , it was confirmed that the ITIH1 protein expression in the liver tissue of the db/db mice was increased compared to the normal group. ITIH1 protein expression was decreased in the metformin-treated group as a positive control compared to db/db mice. It was confirmed that the expression of ITIH1 protein in the mulberry leaf-treated group was decreased compared to the metformin-treated group, and the ITIH1 protein expression was the most decreased in the mulberry leaf and mulberry fruit (1:1)-treated group.

As a result, it can be seen that the mulberry and mulberry complex extracts have significantly superior effects in improving glucose tolerance and reducing ITIH1 protein expression, compared to metformin and the mulberry single extract.

Claims (14)

A pharmaceutical composition for improving insulin sensitivity, comprising a complex extract of mulberry ( Broussonetia kazinoki ) and sputum ( Juglans mandshurica ).
The pharmaceutical composition for improving insulin sensitivity according to claim 1, characterized in that it has effects of increasing insulin sensitivity and decreasing insulin resistance.
According to claim 1, Diabetes, diabetes complications, impaired glucose tolerance, and insulin resistance syndrome (Insulin resistance syndrome) characterized in that for preventing or treating one or more diseases selected from the group consisting of, the pharmaceutical composition.
The pharmaceutical composition according to claim 3, wherein the diabetic complications are at least one disease selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy.
The method of claim 3, wherein the insulin resistance syndrome is one or more diseases selected from the group consisting of insulin resistance-induced obesity, hypertension, arteriosclerosis, hyperlipidemia, hyperinsulinemia, nonalcoholic fatty liver, and type 2 diabetes. , pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the mulberry extract is a mulberry leaf extract.
The pharmaceutical composition according to claim 1, wherein the extract is a shamrock fruit extract.
The pharmaceutical composition of claim 1, wherein the complex extract of mulberry and mulberry trees reduces the expression of ITIH1 (Inter-alpha Trypsin Inhibitor Heavy chain 1) mRNA or protein.
A food composition for improving insulin sensitivity, comprising a complex extract of mulberry and mulberry.
The food composition for improving insulin sensitivity according to claim 9, characterized in that it has effects of increasing insulin sensitivity and decreasing insulin resistance.
According to claim 9, diabetes, diabetes complications, impaired glucose tolerance, and insulin resistance syndrome (Insulin resistance syndrome) characterized in that for preventing or improving one or more diseases selected from the group consisting of, the food composition.
A feed composition for animals for improving insulin sensitivity, comprising a complex extract of mulberry and mulberry.
The feed composition for animals for improving insulin sensitivity according to claim 12, characterized in that it has effects of increasing insulin sensitivity and decreasing insulin resistance.
According to claim 12, Diabetes, diabetes complications, impaired glucose tolerance, and insulin resistance syndrome (Insulin resistance syndrome) characterized in that for preventing or improving one or more diseases selected from the group consisting of, animal feed composition.

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