KR102167112B1 - Composition for prevention or treatment of diabetes comprising phenylalkyl glucopyranoside derived from Brugmansia arborea - Google Patents

Abstract

The present invention relates to a phenylalkyl glucopyranoside compound isolated from angel's morning glory ( Brugmansia arborea ), a composition for preventing, improving or treating diabetes comprising the same, and a method for preparing the compound.
The fraction of the angel's morning glory extract of the present invention and the phenylalkyl glucopyranoside compound isolated therefrom restores pancreatic islets damaged by alloxane in zebrapac larvae, and prevents, improves or treats diabetes pharmaceutical compositions, foods It can be used as a composition.

Description

Composition for prevention or treatment of diabetes comprising phenylalkyl glucopyranoside derived from Brugmansia arborea}

The present invention is angelic morning glory ( Brugmansia arborea ) extract or a phenylalkyl glucopyranoside compound isolated from a fraction thereof; A composition for preventing, improving or treating diabetes containing the angel's morning glory fraction or the compound; And a method for preparing the compound.

Diabetes mellitus is a disease that causes abnormalities in carbohydrate metabolism due to insufficient insulin or low sensitivity to insulin. Insulin, a polypeptide hormone, is made by β-cells of the pancreatic islets (PI) in the pancreas. It is secreted when the concentration of glucose in the blood increases, and when the concentration of glucose decreases, the secretion is suppressed, and thus the proper activity of the energy source. Is adjusted. In diabetic patients, a decrease in the number of β-cells and its dysfunction occurred.

The American Diabetes Association divides the medical classification of diabetes into two categories: insulin-dependent diabetes (type 1) and insulin-independent diabetes (type 2). The pathogenesis of insulin-independent diabetes (type 2), which accounts for more than 95% of diabetes mellitus, is known to be a combination disorder of insulin secretion disorder and insulin resistance. Insulin secretion disorder refers to a situation in which an appropriate amount of insulin is not secreted from the β-cells of the pancreas according to the blood glucose concentration, and this includes both a quantitative decrease in insulin-secreting β-cells or a functional secretion disorder. Insulin resistance refers to a situation in which the secreted insulin reaches the target organ through the bloodstream, but the insulin action and sensitivity in the target cell are reduced.It is generally considered to be a signal transduction disorder after cell membrane receptor binding, and the cause is a genetic predisposition. , Obesity, decreased physical activity, and hyperglycemia or dyslipidemia. If there is insulin resistance, a larger amount of insulin must be secreted to overcome the resistance. Conversely, high blood sugar itself caused by insufficient insulin secretion may worsen insulin resistance again. If diabetes is left untreated, diabetic retinopathy, kidney disease, mastopathy, cardiovascular complications, and microvascular complications may occur. Therefore, it is possible to prevent death from diabetes and reduce mortality through the control of insulin or hypoglycemic drugs.

Therefore, repairing damaged pancreatic β-cells or increasing glucose uptake in pancreatic islet cells can be a meaningful treatment for diabetes (Sunil C et al., Effect of ethanolic extract of Pisonia alba Span. leaves on blood glucose. levels and histological changes in tissues of alloxan-induced diabetic rats.Int J Appl Res Nat Prod 2009;2:4-11). Substances used in diabetes treatments to date are drugs that increase insulin secretion or increase sensitivity to insulin, pioglitazone and rosiglitazone, which enhance insulin response, metformin, which improves insulin resistance, There are sulfonylurea and meglitinide series, which stimulate insulin secretion in the β-cells of the pancreas. However, since these drugs have side effects such as weight gain along with toxicity to the liver, kidneys, muscles and heart, there are few side effects and no weight gain, etc., and effectively treat or prevent diabetes. The necessity of development is urgently required.

Angel's Morning Glory ( Brugmansia arborea ) is an evergreen shrub distributed in the United States, Africa, Australia and Asia, reaching a height of 3 to 11 m. This plant is used as an analgesic, rheumatoid arthritis, decongestant, antispasmodic, etc., and has been reported to show anticholinergic activity. In addition, many alkaloid compounds have been isolated from the entire angel's morning glory. However, few studies have been made on the chemical composition of the angel's morning glory flower and its biological activity.

Under this background, the present inventors have made diligent efforts to find a substance having an antidiabetic effect derived from a natural product, and as a result of separating and identifying 6 kinds of phenylalkyl glucopyranoside compounds from the flower extract of the morning glory of an angel or a fraction thereof, when these are treated Damaged pancreatic islets were recovered and confirmed that they had an antidiabetic effect, and the present invention was completed.

Capasso, A.; De Feo, V.; De Simone, F.; Sorrentino, L. Int J Pharmacogn 1997, 35, 43-48.

One object of the present invention is to provide a compound represented by the following formula (1):

[Formula 1]

In Formula 1, R is glucopyranosyl.

Another object of the present invention is a fraction of the morning glory extract of angels; Or a compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt of the compound, isolated therefrom, as an active ingredient, to provide a pharmaceutical composition for preventing or treating diabetes:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

Another object of the present invention is a fraction of an angel's morning glory extract; Or it is to provide a food composition for preventing or improving diabetes, comprising a compound represented by the following Formula 1 or Formula 2, or a food pharmaceutically acceptable salt of the compound, isolated therefrom:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

Another object of the present invention is to provide a method for preparing a compound represented by Formula 1 or Formula 2, comprising separating a compound represented by the following Formula 1 or Formula 2 from an angel's morning glory extract or a fraction thereof will be:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

One aspect of the present invention for achieving the above object provides a compound represented by the following Formula 1:

[Formula 1]

In Formula 1, R is glucopyranosyl.

The compound represented by Formula 1 is (2 S , 3 S )-1-phenyl-2,3-dihydroxybutyl O - β -D-glucopyranosyl-(1→6)- β -D-glucopy It may be ranoside ((2 S , 3 S )-1-phenyl-2,3-dihydroxybutyl O - β -D-glucopyranosyl-(1→6)- β -D-glucopyranoside).

Specifically, the compound represented by Formula 1 may be isolated from an angel's morning glory extract or a fraction thereof.

The compound is a novel, unknown compound, and was first identified in the present invention.

In an embodiment of the present invention, a methanol extract of dried morning glory flowers of an angel was obtained, an n -butanol fraction was obtained therefrom, and a silica gel column chromatography was applied to the fraction to separate the compound represented by Formula 1 .

In addition, when the compound represented by Formula 1 was treated with the zebrafish larvae in which the pancreatic islet was damaged due to alloxane treatment, it was confirmed that the damaged pancreatic islet was recovered, and the compound had an antidiabetic effect.

Another aspect of the present invention for achieving the above object is a fraction of an angel's morning glory extract; Or it provides a pharmaceutical composition for preventing or treating diabetes, comprising, as an active ingredient, a compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt of the compound, isolated therefrom:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

The'compound represented by Formula 1'is as described above.

In the present invention, the term'Angel 's Morning Glory ( Brugmansia arborea )' is a flowering plant belonging to the Solanaceae family, and can be purchased commercially or harvested or cultivated in nature. Specifically, the angel's morning glory flowers, roots, stems, leaves, etc. may be used without limitation, and flowers may be preferably used, but the present invention is not limited thereto.

In the present invention, the term'extract' refers to an extract obtained by extracting the morning glory of an angel, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a crude purified product or purified product of the extract, or these It includes the extract of all formulations that can be formed using the extract itself and the extract, such as a mixture of the extract.

In the present invention, the type of the extraction solvent used to extract the extract is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C ₁ to C ₄ lower alcohols such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; And hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and specifically, methanol may be used, but is not limited thereto.

In the present invention, a method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, and these may be performed alone or in combination of two or more methods. Any method of producing an angel's morning glory extract to separate a fraction or compound having a diabetic treatment effect may be used without limitation.

In the present invention, the term "fraction" means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. Specifically, the present invention includes the result of fractionation of the angel's morning glory extract by various methods such as solvent fractionation, ultrafiltration fractionation, and chromatography fractionation, and more specifically, It may be an ethyl acetate fraction or an n -butanol fraction, but is not limited thereto.

In an embodiment of the present invention, the dried morning glory flowers of angels were extracted with 80% methanol at room temperature, and concentrated to obtain an extract, and an ethyl acetate (EtOAc) fraction and an n -butanol ( n- BuOH) fraction were obtained from the extract. Obtained.

In addition, when the ethyl acetate fraction and n-butanol fraction were treated with the zebrafish larvae in which pancreatic islets were damaged due to alloxane treatment, it was confirmed that the damaged pancreatic islets were recovered, and the fractions had an antidiabetic effect.

In the present invention, the term'compound represented by Formula 2'

[Formula 2]

And, in Formula 2, R ₁ is benzyl, phenylethyl or phenylpropyl, and R ₂ is H or glucopyranosyl.

Specifically, the compound represented by Formula 2

Benzyl O - β -D-glucopyranoside (benzyl O - β -D-glucopyranoside),

Benzyl O - β -D- glucopyranosyl - (1 → 6) - β -D- gluconic nose Llano side (benzyl O - β -D-glucopyranosyl- (1 → 6) - β -D-glucopyranoside),

2-phenylethyl O - β -D- gluconic nose side pyrano (2-phenylethyl O - β -D -glucopyranoside),

2-phenylethyl O - β -D- glucopyranosyl - (1 → 6) - β -D- gluconic nose side pyrano (2-phenylethyl O - β -D -glucopyranosyl- (1 → 6) - β -D- glucopyranoside) or

3-phenylpropyl O - β -D- gluconic nose Llano side (3-phenylpropyl O - β -D -glucopyranoside) may be.

Specifically, the compound represented by Formula 2 may be isolated from an angel's morning glory extract or a fraction thereof. In addition, the compound represented by Formula 2 may be isolated from other natural sources, chemically synthesized through a method known in the art, or may be a commercially available material, including the present invention regardless of the access route Can be used in

In one embodiment of the present invention, a methanol extract of dried morning glory flowers of an angel was obtained, ethyl acetate fraction and n -butanol fraction were obtained therefrom, and then silica gel column chromatography was applied to the fractions to obtain the five compounds. Was separated.

In addition, when the above five compounds were treated to zebrafish larvae in which pancreatic islets were damaged due to alloxane treatment, it was confirmed that the damaged pancreatic islets were recovered, thereby confirming that the compounds had antidiabetic effects.

In the present invention, the term'diabetes' refers to a disease that occurs when the secretion of insulin is insufficient or the action and function of insulin is not sufficiently performed. In case of diabetes, excessive decomposition of glycogen, protein, and fat causes an abnormal increase in glucose concentration in the liver or blood, resulting in diabetes and ketonuria, and circulation disorders along with blood concentration due to electrolyte loss due to abnormal water and electrolyte metabolism, It leads to pathological conditions such as kidney failure. Insulin is secreted by β-cells of the pancreatic islet present in the pancreas, and is secreted when the concentration of glucose in the blood increases, and when it decreases, secretion is suppressed to regulate the proper activity of energy sources. Diabetes can be diagnosed by measuring blood glucose concentration. In humans, diabetes is usually diagnosed when blood glucose is usually 200 mg/dl or more and 140 mg/dl or more when fasting. Therefore, it can be usefully used for treating or preventing diabetes by recovering damaged pancreatic β-cells or increasing glucose absorption in pancreatic islet cells.

In the present invention, the term'prevention' refers to all actions of inhibiting or delaying diabetes by administering the composition of the present invention.

In the present invention, the term'treatment' means all actions in which symptoms of diabetes are improved or cured by administration of the composition of the present invention.

In the present invention, diabetes prevention or treatment may be achieved by increasing the size of the damaged pancreatic islet.

In the present invention, the term'pharmaceutical composition' means that it is prepared for the purpose of preventing or treating diseases, and can be administered in various oral and parenteral formulations during actual clinical administration. In the case of formulation, usually used fillers, extenders, It can be prepared using diluents or excipients such as binders, wetting agents, disintegrants, and surfactants.

In addition, pharmaceutically acceptable additives may be further included according to each formulation, and in this case, pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , Lactose, mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol, talc, and the like can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are at least one excipient in the composition of the present invention, such as starch, calcium carbonate, and sucrose. (Sucrose), lactose (Lactose) or gelatin can be mixed to prepare. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. I can.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method You can choose. The dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention can be administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health condition, type of disease, severity, The activity of the drug, its sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. In addition, the pharmaceutical composition of the present invention may be used alone or in combination with other pharmaceutically active compounds exhibiting an effect of preventing, treating, or improving cancer, or in a suitable set.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

Another aspect of the present invention for achieving the above object is a fraction of an angel's morning glory extract; Or it provides a food composition for preventing or improving diabetes, comprising a compound represented by the following Formula 1 or Formula 2, or a food pharmaceutically acceptable salt of the compound, isolated therefrom:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

'Angel's morning glory','extract','fraction','compound represented by formula 1','compound represented by formula 2','diabetes' and'prevention' are as described above.

In the present invention, the terms'pharmaceutically acceptable salt' and'food acceptable salt' are in a form that can be used pharmaceutically and food, respectively, among salts, which are substances in which cations and anions are bound by electrostatic attraction. It refers to a salt, and is a concentration that has a relatively non-toxic and harmless effect to the patient, and side effects caused by this salt do not degrade the beneficial efficacy of the compound represented by Formula 1 or Formula 2 of the present invention. Means any organic or inorganic addition salt. Such salts include acid addition salts formed by pharmaceutically or food-acceptable free acids or metal salts formed by bases.

Inorganic acids and organic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid can be used as organic acids. (maleic acid), succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used. , Is not limited thereto.

In addition, a base can be used to make a pharmaceutically or food acceptable metal salt. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-dissolved compound salt, and evaporating and drying the filtrate. At this time, the metal salt is particularly suitable for pharmaceutical or food, but is not limited thereto to prepare sodium, potassium, or calcium salt. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Unless otherwise specified, the pharmaceutically acceptable salt and the food pharmaceutically acceptable salt include acidic or basic salts that may be present in a compound isolated from the angel's morning glory extract or fractions thereof of the present invention. For example, pharmaceutically or food acceptable salts may include sodium, calcium and potassium salts of a hydroxy group, and other pharmaceutically or food pharmaceutically acceptable salts of an amino group include hydrobromide, sulfate, and hydrogen. Sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, It can be prepared through a method for preparing a salt known in the art.

In the present invention, the term'improvement' means all actions that reduce the degree of diabetes symptoms.

In the present invention, the term'food' includes, for example, various foods, beverages, gums, teas, vitamin complexes, health function (sex) foods, and the like, and includes all foods in the usual sense. The food can be prepared in formulations such as tablets, granules, powders, capsules, liquid solutions, and pills according to a known manufacturing method, and the content of the compound according to the present invention or its food pharmaceutically acceptable salt is food depending on the formulation. It can be adjusted to 0.0001% to 100% by weight based on the total weight of the composition. Except for including the compound of the present invention or a food acceptable salt thereof as an active ingredient, there is no particular limitation on other ingredients, and various conventional flavoring agents or natural carbohydrates may be included as additional ingredients.

'Health function (sex) food' is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects that are processed to efficiently display bioregulatory functions in addition to nutrition supply. do. The "function (sex)" refers to obtaining an effect useful for health use, such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of production, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food may be prepared without limitation as long as it is a formulation recognized as a food. The health functional food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, there are no side effects that may occur when taking the drug for a long time using food as a raw material, and has an advantage of excellent portability.

Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, gums, confectionery, or encapsulating, powdering, and suspending. It means bringing about an effect, but unlike general drugs, it has the advantage of having no side effects that may occur when taking the drug for a long time by using food as a raw material.

The health functional food composition may further include a physiologically acceptable carrier, and the type of carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the health functional food composition may include, without limitation, additional ingredients that are commonly used in food compositions to improve smell, taste, vision, etc., and may be selected according to the type of food and used in an appropriate amount.

Another aspect of the present invention for achieving the above object is a compound represented by Formula 1 or Formula 2 comprising the step of separating a compound represented by the following Formula 1 or Formula 2 from an angel's morning glory extract or a fraction thereof Provides a method of manufacturing:

[Formula 1]

[Formula 2]

In the above formula,

R is glucopyranosyl,

R ₁ is benzyl, phenylethyl or phenylpropyl,

R ₂ is H or glucopyranosyl.

The'angel morning glory','extract','fraction','compound represented by formula 1'and'compound represented by formula 2'are as described above.

Fractions of the morning glory extract of the angel of the present invention; The compound represented by Chemical Formula 1 or Chemical Formula 2 isolated therefrom can be used as a bar for recovering pancreatic islets damaged by alloxane, as a pharmaceutical composition for preventing, improving or treating diabetes, a food composition, and the like.

Figure 1 shows the six compounds isolated from the morning glory extract or a fraction thereof of an angel in an embodiment of the present invention.
Figure 2 shows the results confirming that the fraction of the morning glory extract of angel and the compound of Figure 1 have a recovery effect on pancreatic islets damaged by alloxane in zebrafish larvae. At this time, NOR: normal group, AX: alloxane treatment group, GLM: alloxane and glimepiride treatment group, BAFE: alloxane and ethyl acetate fraction treatment group BAFB: Alloxane and n -butanol fraction treatment group, Compounds 1 to 6: Alloxane and Compounds 1 to 6 respectively mean treatment groups. ( ^### p <0.001; compared with the normal group), (* p <0.05, ** p <0.01, *** p <0.001; compared with the alloxane treatment group). Scale bar = 100 μm.
FIG. 3 shows the results of confirming whether or not there is a recovery effect on the damaged pancreatic islet after further treatment of diazooxide (DZ) in the experimental group of FIG. 2 in order to clarify the mechanism of the recovery effect shown in FIG. 2. ( ^## p <0.01, ^### p <0.001; compared with normal group), (* p <0.05, ** p <0.01, *** p <0.001; compared with alloxane treatment group), ( + p <0.05, ++ p <0.01; compared to the non-DZ treatment group). Scale bar = 100 μm.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Materials, reagents and equipment

1-1: plant material

The dried flowers of the angel's morning glory used in this example were supplied from Herb Island in Pocheon, Korea in June 2014, and were confirmed by Professor Dae-geun Kim of Woosuk University in Jeonju. The voucher specimen (KHU2014-0623) is stored in the Laboratory of Natural Products Chemistry, Kyunghee University in Yongin, Korea.

1-2: reagents and instruments

Silica gel (SiO ₂ ) and octadecyl SiO ₂ (ODS) used in column chromatography (CC) were Kysel gel 60 (Merck, Darmstadt, Germany), Lichroprep RP-18 (40-60 μm, Merck) and Sephadex LH- 20 (Amersham Biosciences, Uppsala, Sweden).

Thin layer chromatography (TLC) was analyzed using Kysel gel 60 F ₂₅₄ and RP-18 F ₂₅₄ S (Merck) TLC plates. Spots were detected using a UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, Westbury, NY, USA) and a 10% H ₂ SO ₄ solution.

Nuclear magnetic resonance (NMR) spectrum was measured using a 400 MHz FT-NMR spectrometer (Varian Inova AS-400, Palo Alto, CA, USA).

Deuterium solvent was purchased from Merck and Sigma Aldrich (St, Louis, MO, USA).

IR spectra were measured using a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England).

Electron ionization mass spectrometry (EI/MS) and fast atomic bombardment mass spectrometry (FAB/MS) spectra were measured using JEOL JMS-700 (Tokyo, Japan).

Melting point was measured using a Fisher-John melting point apparatus (Fisher Scientific, Miami, FL, USA) without modification.

Example 2: Preparation of extract and fraction of morning glory of angel

The dried flowers of the angel's morning glory (900 g, dry weight) were extracted with 80% aqueous methanol (27L Х 3) at room temperature for 24 hours. 160 g of the concentrated methanol extract was suspended in 1 L of water, followed by continuous extraction with ethyl acetate (EtOAc, 1 L Х 3) and n -butanol ( n -BuOH, 0.6 L Х 3). The extract was concentrated to yield an EtOAc fraction (BAFE, 12g), an n- BuOH fraction (BAFB, 16g) and an H ₂ O fraction (BAFW, 132g), respectively.

Example 3: Isolation and Identification of Compounds from Fractions of Angel's Morning Glory

3-1: Ethyl acetate fraction of angel's morning glory and n -Isolation of compounds from the butanol fraction

The ethyl acetate fraction was applied to SiO ₂ CC (7.0 Х 15.0 cm) and n -hexane-EtOAc (4: 1 → 2: 1 → 1: 1, 2.0 L each) and CHCl ₃ -MeOH-H ₂ O (50: 3: 1 → 36: 3: 1 → 25: 3: 1 → 18: 3: 1 → 12: 3: 1 → 9: 3: 1 → 7: 3: 1 → 6: 4: 1, 1.5 L each) Eluted with. The eluate was monitored by TLC to obtain 16 fractions (BAFE-1 to BAFE-16). BAFE-10 [966.2 mg, elution volume/total volume (V _e /V _t ) 0.649-0.718] was added to ODS CC (4.5 x 10 cm) and acetone-H ₂ O (1: 2 → 1: 1, both 3.7 L) to give 16 fractions (BAFE-10-1 to BAFE-10-16). Apply BAFE-10-5 (64.2 mg, V _e /V _t 0.020-0.034) to SiO ₂ CC (2.5 x 7 cm) and use CHCl ₃ -MeOH-H ₂ O (30: 3: 1, 1.2 L). Purified compound 5 [BAFE-10-5-4, 13.9 mg, V _e /V _t 0.458- by elution and ultimately with 6 fractions (BAFE-10-5-1 to BAFE-10-5-6) 0.733, TLC(SiO ₂ F ₂₅₄ ) R _f 0.54, CHCl ₃ -MeOH-H ₂ O=9:3:1] was obtained.

n -Butanol fraction (BAFB) was applied to SiO ₂ CC (7.0 Х 13.0 cm) and CHCl ₃ -MeOH-H ₂ O (12: 3: 1 → 9: 3: 1 → 6: 4: 1, each 12.0 L ) To obtain 14 fractions (BAFB-1 to BAFB-14). Apply BAFB-5 (447.5 mg, V _e /V _t 0.063-0.075) to Sephadex-LH-20 CC (2.5 x 50 cm) and elute with MeOH-H ₂ O (4: 1, 296 mL) to Fractions (BAFB-5-1 to BAFB-5-6) were obtained. BAFB-5-3 (151.9 mg, V _e /V _t 0.446-0.500) was added to ODS CC (2.0 Х 17 cm) and eluted with MeOH-H ₂ O (1: 2, 485 mL) to 9 fractions (BAFB -5-3-1 to BAFB-5-3-9), as well as compound 1 [BAFB-5-3-3, 88.0 mg, V _e /V _t 0.155-0.196, TLC(SiO2 F ₂₅₄ ) R _f 0.44 , CHCl ₃ -MeOH-H ₂ O=12:3:1] and compound 3 [BAFB-5-3-6, 20.5 mg, V _e /V _t 0.289-0.330, TLC(SiO ₂ F ₂₅₄ ) R _f 0.44 , CHCl ₃ -MeOH-H ₂ O=12:3:1] was obtained. Apply BAFB-9 (1.2g, V _e /V _t 0.163-0.225) to SiO ₂ CC (4.0x15.0cm) and elute with CHCl ₃ -MeOH-H ₂ O (10: 3: 1, 3.1 L) 14 fractions (BAFB-9-1 to BAFB-9-14) were obtained. BAFB-9-8 (487.7 mg, V _e /V _t 0.308-0.607) was applied to SiO ₂ CC (3.5 x 15.0 cm) and EtOAc- n -BuOH-H ₂ O (22: 3: 1, 2.7 L) Elution with 15 fractions (BAFB-9-8-1 to BAFB-9-8-15) were obtained. BAFB-9-8-15 (33.7 mg, V _e /V _t 1.000) was applied to ODS CC (2.0 Х 5.0 cm) and eluted with MeOH-H ₂ O (1: 3, 430 mL), ultimately resulting in 7 With fractions (BAFB-9-8-15-1 to BAFB-9-8-15-7), compound 6 [BAFB-9-8-15-3, 12.0 mg, V _e /V _t 0.070-0.140, TLC (SiO ₂ F ₂₅₄ ) R _f 0.50, EtOAc- n -BuOH-H ₂ O=4:5:1] was obtained.

BAFB-12 (899.3 mg, V _e /V _t 0.392-0.495) was added to ODS CC (3.5 Х 5.0 cm) and eluted with MeOH-H ₂ O (1: 3, 2.9 L) to obtain 12 fractions (BAFB-12 -1 to BAFB-12-12) were obtained. Apply BAFB-12-5 (182.2 mg, V _e /V _t 0.082-0.149) to SiO ₂ CC (2.5 x 16.0 cm) and use CHCl ₃ -MeOH-H ₂ O (9: 3: 1, 1.1 L) Elution and ultimately with 8 fractions (BAFB-12-5-1 to BAFB-12-5-8), compound 4 [BAFB-12-5-3, 104.3 mg, V _e /V _t 0.407-0.433, TLC(SiO ₂ F ₂₅₄ ) R _f 0.54, CHCl ₃ -MeOH-H ₂ O=6:4:1] was obtained. BAFB-13 (1.8g, V _e /V _t 0.495-0.575) was applied to ODS CC (4.0 Х 9.0cm) and eluted with MeOH-H ₂ O (1: 2, 2.9 L) to 13 fractions (BAFB- 13-1 to BAFB-13-13) were obtained. BAFB-13-5 (128.7 mg, V _e /V _t 0.075-0.097) was applied to ODS CC (2.5 x 7.0 cm) and eluted with MeOH-H ₂ O (1: 2, 300 mL), ultimately 8 Fractions of dogs (BAFB-13-5-1 to BAFB-13-5-8), as well as compound 2 [BAFB-13-5-2, 10.3 mg, V _e /V _t 0.120-0.140, TLC (SiO ₂ F ₂₅₄ ) R _f 0.57, CHCl ₃ -MeOH-H ₂ O=6:4:1] was obtained.

3-2: Identification of isolated compounds 1 to 6

Each compound was identified for the compounds 1 to 6 isolated in Example 3-1.

The results of analyzing compound 1 are as follows:

-White amorphous powder (CH ₃ OH);

-43° (c=0.10, CH₃OH); -

-43° ( c =0.10, CH ₃ OH);

-Positive FAB-MS m/z 293 [M+Na] ⁺ ;

-IR (KBr, v) 3502, 1697, 1530 cm ^-1 ;

^{-The data of 1} H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Tables 1 and 2 below.

As a result of the analysis, it was confirmed that Compound 1 was benzyl O - β -D-glucopyranoside.

The results of analyzing compound 2 are as follows:

-White amorphous powder (CH ₃ OH);

-90° (c=0.20, CH₃OH);

-90° ( c =0.20, CH ₃ OH);

-Positive FAB-MS m/z 433 [M+H] ⁺ ;

-IR (KBr, v) 3510, 1671, 1600 cm ^-1 ;

^{-The data of 1} H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Tables 1 and 2 below.

Analysis, the compound is 2-benzyl-O - was identified as β -D- gluconic nose Llano side - β -D- glucopyranosyl - (1 → 6).

The results of analyzing compound 3 are as follows:

-White amorphous powder (CH ₃ OH);

-35.3° (c=0.50, CH₃OH);

-35.3° ( c =0.50, CH ₃ OH);

-Positive FAB-MS m/z 285 [M+H] ⁺ ;

-IR (KBr, v) 3368, 1683, 1573 cm ^-1 ;

-The data of ¹ H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Tables 1 and 2 below.

As a result of analysis, it was confirmed that Compound 3 was 2-phenylethyl O - β -D-glucopyranoside.

The results of analyzing compound 4 are as follows:

-White amorphous powder (CH ₃ OH);

-82° (c=0.20, CH₃OH); -

-82° ( c =0.20, CH ₃ OH);

-Positive FAB-MS m/z 447 [M+H] ⁺ ;

-IR (KBr, v) 3370, 1685, 1586 cm ^-1 ;

^{-The data of 1} H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Tables 1 and 2 below.

Was identified as β -D- gluconic nose Llano side-analysis results of Compound 4 is 2-phenyl ethyl O - β -D- glucopyranosyl - (1 → 6).

The results of analyzing compound 5 are as follows:

-White amorphous powder (CH ₃ OH);

-29.3° (c=0.75, CH₃OH);

-29.3° ( c =0.75, CH ₃ OH);

-Positive FAB-MS m/z 299 [M+H] ⁺ ;

-IR (KBr, v) 3364, 1687, 1574 cm ^-1 ;

-The data of ¹ H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Tables 1 and 2 below.

As a result of the analysis, it was confirmed that Compound 5 was 3-phenylpropyl O - β -D-glucopyranoside.

The results of analyzing compound 6 are as follows:

-White amorphous powder (CH ₃ OH);

-23.0° (c=0.10, CH₃OH);

-23.0° ( c =0.10, CH ₃ OH);

-HR-positive FAB-MS m/z 491.2125 [M+H] ⁺ (calculated for C ₂₂ H ₃₅ O ₁₂ 491.2128);

-IR (KBr, v) 3417, 1605, 1507 cm ^-1 ;

-The data of ¹ H- and ¹³ C-NMR (400 MHz and 100 MHz, CD ₃ OD, δ _H and δ _C ) are shown in Table 3 below.

As a result of the analysis, compound 6 is (2 S ,3 S )-1-phenyl-2,3-dihydroxybutyl O - β -D-glucopyranosyl-(1→6)- β -D-glucopyranoside. Confirmed. Compound 6 is a novel compound that was first isolated and identified from natural sources.

[Table 1]

¹ H-NMR spectral data of compounds 1 to 5 (CD ₃ OD; 400 MHz).

(δ in ppm, J in Hz)

[Table 2]

¹³ C-NMR spectrum data of compounds 1 to 5 (CD ₃ OD; 100 MHz).

(δ in ppm)

[Table 3]

¹ H-NMR and ¹³ C-NMR spectral data of compound 6 in CD ₃ OD (δ _H , 600 MHz; δ _C , 150 MHz in ppm, coupling pattern, J in Hz)

Example 4: Evaluation of the recovery efficacy of pancreatic islet damaged by alloxan in zebrafish larvae

4-1: Zebrafish (ZF) management and maintenance

Adult zebrafish were maintained in an S-type ZF system (1500[W] Х 400[D] Х 2050[H] mm) (Daejeon, Korea), and repeated photoperiods of light for 14 hours and dark for 10 hours at 28.5°C. To obtain zebrafish larvae, two pairs of adult zebrafish were placed in a spawning box overnight. The zebrafish lay their eggs for 30 minutes while giving the light. For cultivation, zebrafish embryos were harvested 3 hours after fertilization, and maintained in 0.03% sea salt solution at 28.5° C. with light and 10 hours dark photoperiod for 14 hours. Zebrafish were treated according to the standard zebrafish protocol, and all experiments were approved by the Animal Care and Use Committee of Kyunghee University (KHUASP[SE]-15-10).

4-2: statistical analysis

Statistical analysis was performed using GraphPad Prism (version 5, GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as the mean ± standard error of the mean (SEM) for 3 replicates. Statistical significance was determined using an iterative one-way ANOVA followed by Tukey's post-hoc test. P <0.05 was considered statistically significant.

4-3: Evaluation of recovery efficacy of pancreatic islets damaged by alloxane

Zebrafish larvae were normal group (NOR), alloxane treatment group (AX), glimepiride treatment group after alloxane treatment (GLM, positive control), ethyl acetate fraction treatment group of angel's morning glory after alloxane treatment (BAFE), allox Of the n-butanol fraction treatment group (BAFB) of angel's morning glory after acid treatment, compound 1 to compound 6 treatment group (respectively, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6) after alloxane treatment It was classified into a total of 11 groups.

Five days after fertilization, wild-type zebrafish larvae were placed in 96-well plates. The zebrafish larvae were exposed to 100 μM alloxane for 15 minutes to induce pancreatic islet damage.

Thereafter, in order to confirm the recovery efficacy of the angel's morning glory fraction and each compound, each fraction (1 μg/mL) and each compound (1 μg/mL) were treated for 1 hour according to the 11 group classifications. . Glimepiride was treated with 10 μg/mL.

Then, zebrafish larvae were stained with 25 μM 2-NBDG for 1 hour and rinsed with 0.03% sea salt solution for 20 minutes to obtain pancreatic islet images. After staining, pancreatic islet images were captured using a fluorescence microscope and analyzed using Focus Lite and Image J software.

2-NBDG is 2-[ N- (7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, i.e., a fluorescent glucose analog. It is a fluorescent dye used to observe absorption activity. An increase in fluorescence intensity indicates an increase in the amount of 2-NBDG absorbed into the zebrafish, indicating that the test substance has a diabetes treatment effect.

In order to evaluate the recovery effect of the fraction and the compound on the damaged pancreatic islet, the results of analyzing the size change of the pancreatic islet and the fluorescence of the pancreatic islet stained with 2-NBDG are shown in FIG. 2.

Referring to FIG. 2(A), when zebrafish larvae were exposed to alloxane (AX), the size of the pancreatic islet was significantly reduced to 51.8% ( p = 0.0003) compared to the normal group (NOR). The positive control group, glimepiride-treated zebrafish larvae (GLM), showed an increase in pancreatic islet size of 89.5% ( p = 0.0047) compared to the alloxane-treated group (AX).

The size of pancreatic islets in the ethyl acetate fraction treatment group (BAFE) and the n -butanol fraction treatment group (BAFB) was significantly up to 64.0% and 69.4% ( p = 0.0091 and 0.0065, respectively) compared to the alloxane treatment group (AX). Increased. All of the treatment groups of compounds 1 to 6 also increased the size of pancreatic islets compared to the alloxane treatment group (AX). Specifically, compounds 1 to 6 increased the pancreas damaged by alloxane by 82.0, 89.8, 80.0, 97.8, 103.1 and 99.6% ( p = 0.0011, 0.0037, 0.0012, 0.0002, 0.0011, 0.0011 and 0.0011, respectively).

Figure 2(B) shows that the size of pancreatic islets decreased by alloxane (AX) in the ethyl acetate fraction treatment group (BAFE), the n -butanol fraction treatment group (BAFB), and the compounds 1 to 6 treatment groups was increased. This is a picture taken under a microscope. It can be seen that the size of the pancreatic islet was significantly increased in all treatment groups compared to the alloxane treatment group (AX).

Taken together, the fraction obtained from the flower of the angel's morning glory and the compounds 1 to 6 isolated therefrom all showed the effect of restoring the pancreatic islet damaged by alloxane treatment in zebrafish larvae. Both fractions and compounds 1 to 6 exhibited an effect equal to or greater than that of the positive control, and in particular, compounds 2 and 4 to 6 showed remarkably better recovery effects than the positive control (glimepiride-treated group).

Example 5: Action of diazoxide (DZ) on diabetic zebrafish induced by alloxane

In order to elucidate the mechanism of the pancreatic islet recovery effect identified in Example 4, the pancreatic β-cell K _ATP channel was used to open a K _ATP channel (ATP dependent K ⁺ channel) using diazoxide (DZ). It was confirmed whether the active effect was related to this mechanism.

The configuration and experimental method of the experimental group were the same as in Example 4, except that a group further treated with diazooxide was added to the experimental group of Example 4. After alloxane treatment, a fraction or a diazooxide (25 μM) was treated with each compound. The results are shown in FIG. 3.

3(A), the size of pancreatic islets was significantly reduced in the DZ-treated normal group (NOR) compared to the DZ-treated normal group (NOR) (43.4%, p = 0.0052). In addition, the alloxane-treated group (AX) remained in a reduced state of pancreatic islets regardless of whether or not DZ was treated. Alloxane and 10 μg/mL glimepiride treatment group significantly reduced the size of pancreatic islets after further treatment with DZ (48.9%, p = 0.0202).

In the group treated with compounds 1, 2, 4 or 6 and alloxane, there was no significant difference in the size of pancreatic islets after further treatment with DZ, which is the mechanism of recovery of pancreatic islets by compounds 1, 2, 4 and 6 K indicates that there is no relationship with the _ATP channel. On the other hand, in the compound 3 or 5 and alloxane-treated group, the size of the pancreatic islet was significantly reduced after further treatment with DZ (Compound 3: 57.6%, p = 0.0358, Compound 5: 69.3%, p = 0.0358). These results suggest that compounds 3 and 5 can restore pancreatic islets by stimulating insulin secretion by Ca ²⁺ influx through the blockage of K _ATP channels in β-cells.

Figure 3(B) The upper panel is a photograph taken with a microscope of an increase in the size of pancreatic islets reduced by alloxane (AX) in the treatment group of compound 3 or 5. The lower panel shows the results after further treatment with DZ in the experimental group, and the size of the pancreatic islets recovered by treatment with Compound 3 or 5 was significantly reduced by DZ treatment.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

Claims (10)

Compound represented by the following formula (1):

[Formula 1]

In Formula 1,
R is glucopyranosyl.
The compound of claim 1, wherein the compound is isolated from an angelic morning glory ( Brugmansia arborea ) extract or a fraction thereof.
Fractions of Angel's Morning Glory Extract; or
A pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient a compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt of the compound isolated therefrom:

[Formula 1]

[Formula 2]

In the above formula,
R is glucopyranosyl,
R ₁ is benzyl, phenylethyl or phenylpropyl,
R ₂ is H or glucopyranosyl.
However, 2-phenylethyl O - β -D-glucopyranoside is excluded.
The pharmaceutical composition of claim 3, wherein the fraction is an ethyl acetate fraction or an n -butanol fraction.
The method of claim 3, wherein the compound represented by Formula 2 is
Benzyl O - β -D- glucopyranosyl - (1 → 6) - β -D- gluconic nose Llano side,
2-phenylethyl O - β -D-glucopyranosyl-(1→6)- β -D-glucopyranoside or
3-phenylpropyl O - β -D-glucopyranoside, the pharmaceutical composition.
The pharmaceutical composition according to any one of claims 3 to 5, wherein the composition restores damaged pancreatic islets.
Fractions of Angel's Morning Glory Extract; or
A food composition for preventing or improving diabetes, comprising, as an active ingredient, a compound represented by the following Formula 1 or Formula 2, or a food acceptable salt of the compound isolated therefrom:

[Formula 1]

[Formula 2]

In the above formula,
R is glucopyranosyl,
R ₁ is benzyl, phenylethyl or phenylpropyl,
R ₂ is H or glucopyranosyl.
However, 2-phenylethyl O - β -D-glucopyranoside is excluded.
The food composition according to claim 7, wherein the fraction is an ethyl acetate fraction or an n -butanol fraction.
The method of claim 7, wherein the compound represented by Formula 2 is
Benzyl O - β -D- glucopyranosyl - (1 → 6) - β -D- gluconic nose Llano side,
2-phenylethyl O - β -D-glucopyranosyl-(1→6)- β -D-glucopyranoside or
3-phenylpropyl O - β -D-glucopyranoside, food composition.
A method for preparing a compound represented by Formula 1 or Formula 2, comprising the step of separating the compound represented by the following Formula 1 or Formula 2 from the angel's morning glory extract or a fraction thereof:

[Formula 1]

[Formula 2]

In the above formula,
R is glucopyranosyl,
R ₁ is benzyl, phenylethyl or phenylpropyl,
R ₂ is H or glucopyranosyl.
However, benzyl O-β-D-glucopyranoside and 2-phenylethyl O - β -D-glucopyranoside are excluded.

Images