KR101278273B1 - A composition comprising triterpenoids isolated rhododendron brachycarpum for treating or preventing metabolic diseases - Google Patents

Abstract

PURPOSE: A composition containing triterpenoids isolated from Rhododendron brachycarpum for preventing or treating metabolic diseases is provided to suppress protein tyrosine phosphatase 1B(PTP1B), and to suppress or treat diabetes or obesity. CONSTITUTION: A composition for preventing or treating obesity or diabetes contains one or more kinds of triterpenoids selected from the group consisting of rhododendric acid A(compound 1), 2alpha, 3alpha, 23-trihydroxyursa-12,20(30)-dien-28-oic acid(compound 3), and actinidic acid(compound 5) denoted by chemical formulas 1 and 3, which are isolated from Rhododendron brachycarpum. A method for isolating rhododendric acid A denoted by chemical formula 1 comprises: a step of extracting Rhododendron brachycarpum leaves with water, C1-C4 alcohols, or a mixture thereof; a step of sequentially fractioning the extract with n-hexane, chloroform, and ethyl acetate; and a step of isolating the compound denoted by the chemical formula 1 from the chloroform fraction using chromatography. [Reference numerals] (AA) Compound 1; (BB) Compound 3; (CC) Compound 5

Description

A composition comprising triterpenoids isolated Rhododendron brachycarpum for treating or preventing metabolic diseases}

The present invention relates to a composition for the prophylaxis or treatment of metabolic diseases containing triterpenoid compounds derived from Rhododendron. More specifically, the present invention is Rhododendron brachycarpum ) rhododendric acid A (compound 1), rotundic acid (compound 2), 2α, 3α, 23-trihydroxyursa-12,20 (30) -diene-28 Oic acid (2α, 3α, 23-trihydroxyursa-12,20 (30) -dien-28-oic acid, compound 3), 23-hydroxyursolic acid (compound 4) and actinidic acid ( Actinidic acid, compound 5) relates to a composition for the treatment of metabolic diseases containing at least one triterpenoid compound selected from the group consisting of (triterpenoids).

In modern society, quality of life is improving and lifespan is being extended. However, with the increase of lifespan, the incidence of cardiovascular and metabolic diseases including cancer is increasing. According to the recently released statistics of the World Health Organization, it is reported that diseases that cause high mortality rates worldwide include various cancers, cardiovascular diseases, metabolic diseases, and neuropsychiatric diseases.

 Among the metabolic diseases, diabetes mellitus is a chronic wasting disease characterized by an absolute deficiency or resistance to insulin metabolic dysfunction, and causes various complications such as systemic lethargy and reduced resistance, vascular disorders, cerebral infarction, and myocardial infarction. Diabetes mellitus is divided into two types according to its pathogenesis and symptoms. Insulin-dependent type 1 diabetes caused by destruction of beta cells, insulin secreting cells by lymphocyte immunological mechanisms, and the function of beta cells secreting insulin There is type 2 diabetes, which is insulin-independent, caused by depression and increased resistance to insulin in peripheral target organs. With the aging of modern society and changing lifestyles, adult type 2 diabetes accounts for more than 90% of all patients, and the number is increasing steadily.

Insulin resistance refers to a condition in which the response to a certain amount of insulin is less than normal. Generally, it is caused by genetic and environmental factors. It is a major cause of the development of type 2 diabetes. The main target is to improve the sensitivity.

Current drug treatments can be divided into several types according to their treatment mechanisms. The sulfonylurea system indirectly increases insulin secretion and enhances insulin action, but may have side effects such as hypoglycemia and loss of insulin secretion. have. The biguanide system acts to inhibit the formation of glucose in the liver and to reduce the absorption of sugar in the gastrointestinal tract, but has side effects such as gastrointestinal disorders and nephrotoxicity. The alpha-glucosidase inhibitors in the small intestine Inhibit the action of glucose-producing enzymes to suppress the rise of blood sugar, while diarrhea, abdominal pain and other side effects.

In recent years, the development of insulin-increasing agents has been in full swing, and there are representative drugs of thiazolidinedione (TZD) which activates peroxisome proliferator activated receptor γ (PPAR-γ). PPAR-γ binds to the retinoid X receptor (RXR), one of the nuclear hormone receptors, to form a heterodimer and then acts as a transcription factor to adipocytes. Differentiation, carbohydrates, fat metabolism is important. In particular, PPAR-gamma (PPAR-γ) is mainly present in adipocytes and acts as an adipogenesis and glucose uptake (PPAR-γ agonist) is 2 It is widely used as a type diabetes treatment. In addition, thiazolidinedione (TZD) -based drugs are also widely used as a treatment for type 2 diabetes (Diabetes, 1998, 47 (4), 507 ~ 514).

However, the current situation of drug treatment has not reached the treatment of the underlying cause of diabetes, and various side effects have been reported, and it is urgent to develop a drug that can fundamentally solve insulin resistance.

On the other hand, obesity is not just a cosmetic effect due to overweight and lowering the quality of life is a major cause of death, the recognition is changing to a disease that occupies a large proportion in metabolic diseases. According to the World Health Organization (WHO) statistics, the incidence of obesity has increased, with about 6% of the world's population as of 2008, about 400 million people estimated to be obese, and by 2015 it will be more than 700 million. Particularly in the OECD countries, the adult obesity population is increasing year by year, and one in three in the US is obese. In Korea, obesity population is increasing rapidly due to changes in living environment, and the Korean adult obesity population with body mass index (BMI) of 25 or more has increased by 40 years every year, and 3 out of 10 people in 2005 were obese. According to data from the Korea Centers for Disease Control and Prevention, the obesity population in 1995 was 26.3% of the total population, but in 2001 it increased to 30.6% of the total population and in 2005 to 31.5% of the total population.

Meanwhile, protein tyrosine phosphatase 1B (PTP1B) causes dephosphorylation of insulin receptors and insulin receptor substrates, as shown in FIG. It is known as an enzyme that causes insulin resistance by interfering with. In addition, the protein tyrosine dephosphorylase is known to cause obesity by inhibiting the leptin signaling mechanism by inhibiting the phosphorylation occurring at the leptin receptor and Jak also in the signaling process of leptin (see reference 1). Chem., 2004, 47 (17), 4142-4146).

[Reference Figure 1]

Although the study of the protein tyrosine dephosphatase 1B is relatively in its infancy, the biological function and disease-causing mechanism of the protein tyrosine dephosphatase 1B have recently been identified and thus become a target of new drug development. (Med. Res. Revi., 2012, 32, 459-517). In particular, as the role of the protein tyrosine dephosphatase 1B in intractable diseases such as diabetes, obesity, and cancer has been identified, a lot of investment is being made in developing new drugs targeting the enzyme inhibition. As an example of this, insulin resistance was removed in mice from which the protein tyrosine phosphatase 1B (PTP1B) gene was removed did not cause type 2 diabetes and suppressed obesity (Science, 1999, 283, 1544-1548), substances that inhibit protein tyrosine phosphatase 1B (PTP1B) have been shown to exhibit antidiabetic effects (J. Med. Chem., 2000, 43 (5)). , 995-1010).

Meanwhile, inhibitors of various tyrosine dekinases, including protein tyrosine dephosphatase 1B, which have been in clinical trials to date, are as shown in the following Reference 2, which are mostly synthetic compounds, which have been dropped from clinical trials due to low bioavailability, toxicity, and other side effects. There are many cases. Therefore, there is a need for development of a safe natural-derived inhibitor (Nat. Rev. Drug Discov., 2002, 1, 696-709).

[Reference Figure 2]

In this regard, the present inventors have bowed roe pee ( Astilbe) koreana ) (Bioorg.Med. Chem. Lett., 2006, 16, 3273 ~ 3276), Acanthopanax koreanum ) (Bioorg.Med.Chem.Lett. 2006, 16, 3061-3064), Erythrina mildbraedii ) (J. Nat. Prod., 2006, 69, 1572-1576 ; Chem. Pharm. Bull., 2008, 56, 85-88) confirmed that the compounds isolated from the inhibitory activity of the protein tyrosine dekinase 1B.

In addition, the present inventors, while repeating the above studies, triterpenoid compounds derived from Rhododendron brachycarpum also (triterpenoids) The present invention was completed by confirming that the protein tyrosine dephosphatase 1B had inhibitory activity.

Spiegelman, B.M., PPAR-gamma: adipogenic regulator and thiazolidinedione receptor., Diabetes, 1998, 47 (4), 507-514. Hooft van Huijsduijnen, R. et al. Prospects for inhibitors of protein tyrosine phosphatase 1B as antidiabetic drugs., J. Med. Chem., 2004, 47 (17), 4142-4146. Elchebly, M. et al., Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene., Science, 1999, 283 (5407), 1544-1548. Malamas, M.S. et al., New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties., J. Med. Chem., 2000, 43 (5), 995-1010. Thareja, S. et al., Protein tyrosine phosphatase 1B inhibitors: a molecular level legitimate approach for the management of diabetes mellitus., Med. Res. Revi., 2012, 32, 459-517. Johnson, T. O. et al., Protein tyrosine phosphatase 1B inhibitors for diabetes., Nat. Rev. Drug Discov., 2002, 1, 696-709. Na. M. et al., Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana., Bioorg. Med. Chem. Lett., 2006, 16, 3273-3276. Na, M. et al., Inhibition of protein tyrosine phosphatase 1B by diterpenoids isolated from Acanthopanax koreanum., Bioorg. Med. Chem. Lett. 2006, 16, 3061-3064. Na, M. et al., Protein tyrosine phosphatase-1B inhibitory activity of isoprenylated flavonoids isolated from Erythrina mildbraedii., J. Nat. Prod., 2006, 69, 1572-1576. Jang, J. et al., Prenylated flavonoids with PTP1B inhibitory activity from the root bark of Erythrina mildbraedii., Chem. Pharm. Bull., 2008, 56, 85-88. Cui, L. et al., Protein tyrosine phosphatase 1B inhibitors from Morus root bark. Bioorg. Med. Chem. Lett. 2006, 16, 1426.

An object of the present invention is to provide a composition for the prevention or treatment of metabolic diseases containing triterpenoid compounds derived from Rhododendron brachycarpum . More specifically, an object of the present invention is Rhododendron brachycarpum derived from Rhododendric acid A (rhododendric acid A, Compound 1), rotundiic acid (rotundic acid, Compound 2), 2α, 3α, 23-trihydroxyursa -12,20 (30) -diene-28-oic acid (2α, 3α, 23-trihydroxyursa-12,20 (30) -dien-28-oic acid, compound 3), 23-hydroxyursolic acid (23- It is to provide a composition for treating metabolic diseases containing at least one triterpenoid compound selected from the group consisting of hydroxyursolic acid, compound 4) and actinidic acid (compound 5). .

The present invention relates to a composition for the prophylaxis or treatment of metabolic diseases containing triterpenoid compounds derived from Rhododendron. In more detail, the present invention is Rhododendric acid A (Compound 1), Rotundiic acid (Compound 2), 2α, 3α, 23-trihydroxyursa-12 , 20 (30) -diene-28-oic acid (2α, 3α, 23-trihydroxyursa-12,20 (30) -dien-28-oic acid, compound 3), 23-hydroxyursolic acid The present invention relates to a composition for treating metabolic diseases containing at least one triterpenoid-based compound selected from the group consisting of Compound 4) and Actinidic acid (Compound 5).

[Formula 1]

[Formula 2]

(3)

The metabolic disease may be a disease selected from obesity or diabetes.

In addition, the triterpenoid-based compound is characterized by inhibiting the activity of protein tyrosine phosphatase 1B (protein tyrosine phosphatase 1B, PTP1B).

In another aspect, the present invention provides a novel compound Rhododendric acid A of Formula 1, Rhododendron Brachycarpum ) can be provided a method for separating the rhododendric acid A (rhododendric acid A) of the formula (1) from water, C1 to C4 lower alcohol or extracts of a mixed solvent thereof.

[Formula 1]

Hereinafter, the present invention will be described in detail.

The triterpenoid compound is Rhododendron brachycarpum ), water, lower alcohols of C1 to C4, or a mixture of these solvents can be separated. In addition, the triterpenoid-based compound may be obtained by fractionating chromatographic extracts of Rhododendron water, C1 to C4 lower alcohols, or mixed solvents thereof.

Therefore, preferably, the triterpenoid-based compound is a step of extracting the leaves of the Rhododendron leaf with water, a lower alcohol of C1 to C4 or a mixed solvent thereof to prepare a Rhododendron extract; Sequentially fractionating the extract of Panaxaceae using n-hexane, chloroform, ethyl acetate; And separating each of the fractions using chromatography to separate the compounds.

The chromatography can be carried out using silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

Meanwhile, the triterpenoid compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared as a pharmaceutically acceptable salt. Preferably the compounds of the present invention can be used to make a pharmaceutically acceptable metal salt using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of metabolic diseases containing a triterpenoid compound. The pharmaceutical composition comprising the triterpenoid-based compound may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively. It can be formulated and used in the form. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition containing the triterpenoid-based compound of the present invention depends on the age, sex and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration and the judgment of the prescriber. Will depend. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition containing the triterpenoid compound of the present invention can be administered to mammals such as rats, livestock, humans, and the like by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the compound of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.

The present invention relates to a composition for the prophylaxis or treatment of metabolic diseases containing triterpenoid compounds isolated from Rhododendron. Protein tyrosine dephosphatase 1B causes dephosphorylation of insulin receptors and insulin receptor substrates, disrupting insulin signaling mechanisms, leading to insulin resistance, and inhibiting phosphorylation occurring at leptin receptors and Jak By doing so, it is known to play a role in inhibiting leptin signaling mechanism, causing obesity. Accordingly, the compounds derived from Pleurotus eryngii are excellent in inhibitory activity of the protein tyrosine dephosphorase 1B and may be usefully used as a composition for preventing or treating diabetes or obesity.

) 상관 관계(도 4A) 및 NOE (↔) 상관관계(도 4B)를 나타내는 그림이다.1 is also a diagram showing the ¹ H- ¹ H COSY spectrum den trick acid A (Compound _{1) (CDCl 3, 600 MHz} ).
2 is a graph showing the HMBC spectrum results of Rhododendric acid A (Compound 1).
3 is a diagram showing the NOESY spectrum of Rhododendric acid A (Compound 1) (irradiation at H-3, 500MHz).
FIG. 4 shows HMBC (H → C) and ¹ H- ¹ H COSY of Rhododendric Acid A (Compound 1)

) Correlation (FIG. 4A) and NOE (↔) Correlation (FIG. 4B).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

Example 1 Isolation of Triterpenoid Compounds from Rhododendron

The leaves of Rhododendron brachycarpum were purchased in 2011 at the Rwanodendron brachycarpum farm in Gongju, South Korea. 25 kg of the leaf of the Rhododendron was extracted with 250 L of methanol twice a week at room temperature and then filtered. The filtered methanol extract was concentrated to give 6 kg of methanol extract. The methanol extract (3 kg) was suspended in 10 L of water, n-hexane (10 Lx3), CHCl ₃ (10 Lx3) and ethyl acetate (10 Lx3) were sequentially added to n-hexane fraction, chloroform fraction, Ethyl acetate fractions (438g, 140g, 450g, respectively) were obtained, and finally fractions containing the water layer residue were obtained. Thereafter, the triterpenoid compounds of the present invention were isolated from the chloroform fraction (88% inhibitory effect at a concentration of 30 µg / ml) having a relatively strong inhibitory activity against protein tyrosine dephosphatase 1B in the four fractions.

The chloroform fraction (140 g) was prepared by silica gel column VLC and n-hexane-ethyl acetate (gradient from 8: 2, 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8, 1: 9, to 0: 1) was used to obtain nine small fractions (Fr. C1-C9). Of these, Fr. C9 small fraction (30 g) was obtained by using a silica gel column and n-hexane-ethyl acetate (gradient from 8: 2 to 0: 1) to three small fractions (Fr. C91-C93).

Fr. C92 subfractions (2.47 g) were further fractionated into five subfractions using isocratic conditions of MPLC [C18 SNAP Cartridge KP-C18-HS column] and MeOHH ₂ O (80:20). Of these, Fr. A small fraction of C922 (330 mg) was purified using HPLC (Luna C18 (2) column: 250 x 21.2 mm, mobile phase: 80% MeOH, flow rate: 6 mL / min) to obtain Compound 1 (3.7 mg, t _R 54.6 min). ), Compound 2 (4.4 mg, t _R 23.5 min), Compound 3 (39.2 mg, t _R 32.7 min) and Compound 4 (5.8 mg, t _R 66.3 min) were separated.

In addition, Fr. The C93 (8.82 g) small fraction was separated under the conditions of RP-C18 MPLC and methanol-water (from 4: 6 to 0: 1) to obtain seven small fractions (Fr. C931-C937), and compound 5 (18.0 mg). , t _R 30.2 min) is Fr. C932 small fraction (1.56 g) was isolated using HPLC (Luna C18 (2) column: 250 × 21.2 mm, mobile phase: 80% MeOH, flow rate: 6 mL / min).

<Example 2. Confirmation of physicochemical structure of the compound derived from panaxaceae.

Example 2-1. Rhododendric Acid A (Compound 1)

rhododendric acid A;

white amorphous powder;

: +70.4 (c 0.03, MeOH); [ α ]

: +70.4 ( c 0.03, MeOH);

UV λ max (MeOH) 207 nm;

HRFABMS m / z 471.3476 [M + H] ⁺ (calcd for C ₃₀ H ₄₇ O ₄ 471.3474);

¹ H-NMR (pyridine- d ₅ , 600 MHz): δ _H 5.50 (1H, brt, J = 3.1 Hz, H-12), 4.82 (1H, s, H-30a), 4.77 (1H, s, H-30b), 4.24 (1H, dd, J = 5.6, 10.9 Hz, H-3), 4.20 (1H, d, J = 10.6 Hz, H-23a), 3.74 (1H, d, J = 10.6 Hz, H-23b), 2.77 (1H, d, J = 11.3 Hz, H-18), 1.17 (3H, s, H-27), 1.12 (3H, d, H-29), 1.07 (6H, s, H -24, H-26), 0.98 (3H, s, H-25);

¹³ C-NMR (pyridine- d ₅ , 150 MHz): δ _c 179.7 (C-28), 154.2 (C-20), 139.4 (C-13), 126.4 (C-12), 105.5 (C-30), 73.8 (C-3), 68.3 (C-23), 56.1 (C-18), 49.0 (C-5), 48.5 (C-9), 48.7 (C-17), 43.3 (C-4), 43.0 (C-14), 40.4 (C-8, C-22), 39.3 (C-1), 38.1 (C-19), 37.6 (C-10), 33.6 (C-7), 33.2 (C-21 ), 29.1 (C-15), 28.1 (C-2), 25.3 (C-16), 24.1 (C-11, C-27), 19.0 (C-6), 17.9 (C-26), 17.0 ( C-29), 16.5 (C-25), 13.6 (C-24);

Example 2-2. Rotunic Acid (Compound 2)

rotundic acid;

white amorphous powder;

: +34.1 (c = 0.05, MeOH); [ α ]

: +34.1 ( c = 0.05, MeOH);

FAB-MS m / z : 489 [M + H] ⁺ ;

¹ H-NMR (pyridine- d ₅ , 600 MHz): δ _H 5.62 (1H, brt, J = 3.1 Hz, H-12), 4.21 (1H, dd, J = 4.6, 11.3 Hz, H-3) , 4.18 (1H, d, J = 10.4 Hz, H-24b), 3.72 (1H, d, J = 10.4 Hz, H-24a), 3.06 (1H, s, H-18), 1.69 (3H, s, H-27), 1.45 (3H, s, H-29), 1.13 (3H, s, H-25), 1.12 (3H, d, J = 6.6 Hz, H-30), 1.06 (3H, s, H -23), 0.99 (3H, s, H-26);

Example  2-3. 2α, 3α, 23- Trihydroxyursa -12,20 (30)- Dien -28- Oh Iksan  (Compound 3)

2α, 3α, 23-trihydroxyursa-12,20 (30) -dien-28-oic acid;

white amorphous powder;

: +62.4 (c = 0.05, MeOH); [ α ]

: +62.4 ( c = 0.05, MeOH);

FAB-MS m / z : 487 [M + H] ⁺ ;

¹ H-NMR (pyridine- d ₅ , 600 MHz): δ _H 5.46 (1H, br s, H-12), 4.80 (1H, s, H-30a), 4.75 (1H, s, H-30b), 4.28 (1H, broad doublet, J = 10.5 Hz, H-2), 4.16 (1H, s, H-3), 3.93 (1H, d, J = 10.6 Hz, H-23a), 3.77 (1H, d, J = 10.6 Hz, H-23b), 2.74 (1H, d, J = 11.8 Hz, H-18), 1.12 (3H, s, H-27), 1.08 (3H, d, J = 6.3 Hz, H- 29), 1.04 (3H, s, H-26), 1.00 (3H, s, H-25), 0.87 (3H, s, H-24);

Example  2-4. 23- Hydroxyuronic acid  (Compound 4)

23-hydroxyursolic acid;

white amorphous powder;

: +60.0 (c = 0.30, MeOH); [ α ]

: +60.0 ( c = 0.30, MeOH);

FAB-MS m / z : 495 [M + Na] ⁺ ;

¹ H-NMR (pyridine- d ₅ , 600 MHz): δ _H 5.52 (1H, br s, H-12), 4.23 (1H, dd, J = 5.1, 11.2 Hz, H-3), 4.20 (1H, d, J = 10.6 Hz, H-23a), 3.74 (1H, d, J = 10.6 Hz, H-23b), 2.65 (1H, d, J = 11.3 Hz, H-18), 1.20 (3H, s, H-27), 1.10 (3H, s, H-24), 1.08 (3H, s, H-26), 1.01 (3H, d, J = 6.5 Hz, H-29), 0.99 (3H, s, H -25), 0.95 (3H, d, J = 6.5 Hz, H-30);

Example  2-5. Actinidic acid  (Compound 5)

actinidic acid;

white amorphous powder;

: +75.5 (c = 0.1, MeOH); [ α ]

: +75.5 ( c = 0.1, MeOH);

FAB-MS m / z : 487 [M + H] ⁺ ;

¹ H-NMR (methanol-d4, 250 MHz): δ _H 5.23 (1H, br s, H-12), 4.64 (1H, s, H-30a), 4.59 (1H, s, H-30b), 3.67 (1H, m, H-2), 3.45 (1H, d, J = 11.1 Hz, H-23a), 3.32 (1H, d, J = 8.4 Hz, H-3), 3.23 (1H, d, J = 11.1 Hz, H-23b), 2.22 (1H, d, J = 12.2 Hz, H-18), 1.15 (3H, s, H-27), 1.01 (3H, s, H-25), 0.97 (3H, d, J = 6.1 Hz, H-29), 0.81 (3H, s, H-26), 0.66 (3H, s, H-24);

Table 1 below shows the ¹³ C-NMR spectrum results of Compounds 1-5.

position Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 One 39.3 39.2 43.0 39.3 40.7 2 28.1 28.0 66.6 28.1 69.7 3 73.8 73.9 79.3 73.8 78.1 4 43.3 43.2 42.3 43.3 44.1 5 49.0 49.0 43.9 49.0 48.8 6 19.0 18.1 18.7 19.0 19.1 7 33.6 33.6 33.5 33.7 33.3 8 40.4 40.7 40.5 40.4 40.4 9 48.5 48.2 48.4 48.5 49.2 10 37.6 37.6 38.8 37.5 48.2 11 24.1 24.4 24.1 24.1 24.5 12 126.4 128.4 126.2 126.0 127.0 13 139.4 140.3 139.4 139.7 139.6 14 43.0 42.5 43.2 42.9 43.5 15 29.1 29.7 29.0 29.1 29.2 16 25.3 26.8 25.3 25.3 25.3 17 48.7 48.7 48.7 48.5 49.4 18 56.1 55.0 56.0 54.0 56.6 19 38.1 73.0 38.1 39.9 39.0 20 154.2 42.7 154.2 39.8 154.5 21 33.2 27.3 33.1 31.5 33.6 22 40.4 38.9 40.1 37.9 38.5 23 68.3 68.3 71.6 68.3 66.3 24 13.6 13.4 18.2 13.5 13.9 25 16.5 17.6 17.9 16.5 17.7 26 17.9 17.1 17.5 17.9 17.8 27 24.1 25.0 24.1 24.3 24.1 28 179.7 181.1 179.8 180.4 181.0 29 17.0 27.5 16.9 17.9 16.8 30 105.5 16.3 105.4 21.8 105.3 Compound 1-4: Measured at 150 MHz & in pyridine-d5
Compound 5: Measured at 63 MHz & in methanol-d4

< Example  3. Protein Tyrosine Dephosphoryase  Activity measurement of 1B>

By If the substrate, p-nitrophenyl phosphate (p-NPP ) by protein tyrosine dephosphorylation enzymes 1B changed to p-nitrophenol (p-NP) exhibits a yellow measuring the change in absorbance at 405nm was measured for enzymatic activity (Bioorg . Med. Chem. Lett. 2006 , 16, 1426 ~ 1429).

Protein tyrosine dekinase 1B (PTP1B, human, recombinant) was purchased from BIOMOL International LP (Plymouth Meeting, PA). In a 96 wells plate (96 wells in a microtiter plate) 2mM p-NPP and 0.05 ~ 0.1 ㎍ protein tyrosine dephosphatase 1B was added to each experimental group was treated with compounds 1-5. At this time, the composition of p-NPP, protein tyrosine dephosphatase 1B and compounds 1 to 5 in 96-well plate was composed of 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT). And the final volume was 100 μl. Thereafter, the mixed treatment was placed at 37 ° C. for 30 minutes, and then the reaction was stopped by treating 50 μl of 10 M sodium hydroxide (10 MNaOH), and finally, the amount of p-NPP remaining was confirmed at 405 nm. The activity of each compound is expressed as IC ₅₀ (the half maximal inhibitory concentration). As a control, RK-682 (3-hexadecanoyl-5-hydroxymethyl-tetronic acid), one of the inhibitors of tyrosine dephosphatase, was used.

compound IC ₅₀ (μM) Compound 1 6.3 ± 0.6 Compound 2 20.1 ± 1.4 Compound 3 17.4 ± 1.0 Compound 4 7.4 ± 0.6 Compound 5 18.0 ± 0.9 RK-682 4.0 ± 0.3

Referring to the results of Table 2, it was confirmed that the triterpenoid-based compounds of the present invention has a significant inhibitory effect on tyrosine dephosphatase similar to RK-682.

< Example  4. Toxicity Test>

Example  4-1. Acute toxicity

This experiment was carried out to investigate the toxicity to animals in acute (within 24 hours) when the compound of the present invention is ingested in a short period of time and to determine the mortality rate. Twenty ICR mice, which are common mice, were prepared, and 10 were assigned to each group. In the control group, only 30% PEG-400 was administered, and the experimental group was orally administered with rhododetric acid A (compound 1) of the present invention at a concentration of 1.0 g / kg. Each mortality rate was examined 24 hours after administration, and both the control group and the experimental group administered with Compound 1 survived.

Example  4-2. Experimental group  And control organ organs and tissue toxicity experiments

In the long-term toxicity experiment, the study group and the solvent administered with rhododetric acid A (compound 1) of the present invention at a concentration of 1.0 g / kg in order to investigate the effect on each organ (tissue) of the animal in C57BL / 6J mice Blood was collected after 8 weeks from animals in the control group administered with the bay, and blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was carried out with an optical microscope through a conventional tissue section production process. No abnormal abnormalities were observed.

< Examples  1: Pharmaceutical Formulation example >

1-1. Manufacture of tablets

200 g of rhododetric acid A (compound 1) of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2. Injection preparation

1 g of rhododetric acid A (compound 1) of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

Claims (5)

Rhododendric acid A (Compound 1), 2α, 3α, 23-trihydroxyursa-12,20 (30) -diene-28, isolated from Rhododendron brachycarpum At least one tree selected from the group consisting of oic acid (2α, 3α, 23-trihydroxyursa-12,20 (30) -dien-28-oic acid, compound 3) and actinidic acid (compound 5) A composition for the prevention or treatment of obesity or diabetes containing terpenoid-based compounds (triterpenoids).
[Formula 1]

(3)

delete The method of claim 1,
The triterpenoid-based compound is a composition for preventing or treating obesity or diabetes, characterized in that to inhibit the activity of protein tyrosine dephosphoryase 1B (protein tyrosine phosphatase 1B, PTP1B). The new compound of the formula 1 Rhododendric acid A (rhododendric acid A).
[Formula 1]

Extracting Rhododendron brachycarpum leaves with water, C1 to C4 lower alcohol, or a mixed solvent thereof to prepare a Rhododendron brachycarpum leaf;
Sequentially fractionating the extract of Panaxaceae using n-hexane, chloroform, ethyl acetate; And
Separating the compound of Chemical Formula 1 using chromatography from the chloroform fraction obtained through the fraction using chromatography;
Rhododendric acid A (rhododendric acid A) of the formula 1 comprising a.
[Formula 1]

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