KR102066966B1 - A pharmaceutical composition comprising compounds isolated from the root bark of Morus alba L. for preventing or treating diabetes mellitus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diabetes, containing a compound isolated from Mori cortex. The compound, that is morsalvin A (a compound 1), morusalvin B (a compound 2), morusalvin C (a compound 3), morsalvin D (a compound 4), albanin T (a compound 5), and macrourin G (a compound 6), has excellent protein tyrosine dekinase 1B inhibitory activity and alpha-glucosidase inhibitory activity, thereby being able to be usefully used as a composition for preventing or treating diabetes.

Description

A pharmaceutical composition comprising compounds isolated from the root bark of Morus alba L. for preventing or treating diabetes mellitus}

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes, comprising a compound isolated from the epidermis.

Diabetes has problems with insulin secretion, such as decreased insulin secretion and resistance due to genetic and environmental causes, or malfunctions of insulin, causing glucose in the blood to not be transferred / stored to the cells, resulting in too much blood in the blood. It is a metabolic disease with symptoms of hyperglycemia that is much higher than that of normal people. Diabetes ketoacidosis, hyperglycemic coma (Kitabachi, AE et al., Diabetes care, 32 (7), 1335-1345, 2009), It causes complications such as cardiovascular disease, high blood pressure, stroke, chronic kidney failure, diabetic ulcer, and diabetic retinopathy (WHO, Diabetes Fact sheet No. 312, 2013). This diabetes is known to be the fourth highest cause of death in Korea, including complications.

This type of diabetes is caused by inherent genetic factors, type 1 diabetes (T1D), which produces little or no insulin in the body, and environmental factors such as obesity, lack of exercise, and post-pregnancy stress. The disease may be classified into Type 2 Diabets, Gestational Diabetes, and other diabetes, which are caused by insulin resistance in which the body does not produce enough insulin or the cells do not respond to insulin. Type 1 diabetes, which accounts for about 10% of all diabetes, occurs mainly in children, and is caused by the insufficiency of insulin by disruptive lesions of pancreatic β cells. Type 2 diabetes occurs mainly in adults, due to insulin resistance, in which the body does not secrete enough insulin to function properly or the cells do not respond to insulin. Gestational diabetes has never been diagnosed with diabetes, but during pregnancy the body does not produce enough insulin, which means that blood sugar levels gradually increase.

In general, as diabetes mellitus becomes longer, the blindness due to chronic complications, end stage renal failure, nerve disease, lower extremity amputation and infectious diseases increase rapidly. In particular, cerebrovascular disease and cardiovascular disease account for the largest portion of diabetes complications. About 3/4 of the disease deaths are caused by diabetes complications, and the risk of death from cardiovascular complications can also increase the duration of diabetes for 10 years. It is reported to increase by 24% each time. The prevalence of coronary artery disease was two times higher and the prevalence of peripheral vascular disease was more than three times higher in diabetic patients than normal patients (Ko Boram et al., 21 (5), 382-388, 2006; Schneider). , SH et al., Metabolism, 37 (10), 924-929, 1988), are the causes of atherosclerosis in diabetes, including hyperglycemia, lipid metabolism, hyperinsulinemia, hypertension, and changes in blood coagulation mechanisms. (Aronoff, S. et al., Diabetes Care, 23 (11), 1605-1611, 2000). Many of these complications reduce the quality of life of individuals and the social and economic losses are also very large.

The most basic of diabetes management is blood sugar control, exercise therapy or diet therapy to control blood sugar. If blood glucose is not controlled in this way, drug therapy is started. Type 1 diabetes is administered by insulin injection (WHO, Diabetes Fact sheet No. 312, 2013), and type 2 diabetes is administered by oral hypoglycemic agents or insulin administration (Kim Dong-lim, Konkuk University Hospital KRC, 2012) . However, in addition to the positive aspects of continuous oral hypoglycemics, conventional oral hypoglycemic agents not only cause various side effects such as hypoglycemia, diarrhea, bloating, weight gain, lactate, heart toxicity, and liver toxicity. Since pancreatic β cells that function in insulin secretion are irreversibly damaged and destroyed, and insulin resistance occurs, the drug is finally ineffective and requires insulin injection. In addition, insulin, which is used most often as a diabetes treatment, has to be injected subcutaneously 2-3 times daily, causing discomfort and rejection for injections, and this also has a very high possibility of causing hypoglycemia.

Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that causes insulin resistance by dephosphorylation of insulin receptors and insulin receptor substrates, disrupting the signaling mechanisms of insulin. Known. In addition, the protein tyrosine dephosphorylase is also known to cause obesity by inhibiting the phosphorylation occurring at the leptin receptor and Jak and inhibiting the leptin signaling mechanism even in the signaling process of leptin (Malamas, MS et al., J. Med. Chem., 43 (5), 995-1010, 2000).

Although the research on protein tyrosine dephosphatase 1B to date is relatively early, the biological function and disease-causing mechanism of protein tyrosine dephosphatase 1B have recently been identified and thus have been the target of new drug development. (Thareja, S. et al., Med. Res. Rev., 32 (3), 459-517, 2012). In particular, mice with the protein tyrosine dekinase 1B gene eliminated insulin resistance and did not cause type 2 diabetes, and obesity was inhibited (Elchebly, M. et al., Science, 283 (5407). , 1544-1548, 1999), and agents that inhibit the protein tyrosine dephosphatase 1B have been shown to exhibit antidiabetic effects (Klaman, LD et al., Mol. Cell. Biol., 20 (15), 5479-5489, 2000).

Inhibitors of various tyrosine dekinases, including protein tyrosine dephosphatase 1B, which have been in clinical practice until now, are mostly synthetic compounds and have been eliminated in clinical trials due to low bioavailability, toxicity and side effects. Therefore, there is a need for development of inhibitors derived from highly safe natural products (Johnson, T. O. et al., Nat. Rev. Drug. Discov., 1 (9), 696-709, 2002).

More than 1000 plants have been used worldwide to treat type 2 diabetes (Coman, C. et al., Not. Bot. Horti. Agrobo., 40 (1), 314-325, 2012), and a variety of natural It has been reported that the derived compounds exhibit inhibitory activity against protein tyrosine dephosphatase 1B (Jiang, CS et al., Acta. Pharmacol. Sin., 33 (10), 1217-1245, 2012).

Alpha-glucosidase is a digestive enzyme present in the villi of the small intestine that hydrolyzes disaccharides or small sugars into monosaccharides that are digested and absorbed by carbohydrates. Therefore, the alpha-glucosidase inhibitor can inhibit the increase of blood sugar level by inhibiting the hydrolysis of carbohydrates (lowing digestion rate) in the upper part of the plant including duodenum, so it can be used for the treatment of adult diseases such as diabetes, obesity, and fructose. Can be. In addition, many studies have been conducted on the inhibition of alpha-glucosidase, which is essential for carbohydrate metabolism, for use in regulating blood glucose lowering (Tewari, N. et al., Bioorg. Med. Chem., 11 (13), 2911). -2922, 2003).

The root bark of Morus alba L. (Mori Radicis Cortex) is the root bark of the mulberry (Morus alba L.), specifically removing the cork layer of the mulberry or its related plant roots. Since ancient times, it has been known to have antipyretic, anti-convulsion, anti-allergic, anti-inflammatory and anti-diuretic effects.

Epidermis skin contains components such as prenylated flavonoid, benzofuran, and phenolic compounds, and these compounds inhibit cytotoxicity, COX-1, COX-2 and nitric oxide (NO). It has a variety of activities, including the inhibitory effect on production

As a related art related to the composition for the prevention or treatment of diabetes mellitus derived from lettuce extract, Korean Laid-Open Patent Publication No. 10-2015-0102833 includes moleucine, quanone H, chalcoracin and mongolycin among the compounds isolated from the extract of ethanol from lettuce extract. A composition for preventing or treating diabetes mellitus has been disclosed, and Korean Patent Laid-Open Publication Nos. 10-2014-0123264 and 10-2012-0111771 disclose cathachinin B, quaanol A, mulberofuran G, and mulberry isolated from epidermis. Pharmaceuticals for the prevention and treatment of diabetic complications, including verofuran K, quaanone G, quaanone Z, oxyresveratrol, 2 ′, 4 ′, 5, 7-tetrahydroxyflavanone, morusignin L or dihydromorin The composition was disclosed, and in the preceding paper [Park, Soo-yeon et al., Journal of Nutrition and Health (J Nutr Health), 49 (1), 18-27, 2016], the upper limb and epithelial ethanol extracts were elevated in postprandial blood glucose in vivo and in vitro. Mi in suppression regulation Has been disclosed for impact. However, there is no previous report confirming that the epidermis derived compound represented by Formula 1 of the present invention has a therapeutic effect on diabetes.

Korean Unexamined Patent Publication No. 10-2015-0102833, A composition for preventing and treating diabetes, which contains a compound isolated from the extract of lettuce extract, September 08, 2015, published. Korean Unexamined Patent Publication No. 10-2014-0123264, Use of a compound isolated from Sangbaekpi, October 22, 2014, published. Korean Unexamined Patent Publication No. 10-2012-0111771, Use of the compound isolated from baekryepi, October 11, 2012, published.

Correlation between Diabetic Peripheral Vascular Disease and Ankle-Brachial Index, Ko Boram et al. Korean Society of Endocrinology, 21 (5), 382-388, 2006. Kim Dong-rim, Drug Treatment of Type 2 Diabetes, Konkuk University Hospital KRC, 2012. Park, Soo-Yeon et al., Effect of Upper and Upper Epithelial Ethanol Extracts on the Control of Postprandial Glucose Inhibition in Vitro and in Vitro, Journal of Nutrition and Health (J Nutr Health), 49 (1), 18-27, 2016. Aronoff, S. et al., Pioglitazone hydrochloride monotherapy improves glycemic control in the treatment of patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. The Pioglitazone 001 Study Group, Diabetes Care, 23 (11), 1605-1611, 2000. Coman, C. et al., Plants and natural compounds with antidiabetic action, Not. Bot. Horti. Agrobo., 40 (1), 314-325, 2012. Elchebly, M. et al., Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene, Science, 283 (5407), 1544-1548, 1999. Jiang, C. S. et al., Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades, Acta. Pharmacol. Sin., 33 (10), 1217-1245, 2012. Johnson, T. O. et al., Protein tyrosine phosphatase 1B inhibitors for diabetes, Nat. Rev. Drug. Discov., 1 (9), 696-709, 2002. Kitabachi, A. E. et al., Hyperglycemic crises in adult patients with diabets, Diabetes care, 32 (7), 1335-1345, 2009. Kim, D. H. et al., Characterization of the inhibitory activity of natural tanshinones from Salvia miltiorrhiza roots on protein tyrosine phosphatase 1B, Chem Biol Interact., 278, 65-73, 2017. Klaman, L. D. et al., Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice, Mol. Cell. Biol., 20 (15), 5479-5489, 2000. Li, T. et al., A microplate-based screening method for α-glucosidase inhibitors, Chin. J. Clin. Pharmacol. Therapeut., 10, 1129, 2005. Malamas, M. S. et al., New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties, J. Med. Chem., 43 (5), 995-1010, 2000. Na, M. K. et al., Protein tyrosine phosphatase 1B inhibitory activity of anthraquinones and stilbenes, Nat. Prod. Sci., 14, 143-146, 2008. Schneider, S. H. et al., Impaired fibrinolytic response to exercise in type II diabetes mellitus: Effect of exercise and physical training, Metabolism, 37 (10), 924-929, 1988. Tewari, N. et al., Synthesis and bioevaluation of glycosyl ureas as alpha-glucosidase inhibitors and their effect on mycobacterium, Bioorg. Med. Chem., 11 (13), 2911-2922, 2003. Thareja, S. et al., Protein tyrosine phosphatase 1B inhibitors: a molecular level legitimate approach for the management of diabetes mellitus, Med. Res. Rev., 32 (3), 459-517, 2012. WHO, Diabetes Fact sheet No. 312, 2013.

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diabetes comprising a compound isolated from the epithelium.

The present invention relates to an anhydrous morphosarbin A (compound 1), an anhydrous sarvin B (compound 2), an anhydrous sarvin C (compound 3), an anhydrous sarvin D (compound 4), an albanine T (compound 5) and macrourin G It relates to a pharmaceutical composition for preventing or treating diabetes, comprising at least one compound selected from the group consisting of (Compound 6) as an active ingredient.

[Formula 1]

The compound may be synthesized according to a conventional method in the art, may be prepared as a pharmaceutically acceptable salt, or may be obtained by chromatography from an extract of lettuce extract.

The extract of baekpipi is an extract extracted from baekpipi with one or more solvents selected from the group consisting of water, lower alcohols of C1 to C4, n-hexane, dichloromethane and ethyl acetate, the lower alcohols of C1 to C4 include methanol, Ethanol, propanol, isopropanol, butanol and the like can be used.

The chromatography is silica gel column chromatography, flash column chromatography, Sephadex LH-20 column chromatography, RP-18 column chromatography (RP) -18 column chromatography, thin layer chromatography (TLC), medium pressure liquid chromatography, high performance liquid chromatography (HPLC) and the like can be selected and used.

The pharmaceutical compositions according to the invention may be formulated in a suitable form with the pharmaceutically acceptable carriers generally used. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause an allergic reaction, such as a gastrointestinal disorder, dizziness, or the like.

In addition, the pharmaceutical composition may be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. . Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. When formulated, diluents or excipients such as fillers, stabilizers, binders, disintegrants and surfactants are 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, sucrose or lactose in the compound of the present invention. Mixed with gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition comprising the epidermis derived compound disclosed in the present invention as an active ingredient may be administered to mammals such as rats, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Dosage may include the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the time of administration, the route of administration, the absorption, distribution and excretion of the drug, the type of drug used and the prescriber's It will vary depending on judgment. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.001 mg / kg / day to 2000 mg / kg / day. More preferred dosage is 0.01 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In another aspect, the present invention provides a health functional food for preventing or improving diabetes comprising at least one compound selected from the group consisting of Chemical Formula 1 isolated from the extract of lettuce extract and food supplements. The health functional food refers to a food manufactured or processed using raw materials or ingredients having useful functionalities, and includes all of health supplements, functional foods, nutritional supplements, and supplements.

The chlorophyll-derived compound may be added in an amount of preferably 0.001% to 50% by weight, more preferably 0.001% to 30% by weight, and most preferably 0.001% to 10% by weight based on the total weight of the whole food. have. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. Etc.

In another aspect, the present invention,

Preparing lettuce extract by extracting lettuce extract with water, C1 to C4 lower alcohol or a mixed solvent thereof;

Suspending the lettuce extract in water and sequentially fractionating n-hexane, dichloromethane and ethyl acetate to prepare n-hexane, dichloromethane and ethyl acetate fractions; And

The respective fractions were chromatographed, and the first compound of formula 1 was added murusarvin A (compound 1), morusalvin B (compound 2), morusalvin C (compound 3), morusalvin D (compound 4) and albanine T (compound 5). And macrourine G (compound 6).

The present invention also provides novel compounds of the formula (2): Morusalvin A (Compound 1), Morusalvin B (Compound 2), Morusalvin C (Compound 3), Morusalvin D (Compound 4) and Albanine T (Compound) 5) Provide.

[Formula 2]

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of diabetes comprising a compound isolated from the epidermis, wherein the compound, namely, moru salvin A (compound 1), moru salvin B (compound 2), moru salvin C (compound 3) ), Morusalvin D (Compound 4), Albanine T (Compound 5) and Macrourin G (Compound 6) have excellent protein tyrosine dephosphatase 1B inhibitory activity and alpha-glucosidase inhibitory activity to prevent diabetes or It can be usefully used as a therapeutic composition.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

Example 1. Isolation of Epidermis-derived Compound

10 kg of the dry powder of the lettuce powder was refluxed and filtered five times with 20 L of methanol, and then concentrated under reduced pressure to obtain 1.2 kg of methanol extract. The methanol extract was suspended in 2 L of water, and n-hexane, dichloromethane and ethyl acetate were sequentially fractionated to obtain n-hexane fraction (150 g), dichloromethane fraction (215 g) and ethyl acetate fraction (304.3 g).

The ethyl acetate fraction (304.3 g) in the fraction was fractionated by silica gel column chromatography according to the concentration gradient elution condition of methanol: dichloromethane (0 → 100, v / v), and 20 small fractions (F1-). F20) was obtained.

F3 (3.4 g) of the small fractions were subjected to Sephadex LH-20 column chromatography using a mobile phase of methanol: water (1.5: 1, v / v) to obtain 17 (F3.1-F3.17) small fractions. Got it. In addition, F3.7 (154.3 mg) in the small fraction was purified by RP-18 column chromatography under a solvent condition of methanol: water (2: 1, v / v) to obtain Compound 5 (2.0 mg). .15 (170.8 mg) was prepared as semi-purified RP-HPLC (semi) according to the concentration gradient elution condition of methanol: water (75: 25 → 80: 20, v / v) containing 0.05% trifluoroacetic acid. -preparative RP-HPLC, Gilson Trilution System, YMC Pak ODS-A column (20 × 250mm, 5㎛ particle size, UV detection at 254nm, flow rate of 5ml / min)) to give compound 2 (4.5mg, t _R = 23.0 min).

Next, F5 (6.8 g) in the small fractions was subjected to silica gel column chromatography under elution conditions of dichloromethane: methanol (20: 1 → 0: 1, v / v) to obtain 16 small fractions (F5.1-F5. 16). In addition, F5.10 (237.4 g) in the small fraction was subjected to RP-18 column chromatography using a mobile phase of methanol: water (2.5: 1, v / v) to obtain Compound 6 (5.4 mg). .14 (432.3 mg) was purified using semi-preparative RP- according to the concentration gradient elution condition of methanol: water (75: 25 → 80: 20, v / v) containing 0.05% trifluoroacetic acid. HPLC, Gilson Trilution System, YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size, UV detection at 254 nm, flow rate of 5 mL / min)) gave Compound 1 (1.8 mg, t _R = 43.0 min). Got it.

Next, F8 (15.1 g) in the small fractions was fractionated by silica gel column chromatography according to the concentration gradient elution conditions of dichloromethane: methanol (100: 0 → 0: 100, v / v) and 23 small fractions (F8. 1-8.23). In addition, F8.6 (2.5 g) in the small fraction was passed through a Sephadex LH-20 column under a mobile phase of methanol: water (1: 1, v / v) to eleven small fractions (F8.6.1-F8. 6.11). In addition, the purified fraction RP-HPLC (0.05% trifluoroacetic acid containing methanol-water, 70-80%, concentration gradient elution condition) to give F8.6.8 (173.5 mg) in the small fraction compound 3 (5.5 mg, t _R = 24.0 min) and purified RP-HPLC (0.05% trifluoroacetic acid with methanol: water (70: 30 → 85: 15, v / v) gave small fraction F8.6.10 (381.3 mg) , Concentration gradient elution conditions) afforded compound 4 (6.8 mg, t _R = 22 min).

Physical and chemical properties of the novel compounds 1 to 5 of the present invention obtained from the above process are as follows.

Compound 1.Morusalbin A

Yellow amorphous powder;

+48.6 (c 0.07, MeOH);

+48.6 ( c 0.07, MeOH);

IR (KBr) ν _max 3317, 2943, 2831, 1680, 1646, 1550, 1505, 1448, 1116, 1024 cm ⁻¹ ;

UV (MeOH) λ _max (log ε ) 337 (2.28), 327 (1.83), 283 (1.34), 211 (2.71) nm;

ECD ( c 0.0012 mM, MeOH): Δ ε ₂₀₃ +8.10, Δ ε ₂₃₀ -5.68, Δ ε ₂₅₄ +3.25, Δ ε ₂₇₅ -1.41, Δ ε ₂₉₄ +6.20, Δ ε ₃₀₉ +8.28;

¹ H (500 MHz in CD ₃ OD) and ¹³ C NMR (125 MHz in CD ₃ OD) data see Table 1 below;

HRESI-MS m / z 599.1315 ([M + Na] ⁺ , calcd for C ₃₄ H ₂₄ O ₉ Na, 599.1318), m / z 577.1509 ([M + H] ⁺ , calcd for C ₃₄ H ₂₅ O ₉ , 577.1499).

Compound 2. Morusalbin B

Brown amorphous powder;

+237.0 (c 0.11, MeOH);

+237.0 (c 0.11, MeOH);

IR (KBr) ν _max 3327, 2943, 2831, 1550, 1508, 1448, 1116, 1024 cm ^-1 ;

UV (MeOH) λ _max (log ε) 334 (1.74), 324 (1.78), 287 (0.82), 210 (2.61) nm;

ECD (c 0.0016 mM, MeOH): Δε ₂₃₁ +11.26, Δε ₂₈₂ +0.42, (observed as a valley), Δε ₂₉₉ +2.74, Δε ₃₃₄ +3.63;

¹ H (500 MHz in CD ₃ OD) and ¹³ C NMR (125 MHz in CD ₃ OD) data see Table 1 below;

HRESI-MS m / z 583.1366 ([M + Na] ⁺ , calcd for C ₃₄ H ₂₄ O ₈ Na, 583.1369), m / z 561.1548 ([M + H] ⁺ , calcd for C ₃₄ H ₂₅ O ₈ , 561.1549).

Compound 3.Morusalbin C

Brown amorphous powder;

IR (KBr) ν _max 3328, 2945, 2831, 2504, 1646, 1680, 1647, 1550, 1508, 1447, 1117, 1025 cm ⁻¹ ;

UV (MeOH) λ _max (log ε) 337 (3.04), 323 (2.68), 285 (2.53), 224 (3.76) nm;

¹ H (500 MHz in CD ₃ OD) and ¹³ C NMR (125 MHz in CD ₃ OD) data see Table 1 below;

HRESI-MS m / z 583.1371 ([M + Na] ⁺ , calcd for C ₃₄ H ₂₄ O ₈ Na _, 583.1369), m / z 561.1552 ([M + H] ⁺ , calcd for C ₃₄ H ₂₅ O ₈ , 561.1549 ).

Compound 4.Morusalbin D

Brown amorphous powder;

+446.5 (c 0.17, MeOH);

+446.5 (c 0.17, MeOH);

IR (KBr) ν _max 3327, 2943, 2831, 1505, 1447, 1117, 1024 cm ⁻¹ ;

UV (MeOH) λ _max (log ε) 333 (2.95), 324 (2.29), 287 (1.83), 225 (3.18) nm;

ECD (c 0.0025 mM, MeOH): Δε ₂₃₅ +15.02, Δε ₂₆₁ +2.27, (observed as a valley), Δε ₂₉₆ +9.20;

¹ H (500 MHz in CD ₃ OD) and ¹³ C NMR (125 MHz in CD ₃ OD) data see Table 2 below;

HRESI-MS m / z 571.1729 ([M + Na] ⁺ , calcd for C ₃₄ H ₂₈ O ₇ Na _, 571.1733), m / z 549.1903 ([M + H] ⁺ , calcd for C ₃₄ H ₂₉ O ₇ , 549.1913 ).

Compound 5.Albanin T

Brown amorphous powder;

-17.0 (c 0.09, MeOH);

-17.0 ( c 0.09, MeOH);

IR (KBr) ν _max 3327, 2943, 2830, 1447, 1026 cm ⁻¹ ;

UV (MeOH) λ _max (log ε ) 289 (2.45), 262 (3.01), 212 (3.38) nm;

ECD ( c 0.0022 mM, MeOH): Δ ε ₂₀₂ +3.43;

¹ H (500 MHz in CD ₃ OD) and ¹³ C NMR (125 MHz in CD ₃ OD) data see Table 2 below;

HRESI-MS m / z 461.1579 ([M + Na] ⁺ , calcd for C ₂₅ H ₂₆ O ₇ Na _, 461.1576), m / z 439.1761 ([M + H] ⁺ , calcd for C ₂₅ H ₂₇ O ₇ , 439.1757 ).

Position Compound 1 Compound 2 Compound 3 δ _H mult.,
( J in Hz) δ _C , type δ _H mult.,
( J in Hz) δ _C , type δ _H mult.,
( J in Hz) δ _C , type One 2 156.4, C 159.2, C 156.1, C 3 6.95, s 102.2, CH 6.85, s 103.2, CH 6.85, s 102.2, CH 3a 123.3, C 123.0, C 123.2, C 4 7.35, d (8.5) 122.0, CH 7.26, d (8.5) 122.3, CH 7.32, d (8.5) 122.1, CH 5 6.74,
dd (8.5, 2.5) 113.4, CH 6.66,
dd (8.5, 2.5) 113.5, CH 6.72,
dd (8.5, 2.5) 113.3, CH 6 157.4, C 157.4, C 157.4, C 7 6.90, d (2.5) 98.5, CH 6.79, d (2.5) 98.5, CH 6.86, d (2.5) 98.6, CH 7a 157.0, C 157.2, C 156.9, C One' 130.1, C 133.7, C 132.7, C 2' 6.93, d (1.0) 105.1, CH 6.61, d (1.0) 104.9, CH 6.79, s 104.0, CH 3 ' 153.4, C 155.21, C 157.0, C 4' 117.9, C 112.1, C 116.8, C 5 ' 158.2, C 155.23, C 157.0, C 6 ' 7.43, d (1.0) 100.4, CH 6.76, d (1.0) 105.7, CH 6.63, s 103.7, CH One'' 71.1, C 136.4, C 140.3, C 2'' 154.8, C 6.72, s 125.2, CH 7.11, s 132.4, CH 3 '' 114.0, C 77.8, C 134.8, C 4'' 5.13, d (2.0) 51.3, CH 2.18, d (12.0) 43.3, CH 137.4, C 5 '' 3.46, m 36.7, CH 2.75, ddd (12.0, 10.5, 6.0) 28.3, CH 141.8, C 6 '' 1.94, dd (13.0, 4.0)
2.38, dd (13.0, 2.0) 33.1, CH ₂ 2.04, dd, (16.0, 10.5)
2.67, dd (16.0, 6.0) 36.1, CH2 7.21, d (1.0) 130.7, CH 7 '' 1.90, s 23.4, CH ₃ 1.84, s 23.9, CH ₃ 2.47, s 21.6, CH ₃ 8'' 204.0, C = O 95.8, C 204.4, C = O 9 '' 113.3, C 115.5, C 115.7, C 10 '' 166.8, C 156.6, C 166.04, C 11 '' 6.37, d (2.5) 104.2, CH 6.21, d (2.5) 104.2, CH 5.97, d (2.5) 102.8, CH 12 '' 167.4, C 160.9, C 166.08, C 13 '' 6.61, dd (8.5, 2.5) 109.8, CH 6.40, dd (8.5, 2.5) 110.1, CH 6.03, dd (8.5, 2.5) 108.4, CH 14 '' 8.24, d (8.5) 133.9, CH 7.54, d (8.5) 127.8, CH 7.19, d (8.5) 138.0, CH 15 '' 117.7, C 117.9, C 133.7, C 16 '' 155.7, C 153.6, C 7.13, d (8.5) 131.3, CH 17 '' 6.21, d (2.5) 104.6, CH 6.46, d (2.5) 104.88, CH 6.61, d (8.5) 116.0, CH 18 '' 158.9, C 158.4, C 157.8, C 19 '' 6.45, dd (8.5, 2.5) 110.1, CH 6.43, dd (8.5, 2.5) 110.7, CH 6.61, d (8.5) 116.0, CH 20 '' 7.28 d (8.5) 130.5, CH 7.02, d (8.5) 128.4, CH 7.13, d (8.5) 131.3, CH

Position Compound 4 Compound 5 δ _H mult., ( J in Hz) δ _C , type δ _H mult., ( J in Hz) δ _C , type One 130.7, C 2 7.29, d (8.5) 128.9, CH 162.2, C 3 6.70, d (8.5) 116.6, CH 121.4, C 4 158.5, C 183.7, C = O 4a - - 105.4, C 5 6.70, d (8.5) 116.6, CH 163.5, C 6 7.29, d (8.5) 128.9, CH 6.19, d (2.0) 99.7, CH 7 - - 165.7, C 8 - - 6.27, d (2.0) 94.6, CH 8a - - 159.7, C α 6.90, d (16.5) 129.3, CH - β 6.74, d (16.5) 126.6, CH - One' 139.1, C 108.4, C 2' 6.52, d (1.5) 107.2, CH 160.8, C 3 ' 154.8, C 114.6, C 4' 112.9, C 157.7, C 5 ' 158.2, C 6.43, d (8.5) 109.3, CH 6 ' 6.43, d (1.5) 107.1, CH 7.07, d (8.5) 130.8, CH One'' 133.8, C 3.17, d (7.0) 25.1, CH ₂ 2'' 6.31, d (5.0) 123.8, CH 5.12, d (7.0) 123.3, CH 3 '' 3.22, m 35.5, CH 132.9, C 4'' 3.21, dd (11.0, 5.5) 37.6, CH 1.61, s 25.9, CH ₃ 5 '' 2.86, ddd (11.0, 11.0, 5.5) 28.9, CH 1.45, s 17.9, CH ₃ 6 '' 1.94, dd (17.0, 11.0)
2.60, dd (17.0, 5.5) 36.9, CH ₂ 3.16, d, (9.0) 28.9, CH ₂ 7 '' 1.71, s 24.1, CH ₃ 4.69, t, (9.0) 91.7, CH 8'' 103.1, C 72.5, C 9 '' 117.6, C 1.25, s 25.9, CH ₃ 10 '' 158.0, C 1.25, s 24.9, CH ₃ 11 '' 6.24, d (2.5) 104.6, CH 12 '' 160.1, C 13 '' 6.06, dd (8.5, 2.5) 107.0, CH 14 '' 7.04, d (8.5) 130.6, CH 15 '' 118.5, C 16 '' 153.8, C 17 '' 6.25, d (2.5) 104.2, CH 18 '' 157.8, C 19 '' 6.38, dd (8.5, 2.5) 110.1, CH 20 '' 7.02, d (8.5) 128.1, CH

< Example  2. Protein Tyrosine Dephosphoryase  Confirmation of 1B Inhibitory Activity>

The protein p-nitrophenyl phosphate (p-NPP) is changed to p-nitrophenol (p-NP) by the protein tyrosine dephosphatase 1B and becomes yellow. Enzyme activity was measured through changes in absorbance (Kim, DH et al., Chem Biol Interact., 278, 65-73, 2017; Na, MK et al., Nat. Prod. Sci., 14, 143-146, 2008).

To each well (final volume 100 μl) of a 96 well plate was added buffer (50 mM citrate, pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT) containing protein tyrosine dephosphatase 1B, followed by the present invention. Compounds 1-6 were treated respectively. Then 50 μl of 2mM p-nitrophenyl phosphate was added, followed by reaction at 37 ° C. for 15 minutes, and the reaction was terminated by treatment with 10 μl of 10M NaOH. The final amount of p-nitrophenol produced was measured at absorbance (OD) of 405 nm (VERSA max, Molecular Devices, Sunnyvale, Calif., USA) and the protein tyrosine dephosphatase 1B inhibitory activity of each compound was calculated by the following equation (1). Table 3 shows.

[Equation 1]

% Inhibition = {(Ac - As) / Ac} × 100

(Ac is the absorbance of the control, and As is the absorbance of the sample)

Condition IC ₅₀ (μM) Compound 1 6.07 ± 0.12 Compound 2 6.77 ± 0.81 Compound 3 9.67 ± 0.08 Compound 4 1.90 ± 0.12 Compound 6 2.52 ± 0.03 Ursolic acid 9.47 ± 0.47

Looking at the Table 3, because the compound derived from baekryepi of the present invention shows more than equivalent protein tyrosine dephosphatase 1B inhibitory activity compared to ursolic acid, a positive control group, it can be seen that it can be usefully used as a composition for preventing or treating diabetes. there was.

<Example 4. Confirmation of alpha-glucosidase inhibitory activity>

Changes in absorbance (microplate spectrophotometer, Molecular Devices) at 405 nm using p-nitrophenyl α-D-glucopyranoside (p-NPG), a substrate of alpha-glucosidase Enzyme activity was measured (Li, T. et al., Chin. J. Clin. Pharmacol. Therapeut., 10, 1129, 2005).

The activity of each compound is shown in Table 4 as IC ₅₀ (the half maximal inhibitory concentration).

Condition IC ₅₀ (μM) Compound 1 4.53 ± 0.30 Compound 2 5.07 ± 0.34 Compound 3 5.40 ± 0.45 Compound 4 3.55 ± 0.03 Compound 5 35.35 ± 0.42 Compound 6 3.61 ± 0.24 Acarbose 203.97 ± 4.27

Looking at the Table 4, the compounds 1 to 6 of the present invention showed significantly superior alpha-glucosidase inhibitory activity compared to the acarbose which is a positive control group.

Through this, it was found that the compound of the present invention may be useful as a composition for preventing or treating diabetes mellitus derived from the compound and the extract from the extract.

Preparation Example 1 Preparation of Tablet

20 g of compound 4 (morusvin D) of Example 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, respectively. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Preparation Example 2 Preparation of Capsule

100 mg of compound 4 (morusalvin D) of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed, and the above ingredients were mixed according to a conventional capsule preparation method and added to a gelatin capsule. The capsule was prepared by filling.

Preparation Example 3 Preparation of Injection

1 g of Compound 4 (Morusabine D) of Example 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. The solution was bottled and sterilized by heating at 20 ° C for 30 minutes.

Preparation Example 4 Preparation of Health Functional Food

20 g of compound 4 (morusalvin D) of Example 1 of the present invention, a vitamin mixture proper amount, 70 µg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 µg of vitamin B12, Vitamin C 10mg, Biotin 10µg, Nicotinamide 1.7mg, Folic Acid 50µg, Pantothenate 0.5 mg, Mineral Mixture Proper, Ferrous Sulfate 1.75mg, Zinc Oxide 0.82mg, Magnesium Carbonate 25.3mg, Potassium Phosphate 15mg , 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

< Formulation example  5. Manufacturing of Functional Health Drinks>

1 g of Compound 4 (Morusabin D) of Example 1 of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed to prepare a beverage by stirring, heating, filtration, sterilizing, and refrigerating according to a conventional beverage preparation method.

Claims (8)

Morusalvin A (Compound 1), Morusalvin B (Compound 2), Morusalvin C (Compound 3), Morusalvin D (Compound 4), Albanine T (Compound 5) and Macrourin G (Compound 6) A pharmaceutical composition for preventing or treating diabetes comprising at least one compound selected from the group consisting of) as an active ingredient.
[Formula 1]

The method of claim 1,
The compound 1 to 6 is a pharmaceutical composition for preventing or treating diabetes, characterized in that the compound isolated from the epithelium. The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating diabetes, characterized in that it is formulated in a pharmaceutical dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent. Morusalvin A (Compound 1), Morusalvin B (Compound 2), Morusalvin C (Compound 3), Morusalvin D (Compound 4), Albanine T (Compound 5) and Macrourin G (Compound 6) A health functional food for preventing or improving diabetes comprising at least one compound selected from the group consisting of) as an active ingredient.
[Formula 1]

The method of claim 4, wherein
The compound 1 to 6 is a dietary supplement for preventing or improving diabetes, characterized in that the compound isolated from the epithelium. The method of claim 4, wherein
Formulation of the health functional food is a dietary supplement for preventing or improving diabetes, characterized in that selected from the group consisting of tablets, capsules, pills or liquids. Preparing lettuce extract by extracting lettuce extract with water, C1 to C4 lower alcohol or a mixed solvent thereof;
Suspending the lettuce extract in water and sequentially fractionating n-hexane, dichloromethane and ethyl acetate to prepare n-hexane, dichloromethane and ethyl acetate fractions; And
The respective fractions were chromatographed to give the following formula: Morusalvin A (Compound 1), Morusalvin B (Compound 2), Morusalvin C (Compound 3), Morusalvin D (Compound 4), and Albanine T (Compound 5) And macrourine G (compound 6).
[Formula 1]

In the group consisting of Morusalvin A (Compound 1), Morusalvin B (Compound 2), Morusalvin C (Compound 3), Morusalvin D (Compound 4) and Albanine T (Compound 5) having the structure of formula (2) New compound selected.
[Formula 2]