WO2014168458A1

WO2014168458A1 - Use of compounds isolated from morus bark

Info

Publication number: WO2014168458A1
Application number: PCT/KR2014/003163
Authority: WO
Inventors: Hwan Bong Chang; Joobyoung Yoon; Hyunyong Lee; Yang Kook Rho
Original assignee: Dong Wha Pharm Co Ltd
Current assignee: Dong Wha Pharm Co Ltd
Priority date: 2013-04-12
Filing date: 2014-04-11
Publication date: 2014-10-16
Anticipated expiration: 2015-10-12
Also published as: KR20140123264A

Abstract

The present invention relates to pharmaceutical composition for the prevention and treatment of diabetic complications, or health food, comprising Cathayanin B or Kuwanol A isolated from Morus Bark. The compound Cathayanin B according to the present invention is isolated from Morus Bark and inhibits the formation of advanced glycation end-products (AGEs), which cause diabetic complications, and inhibits aldose reductase activity. Kuwanol A, another compound isolated from Morus Bark, inhibits TGF-β1 signaling. Therefore, the pharmaceutical composition or health food comprising Cathayanin B or Kuwanol A according to the present invention can be used to inhibit diabetic nephritis, diabetic retinopathy, and diabetic neuropathy, which are diabetic complications. In particular, Kuwanol A can also be used for pharmaceutical composition for the prevention and treatment of various fibrosis and metastasis conditions as well as health food to improve said conditions.

Description

USE OF COMPOUNDS ISOLATED FROM MORUS BARK

The present invention relates to a novel medical use of Cathayanin B and Kuwanol A, compounds isolated from Morus Bark.

Diabetes is characterized by persistence of hyperglycemia resulting from abnormal secretion or function of insulin. It induces several symptoms due to hyperglycemia and causes excretion of glucose through urination. Recently in Korea, prevalence rate of diabetes is rapidly increasing due to westernization of diet and growth of senior citizen population.

Diabetic complications are divided into acute diseases and chronic diseases; acute diseases include diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome, and chronic diseases include microvascular diseases such as diabetic nephropathy, diabetic retinopathy, diabetic cataract, diabetic neuropathy, and macrovascular diseases such as diabetic cardiomyopathy and cerebrovascular disease.

Advanced glycation end product (AGE) production inhibitors, aldose reductase activity inhibitors and transforming growth factor-β (TGF-β) receptor activity inhibitors are currently being studied as drugs which treat and prevent the above-mentioned diabetic complications.

AGE production inhibitor inhibits formation of advanced glycation end products by suppressing induction of diabetes complications resulting from AGE production through nonenzymatic glycation of protein in persistent hyperglycemia.

Long-term persistence of hyperglycemia leads to structural and functional changes in protein and lipid through nonenzymatic binding and rearrangement of reducing sugars (such as glucose) with proteins and lipids. In this process, irreversible formation of AGEs occurs. AGEs formed in such hyperglycemic conditions cross-link to extracellular matrix proteins such as collagen or fibronectin of long-lived cells, or bind to receptors for AGEs (RAGEs) on the cell’s surface, triggering the signal transduction mechanism, and thus inducing diabetic complications (Schmidt, A. M., et. Al., 2000, Trends Endocrinol. Metab., 11, 368-375).

We can further categorize the different types of diabetic complications induced by advanced glycation end products. Advanced glycation end products activate Smad-2/3 in either TGF-β-dependent or TGF-β-independent way, inducing diabetic nephritis (Li, J.H., et. Al.,2004, FASEB J. 18, 176-178; Fukami K., et.al., 2004, Kidney Int. 66, 2137-2147; Chung, C.K., et. al., 2010, J. Am Soc. Nephrol.. 21, 249-260), and AGEs are known to cause diabetic retinopathy as well as diabetic neuropathy through interaction with receptors for advanced glycation end products (RAGEs) (Barile G. R., et. al., 2005, Invest Ophthalmol Vis Sci. 46(8), 2916-2924; Toth C., et. al., 2008, Diabetes. 57(4), 1002-1017).

Administration of AGE formation inhibitors in animal studies has shown significant inhibition of induction of diabetic nephritis (Osicka T. M., et. al., 2000, Diabetes. 49(1), 87-93; Yang C. W., et. al., 1994, Proc Natl Acad Sci U S A. 91(20), 9436-9440), diabetic retinopathy (Hammes H.P., et. al., 1991, Proc Natl Acad Sci U S A. 88(24), 11555-11558) and diabetic neuropathy (Duran-Jimenez B., et. al., 2009, Diabetes. 58(12), 2893-2903).

The typical AGE production inhibitors include aminoguanidine and pyridoxamine (commercial name: Pyridorin), but the development of aminoguanidine was ceased due to toxicity related with vitamin B deficiency in clinical trial phase Ⅲ experimentation, and pyridoxamine has recently entered clinical trial phase Ⅲ; there are no commercialized drug of pyridoxamine yet.

In hyperglycemic condition, aldose reductase, which is the main mediator of activated polyol pathway, converts glucose to sorbitol. In process of sorbitol production, excessive consumption of NAD+/NADP+ occurs, which leads to decrease in synthesis of myo-inositol, nitrogen monoxide and taurine. Also, as osmotic pressure increases by sorbitol accumulation, it can cause damages in cell and tissue. Fructose, which is the end product of polyol pathway, is more than 10 times more potent than glucose in nonenzymatic glycation of proteins. Activation of polyol pathway leads to increased formation of sorbitol, whose conversion to fructose promotes further AGE production, leading to various diabetic complications. (Nakamura, N., et. Al., 2000, Free Radic Biol. Med., 29: 17-25).

Also, aldose reductase inhibitor was reported as fundamentally effective on treatment of diabetic neuropathy, diabetic retinopathy, cataract and diabetic nephritis in animal experiment or clinical trial(Misawa, S. et. al., Neurology 2006, 66(10): 1545; Fujishima, H. et. al., Br J Ophthalmol 2002, 86(8): 860; Robinson WG JR., et. al., Invest Ophthamol Vis Sci. 1996 May;37(6):1149-56.; Iso K., et. al., J Diabetes Complications. 2001 Sep-Oct;15(5):241-4.).

At the moment, several drugs which inhibit the activity of aldose reductase are undergoing clinical trials. In case of epalrestat (product name: Kinedak), the typical aldose reductase inhibitor, annual sales of the drug recorded approximately 2,500 million in 2003 after being released as diabetic neuropathy medication in 1992 in Japan. However, the drug is approved for sale only in Japan and is not sold in United States and other countries.

TGF-β superfamily consists of TGF, activin, bone morphogenetic protein as well as inhibin, and performs proliferation, differentiation, migration, apoptosis, and other multilateral functions on cells of each tissue. These receptors include type Ⅱ receptors binding with ligand, type I receptors which are called ALK (activin like kinase), and type Ⅲ receptor. When type Ⅱ receptor combines with ligand it induces oligomerization with type Ⅰ receptor, which stimulates specific gene transcription through the phosphorylation of Smad2/3 (Maustakas A. et. al., 1993, J. Biol. Chem. 268(30) 22215-22218).

From the pathophysiologic perspective of several diseases, TGF-β performs an important role. TGF-β induces fibrosis by producing excessive collagen or fibronectin, which are extracellular matrix proteins, or is deeply involved in tubulointerstitial disease or metastasis through EMT (epithelial-mesenchymal transition) in tubular epithelial cell of kidney or all kinds of cancer cells (Bottinger E.P. et. al., 2002, J. Am. Soc. Nephrol. 13: 2600-2610; Roberts A. B. et. al., 2006, Cytokine Growth Factor Rev. 17(1-2) 19-27). Also, in vitro and animal studies have shown that AGEs or angiotensin Ⅱ formed in hyperglycemic conditions increases mRNA of TGF-β1 as well as protein synthesis. In the result, the diabetic complications such as diabetic nephropathy, diabetic retinopathy and diabetic neuropathy were exacerbated as well as atherosclerosis, hypertension (Hills C.E. et. al., 2010, Am. J. Nephrol. 31(1)68-74; Paques M., et. al., 1997, Diabetes Metab. 23(2)125-130; Bobik A., et. al., 1999, Circulation 99(22)2883-2891; Fukami K. Et. Al., 2004, Kidney Int. 66(6) 2137-2147).

Outside of that, TGF-β has been reported in involvement of restenosis, osteoporosis, breast cancer, colon cancer, lung cancer, pancreatic cancer and prostate cancer (Gordon K. J. et. al., 2008, Biochim. Biophys. Acta 1782(4) 197-228).

TGF-β inhibitors can be largely divided into monoclonal antibody and chemical drug. Metelimumab (CAT-192), a TGF-β1 monoclonal antibody, is undergoing clinical trial Ⅰ and Ⅱ as treatment of scleroderma, interstitial lung disease and renal disease (Yingling J. M. et. al., 2004, Nat Rev. Drug Discov. 3(12)(1011-1022), and pirfenidone, a chemical drug, was approved for sale as idiopathic pulmonary fibrosis (IPF) medication. Also LY2157299, a TGF-β I type receptor(ALK5) inhibitor, is undergoing clinical trial phase Ⅱ as treatment of liver cancer and metastasis inhibitor (Bueno L., et. al., 2008, Eur. J. Cancer 44(1) 142-150).

Meanwhile, Morus Bark is a drug which is made from root bark of mulberry, and it is known for medical actions such as antitussive, diuretic, hypotensive, sedative, analgesic, antipyretic, antispasmodic, and antibacterial actions (Doosan encyclopedia Encyber & Encyber.com).

Many components have been isolated from Morus Bark, and there have been many studies on them. The present inventor(s) identified AGE production inhibitor compounds isolated from Morus Bark, including mulberrofuran G, mulberrofuran K, Kuwanon G, Kuwanon Z, Oxyreserveratrol, 2’,4’,5,7-tetrahydroxyflavanone, morusignin L and dihydromorin, which can suppress diabetic complication, and filed Korean Patent Application No. 2011-27789 on this matter.

Also, as Korean Patent Application No. 2011-27789 stated, moracin O, moracin P and mulberrofuran H from Morus Bark are disclosed as having inhibitory action on Hypoxia Inducible Factor-1(HIF-1) activation which induces various types of cancer and diabetic retinopathy (Korea Patent Application No. 2007-78888). Moracin-M has been reported to reduce blood pressure in diabetic rats (Zang M., et. al., 2009, Fitoterapia 80(8) 475-477), and kuwanon-L has been known for inhibitory action on protein tyrosine phosphatase 1B1 (PTP1B1) (Cui L., et. al., 2006, Bioorg, Med. Chem. Lett. 16(5) 1426-1429), and morin (3,5,7,2’4’ -pentahydroxyflavone) has been reported to antagonist TGF receptor Ⅱ and to inhibit the glycation of low density lipoprotein (LDL) (Gaffari M. A., et. al., 207, Iran Biomed. J. 11(3) 185-191; Shimanuki T., et. al., 2007, Oncogene 26(23) 3311-3320). Mulberrofuran G also have inhibitory action on tyrosinase (Zheng Z. P., et., al., 2010, Agric. Food Chem. 58(9) 5368-5373) as well as antioxidative activity (Dai S. J., et. al., 2004, Chem. Pharm. Bull (Tokyo) 52(10) 1190-1193). Mulberrofuran K was known as antioxidative effect(Dai S. J., et., al., 2004, Chem. Pharm. Bull (Tokyo) 52(10) 1190-1193), Kuwanon G showed antibacterial effect(Park. K. M., et. al., 2003. J. Ethnopharmacol. 84(2-3) 181-185) and antagonistic effect on bombesin (Mihara S., et. al., 1995, Biochem Biophys Res Commun. 15;213(2):594-9), oxyresveratol was known for its antioxidative action (Lorenz P., et. al., 2003, Nitric Oxide 9(2):64) and anti-inflammatory action (Jung. K. O., et. al., 2003, J Pharm Pharmacol 55(12):1695), dihydromorin was reported to have inhibitory action on tyrosinase (Kuniyoshi S., et., 1998, Planta Medica 64(5) 408-412).

Compounds isolated from above Morus Bark include Cathayanin B and Kuwanol A. Cathayanin B has a structure of Chemical Formula 1 and is disclosed to have weak anticancer effect on human cancer cell line. (Journal of Asian Natural Products Research, 2010, vol. 12, #6 p. 505-515). Kuwanol A has a structure of Chemical Formula 2 and there are no known function of this compound yet (Heterocycles, 1985, Vol. 23, # 4 P. 819-824).

The present inventors saw various pharmacological activities of compounds isolated from Morus Bark. While studying the various pharmacologic activities of compounds isolated from Morus Bark, the present inventors saw that Cathayanin B showed inhibitory effect not only on formation of AGEs, but also on aldose reductase activity, and that Kuwanol A had inhibitory effect on TGF-β signaling, leading to the completion of the present invention.

The object of the present invention is to provide pharmaceutical composition or the health functional food for the prevention and treatment of diabetic complications, using Cathayanin B or Kuwanol A, which are compounds isolated from Morus Bark.

Also, another object of the present invention is to provide pharmaceutical composition or the health functional food for the prevention and treatment of fibrosis or metastasis, using Kuwanol A isolated from Morus Bark.

The present invention relates to pharmaceutical composition comprising Cathayanin B isolated from Morus Bark as an active ingredient for prevention and treatment of diabetic complications.

Cathayanin B is a compound isolated from Morus Bark. It inhibits the production of AGEs (advanced glycation end products), which is a causative substance of diabetic complications, and also inhibits activity of aldose reductase.

AGEs are a causative substance of diabetic complications. Types of AGEs include florescence substances like pentosidine and argpyrimidine, non-florescence substances like N-carboxymethyl lysine (CML) and N-carboxyethyl lysine (CEL). Thus, well-established experiment using fluorescence spectroscopy analysis (Monnier, V. M., et. Al., 1984, Proc. Natl. Acad. Sci. USA 81: 583-587) or using an antibody specific to AGEs (Horie H., et. Al., 1997, J. Clin. Invest. 100(12), 2995-3004) can be used to measure the degree of AGEs production.

In the present invention, inhibitory concentration (IC₅₀) was obtained by measuring the amount of AGEs using microplate reader for fluorescence analysis (Excitation: 360㎚, Emission: 465㎚). The degree of inhibition was confirmed by the western blot analysis using an antibody specific to AGEs. From the above, it was shown that Cathayanin B inhibits AGEs production.

Also, the above compound was performed on inhibitory effect of aldose reductase activity for each concentration using microplate reader for fluorescence analysis (Excitation: 360㎚, Emission: 465㎚) , and inhibitory concentration (IC₅₀) was determined, it was shown that the compound inhibits aldose reductase activity which also causes diabetic complications.

As mentioned above, Cathayanin B isolated from Morus Bark inhibits AGEs which are causative substances of diabetic complications. And it also inhibits activity of aldose reductase. Therefore, the present invention which comprises this compound as an active ingredient provides prevention and treatment of diabetic complications.

Therefore, the pharmaceutical composition according to the present invention comprising Cathayanin B as an active ingredient can be useful for preventing and treating diabetic complications, such as diabetic nephritis, diabetic retinopathy, and diabetic neuropathy.

Also, the present invention relates to the pharmaceutical composition for prevention and treatment of diabetic complications, various types of fibrosis, and metastasis, wherein the composition comprises Kuwanol A, a compound isolated from Morus Bark, as an active ingredient.

Another compound Kuwanol A isolated from Morus Bark has an inhibitory effect on TGF-β signal pathway, a causative factor of diabetic complication, various types of fibrosis, metastasis.

In the present invention, it is identified that Kuwanol A isolated from Morus Bark has an inhibitory mechanism on TGF-β signal pathway from the following experiments: an experiment analyzing the inhibitory effect on TGF-β 1 transcription, and measuring phosphorylation of Smad-2/3 which is signal mediator of TGF-β1. It is also identified that this compound inhibits Epithelial Mesenchymal Transition (EMT) in Human Renal Proximal Tubular Epithelial Cells (RPTECs) and breast cancer cell line (MCF-7).

Therefore, in the present invention, Kuwanol A, a compound isolated from Morus Bark, inhibits TGF-β signaling which is a causative factor of diabetic complication, various types of fibrosis, and metastasis. Therefore, a compound that comprises this as an active ingredient can be used to prevent and treat in diabetic complications, various types of fibrosis, and metastasis.

Thus, in the present invention, the pharmaceutical composition comprising Kuwanol A as an active ingredient can prevent and treat diabetic complications, such as diabetic nephritis, diabetic retinopathy, diabetic neuropathy, and the fibrosis which is lung fibrosis including idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, myelofibrosis, renal fibrosis, Crohn’s disease, keloid, and arthrofibrosis.

The present invention can be administered in various forms, oral and non-oral administration in the actual clinical administration. The most preferable route is oral administration. Also, in case of formulating, it can be prepared using diluents or excipients such as commonly used filing agent, bulking agent, binders, wetting agent, disintegrating agent and surfactant.

The solid preparation for oral administration includes tablets, pills, powders, granules and capsules. Such solid preparations could be prepared by mixing one or more excipients, for example microcrystalline cellulose, low substituted hydroxypropyl cellulose, colloidal silicon dioxide, calcium silicate, starch, calcium carbonate, sucrose or lactose, and gelatin. Besides the simple excipients, a lubricant such as magnesium stearate talc could be used. Moreover, the liquid preparation for oral administration includes suspension, internal solution, emulsion and syrup as well as various excipients such as wetting agent, sweetening agent, flavoring agent and preserved agent besides commonly used diluents such as water and liquid paraffin. The preparation for non-oral administration includes sterilized aqueous solution, nonaqueous solvent, suspension, emulsion, lyophilized product and suppository. As nonaqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil like olive oil and injectable ester like ethyl oleate could be used. For suppository, witepsol, macrogol, tween 61, cocoa butter, laurinum and glycerogellatin could be used.

Furthermore, dose of Cathayanin B or Kuwanol A varies depends on patients’ age, weight, gender, general health, diet, treatment duration, administration route, excretion rate and severity of disease. It is desirable to take 0.001㎎/㎏ to 1,000㎎/㎏ once or divide it into several times.

Also, the present invention relates to the health functional food that comprises Cathayanin B or Kuwanol A, which are isolated from Morus Bark, as an active ingredient, and comprises sitologically acceptable food supplemental additives.

By taking food which contains this compound, diabetic complication which is the secondary symptom of diabetic patient can be improved. Specifically, this invention provides the health functional food that can improve diabetic complications caused by AGEs or aldose reductase, and various types of fibrosis, metastasis caused by TGF-β.

In the present invention, the health functional food that inhibits diabetic complication is comprising Cathayanin B or Kuwanol A isolated from Morus Bark.

In the present invention, it is identified that Cathayanin B inhibits the production of AGEs and inhibits aldose reductase, which are a causative substances of diabetic complication. It also identified Kuwanol A inhibits TGF-β signaling which causes diabetic complications, various types of fibrosis and metastasis.

Thus, the present invention can be useful for the health functional food that improves the symptoms of diabetic complication, such as diabetic nephritis, diabetic retinopathy and diabetic neuropathy, which are induced by AGEs, aldose reductase, and TGF-β signal transduction mechanism. Besides that, the present invention can be useful for the health functional food that improves the symptoms of various types of fibrosis, and metastasis, etc.

The health functional food comprising Cathayanin B or Kuwanol A according to the present invention includes various foods, for example drink, gum, tea, vitamin complex, dietary supplement. Also, it could be used in the forms of pill, powder, granule, infusion, tablet, capsule or drink. The amount of herb extract in food or drink is commonly 0.001 to 10 weight% of total weight of food in case of the health functional food of the present invention, 0.01 to 1 weight% preferably, and in case of the composition of health drink, 0.001 to 10g, and preferably 0.01 to 1g on the basis of 100㎖.

Similar to a common drink, the composition of this health drink of the present invention could comprise various flavoring agents or natural carbohydrate as supplementary ingredient besides comprising the compound isolated from Morus Bark in the indicated ratio as essential substance.

Examples of said natural carbohydrate are monosaccharide, disaccharide such as glucose and fructose, polysaccharide such as maltose and sucrose, common sugar such as dextrin and cyclodextrin and sugar alcohol such as xylitol, sorbitol and erythritol. As flavoring agents besides mentioned above, natural flavoring agents (thaumatin, stevia extracts (for example revaudioside A, glycyrrhizin etc.)) and synthesized flavoring agents (saccharine, aspartame etc) could be used. The ratio of the natural carbohydrate is commonly approximately 1 to 20g, preferably 5 to 12g, per 100㎖ of the health functional food in the present invention.

Besides ones mentioned above, the health functional food according to the present invention could comprise various nutritional supplements, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, colorants, filing agents (cheese, chocolate, etc.), pectic acid and salt thereof, alginate and salt thereof, organic acids, protective colloid thickening agents, pH adjustors, stabilizing agents, antioxidants, glycerin, alcohol and carbonation agents used for soda. Moreover, the health functional food of the present invention could comprise the fruit flesh for manufacturing natural fruit juice, fruit juice drink and vegetable drink. Such ingredients could be used alone or together. The rate of these additives is commonly selected from 0 to approximately 20 per 100 parts by weight.

The present invention can be used to provide pharmaceutical composition or health food using Cathayanin B or Kuwanol A, to prevent and treat diabetic complications.

In particular, Kuwanol A can be used to provide pharmaceutical composition or health food for the treatment of various fibrosis and metastasis, in addition to diabetic complications.

Figure 1 is a picture showing the inhibition of AGEs production by Cathayanin B compound.

Figure 2 is a picture showing the inhibition of Smad-2/3 phosphorylation by Kuwanol A compound.

Figure 3 is a picture showing the inhibition of Epithelial Mesenchymal Transition (EMT) in Human Renal Proximal Tubular Epithelial Cells (RPTECs) and breast cancer cell line (MCF-7) by Kuwanol A compound.

The present invention is hereinafter explained in detail with examples and experiments, which are for illustrative purposes only and shall not be construed as limiting the scope of the present invention.

Example 1: Preparation and analysis of Cathayanin B from Morus Bark

Cathayanin B was isolated and prepared from Morus Bark according to the method described in the Journal of Asian Natural Products Research (Vol 12 No 6 2010, 505-515).

1-1: Methanol extract of Morus Bark

Dried Morus Bark (3 ㎏) in 10 L of methanol was subjected to reflux extraction repeated 3 times, each time for 4 hours, and was then filtered and concentrated under reduced pressure, generating 150 g of methanol extract.

1-2: Preparing organic solvent fraction from Morus Bark extract

The Morus Bark methanol extract obtained in Step 1-1 above was suspended in 6 L of water, and partitioned in hexane (3 L, 3 times) and ethyl acetate (3 L, 3 times) in order, generating a hexane extract (50 g) and ethyl acetate (50 g). 25 sub-fractions (MAE-01~25) were obtained by applying a gradient solvent system consisting of dichloromethane (CH2Cl2)-methanol (70% : 30%, 50% : 50%, 30% : 70%, 10% : 90%, 0 : 100%) to ethyl acetate extracts in Silica gel column chromatography (silica gel column 500g).

1-3: Preparation of active fraction and compound from organic solvent

13 sub-fractions (MA.E-2201~2213) were obtained by applying Reversed Phase silica gel (RP-18) column chromatography (methanol : water = 1:1 → 3:1, Methanol, gradient solvent system) to ethyl acetate sub-fraction, MA.E-22 prepared by the step 1-2 of Example 1. The fraction MA.E-2212 which was obtained from above was isolated and purified by MCI gel (Supelco) ion-exchange chromatography. Cathayanin B (30 mg) was obtained, which shows yellow spots when color developing with dilute sulfuric acid agent and has Rf value of 0.4 (methanol : water = 4:1) in Thin-Layer chromatography (TLC).

Cathayanin B: Amorphous orange-colored powder.

1H-NMR(500 MHz, acetone-d₆) : 1.37(H-13), 1.46(H-12), 1.69(H-7″), 1.89(H-6″), 2.55(H-9), 2.62(H-5″, H-6″), 3.01(H-9), 3.04(H-3″), 3.24(H-4″), 5.05(H-10), 5.95(H-8), 6.02(H-13″), 6.17(H-2″), 6.18(H-17″), 6.27(H-11″), 6.32(H-3′), 6.38(H-19″), 6.41(H-5′), 6.75(H-14″), 7.04(H-20″), 7.29(H-6′).

13C-NMR(125 MHz, acetone-d₆) : 18.4(C-12), 23.9(C-7″), 25.7(C-13), 27.3(C-5″), 31.1(C-9), 32.8(C-3″), 35.6(C-6″), 35.8(C-4″), 91.7(C-2), 95.8(C-8), 99.1(C-3′), 101.0(C-4a), 102.0(C-3), 102.9(C-8″), 103.3(C-17″), 104.1(C-11″), 105.6(C-6), 106.6(C-13″), 109.3(C-5′), 109.9(C-19″), 114.8(C-9″), 115.9(C-15″), 118.3(C-10), 119.9(C-1′), 121.1(C-2″), 125.6(C-6′), 127.7(C-20″), 129.0(C-14″), 134.0(C-1″), 135.9(C-11), 152.2(C-16″), 156.7(C-12″), 157.5(C-18″), 159.6(C-10″), 160.3(C-2′), 160.4(C-8a), 160.9(C-4′), 162.2(C-7), 162.9(C-5), 188.8(C-4)

ESI-MS (negative mode) m/z[M-H]^- : 689

<Example 2> Preparation and analysis of Kuwanol A from Morus Bark

Kuwanol A which was isolated from Morus Bark and prepared according to the method written on Heterocycles Vol 23 No 4 1985, 819-824.

13 sub-fractions (MA.E-2201~2213) were obtained by applying Reversed Phase slica gel (RP-18) column chromatography (methanol : water = 1:1 → 3:1, Methanol, gradient solvent system) to ethyl acetate sub-fraction, from MA.E-22 prepared by the step 1-2 of Example 1. Obtained fraction from this, MA.E-2205 separated and purified by applying ion exchange chromatography (methanol : water = 1.5 : 1). And the fraction from this was separated and purified by high performance liquid chromatography (HPLC, Xterra　prep C18, 5 ㎛, 19ｘ150 ㎜ i.d., 44% acetonitrile, 210 ㎚, 10㎖/min). Kuwanol A (10㎎) was obtained, of which retention time was 16.6mins, of which Rf value was 0.16 (methanol : water = 2:1) in reverse phase Thin-Layer Chromatography (TLC), and which showed strong blue florescence color in UV 365㎚, and which showed black spots turn up when color developing with dilute sulfuric acid.

Kuwanol A: Amorphous orange-colored powder.

¹H-NMR(500 MHz, acetone-d6) : 1.74(H-7″), 1.98(H-6″), 2.67(H-6″), 3.02(H-5″), 3.26(H-4″), 3.44(H-3″), 6.20(H-13″), 6.32(H-17″), 6.34(H-6), 6.37(H-11″), 6.40(H-2), 6.41(H-2″), 6.46(H-19″), 6.59(H-6′), 6.61(H-2′), 6.85(H-8), 7.09(H-20″), 7.20(H-14″), 7.28(H-7), 7.35(H-5).

¹³C-NMR(125 MHz, acetone-d6) : 23.1(C-7″), 27.7(C-5″), 34.3(C-3″), 35.5(C-6″), 36.4(C-4″), 101.7(C-8″), 102.8(C-2), 103.1(C-17″), 103.8(C-11″), 106.0(C-2′), 106.2(C-6′), 106.3(C-13″), 107.6(C-6), 109.0(C-19″), 111.6(C-4′), 116.4(C-4, C-9″), 116.8(C-15″), 122.5(C-2″), 123.7(C-7), 124.8(C-8), 127.0(C-20″), 127.5(C-5), 129.5(C-14″), 132.5(C-1″), 138.6(C-1′), 152.6(C-16″), 153.4(C-3′), 156.1(C-3), 156.8(C-12″), 156.9(C-18″), 158.3(C-1), 159.1(C-5′).

ESI-MS (negative mode) m/z[M-H]^- : 563

<Experiment 1> Assay of inhibition effect on AGE production

Bovine serum albumin (BSA) of 10 ㎎/㎖ was prepared with 50 mM phosphate buffer (pH 7.4), and then mixed with 0.2M fructose and glucose and cultured at 37℃ or at -20℃ (blank group) for 7 days to induce AGE production. The compound of Example 1 isolated from Morus Bark was treated at 5 different concentrations from 0.1㎍/㎖ to 200㎍/㎖ (all compounds were dissolved in 100% ethanol). As a positive control, pyridoxamine, which is known to inhibit AGE production, was cultured on BSA with only fructose and glucose for 7 days at 37℃. Four different concentrations of pyridoxamine, from 1㎍/㎖ to 1000㎍/㎖, were used.

After 7 days, the Example according the present invention and the positive control were measured with a microplate reader (Excitation: 360㎚, Emission: 465㎚) for the amount of AGE produced. From this measurement, the inhibitory potency value (IC₅₀ value) was computed using SigmaPlot. The results are shown in Table 1.

Inhibition rate of AGE production is calculated as shown below. Experiments were conducted in duplicate, and at least 3 independent experiments were conducted, to calculate the mean and the standard deviation of the IC₅₀ value.

Production inhibition (%) = 100 - (fluorescent intensity of test group - fluorescent intensity of blank test group) / (fluorescent intensity of control group - fluorescent intensity of control blank test group) × 100

In addition, western blot analysis using AGE-specific antibody was performed to identify false positive which can happen in fluorescence assays. The results are shown in Figure 1.

Table 1

As seen in Table 1 above, the compound prepared in Example 1 according to the present invention was approximately 4.7 times more potent than pyridoxamine, the positive control, in inhibiting AGE production, in terms of μM.

Moreover, as seen in Figure 1, the compound prepared in Example 1 according to the present invention showed more potent inhibition than pyridoxamine, the positive control, at 100㎍/㎖, in western blot assay using AGE-specific antibodies.

<Experiment 2> Aldose reductase activity inhibition assay

Lenses isolated from 200-250g Sprague-Dawley Rats were homogenized in 135 mM Na⁺, K⁺-phosphate buffer solution comprising 0.5mM phenylmethylsulfonyl fluoride (PMSF) and 10mM 2-mercaptoethanol. The homogenized sample was centrifuged at 14,000 g force to obtain supernatant, which was quantified with Brad-ford assay. The quantified sample was diluted to a final concentration of 20㎎/㎖. It was sampled in small amounts and stored at -70℃ to be used as aldose reductase extract fractions in this experiment.

The different concentration of Cathayanin B compound (1㎍/㎖ - 50㎍/㎖) was added in 100㎕ of 135 mM Na⁺, K⁺-phosphate buffer solution (pH 7.0) containing 1 ㎕ of 3 mM NADPH and 0.5㎕ of 0.2M DL-glyceraldehyde, and then these solutions were incubated in the presence or absence (blank group) of 2 ㎕ aldose reductase isolated from rats at 37℃ for 10 minutes. After 10 minutes, 100㎕ of 6N sodium hydroxide (NaOH) comprising 10mM imidazole was added. Afterwards, it was heated for 10 minutes at 60℃ so that NADP⁺ produced by aldose reductase reactions can be measured with fluorescence.

For positive control group, kaempferol was used at 0.5-50 ㎍/㎖ concentrations. Aldose reductase activity was measured with a microplate reader (Excitation: 360㎚, Emission: 465㎚). From this, the IC₅₀ value was calculated. The inhibition of aldose reductase activity is calculated with the formula below.

Inhibition rate (%) = 100 - (fluorescent intensity of test group - fluorescent intensity of blank test group) / (fluorescent intensity of control group - fluorescent intensity of control blank test group) × 100

All samples were tested in duplicate, and at least 3 independent experiments were conducted to calculate the mean and the standard deviation of the IC₅₀ value.

[Experiment results]

Table 2

As seen in Table 2 above, the active compound Cathayanin B isolated from Morus Bark inhibits aldose reductase activity at a similar level as kaempferol, the positive control.

Therefore, Cathayanin B prepared in Example 1 according to the present invention can be used for a pharmaceutical composition for prevention or treatment of diabetic nephritis, diabetic retinopathy, and diabetic neuropathy which are caused by AGE production and aldose reductase activities in diabetic patients, or for a health functional food to improve the above mentioned conditions.

<Experiment 3> TGF-β1 transcription inhibition assay

A typical TGF-β responsive element, 12XCAGA oligonucleotides, were inserted to a plasmid vector containing the luciferase gene (miniP-pGL4.17 luciferase plasmid vector), it was injected to C2C12 myoblasts to produce stable transgenic cell lines. (Imman G J. et al., 2002, Mol. Pharmacol. 62(1), 65-74; Byfield S D. et. al., 2004, Mol. Pharmacol. 65(3), 744-752). The transgenic cell lines were placed on DMEM medium (10% FBS, 100 U/㎖ penicillin, 100㎍/㎖ streptomycin, 2 mM L-glutamine, 1mM sodium pyruvate, and nonessential amino acids) on 12-well plates, 800㎕/well each. They were incubated in 5% CO2 incubator at 37℃. When the cell density reached over 80%, the medium was switched with non-FBS DMEM and incubated for 16 additional hours. Afterwards, 5 ㎍/㎖ of TGF-β1 and Kuwanol A prepared in Example 2 were added to DMEM (0% FBS) for each concentration and incubated for 24 additional hours. Then, luciferase assay (Promega) was used to measure the fluorescence induced by TFG-β1. Also, Bradford assay was used to normalize fluorescence value with the quantity of protein. The results are shown in Table 3.

The normal group was not treated with TGF-β1. For the control group, 5 ㎍/㎖ TGF-β1 added DMEM medium was used, and 0.8㎕/well of ethanol was added instead of the compound isolated from Morus Bark. Both groups were treated with more than 5 different concentrations ranging from 1 g/㎖to 10 ㎍/㎖, and all samples were tested in duplicate, at least 3 independent experiments were conducted to calculate the mean and the standard deviation of IC₅₀ values.

C2C12 myoblast cell lines from above were treated with drugs, and western blot analysis using phospho-Smad-2/3-specific antibodies was performed to confirm TGF-β1 signal transduction inhibition effect of Kuwanol A. The results are shown in Figure 2. Kuwanol A also inhibited TGF-β1 induced epithelial-mesenchymal transition (EMT) in human renal proximal tubule epithelial cells (RPTECs) and breast cancer cell lines (MCF-7), and this result is shown in Figure 3.

[Experiment results]

Table 3

As seen in Table 3 above, Kuwanol A prepared in accordance with Example 2 showed inhibition of TGF-β1 transcription.

Moreover, as seen in Figure 2, the group treated with 5 ㎍/㎖ Kuwanol A showed significant decrease in the Smad-2/3 phosphorylation. As seen in Figure 3, the group treated with 5 ㎍/㎖ Kuwanol A on human renal proximal tubule epithelial cells (RPTECs) and breast cancer lines (MCF-7) showed decrease of fibronectin, a mesenchymal cell marker protein. E-cadherin, an epithelial cell marker protein, was decreased by TGF-β1, but was recovered by Kuwanol A. From this, it was seen that Kuwanol A inhibited the TGF-β1 induced process of epithelial-mesenchymal transition (EMT).

Therefore, since Kuwanol A prepared in Example 2 according to the present invention inhibits TGF-β1 signaal transduction, it can be used for a pharmaceutical composition for prevention and treatment of TGF-β1 induced diabetic complications, various fibrosis and metastasis, or health functional food for improving said conditions.

Claims

A pharmaceutical composition for the prevention and treatment of diabetic complications, the composition comprising as active ingredients Cathayanin B or Kuwanol A isolated from Morus Bark, and comprising pharmaceutically acceptable excipients.
The composition of claim 1, wherein the diabetic complication is selected from the group consisting of diabetic nephritis, diabetic retinopathy, diabetic neuropathy.
A pharmaceutical composition for the prevention and the treatment of fibrosis or metastasis, the composition comprising Kuwanol A, a compound isolated from Morus Bark, as an active ingredient and comprising pharmaceutically acceptable excipients.
The composition of claim 3, wherein the fibrosis is selected from lung fibrosis including idiopathic pulmonary fibrosis; cirrhosis; endomyocardial fibrosis; myelofibrosis, renal fibrosis, Crohn’s Disease, keloid, and arthrofibrosis.
A health food inhibiting generation of diabetic complications, comprising Cathayanin B or Kuwanol A isolated from Morus Bark.
A health food inhibiting fibrosis or metastasis comprising Kuwanol A isolated from Morus Bark.