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WO2014168458A1 - Utilisation de composés isolés à partir de l'écorce de mûrier - Google Patents

Utilisation de composés isolés à partir de l'écorce de mûrier Download PDF

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WO2014168458A1
WO2014168458A1 PCT/KR2014/003163 KR2014003163W WO2014168458A1 WO 2014168458 A1 WO2014168458 A1 WO 2014168458A1 KR 2014003163 W KR2014003163 W KR 2014003163W WO 2014168458 A1 WO2014168458 A1 WO 2014168458A1
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Prior art keywords
diabetic
kuwanol
isolated
fibrosis
cathayanin
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Hwan Bong Chang
Joobyoung Yoon
Hyunyong Lee
Yang Kook Rho
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Dong Wha Pharm Co Ltd
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Dong Wha Pharm Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Definitions

  • 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.
  • AGE Advanced glycation end product
  • TGF- ⁇ transforming growth factor- ⁇
  • 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.
  • 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).
  • Advanced glycation end products activate Smad-2/3 in either TGF- ⁇ -dependent or TGF- ⁇ -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 ..
  • AGEs are known to cause diabetic retinopathy as well as diabetic neuropathy through interaction with receptors for advanced glycation end products (RAGEs) (Bearliest G. R., et. al., 2005, Invest Ophthalmol Vis Sci . 46(8), 2916-2924; Toth C., et. al., 2008, Diabetes. 57(4), 1002-1017).
  • 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.
  • aldose reductase which is the main mediator of activated polyol pathway, converts glucose to sorbitol.
  • NAD+/NADP+ In process of sorbitol production, excessive consumption of NAD+/NADP+ occurs, which leads to decrease in synthesis of myo-inositol, nitrogen monoxide and taurine.
  • Fructose which is the end product of polyol pathway, is more than 10 times more potent than glucose in nonenzymatic glycation of proteins.
  • 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.).
  • TGF- ⁇ 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.
  • ALK activin like kinase
  • type III receptor type III receptor.
  • TGF- ⁇ performs an important role. TGF- ⁇ 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).
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ 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- ⁇ inhibitors can be largely divided into monoclonal antibody and chemical drug.
  • Metelimumab CAT-192
  • a TGF- ⁇ 1 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.
  • LY2157299 a TGF- ⁇ 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).
  • 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).
  • 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.
  • 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, Fitorick 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.
  • 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.
  • 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).
  • 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- ⁇ 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.
  • 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
  • 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).
  • CML N-carboxymethyl lysine
  • CEL N-carboxyethyl lysine
  • inhibitory concentration (IC 50 ) was obtained by measuring the amount of AGEs using microplate reader for fluorescence analysis (Excitation: 360nm, Emission: 465nm). 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.
  • the above compound was performed on inhibitory effect of aldose reductase activity for each concentration using microplate reader for fluorescence analysis (Excitation: 360nm, Emission: 465nm) , and inhibitory concentration (IC 50 ) was determined, it was shown that the compound inhibits aldose reductase activity which also causes diabetic complications.
  • 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.
  • 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.
  • 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- ⁇ signal pathway, a causative factor of diabetic complication, various types of fibrosis, metastasis.
  • Kuwanol A isolated from Morus Bark has an inhibitory mechanism on TGF- ⁇ signal pathway from the following experiments: an experiment analyzing the inhibitory effect on TGF- ⁇ 1 transcription, and measuring phosphorylation of Smad-2/3 which is signal mediator of TGF- ⁇ 1. 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).
  • EMT Epithelial Mesenchymal Transition
  • RPTECs Human Renal Proximal Tubular Epithelial Cells
  • MCF-7 breast cancer cell line
  • Kuwanol A a compound isolated from Morus Bark, inhibits TGF- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • a lubricant such as magnesium stearate talc could be used.
  • 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.
  • nonaqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil like olive oil and injectable ester like ethyl oleate could be used.
  • suppository witepsol, macrogol, tween 61, cocoa butter, laurinum and glycerogellatin could be used.
  • 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 0.001mg/kg to 1,000mg/kg once or divide it into several times.
  • 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.
  • 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- ⁇ .
  • the health functional food that inhibits diabetic complication is comprising Cathayanin B or Kuwanol A isolated from Morus Bark.
  • 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- ⁇ signaling which causes diabetic complications, various types of fibrosis and metastasis.
  • 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- ⁇ 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.
  • diabetic complication such as diabetic nephritis, diabetic retinopathy and diabetic neuropathy, which are induced by AGEs, aldose reductase, and TGF- ⁇ signal transduction mechanism.
  • 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 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 100ml.
  • 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.
  • sweetitol erythritol
  • sweetitol erythritol
  • sweetitol and erythritol erythritol.
  • the ratio of the natural carbohydrate is commonly approximately 1 to 20g, preferably 5 to 12g, per 100ml of the health functional food in the present invention.
  • 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.
  • 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.
  • 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.
  • FIG 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.
  • EMT Epithelial Mesenchymal Transition
  • RPTECs Human Renal Proximal Tubular Epithelial Cells
  • MCF-7 breast cancer cell line
  • 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).
  • 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).
  • Cathayanin B Amorphous orange-colored powder.
  • Kuwanol A Amorphous orange-colored powder.
  • Bovine serum albumin (BSA) of 10 mg/ml was prepared with 50 mM phosphate buffer (pH 7.4), and then mixed with 0.2M fructose and glucose and cultured at 37°C or at -20°C (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 ⁇ g/ml to 200 ⁇ g/ml (all compounds were dissolved in 100% ethanol).
  • pyridoxamine which is known to inhibit AGE production, was cultured on BSA with only fructose and glucose for 7 days at 37°C. Four different concentrations of pyridoxamine, from 1 ⁇ g/ml to 1000 ⁇ g/ml, were used.
  • Example according the present invention and the positive control were measured with a microplate reader (Excitation: 360nm, Emission: 465nm) for the amount of AGE produced. From this measurement, the inhibitory potency value (IC 50 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 IC 50 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
  • Example 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 ⁇ M.
  • Example 1 the compound prepared in Example 1 according to the present invention showed more potent inhibition than pyridoxamine, the positive control, at 100 ⁇ g/ml, in western blot assay using AGE-specific antibodies.
  • 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 20mg/ml. It was sampled in small amounts and stored at -70°C to be used as aldose reductase extract fractions in this experiment.
  • Cathayanin B compound (1 ⁇ g/ml - 50 ⁇ g/ml) was added in 100 ⁇ l of 135 mM Na + , K + -phosphate buffer solution (pH 7.0) containing 1 ⁇ l of 3 mM NADPH and 0.5 ⁇ l of 0.2M DL-glyceraldehyde, and then these solutions were incubated in the presence or absence (blank group) of 2 ⁇ l aldose reductase isolated from rats at 37°C for 10 minutes. After 10 minutes, 100 ⁇ l of 6N sodium hydroxide (NaOH) comprising 10mM imidazole was added. Afterwards, it was heated for 10 minutes at 60°C so that NADP + produced by aldose reductase reactions can be measured with fluorescence.
  • NaOH 6N sodium hydroxide
  • kaempferol was used at 0.5-50 ⁇ g/ml concentrations.
  • Aldose reductase activity was measured with a microplate reader (Excitation: 360nm, Emission: 465nm). From this, the IC 50 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
  • the active compound Cathayanin B isolated from Morus Bark inhibits aldose reductase activity at a similar level as kaempferol, the positive control.
  • 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.
  • a typical TGF- ⁇ 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.
  • a plasmid vector containing the luciferase gene miniP-pGL4.17 luciferase plasmid vector
  • the transgenic cell lines were placed on DMEM medium (10% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM L-glutamine, 1mM sodium pyruvate, and nonessential amino acids) on 12-well plates, 800 ⁇ l/well each. They were incubated in 5% CO2 incubator at 37°C. When the cell density reached over 80%, the medium was switched with non-FBS DMEM and incubated for 16 additional hours. Afterwards, 5 ⁇ g/ml of TGF- ⁇ 1 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- ⁇ 1. 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- ⁇ 1.
  • TGF- ⁇ 1 5 ⁇ g/ml TGF- ⁇ 1 added DMEM medium was used, and 0.8 ⁇ l/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/mlto 10 ⁇ g/ml, and all samples were tested in duplicate, at least 3 independent experiments were conducted to calculate the mean and the standard deviation of IC 50 values.
  • the group treated with 5 ⁇ g/ml Kuwanol A showed significant decrease in the Smad-2/3 phosphorylation.
  • the group treated with 5 ⁇ g/ml 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- ⁇ 1, but was recovered by Kuwanol A. From this, it was seen that Kuwanol A inhibited the TGF- ⁇ 1 induced process of epithelial-mesenchymal transition (EMT).
  • Kuwanol A prepared in Example 2 according to the present invention inhibits TGF- ⁇ 1 signaal transduction, it can be used for a pharmaceutical composition for prevention and treatment of TGF- ⁇ 1 induced diabetic complications, various fibrosis and metastasis, or health functional food for improving said conditions.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une composition pharmaceutique destinée à la prévention et au traitement de complications diabétiques, ou des aliments santé, comprenant de la cathayanine B ou du kuwanol A isolés à partir de l'écorce de mûrier. Le composé cathayanine B selon la présente invention est isolé à partir de l'écorce de mûrier et inhibe la formation de produits terminaux avancés de glycation (AGE), qui entraînent des complications diabétiques, et inhibe l'activité de l'aldose réductase. Le kuwanol A, un autre composé isolé à partir de l'écorce de mûrier, inhibe la signalisation TGF-β1. Par conséquent, la composition pharmaceutique ou l'aliment santé comprenant la cathayanine B ou le kuwanol A selon la présente invention peuvent être utilisés pour inhiber la néphrite diabétique, la rétinopathie diabétique et la neuropathie diabétique, qui sont des complications diabétiques. En particulier, le kuwanol A peut également être utilisé pour une composition pharmaceutique destinée à la prévention et au traitement de diverses pathologies de fibroses et métastases ainsi que pour un aliment santé visant à améliorer lesdites pathologies.
PCT/KR2014/003163 2013-04-12 2014-04-11 Utilisation de composés isolés à partir de l'écorce de mûrier Ceased WO2014168458A1 (fr)

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KR10-2013-0040342 2013-04-12

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CN118141853A (zh) * 2024-03-06 2024-06-07 西南医科大学附属中医医院 用于治疗慢性肾脏病的桑白皮提取物及其制备方法和用途
CN120284942A (zh) * 2025-04-22 2025-07-11 西南医科大学附属中医医院 桑辛素类化合物在制备治疗慢性肾脏病的药物中的用途
CN118141853B (zh) * 2024-03-06 2026-01-30 西南医科大学附属中医医院 用于治疗慢性肾脏病的桑白皮提取物及其制备方法和用途

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KR102346863B1 (ko) * 2017-12-28 2022-01-03 대구대학교 산학협력단 모린을 유효성분으로 포함하는 신장질환 예방 또는 치료용 조성물
KR102066966B1 (ko) 2018-09-20 2020-01-16 대구가톨릭대학교산학협력단 상백피로부터 분리된 화합물을 포함하는 당뇨병의 예방 또는 치료용 약학 조성물

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* Cited by examiner, † Cited by third party
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CN118141853A (zh) * 2024-03-06 2024-06-07 西南医科大学附属中医医院 用于治疗慢性肾脏病的桑白皮提取物及其制备方法和用途
CN118141853B (zh) * 2024-03-06 2026-01-30 西南医科大学附属中医医院 用于治疗慢性肾脏病的桑白皮提取物及其制备方法和用途
CN120284942A (zh) * 2025-04-22 2025-07-11 西南医科大学附属中医医院 桑辛素类化合物在制备治疗慢性肾脏病的药物中的用途

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