KR20070023425A - A composition containing timosaponin A-III for prevention and treatment of type 2 diabetes mellitus - Google Patents

Abstract

The present invention relates to a composition for preventing and treating type 2 diabetes containing thymosaponin A-III, and more specifically, unlike the conventionally known, thymosaponin A-III has a low effect of preventing and treating type 1 diabetes. The present invention relates to a composition for preventing and treating type 2 diabetes, which contains thymosaponin A-III as an active ingredient by revealing excellent effects for preventing and treating type 2 diabetes.

Timothy saponin A-III, type 2 diabetes

Description

A composition containing timosaponin A-III for prevention and treatment of type 2 diabetes mellitus}

Figure 1 shows the beta cell destruction process by alloxane and streptozotocin.

Figure 2 is a graph showing the hypoglycemic effect of the sub- EA fraction (AA-EA) and sub EA fraction (EA-I ~ EA-V) in the db / db mouse model.

Figure 3 is a graph showing the hypoglycemic effect of thymosaponin A-III in the db / db mouse model.

4 is a graph showing the hypoglycemic effect of thymosaponin A-III in the KK-Ay mouse model.

5 is a graph showing the hypoglycemic effect of thymosaponin A-III in streptozotocin-induced diabetic rat model.

The present invention relates to a composition for preventing and treating type 2 diabetes containing thymosaponin A-III, and more specifically, unlike the conventionally known, thymosaponin A-III has a low effect of preventing and treating type 1 diabetes. The present invention relates to a composition for preventing and treating type 2 diabetes, which contains thymosaponin A-III as an active ingredient by revealing excellent effects for preventing and treating type 2 diabetes.

According to the evidence report: clinical guideline on the identification, evaluation, and treatment of overweight and obesity in adults, 1999, NIH, the National Institutes of Health (NIH) About 15.7 million people have type 2 diabetes.

In addition, according to the World Health Organization, the number of diabetics is estimated to be about 300 million by 2025, and type 2 diabetics who have lost insulin control by insulin now account for about 5.9% of the US population. It is known to reach [A. S. Wagman et al., Current Pharmaceutical Design, 7, 417, 2001].

Diabetes is a disease associated with carbohydrate metabolism that is characterized by symptoms of hyperglycemia in the blood. Type 1 diabetes (insulin dependent diabetes mellitus; about 10% of diabetics) and type 2 diabetes (insulin independent diabetes mellitus; about 90% of diabetics) Can be classified as [B. Set, Clin Therapeu., 25, 1895, 2003; S. N. Davis, J Diabetes Complications., 18, 367, 2004]. Patients with type 1 diabetes have little or no in vivo insulin secretion due to the destruction of the pancreatic beta cells due to congenital or viral invasion or severe damage to the pancreas, progress to extreme hyperglycemia and are prone to ketosis and ketoacidosis. It is mainly acute in childhood, and glycemic control therapy by insulin administration is essential. On the other hand, patients with type 2 diabetes have abnormal genetic, obesity, stress, and hormone secretion in the adult population. It is caused by factors such as taking medication, and it has insulin secretion ability, but it is very low or unbalanced due to excessive secretion of adverse hormones such as glucagon. It is known to be caused by insulin resistance to basal conditions and stimuli, increased endogenous glucose production in the liver, and insulin resistance, which results in decreased glucose utilization in peripheral tissues. It is not sensitive to ketoosis or ketoacidosis. In the case of type 1 diabetes, blood sugar can be controlled by diet, exercise, and insulin administration. However, in case of type 2 diabetes, an additional hypoglycemic agent should be taken.

The hypoglycemic agents used in patients with type 2 diabetes (insulin-independent) can be divided into three groups depending on their mechanism of action. The first group stimulates the beta cells of the pancreas to promote insulin secretion. Sulfonyl urea and meglitinide drugs are used. The second group is biguanide. And insulin sensitizers represented by thiazolidinedion-based drugs. The third group of drugs inhibits the breakdown of sugars to inhibit the absorption of intestinal sugars, and there are drugs such as acarbose and aglytol. Preferred hypoglycemic agents should be able to express their action quickly to prevent excessive post-prandial blood sugar elevation, after which their effects are lost in a short time to not cause hypoglycemia, and also to improve metabolic symptoms accompanying diabetes. In this regard, the drugs are not completely desirable, and the development of oral hypoglycemic agents with less side effects and safer is needed to properly treat diabetes at home and abroad.

In the diabetic animal model, in case of type 1 diabetes, a drug-induced model using alloxane or streptozotocin, or a non-obese diabetic (NOD) which naturally causes type 1 diabetes by crossing the diabetic animal for generations Mice, and models such as BB (bio breeding) rats. On the other hand, in the type 2 animal model, genetically engineered models are widely used. As representative models, db / db mice (diabetes model due to lack of leptin receptor) and ob / ob mice (by leptin lacking) Obese model), Zucker (zucker) rat, The Otsuka Long-Evans Tokushima fatty (OLETF) rat, KK (obesity diabetes model) mouse, KK-Ay (obesity diabetes, high) Insulinemia model) mice and the like [S. Makino et al., Exp Anim., 29, 1, 1980; A. F. Nakhooda et al., Diabetes, 26, 100, 1977; M. Nakamura et al., Diabetol., 3, 121, 1967; K. Kawano et al., Diabetes, 41, 1422, 1992; D. A. Rees et al., Diabetic Medicine, 22, 359, 2005]. Therefore, it is very important to know the characteristics of each animal model (types 1 and 2) well and use them strictly.

N. Nakashima et al., Pseudotimosaponin A-III (Pseudotimosaponin A-III) and Protomosaponin in diabetic rats using alloxan and streptozotocin, the representative drugs used in experimental diabetes models. The hypoglycemic effect of prototimosaponin A-III and thymosamonine A-III was mentioned [N. Nakashima et al., J. Nat. Prod. vol 56, 345, 1994]. In addition, thymosaponin A-III inhibits platelet aggregation [A. Niwa et al., Yakugaku Zasshi 108 (6), 555, 1988], vascular relaxation [G. Wang et al., Life Sci. 71, 1081, 2002].

Alloxanes and streptozotocin invade pancreatic beta cells by the expression of glucose transport protein (GLUT-2), causing DNA breakdown, NAD + depletion, and pancreatic beta cell death, which is representative of many types of studies. It has been used as a diabetes-causing drug [K. Bloch et al., Diabetes Metab Res Rev 21, 253, 2005; D. A. Rees et al., Diabetic Medicine, 22, 359, 2005; J. Kawada et al., Yakugaku Zasshi, 112, 773, 1992; V. M. F. Rastelli et al., Inflamm Res., 54, 173, 2005; R. K. Cuman et al., Inflamm Res. 50, 460, 2001; 1.

Alloxane (alloxan: 5,5, -dihydroxy-2,4,6 (1H, 3H, 5H) -pyrimidinetrione) is an oxide of uric acid, that is, a pyrimidine derivative. 450 This mediated radical reaction destroys the beta cells of the pancreas [K. Bloch et al., Diabetes Metab Res Rev 21, 253, 2005; Yakugaku Zasshi, 112, 773, 1992]. However, it is pointed out that the toxicity of the drug itself and the mortality of the resulting animals is high, IF Federiuk et al., To find the optimal method for minimizing the mortality in the alloxan-induced diabetes model The type of diabetes induced by overdose was investigated. As a result, the model of intraperitoneally administering alloxane at a high concentration (200 mg / kg) lowered mortality by about 10% and reported that type 1 diabetes was induced by 70% or more [I. F. Federiuk, Compara Med, 54, 252, 2004].

Streptozotocin (streptozotocin: 2-deoxy-2- (3-methyl-3-nitrosoureido) -D-glucopyranose) is a derivative of glucose, beta cells based on strong alkylating action Inhibits mitochondrial ATP production and generates superoxide radicals through indirect activation of xanthine oxidase (XOD) due to high levels of ADP accumulated in cells, or directly Oxidative damage to beta cells by generating superoxide radicals by activating [D. A. Rees et al., Diabetic Medicine, 22, 359, 2005; Yakugaku Zasshi, 112, 773, 1992].

There have been reports of streptozotocin-induced type 2 diabetes, but most of these documents are dexamethasone (SJ Giddings et al., Diabetes, 34, 235, 1985), nicotinamide [S. Ozyazgan et al., Pharmacol., 74, 119, 2005], or in combination with a high fat diet for at least one to two weeks [R. de Souza Santos et al., Clin Chim Acta., 2005, Epub ahead of print; MJ Reed et al., Metabolism., 49, 1390, 2000; F. Zhang et al., Exp Anim., 52, 401, 2003]. In other words, the type of diabetes induced by streptozotocin alone, regardless of the dose or the method of administration, is type 1, which is considered to be indisputable. As a model to be clearly distinguished from the streptozotocin-induced diabetes model, neonatal streptozotocin (hereinafter referred to as 'n-streptozotocin') in diabetic rats of 2 days old. There is a model [I. Degirmenci et al., J Ethnopharmacol., 97, 555, 2005; DN Umrani et al., Clin Exp Hypertens., 25, 221, 2003]. The n-streptozotocin-diabetes-induced model has been used in many studies as a type 2 diabetes-induced model using streptozotocin, but in practice, the streptozotocin-induced model in general adult rats has In contrast to the type 1 diabetes-induced model, the n-streptozotocin-induced diabetes model in neonatal rats is clearly classified as a type 2 diabetes model [BM Kim., Diabetol., 44, 2192, 2001; VMF Rastelli et al., Inflamm Res., 54, 173, 2005; RK Cuman et al., Inflamm Res. 50, 460, 2001; UA Shinde et al., J Cell Mol Med., 7, 322, 2003; UA Shindea et al., J Trace Elem Med Biol., 18, 23, 2004].

In conclusion, N. Nakashima et al. [N. Nakashima et al., J. Nat. Prod. The hypoglycemic activity of thymosaponin reported by vol. 56, 345, 1994 is not related to the hypoglycemic effect on type 2 diabetes. It is feed. In addition, the literature presented by N. Nakashima et al. Mentioned only the relative hypoglycemic effect, but the absolute value of the hypoglycemic effect, namely the effect, is unclear.Thymosaponin A-III is prototymosaponin A-III or pseudotimod. On the basis of its weaker activity than saponin A-III, it is insufficient to infer the superiority of hypoglycemia in thymosaponin A-III type 2 diabetes model.

Accordingly, an object of the present invention is to provide a new use for the therapeutic and prophylactic effect of thymosaponin A-III against type 2 diabetes.

The present invention is characterized by a composition for preventing and treating type 2 diabetes which contains thymosaponin A-III as an active ingredient.

The present invention also includes a health food effective for type 2 diabetes containing thymosaponin A-III as an active ingredient.

The present invention will be described in more detail as follows.

The present invention, unlike the conventionally known thymosaponin A-III has a low effect of preventing and treating type 1 diabetes, while the type 2 diabetes prevention and treatment effect is found to contain thymosaponin A-III as an active ingredient It relates to a composition for preventing and treating type 2 diabetes.

In the present invention, timosaponin A-III represented by the following Chemical Formula 1 (timosaponin A-III, 3-O-β-D-glucopyranosyl- (1 → 2) -β-D-galactopyranosyl salsa saponogenin) Was isolated from Anemarrhena asphodeloides.

Referring to the process of separating and obtaining thymosaponin A-III from the hair,

1) recovering the extract obtained by extracting Jimo with an aqueous alcohol solution in tertiary distilled water;

2) removing the supernatant by separating the recovered extract with an equivalent amount of hexane;

3) washing the remaining water layer with the same amount of methylene chloride and separating the ethyl acetate layer by layer separation with the same amount of ethyl acetate; And

4) The separated ethyl acetate layer is further fractionated using silica gel column chromatography, and separated and obtained by recrystallization of thymosaponin with an aqueous dioxane solution.

Unlike previously known thymosaponin A-III, the prevention and treatment effect of type 1 diabetes is insignificant, while the prevention and treatment effect of type 2 diabetes was confirmed by the present inventors.

Therefore, the present invention is characterized by the composition for preventing and treating type 2 diabetes containing thymosaponin A-III as an active ingredient.

The composition may be administered orally or parenterally during clinical administration, for example, intravenously, subcutaneously, intraperitoneally or topically, and may be used in the form of general medicines and health foods.

The compositions may be formulated for oral administration such as tablets, pills, emulsions, solutions, aerosols, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, Formulated in syrups or elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of the capsule formulation, in addition to the substances mentioned above, it contains a liquid carrier such as fatty oil. In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a parenteral formulation, the composition is prepared in solution by mixing in water with stabilizers or buffers and formulated into unit dosage forms of ampoules or vials.

Examples of suitable carriers, excipients and diluents for use in the compositions of the present invention include aluminum salts, phenoxyethylethanol, water, physiological saline, lactose, dextrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, undetermined. Vaginal cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the formulation of the compound of formula 1 of the present invention may further include a filler, anti-coagulant, lubricant, wetting agent, flavoring, emulsifier, preservative and the like.

On the other hand, the dosage into the human body for the active ingredient of the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, but generally 0.1 ~ 1 day Administration at 15 mg / kg is preferred.

The composition of the present invention may be prepared by food or medicine and divided into one to two times a day in accordance with the daily dose.

In addition, the health food is a food prepared by adding the active ingredient to food materials such as beverages, teas, spices, gums, confectionery, capsules, powdered, suspensions, etc. Means to bring, but unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Preparation Example 1 Preparation of Active Fractionated Hairy EA Fraction from Hair

3 kg of Jimo were immersed twice in 20 L of 50% ethanol for 4 hours. It was run at 80 ° C. for effective extraction and stirred using a stirrer. After 4 hours the solids were allowed to settle and the cotton wool was filtered out of the funnel to remove the final float. The filtrate was evaporated with a rotary evaporator and the amount of extract obtained was measured and recovered with 500 mL of tertiary distilled water per 100 g of extract. The recovered extract was mixed with the same amount of hexane (n-Hexane, Jin chemicals pharmaceutical Co. Ltd., Korea) and placed in a separation funnel, and left for 24 hours after stirring to remove the upper hexane layer. Wash in the same way two to three more times.

The remaining water layer was washed 3 to 4 times in the same manner using the same amount of methylene chloride (methylene chloride, Jin chemicals pharmaceutical Co. Ltd., Korea, MC).

The remaining water layer was mixed with the same amount of ethyl acetate (Ethyl acetate, Jin chemicals pharmaceutical Co. Ltd., Korea, hereinafter EA) into a separator, left for 24 hours after stirring to separate only the EA layer of the upper layer. Two to three additional fractions of EA were concentrated under reduced pressure, and the desired EA fraction (AA-EA) was obtained. The mixture was powdered and stored at -20 ° C.

Preparation Example 2 Preparation of Detailed Fractions from Jimo EA Fraction

290 g of Jimmy EA fractions were sequentially fractionated using silica gel column chromatography (170-230 mesh, 3 kg). Solvent conditions are EA (15 L), EA: MeOH = 20: 1 (20 L), EA: MeOH: Water = 20: 2: 0.1 (20 L), EA: MeOH: Water = 10: 2: 0.2 (20 Five fractions of 15 L in sequence were obtained, each named EA-I, EA-II, EA-III, EA-IV and EA-V. Each fraction was concentrated under reduced pressure, powdered and stored at -20 ° C for use.

Preparation Example 3 Isolation and Structure Identification of Active Ingredients

40 g of a 70% aqueous solution of dioxane was added to 1 g of the EA EAV fraction, followed by complete dissolution through a 50 ° C. water bath and sonication. The material obtained by filtering the white needle-like material obtained by leaving the dissolved EA-V fraction in the dissolved state for 24 hours at room temperature was named Compound I (Rf 0.38, 90% MeOH, sulfuric acid coloring, RP-18 F254s, Merck). .

The isolated compound I was structurally analyzed using H ¹ -NMR, C ¹³ -NMR, and ESI-MS to identify thymosaponin A-III [S. Saito et al., Chem. Pharm. Bull. 2342. 1994], which is consistent with that reported in the literature. In addition, a standard product (SKF 7862, WAKO pure chemical industries.

Example 1: hypoglycemic effect in db / db mice

Male mice of 8-9 weeks old db / db strain (C57BL / Ks-db / db) were used for the experiment. C57BL / Ks-db / db mice (developed by the US Jackson Institute, lacking the hypothalamic leptin receptor and causing diabetes through obesity due to abnormalities in the signaling system by leptin) begin obesity around 4-5 weeks of age. , Blood sugar rises. Based on these characteristics, it is a representative type 2 diabetes animal model that is used for the analysis, identification and screening of the mechanism of the development of obesity, diabetes and diabetic complications, and the drug screening of hypoglycemic agents [D. A. Rees et al., Diabetic Medicine, 22, 359, 2005; G. H. Lee et al., Nature, 379, 632, 1996; S. C. Chua et al., Science, 271, 994, 1996; J. K. Naggert et al., Mamm Genome., 6, 131, 1995]

Negative control of 0.5% carboxymethylcellulose (CMC) solution, positive control (rosiglitazone) dissolved in 0.5% carboxymethylcellulose (CMC) solution and test drug are administered intraperitoneally or orally every afternoon. Measurements were taken with a blood glucose meter just prior to dosing in the afternoon at 3-4 day intervals. The body weight and feed volume of the animals were measured at 2 days prior to drug administration. After 10 days of administration, blood glucose measurement and weight measurement, blood was collected and serum was separated to measure liver function, kidney function, and biochemical parameters for lipid testing.

FIG. 2 shows the EA fraction (AA-EA) prepared by Preparation Example 1 and the detailed fractions obtained by Preparation Example 2 (EA-I, EA-II, EA-III, EA-IV, and EA-V). After oral administration of 300 mg / kg each, the blood glucose lowering effect is measured. As a result, it was confirmed that EA-V showed better activity than the lipid EA fraction, and showed the same hypoglycemic effect as the positive control, and searched for the active ingredient based on this fraction.

3 shows oral administration of thymosaponin A-III obtained by Preparation Example 3 by concentration (100 mg / kg, 200 mg / kg, 3000 mg / kg), and shows a hypoglycemic effect. At a concentration of 200 mg / kg, thymosaponin A-III is the main active index component of EA-V fraction, showing an equal or better effect than rosiglitazone, a positive control, and a stronger hypoglycemic effect at 300 mg / kg. It was confirmed that.

Example 2: Hypoglycemic Effect of Timothysamonin A-III on KK-Ay Mice

Male mice of 9-10 week old KK-Ay strain (C57BL / Ks) were used for the experiment. KK-Ay mice originated from Kasukabe, Saitama, Japan, and were developed by Takeda Pharmaceutical Co., Ltd. for genetic research purposes. has been used as a type 2 diabetes model characterized by hyperinsulinemia [M. Okazaki et al., Exp Anim., 51, 191, 2002; J. Suto et al., Eur J Endocrinol., 139, 654, 1998].

Negative control of 0.5% carboxymethylcellulose (CMC) solution, test drug dissolved in 0.5% carboxymethylcellulose (CMC) is administered orally every afternoon, blood glucose measurement is 3 to 4 days apart by cutting the tail vein The blood glucose meter was measured immediately before administration at a certain time in the afternoon. The body weight and feed volume of the animals were measured at 2 days prior to drug administration. After 8 days of administration, blood glucose measurement and weight measurement, blood was collected and serum was separated to measure liver function, kidney function and biochemical parameters for lipid test.

Figure 4 shows the change in blood glucose after oral administration of thymosaponin A-III obtained by Preparation Example 3 by concentration (100 mg / kg, 200 mg / kg), a strong blood sugar at a concentration of 200 mg / kg The drop effect could be confirmed.

Example 3: Hypoglycemic Effect of Timothy Sammonin A-III on Streptozotocin-induced Diabetic Rats

Diabetes induced by intraperitoneal administration of streptozotocin (STZ: 40 mg / kg) dissolved in 50 mM citric acid buffer (pH 4.5) in 7-week-old male Wistar rats (180-200 g body weight) I was. After administration of STZ, only rats with stable blood glucose (400 mg / dl or more) on day 5 were selected and used in the experiment. Negative control of 0.5% carboxymethylcellulose (CMC) solution, positive control (glibeclamide) and test drug dissolved in 0.5% carboxymethylcellulose (CMC) solution are administered orally every 8 days in the afternoon. Measurements were taken with a blood glucose meter just prior to dosing in the afternoon at 3-4 day intervals. Animal body weight and feed volume were measured immediately before drug administration at two-day intervals.

Figure 5 shows the hypoglycemic effect in the streptozotocin-induced diabetic rat type 1 diabetes model of thymosaponin obtained by Preparation Example 3. N. Nakashima et al. [N. Nakashima et al., J. Nat. Prod. Unlike vol 56, 345, 1994], there was no significant hypoglycemic effect of thymosaponin A-III (150 mg / kg).

Based on the above Examples 1 to 3, the present inventors confirmed that thymosaponin A-III has a potent hypoglycemic effect selectively on type 2 diabetes.

Example 4 Toxicity Experiments of Imos Mice of Timothy Saponin A-III

In order to measure the repeated dose toxicity for 1 g of thymosaponin A-III, 4 to 5 week old ICR mice (5 mice per group) fasted for 16 hours were selected as the test animals. Repeated oral administration of 1 g of thymosaponin A-III dissolved in 0.5% carboxymethylcellulose (CMC) for 5 days showed no mortality and no abnormal findings such as organ damage.

Formulation Example 1 Preparation of Powder and Capsule

100 mg of thymosaponin A-III was mixed with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable apparatus.

Formulation Example 2: Preparation of Ointment

Using thymosaponin A-III ointment was prepared in the following composition.

[Furtherance]

Active ingredient 5 g, cetyl palmitate 20 g, cetanol 40 g, stearyl alcohol 40 g, myristan isopropyl 80 g, monostearic acid sorbitan 20 g, polysorbate 60 g, paraoxybenzoic acid propyl 1 g, para 1 g of methyl oxyanate, phosphoric acid and purified water

Formulation Example 3 Preparation of Injection

Injections were prepared using the thymosaponin A-III in the following composition.

[Furtherance]

Active ingredient 100 mg, mannitol 180 mg, sodium dihydrogen phosphate 25 mg, purified water for injection 2974 mg

Formulation Example 4 Preparation of Health Food

0.3 mg thymosaponin A-III, powdered vitamin E, iron lactate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B1 and vitamin B2 were prepared on a daily dose basis.

The components of the health food is as follows (based on the daily dose of a person).

300 mg of active ingredient

Ginseng Extract 100 mg

Green Tea Extract 100 mg

Vitamin C 100 mg

Powdered Vitamin E 120 mg

Iron lactate 2 mg

Zinc oxide 2 mg

20 mg nicotinic acid

Vitamin A 5 mg

Vitamin B1 2 mg

Vitamin B2 2 mg

Corn starch 200 mg

20 mg magnesium stearate

As described above, in the present invention, unlike the conventionally known thymosaponin A-III, the hypoglycemic effect on the type 1 diabetes disease model is insufficient, and the hypoglycemic effect on the type 2 diabetes disease model is It is expected to be very useful for the use of medicines and health foods for preventing and treating type 2 diabetes by confirming its excellence.

Claims (3)

A composition for preventing and treating type 2 diabetes, comprising the compound represented by Formula 1 as an active ingredient. [Formula 1]

2. A composition according to claim 1, wherein said composition is formulated as a preparation for oral or parenteral administration. The health food effective for type 2 diabetes, comprising the compound represented by the following formula (1) as an active ingredient. [Formula 1]

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