KR100572620B1 - Composition comprising protocathechualdehyde and its pharmaceutically acceptable salt for prevention and treatment of diabetic complication - Google Patents

Abstract

The present invention relates to a composition for preventing and treating diabetic complications containing protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient.

Protocatecualdehyde of the present invention effectively inhibits the production of the final glycated product, has an excellent effect on lowering blood sugar, prevents weight loss, decreases renal hypertrophy, and significantly reduces levels of BUN, triglycerides, creatinine, etc. And deterioration of the lens turbidity significantly reduced, cataracts are delayed and prevented.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of diabetic complications resulting from the production of the final glycated product. In addition, the composition of the present invention can be usefully used for preventing and delaying the aging caused by oxidative stress because the rate of reduction of free radical production and the induction rate of oxidative stress is reduced when inhibiting the production of the final glycosylated product.

Description

Composition comprising protocathechualdehyde and its pharmaceutically acceptable salt for prevention and treatment of diabetic complication             

1 is a diagram showing the effect of inhibiting the production of the final glycated product of protocatecualdehyde.

Figure 2 is a diagram visually observed the eye of the diabetic rats administered with protocatecualdehyde.

Figure 3 is a diagram showing the turbidity of the lens isolated from the eye of the diabetic rats administered protocatecualdehyde.

The present invention relates to a composition for preventing and treating diabetic complications containing protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient.

Diabetes is one of the world's most important adult diseases. Recently, with the rapid economic growth, the prevalence of diabetes reaches 7-8% in Korea, and it is a significant cause of death for people in their 60s and 70s. Diabetes mellitus is a disease caused by the lack of insulin (hormone) that carries glucose into the body's cells. Glucose that was not transported into the cells remains in the blood, so the blood becomes a high blood sugar state and is discharged into the urine. The persistence of hyperglycemia causes abnormalities in protein and lipid metabolism in the body, resulting in physiological and biochemical adverse reactions of tissues in the body, leading to complications.

Drug treatment of diabetes has been developed so far to lower blood glucose levels in general, or the anti-diabetic agents under development include insulin, sulfone urea, alpha glucosidase inhibitors, biagnade agents, chiajoridanedione agents, and the like. have. Insulin agent lowers blood sugar by injecting insulin into the body in the form of injection or pump. Sulfonate is a drug that acts on the pancreas and promotes insulin secretion, thereby lowering blood sugar.Mechanism of promoting insulin secretion by increasing the concentration of Ca ^{2+ in the} beta cells by blocking ATP-dependent K ⁺ channels in the beta cell membrane of the pancreas. (Lebovitz, HE, et al., 1992, Drugs, 44, Suppl. 3, 21-28). However, these compounds are at risk of causing hypoglycemia if the dose is incorrect (Campbell, IW et al., 1993, Clinical and physiological aspects, London, Efward Arnold, 387-392). On the other hand, the alpha glucosidase inhibitor inhibits the action of the alpha glucosidase to reduce the decomposition of the high molecular sugar compound to suppress the rapid increase in blood sugar by ingesting the saccharide compound. However, unabsorbed carbohydrates can cause gastrointestinal side effects such as fart, bloating, and diarrhea. Biagunaide is a drug that lowers blood sugar by increasing insulin utilization in peripheral tissues. It is involved in the action of insulin, inhibits the production of sugar in the liver, activates glycolysis and decreases the blood sugar by lowering blood glucose. . Although the biaguanide is widely used to treat type 2 diabetes, it may cause side effects of lactic acidosis (Sirtori, CR, et al., 1994, Pharmacol. Res ., 30: 187-228). . In addition, a number of dental co-edinideone agents that improve the sensitivity of insulin to improve blood sugar control, and lower the levels of fasting blood sugar and glycosylated hemoglobin have been studied. Since dental preparations are insulin-lowering drugs only when insulin is present, severe hypoglycemic effects do not have a hypoglycemic effect.

These drugs have an excellent hypoglycemic effect, and thus the efficacy as a therapeutic agent for diabetes is recognized, but the direct efficacy of the prevention of diabetic complications described below has not been confirmed.

Sustained hyperglycemia can lead to diabetic complications. Diabetic complications are an important cause of death from diabetes and are indicated by diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, and the like. In particular, diabetic cataracts cause blindness, and chronic diabetic nephropathy is the most important cause of end-stage renal failure, and there is no treatment other than hemodialysis and organ transplantation. These diabetic complications can be seen to proceed even when the normal blood sugar level is recovered by treating diabetes mellitus, which is the final glycated product irreversibly produced as a result of the non-enzymatic glycation of the protein due to the persistence of hyperglycemic state ( Advanced glycation endproducts (AGEs) are known as one of the main reasons.

Nonenzymatic glycation of protein, one of the major mechanisms that lead to diabetic complications, is caused by condensation reactions, or milliard reactions, of enzymes with amino acids such as lysine residues and reducing sugars. The result is a final glycated product. The non-enzymatic glycosylation of the protein is carried out by (1) an amino acid group, such as a lysine residue of the protein, and an aldehyde or ketone of a reducing sugar with a nucleophilic addition reaction without enzymatic action to form a Schiff base, an early product. Schiff base and contiguous ketoamine adduct condensate to produce reversible Amadori-type early glycosylated product, and (2) the hyperglycemic state persists, so that reversible Amadori-type early glycosylated product is not degraded. It can be divided into stages that are rearranged and cross-linked with proteins to produce irreversible final glycosylated products.

Unlike the reversible Amadori type of early glycosylated products, the final glycated products are irreversible reaction products. Once produced, blood sugars do not degrade even when they are restored to normal, but accumulate in the tissues during protein survival to abnormally change the structure and function of the tissues. (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. J. Biochem ., 210: 729-739). For example, glycated albumin, one of the final glycosylated products produced by reacting glucose with various kinds of proteins, is an important factor causing chronic diabetic nephropathy. Glycosylated albumin enters renal glomeruli more easily than normal albumin without glycosylation, and high concentrations of glucose stimulate mesangium cells to increase extracellular matrix synthesis. Excessly introduced glycated albumin and increased extracellular matrix result in fibrosis of the glomeruli. These mechanisms continue to damage the glomerulus, leading to the stage of extreme treatment methods such as hemodialysis or organ transplantation. In addition, chronic diabetes has been reported to accumulate collagen in the arterial wall and basal membrane protein in the renal glomeruli and to accumulate in tissues (Brownlee, M., et al., 1986, Sciences , 232, 1629-). 1632).

As described above, glycosylation occurs in proteins such as basement membrane, plasma albumin, lens protein, fibrin, and collagen by non-enzymatic protein glycosylation, and the resulting glycosylated product abnormally changes the structure and function of the diabetic diabetic retinopathy and diabetes. It causes chronic diabetic complications such as cataracts, diabetic nephropathy and diabetic neuropathy.

It is also known that the end glycosylated product produced by the non-enzymatic protein glycosylation reaction plays an important role in aging (Monnier et al., Proc. Natl. Acad. Sci. USA, 81: 583, 1984; Lee et al., Biochem Biophys Res Comm., 123: 888, 1984; Diabetologia, 38: 357-394).

As described above, the final glycosylated product produced by the non-enzymatic protein glycosylation reaction is a major cause of the progression of diabetes complications and aging, and it is necessary to suppress the production of the final glycation products to prevent the progression of diabetes complications and aging.

Meanwhile, protocathechualdehyde (3,4-dihydroxybenzaldehyde) is a low molecular weight compound having a molecular weight of 138.12 and is used as an intermediate for preparing various drugs. Not only is this compound synthesized simply, but also Salvia miltiorrhiza, Salvia chinensis , Gastrodia elata , Coix lachryma-jobi , Avena fatua , Artemisia apiacea , etc. It is a very common compound that is widely distributed in herbal medicines. Protocatecualdehyde has antioxidant (1,1-diphenyl-2-picrylhydrazyl radical scavenging) efficacy (Kikuzaki, H., et al., J. Nutr. Sci. Vitaminol., 2001, apr, 47 (2), 167-171), apoptosis-inducing effects of CTLL-2 cells (Wang, Y., et al., Anticancer Res., 2001, 21 (2A), 1095-1101) and antirheumatic effects have been reported. (Watanabe, K., et al., Pharmacol. Res., 1993, 28 (1)). However, there is no report on the hypoglycemic activity or the inhibitory effect of the final glycated product production.

Therefore, the present inventors found that protocatecualdehyde is excellent in inhibiting blood glucose lowering effect and production of final glycated product, and then protocatecualdehyde may be usefully used for preventing and treating diabetic complications, preventing aging and delaying. It was confirmed that the present invention was completed.

The present invention is to provide a prophylactic aldehyde and its pharmaceutically acceptable salts for the prevention and treatment of diabetic complications containing as an active ingredient.

In addition, the present invention is to provide a composition for anti-aging and delaying containing protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient.

The present invention provides a prophylactic aldehyde and its pharmaceutically acceptable salts for the prevention and treatment of diabetic complications containing as an active ingredient.

In addition, the present invention provides a composition for anti-aging and delaying containing protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient.

Hereinafter, the present invention will be described in detail.

Protocatecualdehyde used in the present invention can be used commercially available reagents, can be obtained by extraction, separation and purification from the herbal medicines, can be produced and used by any conventional method.

That is, protocatecualdehyde is prepared by reacting 3,4-dimethoxybenzaldehyde (3,4-dimethoxybenzaldehyde) with benzene and aluminum chloride at 130 to 150 ° C (Patent: DE 193958).

In addition, the extract is used from herbal medicines such as Cheongho ( Artemisia apiacea ).

After slicing the dried Cheongho, extracted with 80% ethanol at room temperature for 3-5 days, filtered and concentrated under reduced pressure. This was separated into normal hexane, ethyl acetate, and normal butanol, and the ethyl acetate fraction was pre-separated into 10 starches using silica gel column chromatography in a mixture of chloroform and methanol (7: 3). The third fraction is separated again with a mixture of normal hexane and ethyl acetate (8: 2), and the protocatecualdehyde is separated using silica gel column chromatography.

The protocatecualdehyde may form pharmaceutically acceptable salts according to methods conventional in the art. As the pharmaceutically acceptable free acid, inorganic acid and organic acid can be used. Hydrochloric acid, bromic acid, iodic acid, sulfuric acid or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, Methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid or aspartic acid, and the like can be used.

Protocatecualdehyde of the present invention effectively inhibits the production of the final glycated product, which is one of the factors inducing diabetic complications in vitro experiments, excellent in hypoglycemic effect in animal experiments, weight gain compared to the diabetic group Renal hypertrophy and BUN, triglyceride and creatinine levels are significantly reduced to restore deteriorated renal function, and the lens turbidity is significantly lower than that of diabetic group, which is useful for delaying or preventing cataracts.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of diabetic complications caused by the production of the final glycation end products, namely diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy .

In addition, the composition of the present invention can be usefully used for preventing and delaying the aging caused by oxidative stress because the rate of reduction of free radical production and the induction rate of oxidative stress is reduced when inhibiting the production of the final glycosylated product.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the protocatecualdehyde.

The protocatecualdehyde may be administered orally or parenterally during clinical administration, and may be used in the form of a general pharmaceutical formulation.

Protocatecualdehyde and pharmaceutically acceptable salts thereof of the present invention can be administered in effective amounts through various routes of administration. The use together contains a pharmaceutically acceptable carrier. More specifically, pharmaceutically acceptable carriers can be any standard pharmaceutical carrier that can be used in known formulations such as sterile solutions, tablets, coated tablets and capsules. Typically, the carrier may include excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid, talc, vegetable oils or oils, gums, glycols, or other known excipients, and also flavors, color additives And other ingredients. The formulation for administering the composition containing the protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient can be administered by oral, intravenous, intramuscular, or transdermal administration in a conventional manner. However, it is not limited only to these methods. In actual clinical administration, it can be administered in a variety of oral and parenteral formulations, which are formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, liquid solutions, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin.

In addition, the pharmaceutical compositions of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. In order to formulate into a parenteral formulation, it is prepared in solution or suspension by mixing in water with a stabilizer or buffer, including the protocatecualdehyde, and formulated in unit dosage form of ampoules or vials.

The dosage of the present pharmaceutical composition is appropriately selected depending on the absorption rate, inactivation rate, rate of excretion, the age, sex and condition of the patient, and the severity of the disease to be treated in the body. Can be administered in divided doses. When the pharmaceutical composition of the present invention contains the protocatecualdehyde and its pharmaceutically acceptable salts as an active ingredient, the effective dose is 10 to 200 mg / kg / day, preferably 20 to 100 mg /. Kg / day. The exact amount, route of administration, and frequency of the agent can be readily determined according to the nature of the agent, the weight and condition of the subject to be administered, and the nature of the particular derivative to be used.

In the present invention, the protocatecualdehyde is a result of the acute toxicity test in the rat for the test, the minimum lethal dose (LD ₅₀ ) during oral administration at least 1g / ㎏ does not show any toxic effects at all that the very high bio stability As can be seen, the protocatecualdehyde and pharmaceutically acceptable salts thereof of the present invention can therefore be safely administered to a living body.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of diabetic complications, the prevention and delay of aging.

The composition of the present invention can be added to health foods for the purpose of improving diseases caused by diabetic complications and aging. When the composition of the present invention is used as a food additive, the protocatecualdehyde may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the mixed herbal medicine of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material in the manufacture of food or beverage. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea, and drinks. Alcoholic beverages and vitamin complexes, and includes all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following Examples and Experimental Examples are only illustrative of the contents of the present invention and the scope of the invention is not limited by the Examples.

Example 1  : Analysis on the Inhibitory Effect of Protocatecualdehyde on the Production of Final Glycosylated Products

In order to determine the final glycation product inhibitory effect of the protocatecualdehyde of the present invention, the following experiment was performed.

Bovine serum albumin (hereinafter referred to as 'BSA'; manufactured by Sigma, USA) was used as a protein source. BSA was prepared by adding 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / mL.

As a sugar source, a mixture of 0.1 M fructose and 0.1 M glucose was used.

A mixed solution of fructose and glucose was added to the prepared BSA solution. Protocatecualdehyde was dissolved in distilled water at concentrations of 0.00345, 0.0069, 0.0138, 0.138, 0.276, and 0.552 µg / ml, added to the mixture of protein and sugar, and incubated at 37 ° C for 30 days. At this time 0.02% sodium azide was added as an antibacterial agent.

The control group was cultured with the BSA and the sugar mixture, and blanks of the test group and the control group were prepared, respectively, and then uncultured.

All the culture medium was prepared by 4 each to avoid the error as possible, immediately before the culture was filled with nitrogen gas (purity: 99.999%) to prevent contamination. After 30 days, the content of the final glycation product produced in the culture was analyzed. The final glycosylated product is fluorescent and brown, not only possesses physicochemical properties that can cross-link, but also has ligands that can be recognized by cell membrane receptors. The amount of the final glycated product having these characteristics was measured by spectrophotometer (excitation-350 nm, emission-450 nm) to analyze the degree of inhibition of production.

As a positive control group, the experiment was conducted in the same manner as described above, except that the aminoguanidine HCl was dissolved in distilled water at a constant concentration for 30 days.

The production inhibition rate was calculated by the following equation.

Production Inhibition Rate (%) = 100-[(Fluorescence Intensity of Sample-Fluorescent Intensity of Sample Blank) / (Fluorescence Intensity of Control-Fluorescent Intensity of Control Blank)] × 100

The results are shown in Figure 1 and Table 1.

As shown in Table 1, a very good final result of 17.41 ± 3.513%, 73.24 ± 1.58%, 89.02 ± 1.05% at 0.00345 μg / ml, 0.0069 μg / ml, 0.0138 μg / ml when the protocatecualdehyde was incubated for 30 days Glycolysate production inhibitory efficacy was shown (IC ₅₀ value: 0.00647 μg / ml). And from the concentration of 0.138 ㎍ / ㎖ or more it was confirmed that the efficacy is not proportional to the concentration almost the same level of efficacy. On the other hand, the positive control group aminoguanidine-HCl had the effect of inhibiting the production of the final glycated product at 27.5 µg / ml, 55 µg / ml, 110 µg / ml and 550 µg / ml, respectively, at 45.78 ± 2.40%, 55.43 ± 4.0%, and 73.52 ± 1.75%, 96.41 ± 2.20% and IC ₅₀ was 34.90µg / ml.

Therefore, it can be seen that the production inhibitory effect of the final glycosylated product of protocatecualdehyde is very excellent.

Example 2  : Effects of Protocatecualdehyde on Diabetes Complications

In order to determine the therapeutic effect of diabetic complications of the protocatecualdehyde of the present invention, the following experiment was performed.

1. Experimental Animal

Male rats of 4 weeks of age, 120-140 g, were housed in cages and were freely fed with solid feed and water for one week.

The environmental conditions of the animal room were set to a temperature of 23 ± 3 ℃, a relative humidity of 50 ± 10%, a lighting time of 12 hours (8 am to 8 pm), a ventilation frequency of 10 to 20 times / hour, and an illuminance of 150 to 300 Lux. During the test, the temperature and humidity of the animal room were automatically controlled by a thermo-hygrostat, and the environmental conditions such as illuminance were measured regularly, and there was no change affecting the test.

The experimental animals were 1) normal (NC), 2) diabetic-induced (DC), 3) diabetic-induced protocatecualdehyde (DC + PCA), 4) diabetic-induced positive control ( Palestine) was divided into four groups, such as the group [DC + S11], 9 to 10 animals in the diabetes-induced group, 5 to 6 animals in the normal group, and the weight was evenly distributed.

2. Induction of Diabetic Complications by Streptozotocin

Streptozotocin (N- [methylnitrosocarbamoyl] -D-glucosamine; STZ) is a chemical that causes hyperglycemia due to insulin deficiency by selectively destroying beta cells. In particular, single high dose streptozotocin (SHDS) irreversibly induces rapid hyperglycemia due to massive necrosis of beta cells, and can be considered as a suitable model for diabetic complications because it causes irreversible hyperglycemic conditions.

The animals were fasted for one week after adapting to the animal room for one week, and 60 mg / kg body weight of the streptozotocin solution prepared in 0.1M citric acid buffer (pH 4.5) immediately before administration was intraperitoneally injected. Two days after administration of streptozotocin solution, tail vein blood was taken to check blood glucose level, and rats of 300 mg / dl or more were diabetic rats.

As a positive control group, Epalrestat (ONal-2235; S11), which is used as a diabetic complication, was used.

3. General observations (changes in body weight, feed and water intake)

To see the therapeutic effect of chronic diabetic complications, rats were treated with drugs for about 8 weeks from the 31st day after continuing diabetes for 30 days.

Protocatechualdehyde (PCA) and epalestat (S11) used in this experiment were non-aqueous, so they were dissolved in 1% carboxymethyl cellulose (CMC), and carboxymethylcellulose was used in the normal group. Dosing excludes the effect by carboxymethylcellulose.

In two diabetic groups except the normal group, 25 mg / kg of protocatecualdehyde and 25 mg / kg of Epalestat (S11), a positive control group, were dissolved in 1% CMC and administered orally to rats daily for 8 weeks. It was.

After administration of the sample during the dosing period, the animals were fasted for at least 15 hours before the autopsy and weighed.

 Body weight was measured daily, feed amount was measured once a week, water intake was measured daily.

The results are shown in Table 2.

Initial (g) Terminal (g) Weight gain (g) NC 209.26 ± 28.94 462.09 ± 32.91 252.83 DC 154.33 ± 27.26 220.50 ± 64.28 66.17 DC + PCA 154.50 ± 29.55 309.41 ± 48.16 154.91 DC + S11 154.03 ± 45.46 283.24 ± 42.60 129.21

※ NC + CMC: normal group + carboxymethyl cellulose,

   DC + CMC: diabetes-causing group + carboxymethyl cellulose,

   DC + PCA: diabetes-causing group + protocatecualdehyde,

   DC + S11: diabetes causing group + positive control group (epalestat)

As shown in Table 2, the weight of the normal group was increased by 252.83g, only 66.17g in the diabetes-induced group. In addition, 154.91g increased in the diabetic-induced group, and 129.21g increased in the positive control group, epalestat (S11), in the diabetic-induced group. As described above, it can be seen that the group administered with the protocatecualdehyde of the present invention has an increase in weight gain compared to that of the administration of epalestat (S11).

In addition, the diabetic induction group consumed about twice as much feed as the normal group despite the low weight, and the group receiving the protocatecualdehyde and the positive control group epalestat (S11) received more than that.

In addition, the diabetic induction group consumed about 7 times more water than the normal group. However, the group receiving protocatecualdehyde consumed less water than the diabetic inducing group, and the positive control group, epalestat (S11), ingested more water than the diabetic inducing group.

4. Long-term weight measurement

One day before the autopsy, the animals were fasted for at least 15 hours, weighed, anesthetized with ethyl ether, and collected through the abdominal aorta. Some of the blood was treated with heparin, and some whole blood was centrifuged at 3,000 rpm and 4 ° C. for 15 minutes to separate plasma, respectively, and stored at −80 ° C. to be used for each data analysis.

Kidneys were washed after perfusion, removed and weighed. The cortex and water were separated and quenched and stored at -80 ° C. In addition, liver, lung, pancreas, spleen, heart was separated and weighed.

The results are shown in Table 3.

Heart liver spleen lungs kidney Pancreas NC + CMC Absolute weight (g) 1.16 ± 0.07 10.60 ± 0.72 0.79 ± 0.10 1.84 ± 0.23 2.65 ± 0.18 1.13 ± 0.21 Relative weight (%) 0.25 ± 0.02 2.30 ± 0.12 0.17 ± 0.02 0.40 ± 0.04 0.58 ± 0.04 0.25 ± 0.05 DC + CMC Absolute weight (g) 0.84 ± 0.18 ^*** 10.30 ± 1.99 0.46 ± 0.15 ^*** 1.44 ± 0.24 ^** 2.92 ± 0.45 0.79 ± 0.24 ^** Relative weight (%) 0.37 ± 0.06 ^*** 4.62 ± 0.71 ^*** 0.20 ± 0.03 ^* 0.65 ± 0.14 ^*** 1.35 ± 0.37 ^*** 0.35 ± 0.09 ^** DC + PCA Absolute weight (g) 0.99 ± 0.11 ^{**, #} 11.83 ± 0.97 ^* 0.57 ± 0.10 ^*** 1.71 ± 0.29 3.26 ± 0.33 ^*** 0.97 ± 0.18 Relative weight (%) 0.32 ± 0.03 ^{***, #} 3.87 ± 0.41 ^*** 0.19 ± 0.02 0.56 ± 0.12 ^** 1.07 ± 0.12 ^*** 0.32 ± 0.07 ^* DC + S11 Absolute weight (g) 0.95 ± 0.11 11.63 ± 1.08 0.54 ± 0.09 1.55 ± 0.13 3.18 ± 0.30 0.86 ± 0.11 Relative weight (%) 0.34 ± 0.03 ^*** 4.15 ± 0.40 ^*** 0.19 ± 0.02 0.55 ± 0.06 ^*** 1.14 ± 0.11 ^*** 0.31 ± 0.05 ^*

※ NC + CMC: normal group + carboxymethyl cellulose,

   DC + CMC: diabetes-causing group + carboxymethyl cellulose,

   DC + PCA: diabetes-causing group + protocatecualdehyde,

   DC + S11: diabetes causing group + positive control group (epalestat)

   Statistical comparison with normal group (*: p <0.05, **: p <0.01, ***: p <0.001)

   Statistical comparison with diabetic group (#: p <0.05)

As shown in Table 3, in the relative organ weight, the diabetic induction group enlarged all organs except the pancreas, and the kidney and liver increased more than two times compared to the normal group. However, compared with the diabetic group, the protocatecualdehyde group showed a significant decrease in hypertrophy of the kidneys, liver, and heart (p <0.05), and other symptoms of the spleen, lung, and pancreas also decreased. There was no. In addition, the protocatecualdehyde-administered group showed better efficacy than the positive control group, epalestat (S11).

5. Serum Biochemical Factor Analysis

1) hypoglycemic effect

Blood was taken from the orbital vein at 2 week intervals before necropsy and blood glucose was measured using a glucose kit. Plasma glucose content was measured at 500 nm using glucose oxidase method.

2) renal function

① BUN (Blood Urea Nitrogen)

BUN was measured for absorbance at 580nm using the urease-indophenol method.

② triglyceride

In order to quantify the content of triglyceride (triglyceride) was measured at 550nm using the enzyme method (POD).

③ total cholesterol

For determination of total cholesterol content was analyzed at 500 nm using the enzymatic method (enzymatic method).

④ creatinine

Creatinine was measured for absorbance at 515 nm using the Jaffe method.

⑤ protein

Serum protein was measured using BCA assay. Bisconic acid, Na ₂ CO ₃ , NaHCO ₃ , C ₄ H ₄ O ₆ Na ₂ ㆍ 2H ₂ O mixed with 0.1N NaOH and 4% CuSO ₄ ㆍ 5H ₂ O After reacting together, the absorbance was measured at 562 nm.

The results are shown in Table 4.

Blood glucose level (mg / ㎗) BUN (mg / ㎗) Triglycerides (mg / dl) Total cholesterol (mg / dl) Creatinine (mg / dl) Protein (μg / μl) NC + CMC 124.93 ± 16.25 15.12 ± 0.54 33.38 ± 6.62 63.73 ± 11.05 1.54 ± 0.08 12.63 ± 0.28 DC + CMC 408.79 ± 63.10 ^*** 35.74 ± 8.77 ^*** 70.20 ± 13.14 ^*** 54.76 ± 11.85 1.75 ± 0.17 ^* 12.47 ± 0.42 DC + PCA 193.32 ± 136.67 ^### 26.62 ± 3.51 ^*** 50.35 ± 16.63 ^{*, ##} 53.56 ± 6.18 1.57 ± 0.28 ^# 12.35 ± 1.61 # DC + S11 197.94 ± 76.66 ^{*, ###} 23.60 ± 5.15 ^{***, ##} 46.18 ± 8.26 ^{*, ##} 59.50 ± 13.88 1.22 ± 0.16 ^*** 10.79 ± 0.85 ^{***, #}

※ NC + CMC: normal group + carboxymethyl cellulose,

   DC + CMC: diabetes-causing group + carboxymethyl cellulose,

   DC + PCA: diabetes-causing group + protocatecualdehyde,

   DC + S11: diabetes causing group + positive control group (epalestat)

   Statistical comparison with normal group (*: p <0.05, **: p <0.01, ***: p <0.001)

   Statistical comparison with diabetic group (#: p <0.05, ##: p <0.01, ###: p <0.001)

As shown in Table 4, the blood glucose level of the diabetes-induced group was 408.79 ± 63.10 mg / dL, and the protocatecualdehyde-administered group was 193.32 ± 136.67 mg / dL, which is superior to the diabetes-induced group (p <0.001). .

In addition, the BUN level, which is an indicator of renal function, was about 2.4 times higher in the diabetic group than in the diabetic group, and the protocatecualdehyde group was decreased, although not significant, in the diabetic group.

In addition, triglyceride was significantly increased in the diabetic group (p <0.001) and significantly decreased in the protocatecualdehyde group compared to the diabetic group (p <0.05).

In addition, creatinine levels were significantly increased in the diabetic group (p <0.05) and significantly decreased in the protocatecualdehyde group (p <0.05). Creatinine is also used as an indicator of renal function but is not as sensitive as BUN, so even if glomerular filtration rate is reduced by more than 50%, the level remains in the normal range. Therefore, it can be seen that the new function is very good.

Therefore, it can be seen that the protocatecualdehyde of the present invention significantly reduced renal hypertrophy and BUN, triglyceride, and creatinine levels were significantly reduced compared to the diabetic group, and thus renal function was greatly improved.

6. Anti-cataract Effect

The lens was separated from the eye removed under the microscope, transferred to a 24-well plate containing 2 ml of saline, and photographed with a digital camera attached to the microscope. Turbidity was analyzed by using an image analysis system program.

Take a lens with a camera, weigh it, add phosphate buffer solution (pH 6.9), homogenize at 4 ℃, and centrifuge for 20 minutes at 3,000 rpm for supernatant to measure enzyme activity. Stored at. In order to measure the content of sorbitol and the like, ZnSO ₄ and NaOH were added to the remaining homogenate to remove proteins, and the supernatant was collected by centrifugation at 3,000 rpm for 20 minutes and stored at -80 ° C.

The state of observing the lens is shown in FIG. 2, and the turbidity of the lens is shown in Table 5 and FIG. 3.

Lens turbidity (20 pixel / ㎡) Lens turbidity (%) NC 39.91 ± 23.36 8.31 ± 5.21 DC 200.53 ± 47.88 66.84 ± 14.89 ^*** DC + PCA 145.67 ± 57.23 39.00 ± 15.53 ^{**, ##} DC + S11 134.04 ± 45.00 37.38 ± 13.56 ^{***, ###}

※ NC: Normal group,

   DC: diabetes causing group,

   DC + PCA: diabetes-causing group + protocatecualdehyde,

   DC + S11: diabetes causing group + positive control group (epalestat)

   Statistical comparison with normal group (**: p <0.01, ***: p <0.001)

   Statistical comparison with diabetic group (##: p <0.01, ###: p <0.001)

As shown in FIG. 2, cataracts began to appear in the diabetic group from 6 weeks after the onset of diabetes. On the day of necropsy, the ocular status of all groups was white in 3 of 7 eyes in the diabetic group and 1 of them was very weak.

In the protocatecualdehyde-treated group, two of the nine eyes of both eyes were covered with white color, and three of them began to show mild cataract symptoms. In the positive control group, Epalestat (S11), four of seven eyes were covered with both eyes, and one had mild symptoms in the right eye.

The cataract effect in the positive control group Epalestat (S11) -administered group may be attributed to the fact that there are two rats subjected to streptozotocin second induction. In general, when the second streptozotocin induction is performed, the weight gain is small and the lens turbidity is also known to be severe.

As shown in Table 5, the average lens turbidity was 8.31 ± 5.21% in the normal group, and 66.84 ± 14.89% in the diabetic group, indicating that the lens turbidity in the diabetic group was severe. In addition, when protocatecualdehyde and the positive control soldier Epalestat (S11) were administered to the diabetic group, the cataract phenomenon was significantly 39.00 ± 15.53% (p <0.01) and 37.38 ± 13.56% (p <0.001), respectively. This has been reduced.

In addition, as shown in Figure 2, the lens turbidity of the normal group was found to be 20% or less, the turbidity of about 20% can be seen as a normal state. The lens turbidity of the diabetic group was more than 40%, and most of them showed a lens turbidity of more than 60%. More than 80% of the rats were present in 3 of 8 rats. However, most of the lens turbidity in the group administered with protocatecualdehyde was around 40%, and some of them showed normal levels. Only one eye showed more than 60% turbidity. In the positive control group, Epalestat (S11), the turbidity of the lens was about 40%, and none of the rats showed more than 60% turbidity.

Therefore, the protocatecualdehyde of the present invention can be effectively used for the prevention and treatment of cataract expression due to chronic diabetes.

Example 3  : Oral Acute Toxicity in Rats

In order to determine the acute toxicity of the protocatecualdehyde of the present invention, the following experiment was performed.

Acute toxicity test was performed using 6 week-old SPF rats. Eight animals were dissolved protocatecualdehyde in water and administered once orally at a dose of 1 g / kg / 10 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms were observed in all animals treated with the test substance, no animals died, and no toxicity change was observed in weight change, blood test, blood biochemical test, and side findings. As a result, all of the protocatecualdehydes of the present invention did not show a toxicity change in rats up to 1 g / kg, and the minimum oral dose (LD ₅₀ ) was determined to be a safe substance of at least 1 g / kg or more.

Formulation Example 1  : Preparation of Tablet

Protocatecualdehyde 100 mg, corn starch 90.0 mg, lactose 175.0 mg, L-hydroxypropyl cellulose 15.0 mg, polyvinylpyrrolidone 905.0 mg and a suitable amount of ethanol were mixed homogeneously, granulated by wet granulation method and stearic acid 1.8 mg of magnesium was added and mixed, and then one tablet was compressed to 400 mg.

Formulation Example 2  : Manufacture of Capsule

Protocatecualdehyde 100 mg, corn starch 80.0 mg, lactose 175.0 mg, and magnesium stearate 1.8 mg were mixed uniformly, and one capsule was filled with 360 mg each.

Formulation Example 3  : Preparation of functional drinks

522mg of honey

Chioctosanamide 5mg

Nicotinamide 10mg

Riboflavin Sodium Hydrochloride 3mg

Pyridoxine hydrochloride 2mg

Inositol 30mg

Ortic Acid 50mg

Protocatecualdehyde 100mg

200ml water

With the above composition and content, drinks were prepared using conventional methods.

Protocatecualdehyde of the present invention effectively inhibits the production of the final glycated product, has an excellent effect on lowering blood sugar, prevents weight loss, decreases renal hypertrophy, and significantly reduces levels of BUN, triglycerides, creatinine, etc. And deterioration of the lens turbidity significantly reduced, cataracts are delayed and prevented.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of diabetic complications resulting from the production of the final glycated product. In addition, the composition of the present invention can be usefully used for preventing and delaying the aging caused by oxidative stress because the rate of reduction of free radical production and the induction rate of oxidative stress is reduced when inhibiting the production of the final glycosylated product.

Claims (8)

Composition for the prevention and treatment of diabetic cataract or diabetic nephropathy containing protocatecualdehyde or a pharmaceutically acceptable salt thereof as an active ingredient delete delete delete delete delete delete delete

Images