KR100543341B1 - Composition comprising herbal mixture extract for prevention and treatment of diabetic complication - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of diabetic complications using a mixed herbal composition as an active ingredient, and in particular, 5 to 85% by weight each of the counter electrode, gangbakbak, vinegar and licorice.

The mixed herbal composition of the present invention effectively suppresses 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. It inhibits exacerbation and significantly reduces the lens turbidity, which delays and prevents cataracts.

Therefore, the mixed herbal 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. In addition, the mixed herbal composition of the present invention can reduce the free radical production and reduce the induction rate of oxidative stress when inhibiting the production of the final glycation product, it can be usefully used for preventing and delaying aging caused by oxidative stress.

Description

Composition comprising herbal mixture extract for prevention and treatment of diabetic complication             

1 is a view showing the inhibitory effect of the final glycation product production of the mixed herbal composition of the present invention.

2 is a diagram observing the eye of the diabetic rats to which the mixed herbal composition of the present invention was administered.

Figure 3 is a diagram showing the turbidity of the lens isolated from the eye of the diabetes-induced rat administered the mixed herbal composition of the present invention.

The present invention relates to a composition for the prevention and treatment of diabetic complications using a mixed herbal composition as an active ingredient, and in particular, 5 to 85% by weight each of the counter electrode, gangbakbak, vinegar and licorice.

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 in the case of diabetic treatment to recover the normal blood glucose level, which is the final glycated product irreversibly produced as a result of the non-enzymatic glycation reaction of the protein due to the persistent hyperglycemic state ( Advanced glycation endproducts (AGEs) are known as one of the main reasons.

Nonenzymatic glycation of protein, a major mechanism that causes diabetic complications, is caused by condensation reactions, or milliard reactions, of enzymes with amino acids such as lysine residues and reducing sugars. The resulting glycosylated product is produced. 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. Schiffbase and adjacent ketoamine adducts condense with each other to produce a reversible Amadori-type early glycosylated product; It can be divided into stages of rearrangement and cross-linking with proteins to produce irreversible final glycosylated products.

Unlike reversible Amadori type of early glycosylated products, final glycated products are irreversible reaction products. Once produced, blood sugar is not degraded even after recovery to normal, but accumulates in tissues during protein survival and abnormally changes the structure and function of 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 glycation products produced by the reaction between glucose and various proteins, is an important factor in 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 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 in the non-enzymatic protein glycosylation reaction is a major cause of the progression of diabetes complications and aging, it is necessary to suppress the production of the final glycation products in order to prevent the progression of diabetes complications and aging.

Currently, aminoguanidine-HCl, the only synthetic agent for protein glycosylation inhibitors, is a nucleophilic hydrazine, which binds to the amadori product and prevents cross-linking with the protein, thereby inhibiting the formation of the final glycation product. Delay or prevent progression to (Brownlee, M., et al., 1986, Sciences , 232, 1629-1632; Edelstein, D. et al., 1992, Diabetes , 41, 26-29). Aminoguanidine-HCl is the most promising synthetic drug for the prevention and treatment of diabetic complications, but it has been progressed up to Phase III clinical trials. However, there is a problem that toxicity is induced upon long-term administration.

Meanwhile, Euphorbiae Radix is the root of the perennial herb, Euphorbia pekinensis , and belongs to gallic acid, methylgallate, and 3-O-galloylshimic acid (3-O). -galloylshikimic acid) (Kim, JG, et al., Yakhak Hoeji , 1996, 40, 170-176), and its weakness is high, sour, cold and non- (들어가), lungs, stomach (들어가), and the effect of the saengsyo constriction (음 水 逐 飮), small seed swelling (Shin Min-kyo, primary color clinical herbalism, Younglimsa, 487, 1996 ).

Magnolia Bark is a dried bark of Magnolia obovata, M. officinalis, M. officinalis var. Biloba belonging to the Magnolia family . Efficacy in the lower part of the body, and it treats wet weight, complete earth and sand, food gas, abdominal constipation, and dambing indigestion. Pharmacopoeia Commission, Republic of China Pharmacopoeia, Part I, 204, Chemical Industry Press, Beijing), essential oils such as alpha, beta, gamma-oidesmol, magnool and honokiol (honokiol), alkaloids, and saponins. Pharmacological effects include anti-allergic efficacy (Shin, TY, et al., 2001, Arch. Pharm., Res. , 24: 249-255), apoptosis effect (Park, HJ, et al., 2001, Arch. Pharm , Res. , 24: 342-348), NO synthesis inhibitory effect, TNF-α expression inhibitory effect (Son, HJ, et al., 2000, Planata med ., 66: 467-471), antifungal effect (Bang, KH, et al., 2000, Arch. Pharm, Res ., 23: 46-49), mental stability effects (Kuribara, H., et al, 1999, J. Pharm. Pharmacol ., 51: 97-103) and It has been reported to have various effects such as skin cancer inhibitory effect (Komoshima, T. et al., 1991, J. Nat. Prod. , 54: 816-822).

Puerariae Radix is the dried root of Pueraria thunbergiana ( P. lobata ), a perennial mutant belonging to the legumes, whose weakness is persimmon, sour, flat, and rain. (脾), the stomach (胃) to show the efficacy of Haepyop (解 表), Sujin (透疹), Saengjin (生 津), branch office (止瀉). Pharmacological actions include antipyretic, blood pressure lowering, memory enhancement, increased cerebral blood flow, coronary artery dilation, cardiac function, and antiarrhythmic effects (Kim, Ho-Cheol, Herbal Pharmacology, Moon-Dang, 92-94, 2001).

Licorice (Glycyrrhizae radix) is a dried perennial herbaceous perennial herb ( Glycyrrhiza glabra ) and the roots and rhizomes of G. uralensis and other related plants. Flat, stomach, stomach, heart, lungs, intermediary wing, clear heat detoxification, lubrication, waste bin 효능), Harmonic Pharmaceuticals (調和 諸 藥) and the like. Its main component contains glycyrrhizin, a triterpen saponin, and flavonoid compounds such as liquiritin. Pharmacological actions include adrenal cortex hormone-like action, anti-ulcer effect, smooth muscle relaxation action, liver function protection, anti-inflammatory, anti-allergic action, and antiviral action (Kim Ho-cheol, Pharmacology of Pharmacology, Munmundang, 434-436, 2001).

Recently, due to limitations of the development of disease therapeutic agents by conventional synthetic compounds, and problems related to side effects and toxicity during treatment, development of disease therapeutic agents centering on herbal medicines has been actively conducted.

The present inventors have been studying the herbal medicine for the prevention and treatment of diabetic complications, aging, Daekguk, Kang Hobak, Chok Geun, licorice has the effect of inhibiting the production of the final glycation product, in particular they are 5 to 85% by weight The mixed herbal composition was found to be excellent in the inhibitory effect of the production of the final glycated glycosides was found to be useful not only for the prevention and treatment of diabetic complications, but also for preventing and delaying aging, thereby completing the present invention.

 The present invention aims to provide a composition for the prevention and treatment of diabetic complications comprising a mixed herbal composition of Daegeuk, Ganghwabak, Sigma root and licorice as an active ingredient.

In addition, the present invention is to provide an anti-aging and delaying composition using the mixed herbal composition as an active ingredient.

The present invention provides a composition for the prevention and treatment of diabetic complications, which comprises a mixed herbal composition of diaphragm, ganghwabak, sedum root and licorice.

The present invention also provides an anti-aging and delaying composition comprising the mixed herbal composition as an active ingredient.

Hereinafter, the present invention will be described in detail.

The mixed herbal composition of the present invention is characterized in that it comprises 5 ~ 85% by weight each of the counter electrode, ganghwabak, poncho root and licorice.

Mixed herbal composition of the present invention is 1) 5 ~ 85% by weight of each of the counter electrode, ganghwabak, brown root and licorice extract using 10-90% C ₁ to C ₄ alcohol, filtered and concentrated by vacuum Preparing an extract (first step); 2) is prepared, including the step (second step) of mixing each extract obtained in the first step.

The alcohol in step 1) may use 10 to 90% C ₁ to C ₄ alcohol, 80% ethanol is preferred, it is preferable to add 5 to 10 times (w / v) of the herbal medicine.

The steel foil of the first step is prepared by quantifying the thick foil as the manufacturing method is as follows. That is, after adding 3 parts by weight of health to 100 parts by weight of hickory in a container preheated at 50 to 100 ° C., and adding 5-10 times (w / v) of water to the medicinal herb to completely immersed in water, 70 Heat to maintain a temperature of ˜100 ° C. Take out the gourds when the water is almost evaporated. The ganghwabak thus calculated is more effective in inhibiting the production of the final glycated product than the bakbak.

The first gallbladder of the first step is prepared by the numerical value of the root, roasted 100g of the root of the root for 45 minutes at 120 ~ 130 ° C yellow and brown spots appear and cool off. The gallbladder so numbered is more effective in inhibiting the production of the final glycated product than the root.

The mixed herbal composition 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, has an excellent effect on lowering blood sugar in animal experiments, and gains weight and heights compared to the diabetic group. Reduction of hypertrophy and remarkably reduced BUN, triglyceride and creatinine levels to restore deteriorated kidney function, and the lens turbidity is significantly lower than that in diabetic group, which is useful for delaying or preventing cataracts.

Therefore, the mixed herbal composition of the present invention can be usefully used for the prevention and treatment of diabetic complications, namely, diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy and the like, which are caused by the production of the final glycated product. Can be.

In addition, the mixed herbal composition of the present invention can be useful for preventing and delaying aging due to oxidative stress, by reducing the production of free radicals and reducing the induction rate of oxidative stress when inhibiting the production of the final glycation product. .

The mixed herbal composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the mixed herbal medicine.

The mixed herbal medicine can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation.

The mixed herbal composition of the present invention can be administered in an effective amount 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 mixed herbal composition of the present invention as an active ingredient may be administered by oral, intravenous, intramuscular, or transdermal administration in a conventional manner, but is not limited thereto. 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 form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose or lactose. (lactose), gelatin, etc. are mixed and prepared. 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 herbal composition, and formulated in a unit dosage form of ampoules or vials.

The dosage of the mixed herbal composition of the present invention is appropriately selected according to the absorbency, inactivation rate, excretion rate, age, sex and condition of the patient, and severity of the disease to be treated in the body, but generally once a day It can be administered in three to three times. In the pharmaceutical composition of the present invention, when the mixed herbal herbal medicine is contained as an active ingredient, the effective dose is 500 to 2,000 mg / kg / day, and preferably 500 to 1,000 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 mixed herbal composition shows that the acute toxicity test result for the rats in the laboratory, the minimum lethal dose (LD ₅₀ ) at oral administration at least 6g / ㎏ does not show any toxic effect at all, so that the biological stability is very high. Therefore, the mixed herbal composition of the present invention can be safely administered to a living body.

The mixed herbal composition of the present invention may 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 mixed herbal composition of the present invention may be added to health foods for the purpose of improving diseases caused by diabetic complications and aging. When the mixed herbal medicine of the present invention is used as a food additive, the mixed herbal medicine 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 the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, as in general beverages. 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 and Experimental Examples.

Example 1  : Preparation of Mixed Herbal Composition

100 g of the counter electrode was pulverized into powder and 1 liter (ethanol: distilled water = 80: 20) of 80% ethanol solution was added at room temperature (20 to 30 ° C.), followed by extraction at room temperature for 24 hours. The ethanol extract was filtered with filter paper, and extracted five times in the same manner. All extracts were combined and concentrated under reduced pressure.

On the other hand, 100ml each of Kanghubak, Sigma root and licorice were extracted using 1 L of 80% ethanol aqueous solution, filtered and concentrated in vacuo to obtain respective extracts. Each extract was 20 g of anti-tark extract, 10 g of Ganghwa Park extract, 20 g of Sigma root extract, 20 g of licorice extract, of which 10 g were taken and mixed to obtain a mixed herbal composition of 40 g.

Example 2  : Preparation of Mixed Herbal Composition

After crushing the counter electrode, ganghwabak, black root and licorice into powder, take 100g each and mix them, and then add 1L of 80% ethanol aqueous solution (ethanol: distilled water = 80:20) at room temperature (20 ~ 30 ℃) and add them at room temperature. Extraction for 24 hours. The ethanol extract was filtered with filter paper, and extracted five times in the same manner. All extracts were combined and concentrated under reduced pressure.

Experimental Example 1  : Analysis of Inhibitory Effect on the Production of Final Glycosylated Products of the Herbal Composition of the Present Invention

In order to determine the efficacy of the final glycation product production inhibition of the mixed herbal composition of the present invention, the following experiment was performed.

1. Efficacy of Inhibiting Final Glycation Products of Mixed Herbal Compositions of the Present Invention

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.

The mixture of fructose and glucose was added to the prepared BSA solution and used in the experiment.

After mixing the mixed herbal composition prepared in Example 1 in 15% Tween 80, it was added to the mixture of the BSA and sugar and incubated at 37 ℃ 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 fluorescence, brown, not only has a physicochemical property that can cross-link, but also has a ligand 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.

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

2. Inhibitory Effect on Production of Final Glycosylated Products of Daegeuk, Kanghubak, Chok-Geun and Licorice Extracts

Except for culturing for 90 days using the anti-polar, ganghwabak, chogeeungeun and licorice extract was dissolved in 15% Tween 80 at a constant concentration, the experiment was carried out in the same manner as described above.

As a positive control group, the experiment was carried out in the same manner as in the above 1 except that the aminoguanidine HCl was dissolved in distilled water at a constant concentration for 30 days and 90 days.

The results are shown in Figure 1 and Table 1.

Final glycation product production inhibition rate (%) IC ₅₀ value (μg / ml) Concentration (µg / ml) % Inhibition Mixed Herbal Composition (30 Days of Culture) 5.0 23.245 ± 0.698 18.12 10 44.998 ± 1.396 25 63.548 ± 2.234 50 93.283 ± 5.187 Countermeasure extract (90 days culture) 25 34.680 ± 2.685 32.07 50 56.456 ± 2.422 100 64.260 ± 0.871 200 93.376 ± 0.921 250 96.853 ± 0.982 Kanghu Park Extract (90 Days of Culture) 25 14.922 ± 5.040 27.80 50 94.135 ± 3.192 Black Root Extract (90 Days of Culture) 25 25.155 ± 1.542 42.50 50 64.712 ± 3.069 100 100 200 100 250 100 Licorice Extract (90 Days of Culture) 25 49.37 ± 1.802 28.40 50 62.262 ± 14.68 100 100 200 100 250 100 AminoguanidineHCl 30 day culture 27.5 45.78 ± 2.400 34.90 55 55.43 ± 4.000 110 73.52 ± 1.750 550 96.41 ± 2.200 90 days culture 27.5 39.647 ± 3.406 30.80 55 65.712 ± 3.242 110 65.714 ± 5.394 55 89.873 ± 2.554

As shown in Table 1, the mixed herbal composition of the present invention exhibited a very low IC ₅₀ of 18.12 µg / ml when incubated for 30 days, indicating that the production inhibitory effect of the final glycosylated product was excellent.

On the other hand, the extracts of Daegeuk, Kanghubak, Chok-Geun-Geun and Licorice extract showed low IC ₅₀ when incubated for 90 days, so that the production of the final glycosylated product was excellent. However, when the individual extract was incubated for 30 days, the effect was not so good. Did. That is, it can be seen that each individual extract shows its efficacy only after prolonged administration.

In addition, the mixed herbal composition of the present invention exhibits a much lower concentration of IC ₅₀ than the amino guanidine-HCl which is a positive control group, indicating that the final glycation product production inhibitory effect is very excellent.

Therefore, the mixed herbal composition of the present invention can be seen that the inhibitory effect of the production of the final glycosylated product than the individual herbal extracts.

Experimental Example 2  : Treatment effect of diabetic complication of herbal composition of the present invention

In order to determine the therapeutic effect of the diabetic complications of the mixed herbal composition 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 period, 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 were no changes affecting the test.

The experimental animals were: 1) carboxymethyl cellulose administered to the normal group (NC + CMC), 2) carboxymethyl cellulose administered to the diabetes-induced group (DC + CMC), 3) diabetes Divided into four groups including mixed herbal composition induction group (DC + HMP), 4) diabetic induction group (positive control) (EP + S11), and 9 ~ in diabetic induction group. 10 rats were divided into 5 to 6 rats, and their 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.

Since the mixed herbal composition (HMP) and epalestat (S11) used in this experiment were non-aqueous, it was dissolved in 1% carboxymethyl cellulose, and the normal group was administered with carboxymethyl cellulose. The effect was excluded.

1 g / kg of the mixed herbal composition (HMP) prepared in Example 2 and 25 mg / kg of the positive control group of Epalestat (S11) were dissolved in 1% CMC, respectively, in two groups except the normal group. Daily rats were orally administered for 8 weeks.

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 + CMC 209.26 ± 28.94 462.09 ± 32.91 252.83 DC + CMC 154.33 ± 27.26 220.50 ± 64.28 66.17 DC + HMP 153.50 ± 45.06 251.71 ± 40.01 98.21 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 + HMP: diabetic group + mixed herbal composition,

   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, the group receiving the mixed herbal composition (HMP) in the diabetic induction group increased 98.21g, and the group in which the positive control group epalestat (S11) was administered in the diabetic induction group increased by 129.21g. As such, the group administered with the mixed herbal composition of the present invention is less weight gain than the epalestat (S11) administration group, it can be seen that increases than the diabetes-induced group.

In addition, the diabetic induction group consumed about twice as much feed as the normal group despite the low weight, and the positive control group, the Epalestat (S11) administration group, ate more than that. However, the mixed herbal composition-administered group of the present invention consumed slightly less than the diabetic-induced group, and the diabetic-induced group consumed about 7 times more water than the normal group.

In addition, the mixed herbal composition-administered group of the present invention consumed about 5 times less water than the diabetic-induced group, and the Epalestat (S11) -administered group, the positive control group, consumed more water than the diabetic-induced 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 + HMP Absolute weight (g) 0.88 ± 0.14 ^** 10.71 ± 1.21 0.48 ± 0.10 ^*** 1.58 ± 0.21 ^* 2.95 ± 0.48 0.93 ± 0.14 Relative weight (%) 0.35 ± 0.03 ^*** 4.31 ± 0.48 ^*** 0.19 ± 0.02 0.64 ± 0.08 ^*** 1.18 ± 0.10 ^*** 0.38 ± 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 + HMP: diabetic group + mixed herbal composition,

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

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

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. The mixed herbal composition-treated group of the present invention showed a tendency to reduce the hypertrophy of each organ compared to the diabetic-induced group, but there was no significance, and showed the same trend as the positive control group Epalestat (S11) efficacy.

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 ^*** 33.13 ± 5.13 ^*** 70.20 ± 13.14 ^*** 54.76 ± 11.85 1.75 ± 0.17 ^* 12.47 ± 0.42 DC + HMP 271.22 ± 72.28 ^{***, ##} 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 + HMP: diabetic group + mixed herbal composition,

   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 mixed herbal composition administration group of the present invention was 271.22 ± 72.28mg / dL, which is superior to the diabetes-induced group (p < 0.01). The positive control group, Epalestat (S11), was 197.94 ± 76.66 mg / dl.

In addition, the BUN level, which is an indicator of renal function, appeared to be about 2.4 times higher in the diabetic group than in the diabetic group, and the mixed herbal composition administration group of the present invention was significantly lower than the diabetic group (p < 0.05).

In addition, the triglyceride was increased significantly in the diabetic induction group (33.38 ± 6.62 mg / ㎗-> 70.20 ± 13.14 mg / ㎗; p <0.001), the mixed herbal composition of the present invention significantly compared to the diabetic induction group Decreased (50.35 ± 16.63 mg / dL; p <0.05).

In addition, creatinine levels were significantly increased from 1.54 ± 0.08 mg / dL to 1.69 ± 0.24 mg / dL in the diabetic induction group and dropped to 1.51 ± 0.26 (mg / dL) in the mixed herbal composition administration group of the present invention. Creatinine is also used as an indicator of renal function but is not as sensitive as BUN. Therefore, even if the glomerular filtration rate is reduced by more than 50%, the value remains within the normal range. Considering these characteristics, the value of 1.51 ± 0.26 mg / ㎗ means that the renal function is very improved. As shown in Table 3, it can be seen that the renal function is improved as the hypertrophy of the kidney is reduced compared to the diabetic group by the administration of the mixed herbal composition of the present invention.

Therefore, in the mixed herbal composition administration group of the present invention, the renal hypertrophy and BUN, triglyceride, creatinine levels are significantly reduced compared to the diabetic group, it can be seen that the renal function is very 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 visual observation of the eye 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 + HMP 136.54 ± 73.52 37.20 ± 20.25 ^{**, ##} DC + S11 134.04 ± 45.00 37.38 ± 13.56 ^{***, ###}

※ NC: Normal group,

   DC: diabetes causing group,

   DC + HMP: diabetic group + mixed herbal composition,

   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 <0.01, ###: 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 Epalestat (S11) -administered group, both eyes were covered with white eyeballs in four of the seven, 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. In addition, the mixed herbal composition-administered group of the present invention exhibited cataract symptoms in both eyes of three.

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 the diabetic-induced group was positively treated with Epalestat (S11) and the mixed herbal composition of the present invention, respectively, 37.38 ± 13.56% (p <0.001) and 37.20 ± 20.25% (p <0.01), respectively. Cataracts have been reduced.

In addition, as shown in Figure 3, the turbidity of the lens of the normal group appeared less than 20%, it was also very clear in appearance. A turbidity of about 20% can be seen as a steady 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, in the group receiving the positive control Epalestat (S11), the turbidity of the lens was almost 40%, and none of the rats showed more than 60% turbidity. Most of the lens turbidity in the group to which the mixed herbal composition of the present invention was administered was about 40%, and some of them showed normal levels, and two rats exceeded 60%.

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

Experimental Example 3  : Oral Acute Toxicity in Rats

In order to determine the acute toxicity of the mixed herbal composition of the present invention was carried out the following experiment.

Acute toxicity test was performed using 6 week-old SPF rats. The mixed herbal composition prepared in the above example was suspended in water in two animals per group and administered orally at a dose of 6 g / kg / 15 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 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 mixed herbal compositions of the present invention did not show toxic changes in rats up to 6 g / kg, and the minimum oral dose (LD ₅₀ ) was determined to be a safe substance of at least 6 g / kg or more.

Formulation Example 1  : Preparation of Tablet

Mixed herbal composition prepared according to Example 1 100.0 mg, corn starch 90.0 mg, lactose 175.0 mg, L-hydroxypropyl cellulose 15.0 mg, polyvinylpyrrolidone 905.0 mg and ethanol appropriately mixed raw material wet Granulation was carried out by granulation, and 1.8 mg of magnesium stearate was added and mixed, and the tablet was compressed to 400 mg.

Formulation Example 2  : Manufacture of Capsule

The mixed herbal composition prepared according to Example 1, 100.0 mg, corn starch 80.0 mg, lactose 175.0 mg, and magnesium stearate 1.8 mg were mixed uniformly and filled in one capsule 360 mg.

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

500 mg of herbal extract of the present invention

200ml water

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

The mixed herbal composition of the present invention effectively inhibits the production of the final glycated glycosides, has an excellent effect on lowering blood sugar, prevents weight loss, decreases renal hypertrophy, and significantly reduces levels of BUN, triglycerides, creatinine, etc. It inhibits exacerbation and significantly reduces the lens turbidity, which delays and prevents cataracts.

Therefore, the mixed herbal 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. In addition, the mixed herbal composition of the present invention can reduce the free radical production and reduce the induction rate of oxidative stress when inhibiting the production of the final glycation product, it can be usefully used for preventing and delaying aging caused by oxidative stress.

Claims (8)

Pharmaceutical composition for the prevention and treatment of diabetic complications comprising 5 to 85% by weight, respectively, opposition, gangbakbak, chogeeungeun and licorice. The pharmaceutical composition for preventing and treating diabetic complications of claim 1, wherein the diabetic complication is selected from diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. The pharmaceutical composition for preventing and treating diabetic complications of claim 2, wherein the diabetic complications are diabetic cataracts and diabetic nephropathy. Food composition for the prevention and treatment of diabetic complications comprising 5 to 85% by weight, respectively, opposition, gangbakbak, poncho root and licorice. The food composition for preventing and treating diabetic complications of claim 4, wherein the diabetic complications are selected from diabetic retinopathy, diabetic cataract, diabetic nephropathy and diabetic neuropathy. 6. The food composition for preventing and treating diabetic complications of claim 5, wherein the diabetic complications are diabetic cataracts and diabetic nephropathy. Anti-aging and delaying pharmaceutical composition comprising 5 ~ 85% by weight of opposite pole, gangbakbak, chogeeungeun and licorice. Anti-aging and delaying food composition comprising 5 ~ 85% by weight of opposite pole, gangbakbak, chogeeungeun and licorice.

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