KR100468073B1 - Composition comprising from an extract of Salicornia herbacea or isorhamnetin-3-O-β-D-glucoside isolated therefrom - Google Patents

Abstract

PURPOSE: Provided is a composition containing the extract of Salicornia herbacea or iso rhamnetin-3-O-β-D isolated therefrom which inhibit rat lens aldose reductase and sorbitol accumulation. Therefore, it is used for prevention and treatment of diabetes. CONSTITUTION: The pharmaceutical composition for prevention and treatment of diabetes is characterized by containing, as an active ingredient, dichloroethylene or ethylacetate soluble extract of Salicornia herbacea, or iso rhamnetin-3-O-β-D isolated from the extract of Salicornia herbacea.

Description

Composition comprising from an extract of Salicornia herbacea or isorhamnetin-3-O-β-D-glucoside isolated therefrom}

The present invention relates to a composition comprising a seaweed extract or isolamnetine-3- O- β-D-glucoside isolated therefrom.

Diabetes is a chronic disease in which glucose is released into the urine due to a lack of insulin secretion (glucose), the concentration of glucose in the blood is higher than normal. In normal cases, most of the food you eat is converted to glucose, and blood carries this glucose to each cell. Glucose needs insulin (hormones) to move into cells. Diabetes is caused by the inability of glucose to move into cells because insulin is not made or working properly. Glucose that was not transported into the cells remains in the blood, so the blood becomes a high blood sugar 'high blood sugar' state and is discharged into the urine.

Insulin is a hormone produced by the pancreas. Inside the pancreas is a small clump of cells called Langerhans Island, and inside the Langerhans Island there are beta cells that secrete insulin. But glucose in the blood needs the help of insulin to be transported into the cells. In other words, glucose can enter the cell only by opening the insulin receptor (lock) that is present in the cell membrane with the lock (key).

Abnormal glucose levels in diabetes raise the level of hemoglobin in plasma, chronic hyperglycemia, atherosclerosis, microangiopathy, kidney disease, heart disease, diabetic It causes a series of complications such as diabetic retinopathy and other eye diseases.

Diabetes is characterized by two types of insulin production. Insulin dependent diabetes mellitus (IDDM) is caused by a deficiency of insulin, a glucose-regulating hormone in the blood, mainly 10 to It is called juvenile diabetes because it occurs in young people in their 20s. Type II diabetes (non-insulin dependent diabetes, NIDDM) occurs mainly after the age of 40, and occupies most of the diabetic patients in Korea. Unlike type I, it is called adult-type diabetes and the cause of the disease is not known yet, but it is known to be caused by genetic factors and environmental factors. The etiology of type II diabetes has been observed in both pancreatic beta cells with impaired insulin secretion and in target cells with insulin action (insulin resistance), but it is not yet clear which changes are of primary importance.

Aldose reductase is an enzyme in the polyol pathway that reduces glucose to polar sugar alcohols and sorbitol using NADPH as a cofactor. Aldose reductase is known to be an important cause of diabetic complications such as neuropathy, kidney disease and retinopathy in addition to cataracts in the lens (Van Heyningen R. Formation of polyol by the lens of the rat with sugar cataract, Nature, 184 , pp .194-196,1959), (Beyer-Mears A, et al., Diabetes, 33 , pp. 604-607, 1984). In diabetes, an increase in intracellular glucose that occurs in cells with insulin independent glucose transport results in a significant increase in sorbitol production. Increased sorbitol accumulation leads to cell loss (Engerman RL, et al., Diabetes, 33 , p.97-100, 1984), where structural and functional changes in tissues associated with diabetic complications are arrested by aldose reductase inhibitors Has been reported (Donkor IO, et al., European Journal of Medicinal Chemistry, 34 , pp. 235-43, 1999). This means that certain inhibitors of enzymes can be helpful in the treatment and prevention of diabetic complications.

The therapeutic potential of aldose reductase inhibitors has been demonstrated by the clinical results of several compounds including epilestat and tollestat.

Aldose reductase, an enzyme in the sorbitol pathway of glucose metabolism, promotes the reduction of glucose to sorbitol. Aldose reductase has a low substrate affinity for glucose, so the conversion of glucose to sorbitol is not common in normal tissues. However, increased effectiveness of glucose in tissues that do not respond to insulin in diabetes leads to an increase in sorbitol through the polyol pathway. In this route, the first step is glucose to sorbitol by aldose reductase and NADPH, and the next step is oxidation of sorbitol by sorbitol dehydrogenase and NAD ⁺ , resulting in fructose. Sorbitol accumulation is an important cause of diabetes complications including kidneys, nerves, retina, lens, and heart muscle. The development of cataracts in cataracts and a decrease in the rate of nerve conduction are associated with increased levels of sorbitol and fructose in the lens, leukocytes and peripheral nerves (Malone JI, et al., An indicator of diabetic control. Diabetes , 29 , pp.861). -864, 1980).

Streptozotocin (hereinafter referred to as STZ) causes diabetes by selectively destroying the cells of the pancreatic enzymes that secrete insulin, preventing the activity of pancreatic enzymes (Gilman AG, et al., Goodman and Gilman's the Pharmacological Basis of Therapeutics) , 8th ed.Pergamon Press, New York , pp. 1317-1322, pp. 1463-1495, 1990). Hyperglycemia injected with STZ is used as a useful experimental model for studying pathogenic activity of hyperglycemia (Ledoux SP, et al., Diabetes , 35 : pp.866-72, 1986).

Agents currently used for complications such as type II diabetes and insulin resistance include five groups of compounds: biguanides, thiazolidinediones, sulfonylureas, and benzoic acid. Derivative compounds and α-glucosidase inhibitors are used. Among these, biguanide compounds such as metformin are known to prevent excessive blood gluconeogenesis. Thiazolidinedione compounds are thought to act to increase glucose consumption in peripheral nerves, benzoic acids such as sulfonylureas, repaglinide compounds such as tolbutamide and glyburide A-glucosidase inhibitors, such as derivatives and acarbose, act to stimulate insulin secretion and lower plasma glucose.

The sulfonylurea compound cannot be administered to insulin-dependent type I diabetic patients, and non-insulin-dependent type II diabetic patients reduce insulin secretion, abnormal fetal birth, abortion and May cause side effects such as stillbirth. In addition, most sulfonylurea compounds should be administered carefully to patients with impaired liver and kidney function due to the metabolism of sulfonylureas.

The mechanism of biguanide-containing preparations such as metformin has not been elucidated, but although biguanide compounds are unable to increase pancreatic insulin secretion, they lower blood sugar more effectively than sulfonylureas, and also lower the incidence of hypoglycemia. low. However, it can cause nausea, vomiting, diarrhea and rashes at the beginning of administration, and can cause side effects such as fatal lactic acidosis, so it can only be used as a clinical reagent in the United States.

Since sulfonylurea and biguanide-containing preparations have the above drawbacks and side effects, there is a need for an excellent hypoglycemic agent with low side effects and high safety.

Salicornia herbacea ( Salicornia herbacea ) used in the present invention is a yearly plant belonging to the Cenopodiaceae, and it is said that it is salty, salty, or spiritual grass because it is salty in the new agricultural plant. It is also called a 'stalk' because it grows around the coast, Gaepal, and salt fields. It was designated as a natural monument in 1921. The seaweed grows on land but is enriched with various minerals and enzymes contained in the seawater. Especially, 100g of seaweed contains 671mg of calcium, 661mg of magnesium, 475mg of potassium, 441mg of iron, phosphorus 139mg, iodine 65.8mg, sodium 6.5 %, Salinity 15.7%, etc. are known, and 5,200 mg of vegetable fiber and enzyme group that degrade stool are contained, and about 90 kinds of minerals are evenly contained. Pharmacological action is effective in obesity, promotes the function of the stomach, and is effective in nephritis, hepatitis and hypertension, hypotension, anemia, lowering blood sugar, improving the function of the thyroid gland.

In this regard, the present inventors have no side effects, and in order to develop a therapeutic agent for diabetic disease using a safe natural agent, a composition comprising a soluble extract of organic solvent and isoramnetine-3- O- β-D-glucoside isolated from it The present invention was confirmed to inhibit rat lens aldose reductase and sorbitol accumulation in jotosine-induced hyperglycemic rats, thus confirming its efficacy in preventing and treating diabetes-related diseases. .

OBJECT OF THE INVENTION The present invention aims to provide soluble extracts of organic solvent containing seaweed organic solvents exhibiting inhibitory activity in crystalline aldose reductase and sorbitol accumulation assays in streptozotocin-induced hyperglycemic rats and isorametine-3- O- β-D-glucose isolated therefrom. It is to provide a composition for preventing and treating diabetes comprising a seed.

1 is modified in a streptozotocin-induced rat diabetes model after isoramnetine-3- O- β-D-glucoside or epalestat at 25 and 50 mg / kg / day, respectively, via an intragastric tube. Is a measure of the effect on the accumulation of sorbitol in the body ( ^** P <0.01, ^* P <0.05),

Figure 2 is a sciatic model in a streptozotocin-induced rat diabetic model after isoramnetine-3- O- β-D-glucoside or epalestat injection at 25 and 50 mg / kg / day, respectively, via an intragastric tube. A measure of the effect of sorbitol accumulation on nerves ( ^** P <0.01).

FIG. 3 shows red blood cells in a streptozotocin-induced rat diabetes model after isoramnetine-3- O- β-D-glucoside or epalestat is injected at 25 and 50 mg / kg / day through an intragastric tube, respectively. It is the degree ( ^* P <0.05) which measured the effect on the accumulation of sorbitol within.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetes mellitus containing the organic solvent soluble extract of the seaweed as an active ingredient.

The organic solvent soluble extract includes an extract soluble in an organic solvent of n-hexane, dichloroethylene, ethyl acetate, n-butanol, and preferably includes an extract soluble in ethyl acetate.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diabetes, comprising isoramnetine-3- O- β-D-glucoside represented by the following structural formula A isolated from the organic solvent soluble extract of perilla.

(A)

Hereinafter, the present invention will be described in detail.

Soluble extract of the organic solvent of the present invention of the present invention, and isoramnetine-3- O- β-D-glucoside of the formula (A) isolated therefrom can be obtained as follows.

The soluble organic solvent soluble extract of the present invention is collected, dried and pulverized with the crushed seaweed of the present invention, and then water, methanol having a volume of about 2 to 10 times, preferably about 3 to 5 times, the weight of the seaweed sample. , Organic solvents such as ethanol, butanol or the like, or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably at about 1 to 5 weeks, preferably 1 to 3 weeks at methanolosilone. The supernatant was recovered by vacuum filtration using an extraction method such as reflux cooling extraction, and then the above procedure was repeated 2 to 5 times, preferably 3 times, to collect the supernatant and concentrated under reduced pressure to obtain a crude seaweed extract. Can be obtained.

More specifically, the crude seaweed extract obtained by the above process, preferably the methanol extract, is obtained by suspending it with the same amount of water and then repeatedly repeated several times with an organic solvent such as n-hexane, dichloroethylene, chloroform or ethyl acetate. The supernatant can be concentrated under reduced pressure and dried to obtain an organic solvent soluble part, and an organic solvent soluble part can be obtained by extracting and fractionating an organic solvent such as butanol, propanol or methanol to the remaining water layer (lower layer part).

It is also possible to further carry out conventional fractionation processes (Harborne JB, A guide to modern techniques of plant analysis, 3rd Ed. , Pp . 6-7, 1998).

The ethyl acetate acetate fraction having the highest activity among the soluble fractions of the organic solvent was separated into several small fractions by repeating column chromatography using a mixed solvent of dichloroethylene and methanol as a developing solvent, and then the small fractions were recrystallized or otherwise. Purification through column chromatography may yield the compound of formula (A) of the present invention.

Flavonoid compounds can also be separated by further conventional flavonoid separation processes described in the literature (Manguro LOA, et al. , Phytochemistry., 63 , pp . 497-502, 2003), (Rychlinska I, et al. , Acta Poloniae Pharmaceutica , 59 , pp. 53-6, 2002).

The present invention provides a pharmaceutical composition containing isoramnetine-3- O- β-D-glucoside represented by Structural Formula (A) as an active ingredient, the soluble extract of the organic solvent obtained by the above-mentioned manufacturing process or separated therefrom. to provide.

The extract of the present invention through the experiment to confirm the effect on the effect of diabetes mellitus extract obtained by the above method on the effect of the lens Aldose Reductase (Rat Lens Aldose Reductase) activity and sorbitol accumulation (Sorbitol Accumulation) in rats And the inhibitory activity of the compounds on diabetes could be confirmed.

The pharmaceutical composition for preventing and treating diabetes of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition.

Pharmaceutical compositions comprising the extract or compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The pharmaceutical composition comprising the extract or compound according to the present invention may be prepared according to a conventional method, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions. It can be formulated and used in the form. Carriers, excipients and diluents that may be included in the composition comprising the extract or compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extracts or compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a health functional food comprising the above-mentioned seaweed extract or compound and a food acceptable food supplement additive exhibiting a preventive and improving effect of diabetes. Examples of the food to which the extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing and improving diabetes. At this time, the amount of the extract or compound in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract or compound as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extracts or compounds of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts or compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract or compound of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Reference example. Statistical processing

The results of all experiments were expressed as mean ± standard deviation, and statistical processing was performed by Student's t-test to determine the significance based on p <0.05 (* is P <0.05, ** is P <0.01, *** means P <0.001, which is significantly different from the control).

Example 1 Organic Solvent Soluble Extract or Isoramnetine-3- O Isolation of -β-D-glucoside

1-1. Preparation of Seaweed Water Extract

The grasswort (300g) was ground into a mill and powdered, and then, 1 L of water was added thereto, repeated three times, followed by heating, followed by extraction.

1-2. Preparation of Methanol Extract

After grinding the vinegar (2 kg) into a grinding mill and powdering, 5 L of methanol was added thereto, repeated extraction was performed by heating three times, followed by extraction under reduced pressure to obtain 215 g of methanol extract of ethanol, which was used as a sample in the following experimental example.

1-3. Preparation of Fractions by Solvents

After the methanol extract (215 g) obtained in step 1-2 of the above-described method was suspended in water, the solvents were then polarized, followed by n-hexane, dichloroethylene, ethyl acetate, n-butanol, and water. Solvent fractionation sequentially. First, n-hexane was added to the methanol extract, which was suspended in water, and dissolved. Then, only components dissolved in the n-hexane layer were separated and dried under vacuum. This process was repeated three times to obtain a hexane fraction (128 g). Dichloroethylene was added to the remaining aqueous layer, and only the components dissolved in the dichloroethylene layer were separated and dried under vacuum. This process was repeated three times to obtain a dichloroethylene fraction (20 g), and ethyl acetate was added to the remaining aqueous layer, and only the components dissolved in the ethyl acetate layer were separated and dried under vacuum. This process was repeated three times to obtain an ethyl acetate fraction (10 g). N-butane was added to the remaining aqueous layer, and the n-butane fraction was obtained in the same manner as above (5 g). Each fraction was used as a sample in the following experimental example.

1-4. Isoramnetine-3- from seaweed O Isolation of -β-D-glucoside

The ethyl acetate fractions obtained in Example 1-3 were subjected to silica gel chromatography to separate isoramnetine-3- O- β-D-glucoside. That is, the column was filled with silica gel, equilibrated with dichloroethylene, and 20 g of an ethyl acetate fraction was loaded. The mobile phase was first distilled off with dichloroethylene and increased the proportion of methanol by 1%. After the ratio of methanol was increased by 10% after 10% it was separated into 100 fractions. Among the fractions obtained, the activity tended to be the highest in the 20-30 subfractions, and the active ingredients were identified from these subfractions, and analyzed using ¹ H-NMR and ¹³ C-NMR spectra. It was confirmed by raminetin-3- O- β-D-glucoside.

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 12.61 (1H, s, 50OH), 7.94 (1H, d, J = 2.0 Hz, H-2 ′), 7.49 (1H, dd, J = 8.4 , 2.0Hz, H-6`), 6.91 (1H, d, J = 8.4Hz, H-5`), 6.44 (1H, d, J = 2.0Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-6), 5.57 (1H, d J = 7.4 Hz, Glucosyl H-1``), 3.83 (3H, s, -OCH3)

¹³ C-NMR (100 MHz, DMSO-d ₆ ): δ 177.4 (C-4), 164.3 (C-7), 161.2 (C-5), 156.4 (C-2), 156.3 (C-9), 149.4 (C04`), 146.9 (C-3`), 133.0 (C-3), 122.0 (C-6`), 121.1 (C-1`), 115.2 (C-5`), 113.4 (C-2 `), 104.0 (C-10), 100.8 (C-1``), 98.7 (C-6), 93.7 (C-8), 77.5 (C-3``), 76.4 (C-5``) , 74.3 (C-2``), 69.8 (C-4``), 60.6 (C-6``), 55.7 (-OCH ₃ )

IR ν _max (KBr): 3383 (OH), 1652 (C = O), 1055 (CO) cm ⁻¹

FAB-MS: m / z 479 [M + H] ⁺

Experimental Example 1. Experiment for measuring aldose reductase activity in vitro

In order to confirm the inhibitory activity against the crude extracts, solvent-specific fractions and compounds obtained in Example 1 against diabetes, the experiment was carried out by modifying the experimental procedure described in the literature (Sato S, et al., Biochemical). Pharmacology. , 40 , pp. 1033-1042, 1992).

1-1. Preparation and Activity Measurement of Enzymes

Aldose reductase is a lens removed from frozen rats weighing 250-280 g. Lens homogenates were prepared according to the methods described in the literature (Hayman S, et al., Journal of Biological Chemistry , 240 , pp. 877-882). ), The enzyme was used partially purified enzyme having a specific activity of 6.5U / mg, each divided into 1.0ml and stored at -40 ℃. In the aldose reductase (AR) inhibition experiment of the present invention on aldose reductase, DL-glyceraldehyde (glyceraldehyde) was used as a substrate, the inhibitory activity for the enzyme is reduced absorption of NADPH at 340 nm for 4 minutes Measured by spectroscopic method to measure the process.

The inhibitory effect of aldose reductase on the extracts and individual fractions from the seaweeds was determined by the RLAR enzyme on DL-glyceraldehyde as a substrate, and the inhibition rate of the methanol and water extracts of Example 1 on the enzyme of the RLAR enzyme The values of (%) and the individual IC ₅₀ are shown in Table 1, respectively, and the inhibition rates (%) of the individual fractions separated from the methanol extract and their IC ₅₀ values are shown in Table 2.

sample density Inhibition rate IC ₅₀ (μg / ml) (%) (μg / mL) ^* Tetramethylene 10 94.7 0.51 Glutaric acid One 81.7 0.1 10.8 Methanol extract 10 58.7 4.99 5 45.3 One 38.9 Water extract 100 52.0 13.90 10 23.1 One 10.8 ^* Inhibition ratio was calculated as a percentage according to the ratio compared to the control.

sample density Inhibition rate IC ₅₀ (μg / ml) (%) (μg / mL) ^* Tetramethylene 10 92.7 0.52 Glutaric acid One 68.7 0.1 19.8 n-hexane fraction 10 48.3 - Dichloroethylene 10 94.4 2.65 Fraction One 12.1 0.5 1.0 Ethyl acetate 10 93.6 0.26 Fraction One 72.0 0.1 35.3 n-butanol fraction 10 61.9 6.46 5 38.9 One 22.3 ^*: Inhibition rate was calculated as a percentage according to the ratio compared to the control

As shown in Table 1, the methanol extract and the water extract of the seaweed showed different activities for the enzymes depending on the concentration. IC ₅₀ values of methanol extract and water extract were measured at about 4.99 μg / ml and 13.90 μg / ml, respectively, whereas the IC of tetramethylene glutaric acid (TMG), which is well known as an inhibitor for aldose reductase, is known. The value of ₅₀ was 0.51 µg / ml, and the inhibitory activity of the aldose reductase of each fraction obtained from the methanol extract isolated from seaweed was measured. As shown in Table 2, the IC ₅₀ fraction of the ethyl acetate fraction was shown. 0.26 µg / ml was shown, indicating the largest inhibitory activity against aldose reductase.

IC ₅₀ value (1.44 μM) of isoramnetine-3- O- β-D-glucoside was shown to be an important inhibitor of aldose reductase because it showed similar inhibitory activity to IC ₅₀ value of TMG (1.68 μM) .

In order to compare the effects of structural changes, the inhibitory activity of isoramnetine-3- O- β-D-lutinoside and isoramnetine was compared. Flavonol of the 3- O -monosaccharide body showed stronger inhibitory activity than the flavonol compound having a hydroxyl group at position 3. In addition, isoramnetine-3- O -glucoside was more active than isoramnetine-3- O -lutinoside. Table 3 shows the results of experiments on the inhibitory effect of each compound on aldose reductase. Therefore, it was confirmed that the most inhibitory activity against flavonol AR enzyme of 3- O -monosaccharide.

sample density Suppression rate IC ₅₀ (μM) (%) (μM) ^* Tetramethylene 10 79.1 1.68 Glutaric acid One 46.3 0.5 25.4 Isoramnetine 10 88.98 1.44 3- O- β-D- One 46.06 Glycoside 0.5 25.64 Isoramnetine 10 73.7 2.01 3- O- β-D- 5 58.2 Lutinoside One 42.1 Isoramnetine 10 68.2 3.48 5 53.1 One 31.3 ^* Inhibition ratio was calculated as a percentage according to the ratio compared to the control.

Experimental Example 2 Aldose Reductase Determination in Diabetic-Induced Rats in Animal Experiments

The experimental animals used in this experiment were SD male rats weighing 250 to 280 g and were bred on and off at 12 hour intervals in the animal breeding ground where the humidity was maintained at 10% and 23 ± 0.5 ° C for one week. And water were supplied for free intake. Diabetes was induced by a single intravenous injection of streptozotocin (streptozotocin, STZ, Sigma Co., USA) dissolved in phosphate buffered salts acidified with 0.05 M citric acid (pH 4.5) (85 mg / Kg).

Isoramnetine-3- at 4, 7, 24 hours after administration of STZO-β-D-glucoside or Epalestat (ONO Co. Ltd.) were injected into rats via intragastric tubes at dosages of 25 and 50 mg / kg, respectively. Three hours after the last dose, body fluid samples were collected and the lens and sciatic nerve removed. Plasma glucose levels were determined based on the glucose oxidase method described in commonly used literature (Trinder P.,Annals of Clinical Biochemistry, 6, pp.24-27, 1969), the amount of glucosylated hemoglobin in hemoglobin was measured using the Dragin reagent kit 525 as described in the literature (Drabkin DS,et al.,Journal of Biological Chemistry, 98, p.719, 1932). Sorbitol content in the lens, sciatic nerve and erythrocytes of rats was measured enzymatically.

To examine the effect of glucose accumulation in the lens, sciatic nerve and erythrocytes of rats induced with hyperglycemia by injecting STZ to confirm the efficacy of isorametine-3- O- β-D-glucoside from seaweed in animal experiments Experiment.

Blood glucose levels in the diabetic rats were markedly elevated compared to normal rats, and the administration of isoramnetine-3- O- β-D-glucoside was markedly reduced in STZ-injected diabetic rats. In Table 4, isoramnetine-3- O- β-D-glucoside showed an excellent inhibitory effect in blood glucose accumulation experiments of mice induced with diabetes mellitus by STZ administration.

process Blood glucose (g / ℓ) Non-diabetic rat 189.67 ± 19.42 Diabetic rats 474.20 ± 15.15 Epalestat 413.75 ± 20.60 ^* Isolamnetin-3- O- β-D-glucoside 390.60 ± 8.34 ^{** **} P <0.01, ^* P <0.05

Experimental Example 3. Measurement of sorbitol content in diabetic rats in animal experiments

In order to confirm the inhibitory activity of the crude extract, the solvent-specific fractions and the compound obtained in Example 1 against diabetes, the enzymatic assay as described in the literature was modified to measure the accumulation of sorbitol (Clements RS, et al. , Science , 166 , pp. 1007-1008, 1969).

To 0.5 ml of tissue removal protein filtrate was added a reaction mixture containing 0.05 ml of glycine 1.0 ml (pH 9.4), 0.2 mM NAD and sorbitol dehydrogenase 0.64 U, without adding NAD or sorbitol dehydrogenase. The experiment was performed simultaneously with the filtrate. Relative fluorescence due to NADH was measured in excited state (366 nm) and emission wavelength (452 nm), respectively.

Mean accumulation of sorbitol in the lens, sciatic nerve and erythrocytes is shown in FIGS. Sorbitol accumulation (2.01 ± 0.14 nmol / mg dry weight) in the lens of diabetic rats was higher than that of normal rats (0.94 ± 0.06 nmol / mg dry weight). When isoramnetine-3- O- β-D-glucoside or epalestat was administered, the accumulation of sorbitol in the lens decreased by 34.83% and 32.84%, respectively. Sorbitol accumulation (309.59 ± 21.36 nmol / g Hb) in erythrocytes in rats with diabetes was much higher than that of sorbitol in erythrocytes (45.03 ± 4.78 nmol / g Hb) in normal rats. Sorbitol accumulation in red blood cells was 44.19% and 71.6%, respectively, in rats treated with isoramnetin-3- O- β-D-glucoside or epalestat. Sorbitol accumulation in the sciatic nerve of a rat with diabetes has been able to check that the value generally increases as the value (1.09 nmol / mg dry weight) increase in (0.61 nmol / mg dry weight), and isopropyl person netin -3- O - β-D-glucoside or epalestat showed sorbitol accumulation of 21.80% and 24.52%, respectively.

Hereinafter, an example of the preparation of a pharmaceutical composition including the extract of the present invention will be described, but the present invention is not intended to limit the present invention, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

Seaweed Extract Powder 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Seaweed Extract Powder 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Seaweed Extract Powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Seaweed Extract Powder 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Isolamnetin-3- O- β-D-glucoside 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Drink

Isolamnetine-3- O- β-D-glucoside 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the organic solvent soluble extract of the present invention or isorametine-3- O- β-D-glucoside isolated from the inhibitory effect of aldose reductase and sorbitol accumulation in streptozotocin-induced hyperglycemic rats Since it shows an effect, it can be usefully used as a medicine and health functional food for the prevention and treatment of diabetes.

Claims (7)

A pharmaceutical composition for the prevention and treatment of diabetes mellitus containing dichloroethylene or ethyl acetate soluble extract of Salicornia herbacea as an active ingredient. delete delete A pharmaceutical composition for the prevention and treatment of diabetes mellitus, containing isoramnetine-3- O- β-D-glucoside isolated from seaweed. delete delete delete