KR100857691B1 - Composition for inhibiting development diabetes induced complication containing elsholtzia ciliata extract - Google Patents

Abstract

A composition comprising an extract of Elsholtzia ciliata is provided to show excellent anti-oxidative activity, advanced glycation end product inhibitory activity and aldose reductase inhibitory action, thereby being used for preventing or treating diabetic complications. A composition for inhibiting diabetic complications comprises an extract of Elsholtzia ciliata, wherein the extract is obtained by using an organic solvent such as C1-5 alcohol, ethylacetate, hexane, acetone, chloroform, and methylene chloride. A food or a medicine for inhibiting the diabetic complications comprises the composition.

Description

COMPOSITION FOR INHIBITING DEVELOPMENT DIABETES INDUCED COMPLICATION CONTAINING ELSHOLTZIA CILIATA EXTRACT}

Figure 13 shows the process of fractionating the perfume oil 70% EtOH extract in the final 200g powder form by freeze drying. The EtOH extract was suspended in water, and the same amount of n-Hexane was added and separated for a certain time. This process was repeated three times, and the resulting Hexane layer was filtered and decompressed to obtain a final Hexane fraction. As described above, the final fractions were obtained in the order of Hexane fraction, methylene cloride fraction, ethyl acetate fraction, buthanol fraction, and water fraction in order of nonpolar solvent. The obtained fractions were Hexane fraction (38.97g, 19.49%), methylene cloride fraction (31.28g, 15.65%), ethyl acetate fraction (9.97g, 4.99%), buthanol fraction (25.75g, 12.88%) and water fraction (28.22) g, 14.11%). The intermediate layer (20.65g, 10.33%) was set aside because an intermediate layer was formed between the methylene cloride and the water layer during the fractionation process.

[Industrial use]

The present invention relates to a composition comprising a perfume oil extract excellent in antioxidant activity, final glycation product inhibitory activity, aldose reductase inhibitory activity and the like.

[Private Technology]

Glucose, which is present in high concentrations in the blood of diabetic patients, is combined with free amino groups of protein components in the body by non-enzymatic glycosylation reactions to form advanced glycosylation end-products (AGEs). Final glycation end products are representative of diabetic complications.

The present invention relates to the inhibition of diabetic complications through the inhibition of non-enzymatic glycation reaction of perfumed oil, and more particularly, to identify the separation method of fractions and active ingredients from perfumed oil and the high non-enzymatic glycation inhibition inhibitory activity of the extract. Experimental validation relates to senile disease inhibitors related to diabetic complication inhibition and therapeutic and non-enzymatic glycation reaction inhibition.

Balsam (Elsholtzia ciliate) is an aromatic herbaceous herb that grows naturally on the coasts of Asia and the Mediterranean. .-Y. (1986) Oriental Materia Medica, p. 51, Oriental Healing Art Institute, Long Beach, CA.). In Korea, the flowering plants are Elsholtzia splendens, Elsholtzia saxatilis, and Elsholtzia minima, all of which are used for food, ornamental, wheat, medicinal, sweating, diuretic, species, and antipyretic. And hemostasis (Yeung. Him-Che. Handbook of Chinese Herbs and Formulas. Institute of Chinese Medicine, Los Angeles 1985, Isobe, T and Noda, Y. (1992) Nippon Kagaku Kasshi 4, 423, Hsu, H.-Y. (1986) Oriental Materia Medica, p. 51, Oriental Healing Art Institute, Long Beach, CA.). In particular, the fragrance oil has a good fragrance, so it is used as a fragrance of the bath.Chi, HJ et al. (1992) Kor. J. Pharmacog. 23, 77) citral), limonene (limonene). It is called Noyagi, Soyanggi, Seokyang oil, and fragrance. It is about 30 ~ 60cm high, long oval leaves are facing each other, and pink light branches of small branches are gathered on one side to make flowers. It grows in foothills, roads and fields all over the country, and cuts and stalks are dried in the shade when autumn flowers bloom.

Essential oils are essential oils in the outpost of fragrance oils, the main component of which is elsholtzia ketone, and also contains sterols, phenolic substances and flavonoid glycosides. As well as naginataketone, α-pinene, cineole, p-cymene, isovaleric acid, isobutyl-isovalerate, acetic acid, α-β-naginatene, octanol-3,1-octen-3-ol, linalool, camphor, geraniol, n-caproic It contains acid and isocaproic acid.

There has not been any disclosure or teaching on the prevention of diabetic complications or the inhibition of non-enzymatic glycation reactions through inhibition of non-enzymatic glycosylation of scented oils and inhibition of aldose reductase.

[Etiology of microvascular complications of diabetes]

The pathogenesis of diabetic vascular complications has not yet been clearly identified, but the longer the disease duration of diabetes and the poorer the glycemic control status, the more likely the occurrence of hyperglycemia is known to play an important role in the development of microvascular complications of diabetes. It has been estimated to do. The recent US Diabetes Control & Complication Trial (DCCT) study reports that thorough blood glucose control can significantly reduce the incidence of microvascular complications in patients with insulin-dependent diabetes (IDDM), suggesting that hyperglycemia is important for the development of microvascular complications. Proved to play a role.

On the other hand, microvascular complications do not occur at all in patients with long-term uncontrolled diabetes mellitus, and DCCT studies show that it is true. In addition, other metabolic disorders of diabetes besides hyperglycemia may be the cause of microvascular complications, and some genetic factors that have not been identified yet are considered to determine the susceptibility of complications. (Giugliano D, Paolisso G, Ceriello A: Oxidative stress and diabetic vascular comlications. Diabetes Care 19: 257-267, 1996)

[Mechanism of Microvascular Complications Caused by Glucose Hypothesis]

The mechanism by which hyperglycemia causes vascular complications has not yet been fully identified. (1) Chronic hyperglycemia may increase the glycation of various proteins in the body, thereby reducing the function of these proteins. (2) Possibility of raising intracellular sorbitol concentration by the polyol pathyway and decreasing myoinositol to promote intracellular osmotic pressure and denaturation, (3) possibility of changing intracellular redox status, and (4) cell It is thought that these mechanisms are not mutually exclusive, but are complex in nature, as several mechanisms may be involved at the same time or other mechanisms may occur as a result of one mechanism. .

It has been reported that abnormal glycation metabolism occurs during hyperglycemic state and the defense system functions against harmful oxygen free radicals that are produced at the same time, leading to oxidative stress (Yokozawa, T., et al. , 2001, J. of Trad. Med., 18: 107-112. As such, the mechanism of non-enzymatic glycosylation and oxidative stress is correlated.

The polyol pathway is a process that is reduced by aldose reductase (AR) action from aldose or ketose to form sorbitol and sorbitol is oxidized by sorbitol dehydrogenase to produce fructose. The hyperglycemia activates the polyol pathway, resulting in excessive production of sorbitol and fructose and accumulation in tissues resulting in increased osmotic pressure. Due to the increased osmotic pressure, water is introduced and progresses to diabetic retinopathy, diabetic cataract, and diabetic neuropathy (Diabetes, Eungjin Kim, et al., Korean Diabetes Society, Korea Medicine, p. 483; Soulis-Liparota, T., et al., 1995, Diabetologia, 38: 357-394).

Final glycation end products have been reported to activate aldose reductase (AR), the main enzyme of the polyol pathway, in human microvascular endothelial cells (Nakamura, N., et al., 2000, Free Radic Biol, Med. , 29: 17-25). In a steady state, aldose reductase has a very low affinity for glucose, but at high concentrations of glucose, aldose reductase is activated to excessively reduce glucose to sorbitol, and this sorbitol is converted to fructose by sorbitol dehydrogenase. This fructose is about 10 times faster in protein-enzymatic glycosylation than glucose. Thus, high concentrations of fructose bind to the protein and eventually accelerate the formation of the final glycated product. As such, non-enzymatic glycosylation, polyol pathways and oxidative stress mechanisms of action are linked to each other to cause diabetic complications. Therefore, in order to delay, prevent or treat the development of diabetic complications, it has been found that it is very important to suppress the formation of final glycation end products (Brownlee, M., et al., 1988, N. Engl. Med., 318, 1315). -1321).

(1) self-oxidation of glucose and production of free radicals

Glucose enolates itself, reducing oxygen molecules and producing oxides. If the self-oxidation (autooxiation) of glucose by high blood sugar increase this time to form superoxide anion being ^{(O 2 -) (. OH} ), hydroxyl radical, such as hydrogen peroxide (H ₂ O ₂₎ is formed which such cross-linking, fragmentation The free radicals that damage lipid peroxidation and protein also promote the formation of advanced glycation endproducts (AGEs), which in turn promote autooxidation. (Baynes JW, Thorpe SR: The role of oxidative stress in diabetic complications.Current Opinions Endocrinol 3, 277-284, 1996)

(2) non-enzymatic glycosylation pathway

Microvascular disorders caused by hyperglycemia have been reported since the onset of hyperglycemia persists or progresses even after a normal blood glucose condition has been reported.In the absence of hyperglycemia, high blood sugar causes irreversible and persistent changes in long-term substances. The possibility of causing continuous pathological function has been suggested. Therefore, the mechanism of cell damage caused by hyperglycemia is the glycation of irreversible proteins. The aldehyde or ketone groups of glucose react with the free amino groups of the protein to form Schiff base, a reversible glycosylating substance. The formed Schiff base is rearranged into a more stable, yet reversible form of ketoamine (Amadori products), and if left in this state for longer, dehydration and concentration lead to the formation of advanced glycation end-products (AGEs). . Glycation products exhibit specific absorbance and fluorescence, and in particular, amino groups of proteins have cross-link reactions.

Glycosylated products cause browning of proteins such as collagen and basal membrane thickening and altered collagen induce the deposition of lipoproteins or IgG. Brownlee has receptors that recognize and eliminate glycosylated products in macrophages and stimulates secretion of interleukin-1, tumor necrosis factor (TNF), and insulin-like growth factor-1 (IGF-1). Promotes proliferation of cells, mesangial cells and endothelial cells, and glycosylated matrix reduces the ability to fix proteoglycans, thereby reducing the negative charge of microvessels and contributing to the development of diabetic vascular complications.

(3) activation of the polyol pathway

Along with glycosylation of proteins, the most recent development is the mechanism by which the polyol pathway increases cell damage. In the hyperglycemic state, changes in intracellular glucose metabolism occur. When excess glucose enters the cell, it is converted into sorbitol by aldose reductase and by fructose by sorbitol dehydrogenase. In normal blood glucose state, the high Km value of aldose reductase to glucose (70mM) is very low in the sorbitol concentration, but when the intracellular glucose concentration is increased by hyperglycemia, the sorbitol concentration is increased and the sorbitol dehydrogenase reaction is slow. It will accumulate within. The high osmotic pressure of sorbitol accumulated in the cells causes the cells to expand and damage the cells. A typical complication of this principle is cataracts.

The degree of accumulation of sorbitol in cells by hyperglycemia varies greatly from tissue to tissue, and especially in neural and vascular tissues, the increase in sorbitol concentration is not sufficient to show a serious osmotic change. In addition to the mechanism, other damage mechanisms are being studied.

The hypothesis currently being discussed is that (1) myoinositol decreases as the polyol pathway increases, (2) the increase in the polyol pathway decreases the redox status in the cell, and (3) the increase in the polyol pathway is free. It is said that it increases cell damage by radical.

The mechanism by which polyol pathways increase oxidative damage can be largely divided into two mechanisms. The first is that increasing the polyol pathway decreases intracellular redox status, leading to increased production of free radicals and increased oxidative damage. The characteristic changes in blood vessels in hypoxia are the expansion of blood vessels and the increase in blood flow, which is known to be closely related to the increase of NADH / NAD + ratio by inhibiting oxidation of NADH to NAD + in low oxygen state. In the absence of hypoxia, hyperglycemia increases the NADH / NAD + ratio in the cytoplasm as in hypoxia (“hyperglycemic peudohypoxia”). Increased NADH / NAD + ratio in hyperglycemia is associated with increased polyol pathways and increased glycolysis. When hyperglycemic, glucose metabolism to the sorbitol pathway is increased. Sorbitol is converted back to fructose by sorbitol dehydrogenase. At this time, NAD + is reduced to NADH, and the intracellular NADH / NAD + ratio is increased.

As the NADH / NAD + ratio increases, O ₂ is a by-product when NADH is oxidized to NAD + ^. Can come out. Further, when NADH is increased by prostaglandin sikimyeo hydroperoxidase using NADH as a coenzyme promotes prostaglandin H ₂ formed from prostaglandin G _2, wherein O ^_2. Can cause cell damage.

Second, the activation of the polyol pathway weakens the defense against various oxidative damage and damages cell membranes. Under normal conditions, free radicals are rapidly removed by intracellular antioxidants such as glutathione, vitamin E and vitamin C. In the diabetic state, free radical production is increased by hyperglycemia, such as glucose autooxidation and non-enzymatic protein glycosylation, and antioxidants, which act as antioxidants, are reduced. It is supposed to be very vulnerable to oxidative damage by. In addition, if the hyperglycemic state persists due to diabetes, NADPH is consumed when the polyol pathway is activated and glucose is converted to sorbitol by aldose reductase. Aldose reductase has a Kf value of 0.02 mM, which is lower than glutathione reductasse's Km value of 0.23 mM, and aldose reductase has much stronger affinity for NADPH. Therefore, the increased polyol pathway consumes NADPH, which is required for GSSG to be reduced to GSH. Therefore, it is not possible to maintain a sufficient amount of GSH in cells. Therefore, the defense mechanism against various oxidative damage is weakened, thereby damaging the cell membrane. Free radicals damage proteins through lipid peroxidation and cause vascular endothelial or platelet disorders. Oxidized proteins, like glycosylated proteins, cause cross-linking. In damaged vascular endothelial cells, the synthesis of heparan sulfate decreases, leading to the outflow of proteins. The synthesis of prostacyclin also decreases, leading to impaired vasodilation and platelet coagulation. do. Phospholipase A2 is also activated in platelets, increasing the synthesis of thromboxane A2, resulting in increased blood vessel constriction and platelet coagulation.

Jain et al. Reported that GSH levels in insulin-dependent diabetic patients were significantly decreased compared to controls, and that GSH concentrations were negatively correlated with the level of hyperglycemia. Reduction of concentration would play an important role. Mattia et al. Reported that GSH levels, GSSG levels, and GSH / GSSG ratios were decreased in insulin-independent diabetic patients compared to controls. One week of treatment with the aldose reductase inhibitor tolrestat increased GSH levels and GSH / GSSG ratios, suggesting that polyol pathways may play an important role in impairing glutathione redox status in diabetic patients. (Nadler J and Natarajan R: Oxidative stress, inflammation, and diabetic complications.In LeRoith D, Taylor, Olefsky JM: Diabetes Mellitus.A fundamental and clinical text.2nd Eds. 1008-1016)

The present invention is to solve the above problems, the composition comprising the perfume oil extract of the present invention is to provide a composition for preventing or treating diabetes complications.

In order to achieve the above object, the present invention provides a composition for inhibiting diabetic complications comprising a perfume oil extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while searching for a component having an activity that inhibits the complications of diabetes in the herbal medicine that ensures the stability of the human body, the perfume oil extract inhibits the antioxidant effect and non-enzymatic glycation reaction, and exhibits the inhibitory effect of aldose reductase After confirming this, the present invention was completed.

The present invention relates to a composition for inhibiting or treating diabetic complications, and includes perfume extract as an active ingredient. Perfume oil extract may be prepared by extraction and / or fractionation with water or an organic solvent, preferably by extraction and / or fractionation with an organic solvent. The organic solvent may be at least one selected from the group consisting of C1-5 low-cost alcohol, hexane, chloroform, ethyl acetate, acetone and methylene chloride, the low-cost alcohol may be ethanol, methanol, butanol, propanol and isopropanol It is not limited to this. The alcohol dilution water may be one obtained by diluting the alcohol to 50 to 97% by volume of water.

The perfume oil extract of the present invention is preferably a perfume oil ethanol extract and water extract extracted with an aqueous ethanol solution, and most preferably the perfume oil hexane fraction extract extracted from the perfume oil ethanol extract and water extract with hexane, and extracted with methylene chloride After removing the methylene chloride fraction extract, the non-polar fraction and the ethyl acetate fraction extract obtained by extraction with ethyl acetate, and then the butanol fraction extract obtained by extraction with butanol.

For example, the perfume oil extract is carried out by adding 50 liters of water and 0.5 liter to 2 liters of 70% ethanol and then extracting. Extraction temperature may be 50 to 80 ℃, but is not limited thereto. The extraction time is not limited, but may be 30 minutes to 1 month, and a conventional extraction device or an ultrasonic grinding extractor may be used. The prepared extract is then subjected to reduced pressure filtration and lyophilization to remove the solvent.

In addition, as an example of the present invention, the fragrance oil acetyl chloride fraction extract contains 10 to 15 g of the concentrate obtained by concentrating the fragrance oil ethanol extract obtained by adding 0.5 to 2 L of water and 70% ethanol per 100 g of fragrance starch to 100 to 1000 ml of water. Suspension, 100 to 1000 ml of hexane was added and extracted to obtain a fragrance oil hexane fraction (hereinafter referred to as 'hexane fraction extract'). After removing the fragrance hexane fraction extract, 100 to 1000 ml of methylene chloride is added again to the remaining water layer, followed by extraction to obtain a fragrance oil methylene chloride fraction (hereinafter, 'methylene chloride fraction extract'). After removing the methylene chloride fraction extract, ethyl acetate is added to the remaining water layer 100 to 1000 ml again, followed by extraction to obtain a fragrance oil ethyl acetate fraction (hereinafter referred to as "ethyl acetate fraction extract"). When extracted with hexane, methylene chloride or ethyl acetate, the extraction temperature may be 20 to 40 ℃, but is not limited to this, the extraction time is not limited, but may be 30 minutes to 20 hours, conventional extraction equipment or ultrasonic mill extractor Can be used. The prepared extract is then filtered under reduced pressure and lyophilized to remove the solvent.

In addition, as an example of the present invention, the perfume oil butanol fraction extract is suspended in 100 to 1000 ml of 10-15 g of the concentrate obtained by concentrating the perfumed ethanol extract obtained by adding 0.5 to 2 L of water and 70% ethanol per 100 g of fragrance starch. Then, 100 to 1000 ml of hexane is added and extracted to obtain a hexane fraction extract. After removing the fragrance hexane fraction extract, 100 to 1000 ml of methylene chloride was added to the remaining water layer, followed by extraction to obtain a methylene chloride fraction extract. After removing the methylene chloride fraction extract, 100 to 1000 ml of ethyl acetate was added to the remaining water layer, followed by extraction to obtain an ethyl acetate fraction extract. After removing the ethyl acetate fraction extract, 100 to 1000 ml of butanol was added to the remaining water layer, followed by extraction to obtain a butanol fraction extract.

Perfume oil extract or fragrance oil extract according to the present invention inhibits the activity of aldose reductase and the formation of the end glycated product that is increased in the occurrence of diabetic complications, has an antioxidant activity by increasing the free radical scavenging ability, binding lipid peroxide and protein Has the effect of suppressing.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting diabetic complications comprising the perfume oil extract. The perfume oil extract is preferably perfume oil and 70% EtOH extract, and most preferably the perfume oil ethyl acetate and butanol fraction extract.

The pharmaceutical composition for inhibiting diabetic complications of the present invention may include the active ingredient alone, and may further include a pharmaceutically acceptable carrier or excipient according to the formulation, method of use, and purpose of use. When provided in a mixture, the active ingredient may be included in 0.001 to 99.9% by weight in the pharmaceutical composition for inhibiting diabetic complications, but is usually included in an amount of 0.01 to 50% by weight. The pharmaceutical composition for inhibiting diabetic complications can be used for the purpose of preventing or treating various diabetic nephropathy, diabetic cataracts, diabetic neurosis, diabetic heart disease and the like.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, which may be used one or more, but are not limited to these, and are common carriers And excipients can be used. In addition, when formulating a composition for inhibiting complications of diabetes, conventional fillers, extenders, binders, disintegrating agents, surfactants, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers or preservatives, etc. may be further included, oral or parenteral All may be used, but may be preferably used for oral administration.

The formulation of the composition for inhibiting diabetic complications may be in a preferred form depending on the method of use, and is particularly formulated by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Good to do. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.

The dosage of the composition for inhibiting diabetic complications according to the present invention may be determined in consideration of the administration method, the age, sex and weight of the recipient, and the severity of the disease. For example, the composition for inhibiting diabetic complications of the present invention may be administered at least once at 0.1 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is only one example to illustrate and is not limited to the above range.

In addition, the pharmaceutical composition for inhibiting diabetic complications of the present invention may further include a compound or plant extract having a known diabetic complication inhibitory activity in addition to the perfume oil extract, and may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the perfume oil extract. .

The composition for inhibiting diabetes complications of the present invention can be used as food, food additives, beverages or beverage additives. When used as a food, food additive, beverage or beverage additive, the composition for inhibiting complications of diabetes mellitus, various foods, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages, vitamin complexes , Liquor and other health supplements, but are not limited thereto. At this time, the composition for inhibiting diabetes complications may be included in 0.001 to 50% by weight in the final food or beverage, but is not limited thereto.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred embodiments of the present invention, and the present invention is not limited by the following examples.

Example 1: Preparation of Perfumed Water Extract

50 g of dried crushed perfumed starch was placed in 1 L of distilled water, and extracted with warming while stirring three times at 100 ° C. for 2 hours. The extract was filtered through a filter paper (Watman Paper No. 2) and freeze-dried to obtain a final 11 g of a powdered water extract.

Example 2: Preparation of Perfume 70% EtOH Extract

With a total of 4.5kg dried crushed balm starch, 50g each put in 1L of 70% EtOH and extracted with agitation while stirring three times at 70 ℃ 2 hours. The extract was filtered through a filter paper (Watman Paper No. 2), concentrated under reduced pressure, and lyophilized to obtain a final 200 g of a 70% EtOH extract in powder form.

Example 3: Preparation of Perfumed Hexane Fraction Extract

Water was added to 200 g of the perfumed oil 70% EtOH extract obtained in Example 2 and completely dissolved. Then, the same amount of normal hexane was added and shaken to separate the normal hexane layer. The n-Hexane layer obtained by repeating this process three times was filtered and concentrated under reduced pressure to obtain 38.97 g (19.49%) of a dry fraction extract (hereinafter referred to as "fragrant hexane fraction extract").

Example 4 Preparation of Perfumed Methylene Chloride Fraction Extract

The same amount of methylene chloride was added to the fragrance oil extract from which the fragrance hexane fraction of Example 3 was removed, followed by shaking to separate the methylene chloride layer. This process was repeated three times to obtain a fraction extract, which was filtered and concentrated under reduced pressure to obtain 31.28 g (15.65%) of a dry fraction extract (hereinafter referred to as 'fragrant methylene chloride fraction extract').

Example 5: Preparation of Perfume Interlayer

20.65 g (10.33%) was obtained as a non-mixing layer of the intermediate layer (hereinafter referred to as 'fragrant oil intermediate layer') between the perfume oil methylene chloride fraction of Example 4 and the water layer.

Example 6: Preparation of Perfumed Ethyl Acetate Fraction Extract

After removing the fragrance oil methylene chloride fraction of Example 4, the same amount of ethyl acetate was added to the remaining fragrance oil extract, and shaken to separate the ethyl acetate layer. This process was repeated three times to obtain a fraction extract, which was filtered and concentrated to obtain 9.97 g (4.99%) as a fraction extract in the form of a dry matter (hereinafter referred to as 'fragrant ethyl acetate fraction extract').

Examples 7 and 8: Preparation of perfume oil butanol fraction extract and water fraction extract

The same amount of butanol was added to the remaining perfume oil extract of the perfume oil ethyl acetate fraction of Example 6 and shaken to separate the butanol layer. This process was repeated three times to obtain a fraction extract, which was filtered and concentrated to obtain 25.75 g (12.88%) of a dry form (hereinafter, referred to as a 'fragrant butanol fraction extract'), wherein the water layer separated from the butanol layer was concentrated. 20.65 g (10.33%) was obtained in the form of a dry matter (hereinafter, referred to as a 'fragrant extract').

Example 9 Preparation of a Formulation Containing Perfume Extract

9-1: Preparation of Tablets

The raw materials were mixed homogeneously with the ingredients shown in [Table 1], and the tablets were tableted to 200 mg according to the tablet manufacturing method in the pharmacopeia formulation.

ingredient Content (mg) Example 1 or 2 60 Lactose 81 Microcrystalline cellulose 50 Hard silicic anhydride One Crospovidone 6 Magnesium Stearate 2

 9-2: Preparation of capsule

The raw materials of Table 2 were mixed homogeneously and filled so as to contain 270 mg in one capsule according to the capsule preparation method in the pharmacopeia formulation.

ingredient Content (mg) Example 1 or 2 60 Lactose 204 Hard silicic anhydride 4.8 Magnesium Stearate 1.2

9-3: Preparation of soft capsule

The raw materials of Tables 3 and 4 were homogeneously mixed and filled to contain 695 mg of 1 capsule according to the method for preparing soft capsules in the pharmacopeia formulation.

 1) Filling composition

ingredient Content (mg) Example 1 or 2 60 lecithin 20 Soybean oil 149.4 Coconut Cured Oil 38 Beeswax 12

2) dry skin composition

ingredient Content (mg) gelatin 247.2 Concentrated glycerin 78 Dissorbitol 70% 90.05 Paraoxybenzoic Acid Methyl 0.28 Paraoxybenzoic acid propyl 0.07 Echilbanilan Quantity Blue 1 Quantity Yellow 203 Quantity Titanium oxide Quantity Fractional palm oil Quantity

9-4: Preparation of Liquid

The raw materials shown in Table 5 below were prepared according to the liquid preparation method of the pharmacopeia formulations, and filled and sealed in a 100 ml brown bottle and pasteurized.

ingredient Content (mg) Example 1 or 2 100 Citric acid 4 honey 20 Liquid fructose 58 Nicotinic acid amide 20 Sodium benzoate 70 Povidone Quantity Yellow 203, blue 1 Quantity Menthol flavored flavor Quantity Purified water Quantity

Experimental Example 1 Inhibitory Effect of Rhodes Extract on Aldose Reductase

1-1: Preparation of Enzyme Source

The lens of the rat was extracted and hybridized by adding a certain amount of phosphate buffer solution according to its wet weight. After centrifugation at 4 DEG C, the supernatant was taken up and saturated with ammonium sulfate to 40%. The supernatant was centrifuged again, and stirred for about 1 hour by adding ammonium sulfate to 70%. It was suspended in a minimum amount of buffer and dialyzed for about one day to serve as an enzyme source.

1-2: aldose reductase activity measurement

Perfumed oil fraction extracts were treated in 100 μL of the prepared enzyme source and 10 μL of DL-glyceraldehyde, and the rate of decrease of NADPH absorbance was measured at 340 nm. In addition, compared to the Quercetin known to have an aldose reduction effect as a standard material was compared to the treatment as described above to measure the rate of decrease in absorbance.

Sample Concentration (μg / ml) Inhibition Rate (%) Comparative Example 1 1.1 77.72 Example 1 1.1 34.86 5.5 66.43 11 72.05 Example 2 1.1 43.85 5.5 76.49 11 88.93

As shown in Table 6, the comparative results of aldose reductase of Example 1 (perfume extract) and Example 2 (70% EtOH) showed that the inhibitory effect of Example 2 was slightly higher for each concentration of 1.1, 5.5, and 11 µg / ml. appear. And Comparative Example 1 was compared using quercetin that appears as the most active substance in natural products.

Figure 1 shows the results of the aldose reductase measurement of the fragrance fractions in a bar graph. The measurement results were in the order of Examples 5, 6, 7, 3, 4, 8. Quercetin was used as a standard and three fractions of Examples 5, 6 and 7 showed the same high inhibitory effect as Quercetin (Comparative Example 1).

Experimental Example 2: Inhibitory Effect on Formation of Final Glycosylated Product (AGE ′s) Using Methylglyoxal (hereinafter referred to as “MG”) as an Accelerator

MG and BSA were used to inhibit the formation of advanced glycation endproducts. Samples (11, 110 mg / mL) dissolved in dimethyl sulfoxide (DMSO) were added to BSA (5 mg / mL) and MG (2 mM), and the total volume was adjusted to 5 mL with 50 mM phosphate buffer (pH 7.4). At this time, 0.02% sodium azide was added as an antibacterial agent and reacted at 37 ° C. for 24 hours. The fluorescence of the sample after reacting with the sample before the reaction was measured using a spectrofluorometer (Excitation: 355 nm, Emission: 460 nm). The inhibition rate of fluorescence was calculated by comparing the fluorescence increase value of the fractions based on the fluorescence increase value of the control.

Calculation = {1- (S ₁ -S ₀ ) / (C ₁ -C ₀ )} * 100

(S ₀ : sample 0hr fluorescence value, S ₁ : sample 24hr fluorescence value, C ₀ : control 0hr fluorescence value, C ₁ : control 24hr fluorescence value)

Non-enzymatic Glycation Fluorescence Inhibition Concentration (µg / ml) Methylglyoxal-BSA (%) Comparative Example 2 20 94% Example 1 20 37% Example 2 20 43%

As a result of measurement of fluorescence inhibitory activity of the final glycation glycosides of the perfumed water extract and the 70% EtOH extract, the difference between the blank and the control was obvious, and aminoguanidine showed a high inhibitory effect of 94% in the experiment using MG. In the case of perfumed oils, the 70% EtOH extract had a 43% inhibition rate and a higher fluorescence inhibitory effect than the water extract.

Non-enzymatic Glycation Fluorescence Inhibition Sample Concentration (μg / ml) Inhibition Rate (%) Comparative Example 2 20 94 Example 3 20 18 200 34 Example 4 20 20 200 70 Example 5 20 36 200 80 Example 6 20 67 200 90 Example 7 20 17 200 70 Example 8 20 16 200 70

As shown in FIG. 3, FIG. 4 and [Table 8], the inhibitory effect of the final glycosylated product (AGE's) was examined for each fraction of the fragrance oil by a 24-hour short-term search using MG. As a result of measuring the increase in fluorescence based on the blank and control, the fragrance oil ethyl acetate fraction of Example 6 was found to be the highest among the fractions with 67% inhibition at a concentration of 20 ㎍ / mL. As a result of increasing the concentration of the sample 10 times, most of the other fractions except for the hexane fraction of Example 3 showed the most high effect, but Example 6 also showed the highest effect of 90% and almost the same effect as aminoguanidine of Comparative Example 2. Showed.

Experimental Example 3: Determination of 47-day binding inhibition effect of MG and BSA by fluorescence spectrometer

When AGE's are formed, the fluorescent compound is often contained, and thus the formation of AGE's can be predicted by confirming the detection of the fluorescent substance. When the light source is exposed to the solution to be searched (Excitation), the long-wavelength light is emitted from the irradiation light (Emission).

(Numbers from top to bottom)

1: Control, 2: Example 3-500 μg / ml, 3: Example 7-500 μg / ml, 4: Example 1-500 μg / ml, 5: Example 4- 500 μg / ml, 6: Comparative Examples 2-100 μg / ml, 7: Example 6-500 μg / ml, 8: Comparative Example 2-250 μg / ml, 9: Comparative Example 2-500 μg / ml, 10: Blank

In order to confirm the result, the fluorescence was measured after reacting for 47 days using MG. As shown in FIG. 5, the difference between the blank and the control was clearly observed, and the degree of fluorescence inhibition was increased according to the concentration of the active standard aminoguanidin of Comparative Example 2. Among the fragrance oil fractions, Examples 6, 4, 8, 7, and 3 were in order, and Example 6 had the highest effect, and the same inhibitory effect was obtained when 100 µg / ml aminoguanidin of Comparative Example 2 was added.

Experimental Example 4: Inhibition Effect of Lipid Peroxide and Protein Binding

4-1: Manufacture of Malondialdehyde (hereinafter referred to as 'MDA')

According to the method of Gomez-Sancheza (Gomez-Sancheza et al., 1990), 5 ml of 1,1,3,3-tetramethoxy-propane was added to 40 ml of an aqueous solution containing 5 g of Dowex 50WX8-200 resin (supelco). After shaking vigorously, the mixture was left at room temperature for 15-20 minutes to disperse 1,1,3,3-tetramethoxy-propane. The dispersion was filtered to remove the resin, neutralized with NaOH to pH 7.0, and the neutral 1,1,3,3-tetramethoxy-propane dispersion was lyophilized. Methanol was added thereto to make a saturated solution. Droplets were then added to 300 ml of ethyl ether to give a pale yellow MDA precipitate. The ether was removed using a vacuum evaporator to obtain MDA powder.

Malondialdhyde (MDA), a representative lipid peroxide in the body, has two aldehyde groups, which are very unstable and can cause cross-linking not only between amino acid residues in surrounding proteins but also between residues of foreign protein molecules. (Monnier, 1990) If lipid peroxides produced in the body bind to an enzyme, it can induce disease by lowering or changing the activity of the enzyme, and if it is involved in a protective function such as correcting DNA mutations, Damage is known to cause cancer (Hong et al., 1990).

4-2: Inhibitory Effect of Lipid Peroxides and Proteins

MDA and BSA were used to inhibit the formation of advanced glycation endproducts. According to the Park method, add samples (11, 110 mg / mL) dissolved in DMSO (dimethyl sulfoxide) in BSA (2 mg / mL) and MG (1 mM) and adjust 5 mL of total volume with 50 mM Sodium phosphate buffer (pH 7.4). It was. At this time, 0.02% sodium azide was added as an antibacterial agent and reacted at 37 ° C. for 24 hours. The fluorescence of the sample after reacting with the sample before the reaction was measured by a spectrofluorometer (Excitation: 355 nm, Emission: 460 nm). The inhibition rate of fluorescence was calculated by the same method as the inhibitory effect of binding of MG and BSA.

Inhibitory Effect of Lipid Peroxide and Protein Binding Sample Concentration (μg / ml) Inhibition Rate (%) Comparative Example 3 20 40 Example 1 20 25 Example 2 20 37 Example 3 20 39 200 80 Example 4 20 31 200 81 Example 5 20 39 200 93 Example 6 20 60 200 96 Example 7 20 14 200 73 Example 8 20 - 200 54

6, 7, 8 and Table 9 to examine the inhibitory effect of the final glycated glycosides (AGEs) of a 24 hours short-term screening method using MDA, the final form of lipid peroxide in order to promote the formation of the final glycosylated product. In Comparative Example 3, 20 μg / ml of aminoguanidin was used and the inhibitory effect was 40%. In the case of perfume oil, Example 2 (70% EtOH extract) was found to have 12% more fluorescence inhibitory effect than (Water extract) than Example 1.

 As a result of measuring the increase of fluorescence based on the blank and control, Example 6 (fragrant ethyl acetate fraction) was found to have the highest fluorescence inhibition in the fraction as 60% at a concentration of 20 ㎍ / mL. Next, Example 3 (Hex fraction) and Example 5 (interlayer material) were 39%, and the inhibitory effect of Comparative Example 3 was 40%, which was lower than that of Example 6. The experiment was carried out by increasing the concentration of the sample 10 times, and as a result, all the fractions exceeded the fluorescence inhibition rate of 50% and Examples 6, 5, 4, 3, 7, 8 (Fragrance EA fraction, Interlayer, MC, Hex, BuOH, Water fraction). In the same manner as the experimental results at the concentration of 20 ㎍ / ㎖) Example 6 showed that the most effective inhibition rate of 96%.

Experimental Example 5: Determination of free radical scavenging activity

The DPPH radical scavenging effect of the sample was measured by reducing power against DPPH. Dissolve the fragrance oil extract in DMSO, dilute it by concentration, add 1 mL of the sample in 1 minute increments into a test tube containing 2 mL of DPPH (1,1-Diphenyl-2-picyl hydrazyl) solution and 2 mL of ethanol, mix, and leave for 30 minutes, then 517 mm. After measuring the absorbance at, the radical scavenging rate was calculated from the reduced absorbance compared to the control. Vitamin C (ascorbic acid), which is known to have an antioxidant effect, was treated in the same manner as described above to measure DPPH scavenging ability and described the results.

DPPH Radial Scavenging Performance Measurement Sample Concentration (μg / ml) Inhibition Rate (%) Comparative Example 4 100 93.93 50 55.32 10 11.11 Example 3 100 3.68 50 2.24 10 - Example 4 100 3.40 50 - 10 - Example 5 100 7.84 50 3.68 10 - Example 6 100 27.91 50 11.48 10 3.87 Example 7 100 6.86 50 2.24 10 - Example 8 100 - 50 - 10 -

DPPH free radical scavenging ability was measured according to the concentration of the perfume oil fraction samples. As shown in FIG. 9 and Table 10, Comparative Example 4 (ascorbic acid) was used as a comparative material and showed a high inhibitory capacity of 93.9% at 100 µg / ml. Experimental results showed that the fractions of Examples 6, 5, 7, 3, and 4 (Fragrance Ethyl Acetate, Interlayer, BuOH, Hex, MC) were in the order of Example 6. Antioxidant efficacy was shown to be lower than the standard.

As shown in FIG. 10, the results of sulfuric acid coloration after TLC development of Examples 1 and 2 showed that Example 2 had more development materials than Example 1, and the DPPH radical scavenging effect was also compared to Example 1 The extract of 2 was shown to be larger.

Experimental Example 6: Antioxidation Measurement of Fragrant Oil Fraction Using Photochem

The scavenging capacity of each of the examples for free radical (superoxide radical) generated from photosensitizer was measured using PCL method. The reaction was performed using a photochem device and measured after reacting for 5 minutes by mixing buffer solution pH 10.5, 1.5ml and water 1ml, photosensitizer 25ul and sample 10ul. Photochem measured the scavenging ability of chemiluminescence using luminol. 검 To obtain a calibration curve using tocopherol, a comparative material, the antioxidant capacity of the sample was expressed as equivalent tocopherol equivalent (equivalent).

Sample Concentration (μg / ml) Quantity (nmoL) Example 3 0.386 0.875 Example 4 0.386 1.425 Example 5 0.386 2.426 Example 6 0.193 2.194 Example 7 0.386 2.414 Example 8 0.386 0.907

Antioxidant measurement results of each Example using Photochem are shown in FIGS. 11, 12 and Table 11. Tocopherol was used as a standard and was represented graphically according to the concentration. Measurement results Example 6, 5, 7, 4, 8, 3 (Fragrance ethyl acetate, perfume oil intermediate layer, butanol, methylene chloride, water layer, hexane) and Example 6 was the same as tocopherol 2.194nmol at 0.193㎍ / ㎖ concentration It has antioxidant activity and showed the highest efficacy among the embodiments.

The composition comprising the perfume oil extract of the present invention has the effect of inhibiting or preventing diabetes complications because it inhibits antioxidant efficacy and non-enzymatic glycation reaction, and has excellent aldose reductase inhibitory action.

Claims (6)

Composition for inhibiting diabetic complications comprising a perfume oil extract. The method of claim 1, The perfume oil extract is a composition for inhibiting diabetic complications that extract the perfume oil with an organic solvent. The method of claim 2, The organic solvent is one or more selected from the group consisting of C1-5 alcohol, ethyl acetate, hexane, acetone, chloroform and methylene chloride composition for inhibiting diabetes complications. The method of claim 1, The fragrance oil extract is a fraction obtained by extracting the fragrance oil ethanol extract obtained by extracting the fragrance oil with an ethanol aqueous solution with hexane, removing the hexane fraction extract, re-extracted with methylene chloride, fractionated with ethyl acetate, diabetes mellitus Composition for inhibiting complications. Food for inhibiting diabetic complications comprising the composition of any one of claims 1 to 4. A drug for inhibiting diabetes complications comprising the composition of any one of claims 1 to 4.

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