KR100356148B1 - Composition comprising aralia extracts for cataract - Google Patents

Abstract

The present invention relates to an extract and a therapeutic agent comprising the extract as an active ingredient, the composition and the therapeutic agent of the present invention has the effect of preventing, delaying the progression and treatment of cataracts. According to the present invention, the two water extracts are extracted with four solvents-chloroform, ethyl acetate, butanol and water. Adding myo-inositol or taurine to this extract can provide a synergistic effect of cataract treatment. In addition, beverages, herbal preparations, and dietary supplements containing the extract as an active ingredient can obtain the effects of preventing, delaying, treating and restoring cataracts caused by sugar by oral administration.

Description

COMPOSITION COMPRISING ARALIA EXTRACTS FOR CATARACT}

The present invention relates to a composition for treating cataracts, in which arbor is extracted with a solvent, and more particularly, to a composition for treating cataracts by extracting arbor with ethyl acetate, cloform, butanol, or water to inhibit aldose reduction pathways and oxidative stress. It is about.

Diabetes in our country was ranked 6th with 2.8% of all causes of death, according to a special investigation report by the National Statistical Office in 1991 (National Statistical Office, 1992), and the cause of death due to complications from hyperglycemia was 15 After 20 years, various organs in the body are damaged, resulting in chronic diabetic complications such as diabetic cataracts, kidney disease, peripheral neuropathy, and hyperlipidemia (Vlassara H. (1990), Chronic diabetic complications and tissue glycosylation.Diabetes Care 13 (11) ): 1180-1185). Diabetics are 25 times more likely to have blindness, 20 times more likely to have kidney failure, and 2 to 6 times more likely to have coronary heart disease than normal patients.

On the other hand, blindness due to cataract accounts for about 35% of the total blindness (Chylack LT (1984), Mechanism of senile cataract formation. Ophthalmol. 91; 596-602.), Which is an important cause of blindness. Cataracts are found in 50% of people in their 60s, 60% in their 70s, and over 70% in their 80s. Cataracts develop rapidly in people in their 40s and 50s when they are diabetic, and their eyesight deteriorates rapidly. Especially in insulin-dependent diabetes, cataracts are seen in people in their 20s and 30s. Cataract is a disease that causes visual impairment due to the clouding of the lens and blocks the light from reaching the retina. It can be treated as an operation to insert an intraocular lens, but if there is diabetes, the postoperative inflammation occurs, the wound healing period is slow, and the bleeding tends to occur. There is this. In particular, cataracts caused by insulin-dependent diabetes often develop late cataracts, which improve eyesight after surgery.

Aldose reductase inhibitors, which have been developed for the treatment of diabetic complications, are not currently used as side effects, but in order to overcome the side effects and to develop more effective drugs, the structure and aldose reductase inhibitors have recently been developed. Investigations have been actively conducted to investigate the relationship between activities and to characterize the structures that are more active and can act continuously, and to synthesize inhibitors of aldose reductase of such structures (Potier N., Barth P., Tritsch D., Biellmann JF. And Van Dorsselaer A. (1997), Study of non-covalent enzyme inhibitor complexes of aldose reductase by electrospray mass spectrometry, Eur. J. Biochem. 243: 274-282.).

In Korea, studies have been attempted to effectively inhibit aldose reductase by using flavonoids extracted from the roots of Scutellaldose reductaseia baicalensis. Currently, the ones used as cataract drugs are vitamin C and vitamin C. E, including catalin, baineiting, catachrome-OFTAN, Vita-iodurol, quinax, including glutathione, and the like are known. . (Chasovnikova LV, Formazyuk VE, Sergienko VI, Boldrev AA, and Severin SE (1990), The antioxidative properties of carnosine and other drugs Biochem International 20 (6):.. 1097-1103).

Currently, catalin developed in Japan is widely used as a treatment for cataracts in Korea, but treatment with these drugs is impossible to make the denatured lens protein in its original transparent state, and its value is not recognized. . Therefore, the treatment of cataracts is currently in a situation that depends substantially on surgery.

Korean Patent Publication No. 10-195886 is 17 herbal ingredients including Cordyceps sinensis, cow sulfur, yellow white, etc., which lowers blood sugar level and lowers blood lipid level of diabetic patients to prevent diabetes, prevent complications, and treat . This invention is a combination of known herbal ingredients and does not suggest compounds or extracts for the treatment of new diabetes.

Accordingly, it is an object of the present invention to provide a composition comprising arbor extract which is effective in preventing, delaying and treating cataracts caused by sugar.

Another object of the present invention is to provide a mixed composition comprising other active ingredients in the extract of the present invention.

Still another object of the present invention is to provide a dietary supplement comprising the extract of the present invention.

Still another object of the present invention is to provide a herbal preparation comprising the extract of the present invention.

Another object of the present invention is to provide a cataract prevention and treatment agent formulated in a unit dosage form comprising the extract of the present invention.

1 is a schematic diagram showing a polyol path;

Figure 2 is a schematic diagram showing the step of separating the fractions from the ar according to the present invention,

Figure 3 is a photograph classifying the turbidity of the lens induced by 20 mM xylose,

Figure 4 is a graph showing the turbidity density of the lens incubated for 72 hours in 20 mM xylose medium,

5 is a graph showing the antioxidant activity by CL analysis using ABEI-microperoxidase,

Figure 6 is a graph showing the density of turbidity when treated with each extract for cataracts induced with 20 mM xylose,

7 is a graph showing the antioxidant activity of each extract by CL analysis using ABEI-microperoxidase,

8 is a graph showing the density of turbidity of cataract lens treated with each fraction,

9 is a graph showing the delay effect of cataract development of AC fractions,

10 is a graph showing the prophylactic effect of cataract development of AC fractions,

11 is a graph showing the effect of cataract recovery of AC fractions,

12 is a graph showing the effect of the chloroform fraction of the Aralia extract on xylitol after 48 hours of incubation of the lens in xylose treatment medium,

FIG. 13 is a graph showing the effect of chloroform fraction of Ardum extract on aldose reductase activity after 48 hours of lens incubation in xylose treatment medium;

14 is a graph showing the effect of AC fractions on TBARS formation,

15 is a graph showing the effect of AC fractions on conjugate diene formation,

16 is a graph showing the effect of the AC fraction on the glutathione content,

17 is a graph showing the effect on cataracts with the addition of AC fraction and myo-inositol,

18 is a graph showing the effect on cataracts with the addition of AC fraction and taurine,

19 and 20 are graphs showing the effect of the AC fraction and the recovery of cataracts added with myo-inositol and taurine;

21 is a graph measuring the prophylactic effect of cataracts by supplementing 2.5 mM each of myo-inositol and taurine to AC fractions and pre-culture.

Such an object of the present invention can be achieved by extracts extracted with a solvent.

The eyes are largely divided into eyeballs and accessary organs. The eye is composed of three outer membranes: the sclera, the media, and the retina, and the intraocular lens, the vitreous body, and the aqueous humor. The lens of the eye is the only transparent organ in the body and is composed of approximately 35% protein and 63% water. Unlike other tissues, blood vessels and protein metabolism rate is very slow. The nutrient supply of the lens comes from the waterproofing and the waterproofing is connected to the blood vessels, which indirectly affects the blood.

Sugar cataract is known to be involved in the polyol pathway, a mechanism that causes diabetic cataracts, and the increase in oxidative stress, and has been widely used as an experimental model for diabetic cataracts (Kubo E., Miyoshi N., Fukuda M. and Akagi Y. (1999), Cataract formation through the polyol pathway is associated with free radical production.Exp . Eye Res. 68; 457-464). In addition, sugar cataracts are cataracts produced by glucose (glucose, galactose, and xylose), and galactose cataracts consume 30-50% of the total diet as galactose in in vivo experiments. Since turbidity is seen. Xyloid cataracts are induced by ingesting 20-35% of the total diet with xylose. Sometimes, cataracts recover spontaneously from galactose cataracts in vivo.

The best known mechanisms for the development of glucose cataracts are increased polyol pathways and increased oxidative stress (Beyer TA and Hutson NJ (1986), Introduction: Evidence for the role of the polyol pathway in the pathophysiology of diabetic complications, Metabolism 35 (4): 1-3).

The polyol pathway in the lens has been well known as a key to understanding the change in osmotic pressure causing cataracts since the discovery of excess sorbitol in diabetic mice (Chylack LT and Kinoshita JH (1969), A biochemical evaluation of a cataract induced in a high) -glucose medium.Invest.Ophthalmol . 8: 401-406). In the case of diabetes, the glucose level in the waterproofing rapidly increases to 90% of the blood glucose following the increase in blood sugar. Waterproofing glucose is absorbed more quickly than it can enter by diffusion by facilitated transportation into the lens. Insulin is not known at this time. Glucose that enters the lens is metabolized by hexokinase (HK) or aldose reductase. As shown in Figure 1, the polyol pathway consists of two enzymes and the dual rate limiting enzyme is aldose reductase. This enzyme converts sugar aldehydes (glucose, galactose) to the corresponding alcohols (sorbitol, galacitol), which are identical to low Kmaldehyde reductase and glycerol dehydrogenase It is known.

These polyol pathways are not activated under normal physiology, but in diabetes, the activity of aldose reductase is increased and the activity of sorbitol dehydrogenase is decreased to activate polyol (sorbitol). This is because the Km of the aldose reductase is 70-150 mM for glucose, so the polyol pathway is more prevalent in hyperglycemia (Raskin and Rosenstock, 1987). In the case of white lens, the Km of HK to glucose was 0.01-0.03 mM, whereas the Km of aldose reductase was 100 mM. This change in enzyme activity is a mechanism for inducing accumulation of sorbitol with increasing glucose concentration in cells.

Furthermore, aldose reductase reacts with various substrates to reduce other sugar alcohols or aldehydes, while polyol dehydrogenases react characteristically with polyols, resulting in intracellular accumulation of polyols that are no longer metabolized. Polyols are polar in nature and can't escape the cell membrane, causing them to accumulate in the cell and cause osmotic damage. Increasing osmotic pressure increases intracellular fluid, causing the lens to expand and the permeability of the lens membrane to increase. As a result, the concentrations of intracellular K +, amino acids, glutathione, myo-inositol, ATP, Na + -K + -ATPase activity decreased, Na, and Cl increased, and the fibrous tissue of the lens continued to swell. It ruptures to form a visible vacuole. Increased vacuole formation leads to clouding of the lens, resulting in cataracts, and an increase in the osmotic stress due to increasing Na and Cl, which exacerbates lens expansion. In addition, changes in the redox state of NADP + / NADPH and NAD + / NADH due to increased polyol pathways affect glucose metabolism and promote the oxidation of glutathione or protein sulfhydryl groups. An increase in the polyol pathway, ie, sorbitol accumulation, has been observed in tissues with diabetes complications such as nerves, retina, kidney, and lens, and is known to cause diabetic cataracts, peripheral neuropathy, retinopathy, and kidney disease.

On the other hand, it has been found that oxidative stress is involved in the progression of glucose cataract. That is, when the polyol pathway is increased NADPH correspondingly required to become a glutathione (GSSG) a reduced form (GSH) oxidation to increase the consumption of NADPH it is relatively lacking GSH for the reduction of the H ₂ O2 in H ₂ O The lack of increased hydroxyl radical formation, so cataract formation is a combination of osmotic pressure and oxidative stress.

As such, aldose reductase inhibitors that inhibit the production of polyols (sorbitol) in the polyol pathway may be effective to prevent, alleviate or treat cataracts caused by diabetes mellitus, such as quercetin, quercitrin, Sorvinyl (sorbinil), AL1576, TMG, statyl, tollestat, torphotine (aporphine type), benzyl isoquinoline (benzylisoquinoline type), berberine (berberine type) and the like are known. However, many of these compounds are suspected to be carcinogenic, causing side effects such as nausea, and side effects such as liver have been reported.

Based on the mechanism of the development and progression of sugar cataracts, the present inventors have found that the extract of Aralia elata acts as an inhibitor of aldose reductase in the polyol pathway and reduces oxidative stress, thereby preventing glucose cataracts from diabetes and The present invention was found to be able to exhibit a therapeutic effect.

The elm is a plant belonging to the genus Aralia sp. And is a perennial herb native to East Asia. The shell of the shell contains several types of triterpenoids, including saponins, and the bark contains elatoside E, which has a hypoglycemic effect, such as elatoside F and olenolic acid glycosides. It contains glycosides and elatosides A and B that inhibit ethanol absorption (Yoshikawa M., Harada E., Matsuda H., Murakami T., Yamahara J. and Murakami N. (1993), Elatosides A and B, potent inhibitors of ethanol absorption in rats from the bark of Aralia elata See ,: the structure-activity relationships of oleanolic acid oligoglycosides.Chem . Pharm. Bull . (Tokyo) 41: 2069-2071). Oleanoic acid, a type of saponin, prevents acute liver damage caused by various chemicals.

In the present invention, the turmeric was extracted with a solvent and treated with the extract in a lens culture medium extracted from the rats, resulting in a decrease in turbidity. In addition, a variety of solvents may be used to obtain the extract from the arbor, and ethyl acetate, clofoform, butanol or water may be used. Of these, chloroform extract shows the best effect.

Aralia extract of the present invention may be used alone, or may be used in combination with the extract of Pyeongdendron sp. In order to enhance the therapeutic effect of sugar cataracts, it is particularly preferable to use these compounds in admixture of each or at the same time so as to supplement myo-inositol or taurine, which has a decreased intracorporeal content in cataract formation.

The present invention provides a beverage containing water, sweeteners and suitable additives as an active ingredient. Myo-inositol or taurine may be added to the beverage, and the two components may be mixed together. Dulm is a food that has been edible by itself since ancient times, and the extract extracted from it is non-toxic, so it is provided as a drink containing suitable sweeteners and preservatives, so that patients with cataracts, diabetes mellitus or glucose intolerance often drink this drink to obtain a beneficial effect. .

The present invention provides herbal preparations, health supplements, beverages containing the extract as an active ingredient. Administration of arbor extract as a herbal or health supplement may be administered subcutaneously, orally, intramuscularly, in the form of a therapeutically effective amount alone or in a form containing a pharmacologically acceptable carrier, excipient or diluent. It is administered in a variety of ways, including other suitable methods known to. Formulations for oral administration suitable for extracts of the present invention may be in the form of tablets, capsules, solutions, suspensions, syrups, or beverages. Formulations comprising arbor extract for extraoral administration may be in the form of sterile injectables such as sterile, injectable liquids or oily suspensions. The suspension is prepared according to methods well known in the art, using suitable dispersing or wetting agents and suspending agents. The sterile injectables may also be sterile injectable solutions or suspensions containing an orally acceptable diluent or solvent, such as 1,3-butanediol. Suitable diluents include, for example, water, Ringer's solution, and isotonic saline. In addition, sterile, fixed oils can be readily used as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables.

Aralia elata extract of the present invention can be used as a beverage containing water and sweeteners and suitable additives. Myo-inositol or taurine may be added to the beverage, and the two components may be mixed together. Aralia is a food that has been edible by itself since ancient times, and the extract extracted therefrom is not toxic, and is provided as a beverage containing appropriate sweeteners and preservatives, so that cataract patients by sugar can often drink to help treat cataracts.

Dosage when using the extract according to the present invention as a herbal preparation may vary depending on the severity of the disease, sex, age, weight, and the desired effect of the patient to be administered. Generally, when administered orally to an adult, a dose of 20-70 mg, preferably 30-50 mg, per kg of body weight per day may be administered, and when administered orally by injection, a person weighing 60 kg may be used. Doses of 10-50 mg may be administered as a reference.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Induction of Glucose Cataracts in Laboratory Animals

1-1. Lens organ culture

After the sacrifice of 180-200g male mice (SD-rats) with CO ₂ gas, eyeballs were immediately removed and put in iodine solution for a while to disinfect the lens in the posterior approach (Spector A. and Garner WH (1981)). , Hydrogen peroxide and human cataract.Exp. Eye Res. 33 (6): 673-681). At this time, the metal was not in direct contact with the lens. The extracted lens was placed in M199 medium (Sigma, USA) and cultured in a cell incubator maintained at 5% CO ₂ , 95% air, and 37 ° C. Mgent medium contained gentamycin (Gibco, USA) at a concentration of 5 mg / l and fungizone (Gibco, USA) at a concentration of 0.5 ug / ml.

All lenses were incubated in M199 control medium without treatment for the first 24 hours, after which the injured lens was removed.

1-2. Determination of damage to the lens

When the lens was damaged, the amount of protein coming out and microscopic observation of the damage was determined. Only 1 hour of tissue culture according to the method of Tuminia et al. (Tumminia SJ, Qin C., Zigler JS and Russel P. (1994), The integrity of mammalian lenses in organ culture.Exp. Eye Res. 58: 367-374.) The lens was discarded and the protein in the culture medium was more than 7 ug / ml, and only the lens was found in the rest of the lens that had not been found to be damaged by microscopic observation until 24 hours of culture.

1-3. Cataract Induction

Glucose cataract, which is used as an experimental model of diabetic cataract, is a cataract model that is widely used because it is induced by a mechanism similar to diabetes (polyol pathway, oxidative stress). In this experiment, 20 mM of d (+)-xylose was used among various sugar types.

Example 2: Classification of Cataracts

2-1. Observation of turbidity of the lens

Dark field photography technique (Chand D., EL-Aguizy H., Richards RD and Varma SD (1982), Sugar cataracts in vitro: Implication of oxidative stress and aldose reductase in fluorescent microscopes equipped with Olympus, Japan Ⅰ. Exp. Eye Res . 35: 491-497) was used to take photographs with a CCD camera to observe the turbidity and to set the grade according to the degree of turbidity. It was divided into six grades according to the degree of lens turbidity.

Grade 0: Clear with no turbidity

Class 1: turbidity only in the equator

Grade 2: turbidity about 1/4 of the lens radius

Grade 3: turbidity about 1/2 of the lens radius

Grade 4: turbidity at least 1/2 of the lens radius

Class 5: turbidity in front of the lens

2-2. Density Determination of Lens Turbidity

Using an image analyzer loaded with Image pro-plus 4.0 (Media cybernetics, USA) software, the density of the lens turbidity captured by a CCD camera was determined as an arbitrary unit per pixel. The value in arbitrary units is represented by 0 for the darkest and 255 for the brightest.

Example 3 Extraction of Elm and Yellow Yolk

Aralia Canescens Sieb . Et ZUCC. And Phellodendron Amurense RUPR. Purchased in 1998. Branches of bark and yellow bark were pulverized and heated to extract each extract (A extract and P extract) with water, and then mixed with a weight ratio of 1: 1 to extract water (AP extract). The extraction method is as follows.

600 g of each mixture was mixed with 1: 1 mixture of the bark of the arbor and the yellow bark, and 5 L of distilled water was added thereto, followed by extraction, filtration and centrifugation with stirring for 40-50 hours. After centrifugation, 2 L of chloroform was added to the filtrate, and the resin, fiber, protein, and the like were eluted, and then the chloroform layer was removed. To this, n-hexane was added again to elute the remaining resin, fiber, n-hexane soluble substance, and the like. The aqueous layer was recovered, and talc was added to filter and purify to remove talc. The remaining talc was removed by filtration in a membrane filter device and the purified filtrate was lyophilized to powder. By this method, 36g (yield about 6%) of AP extract and 25.8g (yield about 4.3%) and 46.8g of Pleurotus japonica extract (AP extract) (Yield about 7.8%) was obtained. In the AP extract, arbor and baekbaekpi were present in a ratio of about 1: 2.

In addition, AP extract and A extract were divided into four fractions (CHCl ₃ layer, ethylacetate layer, BuOH layer, water layer) by the solvent extraction method as shown in FIG. In the solvent extraction method shown in FIG. 2, the arbor extract was dissolved in water, and then divided twice with petroleum ether to remove chlorophyll and other impurities, discarding the petroleum ether layer, and then distributing the remaining water layer with chloroform. Recovery was performed 92 times with TLC to ensure complete transfer of the material to the chloroform layer. Then, the remaining water layer was recovered 35 times with ethyl acetate and the remaining water layer was recovered 11 times with butanol.

As a positive criterion, quercetin (Sigma, USA), which has strong aldose reductase inhibitory activity and excellent antioxidant activity, was used.

Example 4 Formulations Including Arbor Extract

1. Tablet

250 mg of the freeze-dried powdery extract of the present invention prepared in Example 3, 260 mg of lactose for excipients and 35 mg of Avicel (microcrystalline cellulose), 15 mg of sodium starch glyconate as a disintegration aid, L- for direct use, binder 80 mg of HPC (Low-hydroxyprophylcell ulose) was mixed and mixed in a U-type mixer for 20 minutes. After completion of the mixing, 10 mg of the suspending agent magnesium stearate was further added and mixed for 3 minutes. Tablets containing 250 mg of extract per tablet were prepared by tableting and film coating after a quantitative test and a humidity test.

2. Syrup

An appropriate amount of sucrose was dissolved in a predetermined amount of water, and 80 mg of paraoxymethylbenzoate and 16 mg of paraoxypropylbenzoate were added thereto as a preservative, and 4.5 g of the lyophilized powdery extract of the present invention prepared in Example 3 was added. The solution was dissolved completely while maintaining at 60 ° C., cooled, and distilled water was added to make 150 ml to prepare a syrup.

3. Capsule

300 mg of the freeze-dried powdery extract of the present invention prepared in Example 3 was mixed with 200 mg of lactose as a carrier to fill a hard gelatin capsule to prepare a capsule.

4. Drink

After dissolving 500 mg of the lyophilized powdery extract of the present invention prepared in Example 3 in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and high fructose as an auxiliary component were added, and an appropriate amount was used as a preservative. After adding sodium benzoate, water was added to make the whole amount 100 ml to prepare a beverage composition.

5. Injection

Injectable preparation containing 200 mg of the freeze-dried powdery extract of the present invention prepared in Example 3 in 200 mg of physiological saline containing 1% polyoxyethylene hydrogenated Castro oil and containing the extract in a concentration of 0.1% Was prepared.

Example 5 Capability Test on Cataract Formation of Extracts

I) Delay of cataract formation; The extract was administered simultaneously with the induction of cataracts.

II) Prevention of Cataract Formation; Extracts were administered 24 hours or 48 hours before cataract induction.

III) Reversion after cataract formation; The extracts were administered 24 hours or 48 hours after cataract induction.

The fat-soluble one of the extracts was administered to the medium using dimethyl sulfoxide within 0.1% (v / v). The concentration of the extract was used in the range that the osmotic pressure of the medium in which the extract was administered did not exceed 320 mOsm. Osmotic pressure of the medium was measured by an osmometer (Precision System Inc., USA) using the freezing point drop.

Example 6 In Vitro Biochemical Characterization of Extracts

6-1. Find the IC ₅₀ value for aldose reductase for each extract

Preparation of the aldose reductase enzyme source was followed by the methods of Hayman S. and Kinoshita JH (1965), Isolation and properties of lens aldose reductase. J. Biol. Chem. 240 (5); 877-882. After homogenizing by adding 100 mM potassium phosphate buffer (pH 7.0) corresponding to 10 times the volume of the wet weight of the lens of the white paper, the supernatant was centrifuged at 4 ° C and 10,000 rpm for 20 minutes to serve as the enzyme source.

The activity of aldose reductase was determined by Das B. and Srivastava SK (1985) Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte.Anal . Biochem. Biophys.238 (2); 670-677 Followed. The enzyme source prepared by the above method, 10 mM DL-glyceraldehyde, 0.16 mM NADPH was dissolved in 0.1 M phosphate buffer (pH 6.2), and the absorbance was measured at 340 nm for 5 minutes at 1 minute intervals to determine the enzyme activity of the control group. . In this case, only the buffer solution was added instead of glyceraldehyde as a blank to react with each other to measure the change in absorbance. The blank was subtracted from the control so that the change in absorbance was 0.100 ± 0.005 for 5 minutes. Each extract was put at various concentrations in the reaction solution to measure the change in absorbance, and the value of the control group was obtained by subtracting the absorbance of the extract administration group, and the percentage of the absorbance ratio of the control group was expressed as the enzyme inhibition rate of the extract. 50% enzyme inhibition concentration was determined by plotting the enzyme inhibition rate (%) against the concentration of the extract (log value).

Percent Inhibition = [(Control-Sample) / (Control-Blank)] × 100

The fat-soluble extract was dissolved in DMSO to be within 1% (v / v) of the total reaction solution.

6-2. Determination of Antioxidant Capacity of Extracts

It was measured using chemiluminescence (CL) (Birks JW (1989), Chemiluminescence and photochemical reaction detection. In; Chromatography. VCH Publisher, pp: 10-13). Chemiluminescence is a light energy emitted during a chemical reaction, and cyclic diacylhydrazides such as luminol and aminobutylethyl isoluminol (ABEI) are used as amplifying agents. This amplifying agent is also used as a substrate of peroxidase and causes oxidation in the presence of H ₂ O ₂ . Antioxidants inhibit this oxidation reaction, reducing the CL released during the reaction.

200 ul of 0.6 uM ABEI and 200 ul of extract were placed in a polystyrene tube and 35% hydroperoxide and 10 mg / ml microperoxidase were diluted 1: 100 and injected automatically into the luminometer. The intensity of light by the chemical reaction was detected by a luminometer (LB9501, Clilumat, Germany) for 2 seconds. The antioxidant capacity of the extract is expressed as percent inhibition of light intensity during the reaction.

Example 7: Collection and Pretreatment of Lens

The lens incubated for a period of time was carefully taken out of the tweezers covered with paraffin to remove the water from the filter paper to remove the iris cells attached to the lens. After quick freezing with liquid nitrogen it was stored at -80 ° C and used for analysis within a week. The lens is weighed correctly before analysis, and then pooled 3-4 each to add nitrogen saturated 100 mM potassium phosphate buffer (pH 7.0), homogenized at 4 ° C, and part of the homogenate at 10,000 × g for 20 minutes. After centrifugation, the supernatant was stored at -80 ° C and used for aldose reductase activity measurement and Western blotting experiment.

In addition, some of the lens was homogenized with physiological saline solution, and some of the homogenate was centrifuged at 4,000 rpm for 5 minutes, and then the supernatant was frozen for taurine analysis. The remaining homogenate was mixed with 10% trichloroacetic acid and mixed vigorously at 4,000 rpm. Centrifuged for 5 minutes, the supernatant thereof was neutralized with 2.5 M KOH to pH 6.8-7.6 and stored at -80 ℃ was used for xylitol and myo-inositol analysis.

Example 8: Biochemical Analysis of Lenses

8-1. Activity measurement of aldose reductase

To measure the aldose reductase activity of the lens, the sample was added to 1 ml of reaction system (50 mM phosphate buffer (pH 6.0), 400 mM LiSO 4, 10 mM DL-glyceraldehyde, 0.1 mM NADPH) and absorbance at 340 nm, 37 ° C. The change of was observed for 5 minutes.

8-2. TBARS measurement

Method of Okawa (Ohkawa H., Ohish N. and Yagi K. (1979), Assay for lipid peroxdation in animal as lipid peroxide, an indicator of oxidative stress in the lens tissue by thiobarbituric acid reaction.Anal. Biochem. 95: 351-356). Each sample was diluted with 10 mM potassium phosphate buffer (pH7.4) so that the protein concentration was about 2 mg / ml, and then, 1 ml each was added to a screw-cap tube, and the mixture was warmed at 37 ° C. for 60 minutes. 1 ml of 10% (w / v) trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid were added thereto, followed by vigorous mixing for 30 seconds, closing the stopper with boiling water, and bathing for 15 minutes. After the hot water bath, it was rapidly cooled in iced water, 2 ml of butanol was added to each tube, mixed vigorously, and centrifuged at 4,000 rpm for 10 minutes, and the absorbance was measured at 535 nm with a butanol layer. At this time, tetraethoxypropane was dissolved in methanol as a standard solution.

8-3. Conjugate diene content determination

Fat was first extracted from the homogeneous solution of the lens, and then the content of conjugate diene in the extract was measured. 2 ml of chloroform: methanol (2: 1) mixture and 0.5 ml of homogenate were mixed vigorously, followed by centrifugation at 2,000 × g for 10 minutes, and then the lower layer of glofoform was removed and transferred to a tube. The fat layer was separated and the total volume recorded. 100ul of the fat extract was dried under nitrogen gas to measure total fat concentration and sealed well and stored at -85 ° C. 500 ul of the fat extract was dried well under nitrogen gas, dissolved in 1 ml of cyclohexane, and then absorbance was measured at 233 nm and expressed as absorbance per 1 mg of fat.

8-4. Total fat content

Of the total fat concentration in homogenates of the lens measurement stand Popo Spokane - vanillin Reaction (Fring CS and Dunn RR (1970 ), A colorimetric method for determination for total serum lipids based on the sulfo- phosphovanillin reaction Am J. Clin. Path. 53; 89-91). 0.2 ml of concentrated sulfuric acid was added to the dry fat extract well under nitrogen gas, and the mixture was boiled in boiling water for 10 minutes. After cooling, 5 ml of phosphovaniline reagent was added and activated in a constant temperature bath at 37 ° C. for 15 minutes, and the absorbance was measured at 540 nm within 10 minutes. Olive oil was used as a standard solution.

8-5. Determination of Glutathione (GSH) Content

GSH content was determined by Elman's method (Ellman GL (1959), Tissue sulphydryl groups.Arch . Biochem. Biophys . 82; GSH reacted with DTNB (5,5'-dithiobis; 2-nitrobenzoic acid) to exhibit absorption). 70-77). 0.5 ml of 20% trichloroacetic acid was added to 0.5 ml of the homogeneous solution of the lens, followed by vigorous mixing and centrifugation at 2700 rpm for 20 minutes. After centrifugation, ₄ ml of 0.3 M Na ₂ HPO ₄ and 0.5 ml of DTNB were added to 0.5 ml of the supernatant, and the absorbance was measured at 412 nm. As a standard solution, glutathione (reduced form) was dissolved in distilled water and used.

8-6. Myo-inositol and Xylitol Content

The measurement of the content of myo-inositol and xylitol was measured using HPLC by filtering the pretreated samples with 0.45um filter (millipore). Increase the temperature of the calcium cation exchange column (300 × 6.5mm ID) to 90 ° C., stabilize the baseline by flowing 50 mg / l calcium disodium EDTA aqueous solution as a mobile phase at a rate of 0.5 ml / min, and then inject 50ul of samples in each sample. It was.

8-7. Taurine content

After homogenizing the lens with 0.9% NaCl solution, remove the protein from the centrifuged supernatant using acetonitrile, and add Dabsyl chloride, a derivative, to the centrifuged supernatant, and react with it at 70 ° C for 10 minutes for filtering. It was. The column used water symmetry C18 (3.9 × 150mm) at 40 ℃ and the mobile phase was mixed with acetonitrile 73:27 with a buffer solution (20 mM sodium acetate, 0.05% triethylamine, pH 6.5) and the rate was 1.0 ml / min. It was.

8-8. Electrophoresis (SDS-PAGE)

The amount of this protein was quantified to determine whether the extract also affected the synthesis of aldose reductase protein. Since the molecular weight of aldose reductase was about 38 kd, 10.0% separating gel was used for SDS-PAGE.

After quantifying the protein in the homogeneous solution of the lens, 25 ul of sample containing the same amount of protein was added to each well. 5 × Laemmli's sample buffer was added to each sample, boiled for 5 minutes, loaded and electrophoresed under 120V constant voltage.

8-9. Western blotting

After SDS-PAGE, proteins were transferred from the gel to the nitrocellulose membrane for 30 minutes at 15 V using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell from Bio-Rad. The nitrocellulose membrane was stored in a 4 ° C. refrigerator overnight in a blocking solution in which 5% skim milk was dissolved in a solution containing 0.1% Tween 20 in PBS (phosphate-buffered saline) (PBS-T). The primary antibody was diluted 1: 2,000 in PBS-T, left for 1 hour, washed once for 15 minutes and three times for 5 minutes, followed by alkaline anti-goat IgG (Sigma, USA). ) Was diluted 1: 30,000 and reacted for 40 minutes. The membrane was washed again as above and the solution of pBT (nitro blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3) in developing solution (0.1 M Tris, 0.1 M NaCl, 5 mM MgCl ₂ , pH9.5). -indolyl phosphate) was mixed 2: 1 and developed in the dark for 5 minutes, and dried and photographed.

8-10. Histological examination of the lens

The lens was fixed overnight in 4% glutaldehyde prepared in 0.1 M phosphate buffer (pH 7.4) and then again fixed with 1% osmium detaoxide (OsO ₄ ) for 1 hour. After dehydration by ethanol series and embedded in 100% EPON section (1 micron). Staining with 0.1% toluidine prepared in 1% sodium borate was observed by light microscopy (× 200).

8-11. Protein Quantitation

Protein quantitation was measured using a DC protein analysis system (Bio-Rad, USA) using BSA (Sigma) as standard solution.

Experiment result

1. Measurement of turbidity of sugar cataracts

The degree of turbidity formed in the lens incubated in 20 mM D-xylose medium was observed with a stereomicroscope to photograph the change in turbidity with time. The photograph is shown in FIG.

Over time, turbidity progressed to the cortex and suture lines began to appear slightly at 24 hours of culture. Up to 24 hours of culture, cortical turbidity was clearly seen as the early stage of glucose cataract, and mature cataract was formed at 48 hours of culture. Three-dimensional observation of the lens revealed that clouding occurred only at the equator and in the first half.

In order to quantify the degree of turbidity of the lens observed under the microscope, the degree of turbidity was expressed by an average density value of the lens turbidity using an image analyzer. Figure 4 shows the turbidity of the mouse lens cultured for 72 hours after treatment with 20 mM xylose medium. This experiment averages three or more experiments for four to five lenses. Turbidity density increased significantly with the observation time up to 48 hours of culture, but did not show a significant increase from 48 hours after culture.

2. Inhibitory Activity of Aldose Reductase from Ar (A), Yellow and White (P), and Mixed and AP Extracts

Inhibitory activity of the extracts on the activity of aldose reductase was expressed as IC ₅₀ value.

Inhibitory Activity of Aldose Reductase on Ar (A), Yellow and White (P), and Mixed and AP Extracts extract IC ₅₀ (μg / ml) Aralia Extract (A Extract) 11.3 Yellow White Extract (P Extract) 29.4 Armur (A) Extract + Yellow White (P) Extract 16.8

3. Antioxidant Activity of Extracts of Aralia (A), Yellowish White (P) and Mixed Arrhythmia (AP)

Antioxidant activity of the above extracts was indicated as the degree of inhibition of this reaction by measuring the chemical luminescence (CL) released during the oxidation reaction by H ₂ O ₂ using aminobutylethyl isoluminol (ABEI) as a substrate in vitro. Based on the count of CL released when no extract was added, the extract expressed the antioxidant capacity as a percentage of decreasing this count. A negative value for this percentage indicates the extent to which oxidation is promoted. The results are shown in FIG.

As shown in the graph of Figure 5 A extract was shown to have stronger antioxidant activity than AP or P extract, but weaker than the positive control ascorbic acid.

4. Effects of Ar (A), Yellow and White (P) Extracts, and Ar and Yellow and White (AP) Extracts on the Delay of Glucose Cataract Formation in the Lens

To determine the period of time in which the lens can be cultured and tested, it was observed how many days the clear state was maintained under a microscope in the control medium. At 8-9 days, the lens had a slight turbidity, and the experimental period was set within 8 days. On day 8, histological examination of the lens without any cloudiness was almost identical to that of culture 1 day.

Three extracts were added at the same 1 mg / ml concentration. How much inhibition of cataract formation was suppressed for 48 hours was measured by the density of turbidity and is represented by the graph of FIG. 6. A, AP, and P extracts showed good delay in cataract formation. However, P extract showed little effect at 1mg / ml, and AP extract showed no synergistic effect compared to A and P extract. AP extract significantly decreased turbidity density by 15.7% and 8.1% at 24 and 48 hours, respectively, and A extract significantly decreased turbidity density by 36.4% and 31.3% at 24 and 48 hours, respectively. . AP extract showed grade 3 and 100% grade 4 at 24 and 48 hours of culture, respectively, and extract A had 75% grade 2 and 25% grade 3 at 24 hours, and 48 hours at 48 hours. 95% had grade 3 and 5% had grade 4. The cataract delaying effect in the lens was consistent with the results of in vitro experiments (aldose reductase activity inhibition and antioxidant effect).

5. Inhibitory Activity of Aldose Reductase Activity of Four Fractions of Aralia elata Extract

As a result of the delayed effect of the formation of sugar cataracts, the effect of Ardum extract was the best, and four kinds of solvent extracts of Ardum extract-chloroform (AC), ethylacetate (AE), butanol (AB), water (AW) fractions Proceeded. The chloroform (AC) fraction was dark brown semisolid and insoluble in water. The ethyl acetate (AE) fraction was dark brown semisolid, highly hygroscopic and well soluble in hot water. The butanol (AB) fraction was light brown powdery and soluble in water. The water (AW) fraction was also dark brown powdery and well soluble in water. The results are shown in Table 2.

IC _{50 of the} fractions (ug / ml) Fraction IC ₅₀ (μg / ml) AE Fraction 5.92 AC fraction 2.11 AB fraction 27.2 AW fraction 16.9

Therefore, the inhibitory ability against aldose reductase activity was higher in the order of AC, AE, AW, AB fractions.

6. Antioxidant Activity of Four Fractions of Aralia Extract

The antioxidant capacity of the four fractions of the arbor extract, measured using CL, was tested. Using aminobutylethyl isoruminol as the substrate of peroxidase, oxidation of peroxidase in the presence of H ₂ O ₂ was added to each fraction to inhibit the oxidation reaction and show the inhibition rate (see FIG. 7). The AE fraction showed the highest inhibitory activity of CL over 95% up to 0.01 mg / ml concentration, and the AC and AB fractions showed similar antioxidant activities, and the AW fraction had the lowest antioxidant capacity.

7. Effects of Four Fractions of Aralia Extract on Delayed Glucose Cataract Formation

Four fractions of Aralia elata extract were added to the medium to determine the effect on the formation delay of sugar cataract. The lens was incubated in 20 mM xylose medium and each fraction added 1 mg / ml. Referring to FIG. 8, AC fractions significantly delayed cataract formation (P <0.05) when the four fractions were treated at a concentration of 1 mg / ml. The AC fractions were 90% Grade 2 at 24 hours of culture, 10% Grade 1, and 75% Grade 1 and 25% Grade 2 at 48 hours.

Figure 9 shows the test results for cataract delay of the lens by incubating the lens for 96 hours in 20 mM xylose medium. It has been shown that the AC fraction can incubate cataracts up to 96 hours. T-test between the control group and the AC group to which 20 mM xylose was added significantly delayed cataract formation up to 96 hours (P <0.05). The degree of inhibition of cataract formation peaked at 66.4% at 48 hours and gradually decreased over time, indicating a 36.9% inhibition at 96 hours.

The results of the prophylactic effect on the formation of sugar cataracts of AC fractions are shown in FIG. 10. Cataracts were induced by placing the AC fractions in control medium and incubating for 24 or 48 hours and then changing to xylated medium. As a result, there was almost no difference in the degree of inhibition of cataract formation between 24 and 48 hours of pre-incubation, and the AC fraction preculture significantly inhibited cataract formation up to 72 hours after cataract induction.

Regarding the ability of the AC fraction to recover the sugar cataracts already formed, the change in turbidity after treatment of the cultured lens with chloroform fractions for 24 hours, 48 minutes in 20 mM xylose medium is measured in FIG. 11. It was. When the AC fraction was administered 24 hours after cataract induction, there was no significant change in turbidity without significant difference until day 2, but significantly decreased turbidity on day 3. Turbidity at day 3 was 35.7% less than that at the time of AC fraction administration, and a reduction of turbidity over a three-day day accounted for about 80% of the total reduction over three days. The AC fraction administered 48 hours after cataract induction showed a slight decrease (approximately 2.4%) for 3 days, but was not significant and slightly turbidity increased on day 1 of cataract induction.

12 shows the results of the AC fraction on the xylitol content at 48 hours of culture for each concentration. As the concentration of the AC fraction increased, the xylitol content significantly decreased. The AC fraction was reduced by 16.8% at 0.1 mg / ml, 51.0% at 0.5 mg / ml, and 87.7% at 1 mg / ml. Each bar in the graph represents the average of four experiments for four to five lenses in each group. The experiment was to measure the effect of the chloroform fraction of the Aralia extract on the formation of xylitol after culturing the lens in a medium treated with 20 mM xylose.

FIG. 13 shows the effect of 1 mg / ml AC fractions on the activity of aldose reductase at 48 hours after cataract induction. This experiment measured the effect of chloroform fraction of Ardum extract on the activity of aldose reductase after 48 hours of incubation of the lens in media treated with 20 mM xylose. The control is incubation of the lens with gyros treatment medium, and AC represents the experimental result of the xylose medium in which the lens was treated with the AC fraction. Each bar in the graph represents the average of four experiments for 4-5 lenses in each group. As the concentration of AC fraction increased, the activity of aldose reductase significantly decreased. Therefore, the AC fraction has been shown to lower the polyol pathway by inhibiting the activity of aldose reductase not only in vitro but also in the lens.

8. Effect on Oxidative Stress

MDA, a major lipid peroxide that reacts with thiobarbituric acid (TBA), is an intermediate of lipid peroxidation. Polyunsaturated fatty acids with three or more double bonds are the result of oxidative degradation by free active radicals and are the best indicators of lipid peroxidation. Widely used (Ohkawa H., Ohish N. and Yagi K. (1979), Assay for lipid peroxdation in animal tissue by thiobarbituric acid reaction.Anal . Biochem. 95: 351-356).

In FIG. 14, AC shows the results of the formation of TBARS in the lens cultured in AC treated 20 mM xylose medium and in the untreated group of lenses. Each figure represents the average of four experiments on four or five lenses. The addition of the AC fraction showed a slight decrease in the TBARS content as shown in FIG. 14, but had no significant effect regardless of the concentration. However, in vivo experiments with CL, AC fraction had an antioxidant effect.

FIG. 15 shows the results of the formation of conjugate dienes after 48 hours of incubation with the AC fraction and the control group without treatment in the lens cultured in 20 mM xylose medium. Conjugated diene content was significantly lowered by only 1 mg / ml of AC fraction. Therefore, the AC fraction is thought to have the effect of inhibiting the formation of conjugated diene, an early product of lipid peroxidation in the lens.

Glutathione (GSH) is the first oxidized substance in cataract induction with H ₂ O ₂ and reacts most sensitively to external stress, and GSH content is reduced by reduction of glutathione reductase inhibitor to GSH due to NADPH consumption when polyol pathway is increased. This is known to decrease.

Figure 16 shows the change in total GSH content of the lens upon induction of glucose cataract. Changes in GSH content of the lens incubated for 48 hours in 20 mM xylose medium are shown. Significant decrease in GSH continued up to 48 hours with cataract induction time. The decrease in GSH also showed a significant decrease in the early stage of cataract induction, which appears to be affected by the initially increasing polyol pathway (Aldose Reductase activity). It is also believed that the sustained decrease in GSH is due to the effect of increasing oxidative stress on the formation of glucose cataract. In this experiment, the decrease of GSH and the increase of cloudiness showed a significant high negative correlation (r = -0.891, p <0.001). The AC fraction tended to increase the GSH content with increasing concentration, and at 48 hours after cataract induction, the AC fraction significantly increased the GSH content above 0.5 mg / ml. However, 1 mg / ml of AC fraction showed 28.6% higher GSH content than the control (20 mM xylose). However, when treated with 1 mg / ml of AC fraction, the GSH content corresponds to 38.3% of the GSH content before cataract induction, and thus the level was not restored to the level before cataract induction.

9. Effect on Myo-inositol and Taurine Contents of the Lens

In the case of diabetic cataracts and glucose cataracts, the concentration of myo-inositol or taurine in the lens was decreased in several studies. Therefore, in this experiment, the changes in the amounts of myo-inositol and taurine were measured as glucose cataract progressed. The effect of the AC fraction was examined.

17 and 18 show the effect of AC fraction 1 mg / ml on the content of myo-inositol and taurine according to the cataract induction time in 20 mM xylose medium. AC fraction treatment increased 35.6%, 434.6% and 296.7% of control content at 6, 24 and 48 hours for myo-inositol and 77.9%, 403.0% and 136.8% for taurine at the same time, respectively. Showed. Both of these materials showed the largest increase in content during 24 hours of AC fractionation.

10. Effect of Myo-inositol and Taruine Supplementation on the Prevention and Recovery of Glucose Cataracts in Treatment of Two Chloroform (AC) Fractions

The effect of supplementation of 2.5 mM myo-inositol and taurine on the recovery rate and the changes of myo-inositol and taurine content in the lens were measured.

19 and 20 show the effect of supplementation of myo-inositol and taurine on cataract recovery in turbid density. Incubation of the lens for 24 hours in 20 mM xylose medium added 2.5 mM of myo-inositol and taurine in relation to recovery of turbidity of the released lens. . FIG. 19 shows the effect of adding myo-inositol and taurine and an AC fraction to turbid lens induced for 24 hours in xylose medium, and FIG. 20 shows the effect on lens induced with turbidity for 48 hours. . Recovery at 24 hours of turbid cataract induction was significantly lower in turbidity than supplementation of myo-inositol and taurine on day 1 and 2 of the recovery, but only on the day 3 of recovery. It was similar. Treatment with AC fraction alone did not significantly reduce turbidity until day 2, but significantly decreased on day 3, and when AC fraction was supplemented with myo-inositol and taurine, it began to decrease significantly from day 1.

In the end, myo-inositol and taurine supplementation speeded up the recovery of turbidity. Recovery in the control medium showed a significant reduction in turbidity from day 1 of recovery, but when the turbidity density dropped to about 60 AU / pixel, the recovery rate of turbidity was greatly reduced. Treatment with myo-inositol and taurine in the AC fraction appeared to reduce the recovery rate at higher turbidity densities (80-90 AU / pixel). Recovery at 48 hours after cataract induction increased slightly with the same turbidity density as with myo-inositol and taurine supplementation on day 1, and supplementation of myo-inositol and taurine supplementation on day 2 and 3 significantly increased AC fraction. Only when treated, the turbidity density was reduced.

In FIG. 21, the AC fraction was supplemented with 2.5 mM of myo-inositol and taurine, respectively, to determine the effect of cataracts on preculture. The graph shows the average of three or more experiments on four or five lenses. O indicates a control medium, o indicates an AC medium, o indicates a myo-inositol, taurine, and an AC-treated medium. To the lens incubated for 72 hours in 20 mM xylose medium, 1 mg / ml AC fraction of Aralia extract, 2.5 mM myo-inositol and 2.5 mM taurine were added. On day 1 and 2 of cataract induction, there was little difference in turbid density due to AC fraction treatment and addition of 2.5 mM of the above two substances, and even on day 3, turbid density was reduced by only about 13.8%, which did not have a significant effect. More than 48 hours after cataract induction, there was a deficiency of both substances in the lens, making it difficult to measure. In this experiment, when taurine was supplemented with 10 mM and 30 mM in crystalline culture medium, it was observed that the taurine content in the lens increased in proportion to the concentration. In the present study, on the third day of cataract induction, the content of both substances was measured in the lens and myo-inositol was not measured and the taurine was about 0.01 ± 0.008 mM when only the AC fraction was preincubated. In the pre-incubation of both materials with the AC fraction, taurine was about 0.08 ± 0.012 mM and myo-inositol was about 0.40 ± 0.023 mM.

Example 9 Toxicity Experiments on Aralia elata Extract According to the Invention

LD ₅₀ (an amount capable of killing 50% of experimental animals) is an important value that is an indicator of acute toxicity of drugs in order to confirm the safety when using the extract of arbor according to the present invention as a herbal or dietary supplement. Was obtained by the following method.

Sixty normal ICR mice (male, 22 ± 1 g) were divided into six groups of A to F, each six in a group, and in the group A, the persimmon extract 5 prepared in Example 3 according to the present invention per 1 kg of body weight. Equivalently from the administration of g, 7.5 g in group B, 10 g in group C, 12.5 g in group D and 15 g in group E were administered orally, respectively, and the Berrens-Karber method (Ref. : 高木 敬 次 郞 外: 藥物 實驗, 南山 堂, Japan, p131, 1960) LD ₅₀ value was determined by oral (po) administration of the extract of the present invention. The results are shown in Table 3 below.

Lethal dose by oral administration of Aralia elata extract (LD ₅₀ ) Experimental group Capacity (g / kg) Oral administration group (p.o.) * Z ** d Number of dead animals A 5 0/6 - - B 7.5 0/6 0 2.5 C 10 0/6 0 2.5 D 12.5 0/6 0 2.5 E 15 0/6 0 2.5

* Z: ½ of the number of dead animals in two consecutive doses

** d: difference between two consecutive doses

As shown in the results shown in Table 3, the LD ₅₀ value of oral administration of the Arm extract according to the present invention was 15 g / kg or more, and thus it was found to be a very safe material. In other words, it can be clearly seen that the arthropod extract according to the present invention can be safely administered with little toxicity.

In addition, autopsies and histopathological examinations were performed on the test animals used to measure the LD ₅₀ value in the following manner. At the end of the test, all surviving animals were bled after ether anesthesia, and the organs were extracted and visually examined for all organ abnormalities. Autopsied whole organs were fixed in 10% neutral formalin solution for more than 10 days for pathological examination, and then dehydrated and embedded in a paraffin embedding machine (Fisher, Histomatic Tissue Processor, 166A), followed by an AO Rotary Microtome. 5 μm sections were prepared and observed by hematoxylin and eosin staining.

All animals in each group were dissected, and histologic findings examined under a microscope after HE staining were as follows. When the extract of the present invention was administered up to 15 g / kg of mouse body weight, ideal findings by drug administration were not observed in liver tissue and kidney. In addition, no abnormal findings were observed in cardiomyocytes of the heart, gastrointestinal tract, pancreas, lung, spleen, adrenal gland, brain, testes, ovaries, bone marrow, and the like.

Therefore, the Argul extract according to the present invention had no side effects due to acute toxicity to all organs even when 15 g per 1 kg of body weight, which is the maximum dose that can be administered to mice, and does not cause toxicity such as any organ damage. Was determined to be a safe substance.

Example 10 Properties of Extracts of the Invention According to HPLC (High Performance Liquid Chromatography) Analysis

The extract was dissolved in water at a concentration of 10 mg / ml in order to analyze the extract of the inventors Arum by HPLC. 10 μL of the dissolved extract solution was taken and analyzed using HPLC (HP1090).

(Analysis condition)

Column: LuNa 5u C18 (2); 4.6 mm i.d. x 250 mm L

Mobile phase: 2% acetic acid and methanol gradient system

Flow rate: 1 ml / min

Detection: measured at 254 nm with a photodioid array detector

Repetitive experiments showed characteristic peaks of the extract. FIG. 20 is an HPLC profile of the Aralia extract, which was found to contain a considerable amount of phenolic substances. In particular, phenolic substances with a high content of 3,4-dihydroxybenzoic acid (retention time: RT = 10.941 minutes) were contained in the extract of about 1%, and appeared in caffeic acid (caffeic acid, RT = 18.824 minutes) and the content was 0.16. %, Ferulic acid (RT = 25.53 min) and the content was around 0.04%. However, arbor extract contains many substances that have not yet been identified.

Since phenolic substances are generally known to have excellent antioxidant activity, the antioxidant activity of arbor extract seems to be due to the phenolic substances contained in the extract.

Although the present invention has been described in detail above based on the preferred embodiments, the scope of the present invention is not limited thereto, and substitutions, modifications, and alterations apparent to those skilled in the art within the spirit and scope of the present invention are not limited thereto. It should be understood that it belongs to the scope.

As is evident from the above description, the composition comprising the arbor extract according to the present invention has an effect on the prevention, delay of progression, treatment and recovery of cataract induced by sugar. In particular, the composition of myo-inositol and taurine added to the composition of the present invention has the effect of increasing the therapeutic effect on cataracts by supplementing these compounds which decrease with progression of cataracts. Beverages, herbal preparations, dietary supplements and therapeutic agents containing the extract of Argul according to the present invention are effective in the prevention and treatment of cataracts.

Claims (9)

A composition for preventing or treating cataracts, which is extracted with a solvent selected from the group consisting of ethyl acetate, chloroform, and butanol. delete The composition of claim 1, further comprising one or more compounds selected from the group consisting of myo-inositol or taurine. delete delete delete delete delete delete