[DESCRIPTION]

[invention Title]

THE COMPOSITION COMPRISING THE EXTRACTS, FRACTIONS AND THE ISOLATED COMPOUNDS OF RHUS VERNICIFLUA FOR PREVENTION AND TREATMENT OF DIABETIC COMPLICATIONS

[Technical Field]

The present invention relates to a composition for prevention and treatment of diabetic complications which comprises the extracts of Rhus verniciflua, more precisely a composition for prevention and treatment of diabetic complications which comprises the extracts of Rhus verniciflua, fractions thereof and isolated compounds therefrom by using a solvent such as water, alcohol or a mixed solvent of them as an active ingredient.

[Background Art]

Diabetes is the disease that is caused by- insufficient insulin secretion or mal-function of insulin secretion with increasing blood glucose that is discharged in urine. Diabetes is a serious disease that draws public attention because of its complications. Before insulin was developed, such complications as lethargy and infection threatened human lives. But, these days, according to the advancement of modern chemistry and biochemistry, dietetic

therapy, hypoglycemic agent therapy and insulin therapy have been introduced and thus sudden life-taking risk can be prevented. The highest threat to diabetics now is diabetic complications, which are retinopathy, nephropathy and neuropathy.

More than half of the diabetic complication cases are diabetic retinopathy, which is the leading cause of adult visual loss. Symptoms of diabetic retinopathy are visual disturbance, glaucoma and visual loss, etc. Diabetic neuropathy results from abnormality of carbohydrate metabolism and lipid metabolism, causing peripheral neuropathy. This disease shows the symptoms of tendon reflex decrease, disappearance, perception disorder, and limb pain, etc. In particular, muscle pain occurs during the night and burning sensation on the sole of the foot, hyperalgesia and amblyopia could be observed. In general, such symptoms are bilateral symmetry and motor disorder is rare. In addition, autonomic nervous system dysfunction can be developed, which includes asymmetric pupil, perspiration disorder, impotency, residual urine, hesitancy, urinary retention, constipation and diarrhea, etc. Nephropathy is also one of the most common diabetic complications and is regarded as the leading cause of death by diabetic complications. As diabetes progresses, blood vessels of the whole body are damaged, particularly damage

in kidney blood vessels results in disorder of blood filtering activity of kidney, which is the major function of kidney, resulting in the accumulation of excessive body fluid and toxic materials. Symptoms are glomerulosclerosis, proteinuria and renal failure. Nephropathy is known to be caused by excessive aldose reductase or oxidative stress.

The mechanism causing diabetic complications is explained mainly by non-enzymatic glycation of protein, osmotic stress caused by changes of polyol pathway and oxidative stress caused by free radical.

In non-enzymatic glycation of protein, an amino acid group such as lysine residue is condensated and cross reacted with a reducing sugar without enzyme activity to produce advanced glycation end procucts (AGEs). The advanced glycation end product is an irreversible reaction product. Once it is produced, it is not degraded and instead deposited in tissues even after the blood sugar level returns to normal, resulting in abnormal changes in structures and functions of tissues (Vinson JA et al., J Nutritional Biochemistry, 7, 559-663, 1996; Smith PR et al., Eur. J Biochem., 210, 729-739, 1992). The non- enzymatic glycation of protein causes glycosylation of such proteins as basal membrane, plasma albumin, cataractous lens protein, fibrin and collagen, and the produced advanced glycation end product induces abnormal

changes in structures and functions of tissues to cause diabetic complications such as retinopathy, cataract, nephropathy and neuropathy. When the advanced glycation end procuct is generated under high blood glucose condition, abnormality is induced in lipid metabolism and at the same time defense system against the generated toxic oxygen free radical is weakened, which causes oxidative stress (Yokozawa T et al., J of Trad. Med., 18, 107-112, 2001). As explained above, non-enzymatic glycation and oxidative stress are closely related to each other.

Polyol is an alcohol reduced from aldose or ketose by aldose reductase (AR). In general, most of intracellular glucose is phosphorylated to glucose-6-phosphate by hexokinase under the normal blood glucose condition and non-phosphorylated glucose (approximately 3%) proceeds to polyol pathway. Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are involved in polyol pathway. These enzymes are distributed in peripheral nerves, retina, cornea, iris, lens, kidneys, and erythrocytes, and they flow in the cells by dispersion without insulin when glucose is uptaken. When glucose level in the cells is increased, the activity of aldose reductase (AR), the rate-limiting enzyme that converts glucose into sorbitol via polyol pathway strongly depending on the concentration of a substrate, is increased. Accordingly, the products

sorbitol and fructose are accumulated in the cells. The accumulation of such polyols results in the decrease of myoinositol (MI) uptake in cells by pathogenic osmority and as a result the activity of Na+, K+/ATPase is reduced. Then, motor nerve conduction velocity (MNCV) is decreased to cause diabetic complications including neuropathy. Among the complications, nephropathy is the most common, making it as the leading cause of death by diabetic complications. It is known that diabetics have 20 times higher risk of renal failure, compared with normal people. AS-3201, Tolrestat, Ponalrestrat and Epalrestat, the aldose reductase inhibitors, were developed as nephropathy treatment agents. However, the treatment effect of these medicines is still under examination. To develop a therapeutic agent for diabetic complications, studies on natural materials having aldose reductase inhibitory effect and at the same time having anti-diabetes, anti-oxidation and blood glucose reducing effect are actively undergoing. When blood glucose level is not going down because of diabetes, a diabetic complication preventive agent has to be administered to prevent diabetic complications. Aminoguanidine HCl, which is the only synthetic glycosylation inhibitor up to date, is a nucleophilic hydrazine to be bound to the product of condensation to

prevent cross-linking with a protein, and thereby aminoguanidine HCl inhibits the generation of the advanced glycation end procuct to retard or prevent the progress of diabetes to diabetic complications (Brownlee M et al . , Sciences, 232, 1629-1632, 1986; Edelstein D et al., Diabetes, 41, 26-29, 1992). Aminoguanidine HCl is the most promising synthetic medicine for prevention and treatment of diabetic complications, which proceeded to the third phase of clinical test. However, when it is administered for a long term, toxicity is caused. So, a safer medicine for prevention and treatment of diabetic complications needs to be developed.

Rhus verniciflua is a deciduous hardwood tree belonging to Anacardiaceae, which includes 60 genus 400 species found in subtropical or tropical areas. In Korea, 5 species including Rhus verniciflua, Rhus trichocarpa and Rhus chinensis are found. Its sap, called as lacquer or dried lacquer, has been used for industrial and medicinal purposes. Particularly in Oriental medicine and folk remedies, it has been used as a drug accelerating human energy and blood circulation in human, to make strong and to relieve pain and tussis, and it is also used in traditional food, for example lacquer chicken. Some of the physiological activities of Rhus verniciflua extracts have been reported. As well as the anti-bacterial activities of

materials isolated from Rhus verniciflua or anti-oxidative, anticancer activities of flavonoid and urushiol included in its sap have been reported.

However, a composition containing the extract of Rhus verniciflua which is useful for the prevention or treatment of diabetic complications by inhibiting aldose reductase and production of advanced glycation end procuct has not been reported, yet.

Thus, the present inventors screened a material capable of inhibiting aldose reductase and the production of advanced glycation end procuct from natural materials to overcome the side effects of the conventional drugs for diabetic complications. And the present inventors finally completed this invention by disclosing that the extracts and fractions of Rhus verniciflua could inhibit aldose reductase and the production of advanced glycation end procuct, suggesting that they could be effectively used as a drug for prevention and treatment of diabetic complications.

[Disclosure] [Technical Problem]

It is an object of the present invention to provide a composition containing the extracts of Rhus verniciflua, fractions thereof and isolated compounds therefrom as an

active ingredient that is functioning to inhibit aldose reductase and the production of advanced glycation end procuct for prevention and treatment of diabetic complications caused by changes of polyol pathway and non- enzymatic glycation.

[Technical Solution]

To achieve the above object, the present invention provides a composition for prevention and treatment of diabetic complications which comprising the extracts of

Rhus verniciflua extracted with water, alcohol or a mixture thereof as a solvent as an active ingredient.

The present invention also provides a composition for prevention and treatment of diabetic complications comprising the fractions obtained from each stage of the extraction of Rhus verniciflua with n-hexane, methylene chloride, ethyl acetate and n-butanol stepwise.

The present invention further provides a composition for prevention and treatment of diabetic complications comprising butein, sulfuretin or their pharmaceutically acceptable salts as an active ingredient.

The present invention also provides a treatment method for diabetic complications containing the step of administering a pharmaceutically effective dose of the extracts of Rhus verniciflua, fractions thereof, butein or

sulfuretin to a subject with diabetic complications.

The present invention also provides a prevention method for diabetic complications containing the step of administering a pharmaceutically effective dose of the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin to a subject with diabetic complications.

The present invention also provides a use of the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin for the production of a composition for prevention and treatment of diabetic complications.

The present invention also provides a health food for prevention and improvement of diabetic complications which contains the extracts of Rhus verniciflua extracted with water, alcohol or a mixture thereof as a solvent as an active ingredient.

The present invention also provides a health food for prevention and improvement of diabetic complications containing the fractions obtained from each stage of the extraction of Rhus verniciflua with n-hexane, methylene chloride, ethyl acetate and n-butanol stepwise.

In addition, the present invention provides a health food for prevention and improvement of diabetic complications comprising butein, sulfuretin or their salts as an active ingredient.

Hereinafter, the present invention is described in detail.

The active ingredients of the present invention, which are the extracts of Rhus verniciflua, fractions thereof and isolated compounds therefrom, are prepared by the separation/purification method comprising the following steps :

1) extracting dried Rhus verniciflua with an extraction solvent; 2) filtering and concentrating the extract of Rhus verniciflua of step 1);

3) dissolving the concentrate of step 2) in organic solvents ;

4 ) obtaining fractions by fractionating the dissolved solution of step 3);

5) dissolving the fractions of step 4) in an organic solvent; and

6) obtaining active compounds from the dissolved solutions of step 5) by column chromatography. In the above method, the Rhus verniciflua of step 1) can be any of its parts such as roots, stems, leaves and heartwood, but heartwood and barks are more preferred and either cultured or purchased ones can be used.

In the above method, the extraction solvent of step 1) is water, alcohol or a mixture thereof. At this time,

the alcohol is C1-C4 lower alcohol and the lower alcohol is preferably ethanol. It is preferred for the extraction to add alcohol aqueous solution 5 - 15 times the volume of dried Rhus verniciflua, and more preferably 10 times the volume of dried Rhus verniciflua. After adding ethanol aqueous solution, reflux-cooling is performed preferably for the extraction. Temperature of water bath is preferably 60 ~ 100 °C and more preferably 80 °C , but not always limited thereto. Hours for the extraction are preferably 2 - 6 hours and more preferably 4 hours, but not always limited thereto. Extraction is preferably repeated 1 - 5 times and 4 times is more preferred, but not always limited thereto. After extracting Rhus verniciflua by the above method, the extract is filtered, concentrated and freeze-dried to give Rhus verniciflua extract.

In the above method, the organic solvents of step 3) are added stepwise in the order of n-hexane, methylene chloride, ethyl acetate and n-butanol.

In the above method, the fractions of step 4) are preferably fractionated by using separatory funnel, but not always limited thereto. Every fraction from each organic solvent layer are preferably used and the fraction from acetate layer is more preferred.

In the above method, the organic solvent of step 5) is dichloromethane-methanol (CH2Cl2-MeOH) or hexane-

ethylacetate (Hexane-EtOAc ) , and water-acetonitrile (H2O- Acetonitrile ) is also available if reverse phase chromatography is performed.

In the above method, the column chromatography of step 6) is performed using a filler selected from the group consisting of silica gel, sephadex, RP-18, polyamide,

Toyopearl and XAD resin to separate and purify the active compounds. If necessary, column chromatography is performed several times with different fillers. In a preferred embodiment of the present invention, silica gel column chromatography was performed.

In the above method, the isolated active compounds of step 6) are butein (2', 3, 4, 4'-Tetrahydroxychalcone ) and sulfuretin. In a preferred embodiment of the present invention, the structures of butein and sulfuretin were determined by instrumental analysis (see Formula 1 and Formula 2 ) . [Formula l]

[Formula 2 ]

The present invention provides a preventive and therapeutic agent for diabetic complications containing the extracts of Rhus verniciflua, fractions thereof or isolated compounds therefrom as an active ingredient.

In an example of the present invention, the extracts of Rhus verniciflua, fractions thereof and isolated compounds therefrom, which are butein and sulfuretin, were tested for their effects on the inhibition of aldose reductase. Particularly, the experiment was performed using enzyme precipitates of mouse lens. In addition, to measure the effect on the inhibition of advanced glycation end procuct, the byproduct generated by non-enzymatic cross-linking of bovine serum albumin and glucose was measured by fluorimeter. Particularly, the mouse lens was extracted to prepare aldose reductase. The extracts of Rhus verniciflua, fractions thereof and isolated compounds butein and sulfuretin were added to the aldose reductase, followed by reaction using DL-glyceraldehyde as a substrate. Then, OD ₃₄₀ was measured to calculate the decrease of NADPH.

The advanced glycation end procuct was reacted with bovine serum albumin as a protein source and glucose as a sugar source for 2 days at 60°C. Then, fluorescence analysis was performed with 360 nm of excitation and 440 nm of emission by fluorimeter. As a result, the extracts of Rhus verniciflua, fractions thereof and isolated compounds therefrom (butein and sulfuretin) were all confirmed to have aldose reductase and advanced glycation end procuct inhibitory effect. Among fractions prepared by using different organic solvents stepwise, fractions from ethyl acetate layer demonstrated the highest inhibitory effect (see Tables 1, 2 and 3).

Therefore, the extracts of Rhus verniciflua, fractions thereof and butein and sulfuretin isolated therefrom can be used as a drug for prevention and treatment of diabetic complications caused by osmotic stress resulted from changes of polyol pathway and non- enzymatic glycation of a protein.

The diabetic complications are exemplified by diabetic retinopathy, diabetic neuropathy and diabetic nephropathy.

The diabetic neuropathy is characterized by peripheral neuropathy or autonomic disturbance, and the diabetic nephropathy is exemplified by glomerulosclerosis, proteinuria or renal failure (Michael Brownlee, Nature,

414 , p813 , 2001 ) .

The preventive and therapeutic agent for diabetic complications of the present invention can contain one or more compounds selected from the group consisting of the extracts of Rhus verniciflua, fractions thereof and butein and sulfuretin isolated from those fractions, and additionally one or more active ingredients having the same or similar functions to the above. The extracts of Rhus verniciflua, fractions thereof and butein and sulfuretin isolated from those fractions of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation. The preventive and therapeutic agent for diabetic complications of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing the pharmaceutical composition of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Liquid formulations for oral administrations are suspensions,

solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, lyophilized preparations, suppositories and injections. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, etc. The therapeutic agent of the present invention can be administered by parenterally and the parenteral administration includes subcutaneous injection, intravenous injection and intramuscular injection.

The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose. An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose. The effective dosage of the therapeutic agent of the present invention is 0.0001 ~ 10 g/kg, preferably 0.0001 g

- 5 g/kg, and administration times are 1~6 per day

The preventive and therapeutic agent for diabetic complications of the present invention is evaluated to be a safe substance since its estimated LD ₅₀ value is much greater than 2 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration.

The preventive and therapeutic agent for diabetic complications of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators to prevent and treat diabetic complications.

The present invention also provides a treatment method for diabetic complications containing the step of administering a pharmaceutically effective dose of the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin to a subject with diabetic complications.

The present invention also provides a prevention method for diabetic complications containing the step of administering a pharmaceutically effective dose of the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin to a subject with diabetic complications.

The present invention also provides a use of the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin for the production of a composition for

prevention and treatment of diabetic complications.

The extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin can inhibit aldose reductase and the production of advanced glycation end procuct and has no toxicity, so that they can be effectively used for prevention and treatment of diabetic complications caused by osmotic stress resulted from changes of polyol pathway mechanism and non-enzymatic glycation of a protein.

The present invention also provides a health food for prevention and improvement of diabetic complications which contains the extracts of Rhus verniciflua, fractions thereof or isolated compounds therefrom as a solvent as an active ingredient. The extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin isolated therefrom of the present invention can be used as food additive. In that case, the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin isolated therefrom can be added as it is or as mixed with other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use (prevention, health enhancement or treatment). In general, to produce health food or beverages, the pharmaceutical composition of the present invention is

added preferably by up to 15 weight part and more preferably by up to 10 weight part. However, if long term administration is required for health and hygiene or regulating health condition, the content can be lower than the above but higher content can be accepted as well since the composition has been proved to be very safe.

The food herein is not limited. For example, the composition of the present invention can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included. The composition for health beverages of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. Besides, natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent. The content of the natural carbohydrate

is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 ml of the pharmaceutical composition of the present invention.

In addition to the ingredients mentioned above, the extracts of Rhus verniciflua, fractions thereof, butein or sulfuretin isolated therefrom of the present invention can include in variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The extracts of Rhus verniciflua, fractions thereof or compounds isolated therefrom of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 0.01-0.1 weight part per 100 weight part of the composition of the present invention.

[Advantageous Effect]

The pharmaceutical composition containing the extracts of Rhus verniciflua, fractions thereof or compounds isolated therefrom of the present invention as an

active ingredient can be effectively used as a preventive and therapeutic pharmaceutical composition for diabetic complications and related diseases because they can inhibit aldose reductase to prevent the accumulation of sorbitol which is the fundamental cause of renal failure and neuropathy, common complications of diabetes, inhibit non- enzymatic glycation to suppress advanced glycation end procuct without toxicity.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1; Preparation of Rhus verniciflua extracts <!-!> Preparation of Rhus verniciflua ethanol extract

Dried Rhus verniciflua was purchased from Kyungdong market, Seoul, Korea and then pulverized. 1.5 kg of the dried Rhus verniciflua and 16.0 I of 95% ethanol aqueous solution were added to the extraction vessel, followed by reflux-cooling (temperature of water bath was 80°C). The

solution was filtered with a filter paper to give the extract. The extraction was repeated 4 times, for 4 hours per each extraction. The solvent was concentrated under reduced pressure to give 130.0 g of ethanol extract.

<1-2> Preparation of Rhus verniciflua water extract

1.5 kg of the dried Rhus verniciflua and 16.0 l of water were added to the extraction vessel, followed by extraction at 90 ~ 100 °C two times, for 4 hours per each extraction. The mixture was filtered with a filter paper to give the extract. The solvent was concentrated under reduce pressure to give 120.0 g of water extract.

<1-3> Preparation of Rhus verniciflua methanol extract 1.5 kg of the dried Rhus verniciflua and 16.0 l of methanol were added to the extraction vessel, followed by reflux-cooling (temperature of water bath was 80°C). The solution was filtered with a filter paper to give the extract. The extraction was repeated 4 times, for 4 hours per each extraction. The solvent was concentrated under reduced pressure to give 128.0 g of methanol extract.

Example 2 : Preparation of fractions from the Rhus verniciflua extracts The Rhus verniciflua extracts prepared in Example 1

were fractionated into n-hexane (34.0 g) , methylene chloride (13.3 g), ethyl acetate (21.9 g), n-butanol (7.5 g) and water (2.9 g) fractions.

Example 3 ; Preparation of compounds from the fractions

9 g of the ethyl acetate fraction prepared in Example 2 proceeded to silica gel column chromatography (Kiesel 60, Merck) of 6 cm in the inside diameter. Dichloromethane- methanol (CH2Cl2-MeOH) gradient was used as a developing solvent. Crystallization of the eluted fraction was performed at dichloromethane:methanol = 8:2. The structure was determined by nuclear magnetic resonance (NMR) . As a result, the compound was identified as butein, further confirmed by comparing it with the standard compound (see Formula 1 ) .

In addition, 9 g of the ethyl acetate fraction prepared in Example 2 proceeded to silica gel column chromatography (Kiesel 60, Merck) of 6 cm in the inside diameter. Dichloromethane-methanol (CH2Cl2-MeOH) gradient was used as a developing solvent. Crystallization of the eluted fraction was performed at dichloromethane:methanol = 8.5:1.5. The structure was determined by nuclear magnetic resonance (NMR) . As a result, the compound was identified as sulfuretin, further confirmed by comparing it with the standard compound (see Formula 2).

Experimental Example 1 : Investigation of aldose reductase inhibitory effect of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom To investigate aldose reductase inhibitory effect of the composition of the present invention, experiments with enzyme precipitates of the mouse lens were performed. First, to obtain aldose reductase, the lens was extracted from a mouse. Phosphate buffer was added to the extracted lens according to the wet weight, followed by homogenization. The homogenate was centrifuged at 4 °C to obtain supernatant, to which ammonium sulphate was added at the final concentration of 40%. The mixture was centrifuged again to obtain supernatant, to which ammonium sulphate was added at the final concentration of 70%, followed by mixing for 1 hour. Then, the mixture was centrifuged to obtain precipitate. The obtained precipitate was suspended in PBS, followed by dialysis for one day. As a result, aldose reductase was obtained. The extracts, fractions thereof, butein and sulfuretin isolated therefrom were added respectively to the prepared aldose reductase, which were reacted with DL-glyceraldehyde as a substrate. OD ₃₄₀ was measured to calculate NADPH level. At this time, tetramethylene glutarate (T3385, Sigma) was used cis a control. OD ₃₄₀ was measured at 1 minute

intervals for 7 minutes of reaction.

Calculation method of inhibitory effect: (δsample treated group-δsample not-treated group) / (δsample not- treated group-δsubstrate not-treated group) x100

As a result, as shown in Table 1 and Table 3, the inhibitory effect of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom was confirmed. Among the fractions prepared in Example 2, the ethyl acetate fraction demonstrated the highest inhibitory effect.

[Table 1] Aldose reductase inhibition rates of the extracts of Rhus verniciflua and organic solvent fractions thereof

(%)

Inhibition

Composition Concentration (μg/mL) IC ₅₀ rate(%) (μg/mL)

Control TMG 10 96.00 0.02

Example <1- Ethanol

10 95.65 1.61 1> extract

Example <1- Water

10 92.05 2.08 2> extract

Example <1- Methanol

10 93.56 1.70 3> extract n-hexane 10 32.24 -

Example 2

Methylene 10 86.96 4.47 chloride

IC ₅₀ : 50% inhibition concentration of aldose reductase

TMG : control ( tetramethylene glutaric acid)

Experimental Example 2 ; Investigation of advanced glycation end procuct inhibitory effect of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom The advanced glycation end procuct was reacted with bovine serum albumin as a protein source and glucose as a sugar source for 2 days at 60°C. Then, fluorescence analysis was performed with 360 nm of excitation and 440 nm of emission by fluorimeter (see Tables 2 and 3) (C-H. Wong, et al., Bioorganic & Medicinal Chemistry, 11, p5317, 2003). Aminoguanidine was used as a control. The inhibition rate was 78.4% at 2.5 mM and IC ₅₀ was 960 μM.

[Table 2] Advanced glycation end procuct inhibition rates of

the extracts of Rhus verniciflua and organic solvent fractions thereof (%)

IC ₅₀ : 50% inhibition concentration of advanced glycation end procuct

[Table 3]

Aldose reductase and advanced glycation end procuct inhibition rates of butein and sulfuretin

Experimental Example 3: Cytotoxicity test of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom

Cytotoxicity test was performed according to the conventional method well-informed to those in the art (Korba, B, E, et al., 19, 55-70, 1992) to investigate cytotoxicity of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom prepared in the above examples by examining their activities in cells.

Particularly, 100 μl of cells (2 X 10 ⁴ ) were distributed in each well of a 24-well plate, followed by culture in a 37 °C incubator for 24 hours. Different concentrations of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom were treated to the cells for 3 days. Then, the culture medium was removed and 20 μl of staining solution (tetrazolium compound, inner salt and phenazine ethosulfate) was assed

to each well, followed by reaction in the incubator for 1 hour. The plate was washed with phosphate buffer twice to eliminate the remaining staining solution. 20 μl of fixing solution (50% ethanol and 1% glacial acetic acid) was added thereto, followed by stirring for 30 minutes. OD _5I0 was measured by spectrophotometer, which was compared with OD of the control not treated with the said extracts to determine CC ₅₀ (50% cytotoxic concentration) of the extracts by detecting the concentration of the extract that reduced OD 50%. As a result, the concentration that caused cytotoxicity was very low (see Table 4).

[Table 4]

Experimental Example 4 ; Acute toxicity test of the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom in rats via oral administration

The following experiments were performed to see if the extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom have acute toxicity in rats. 6-week old SPF SD line rats were used in the tests for acute toxicity. The extracts of Rhus verniciflua, fractions thereof and compounds isolated therefrom were suspended in 0.5% methylcellulose solution and orally administered once to rats at the dosage of 2 g/kg/15 ml. Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with naked eyes during autopsy.

The results showed that the test samples did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy.

The compounds used in this experiment were evaluated to be safe substances since they did not cause any toxic change in rats up to the level of 2 g/kg and their estimated LD ₅₀ values were much greater than 2 g/kg in rats.

The Manufacturing Examples of the composition of the present invention are described hereinafter.

Manufacturing Example 1 : Preparation of pharmaceutical formulations

<1-1> Preparation of powders

Rhus verniciflua extract 2 g Lactose 1 g

Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders .

<1-2> preparation of tablets

Rhus verniciflua extract 100 rag

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg Tablets were prepared by mixing all the above components by the conventional method for preparing tablets.

<1-3> Preparation of capsules

Rhus verniciflua extract 100 mg Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules.

<1-4> Preparation of pills

Rhus verniciflua extract 1 g

Lactose 1.5 g Glycerin 1 g

Xylitol 0.5 g

Pills were prepared by mixing all the above components according to the conventional method for preparing pills. Each pill contained 4 g of the mixture.

<1-5> Preparation of granules

Rhus verniciflua extract 150 mg

Soybean extract 50 mg

Glucose 200 mg Starch 600 mg

All the above components were mixed, to which 100 mg of 30% ethanol was added. The mixture was dried at 60°C and the prepared granules were filled in packs .

Manufacturing Example 2 : Preparation of food

Foods containing the Rhus verniciflua extract were prepared as follows.

<2-l> Preparation of spices for cooking Health enhancing spices for cooking was prepared with

20 ~ 95 weight part of the Rhus verniciflua extract according to the conventional method.

<2-2> Preparation of tomato ketchup and sauce Health enhancing tomato ketchup or sauce was prepared by mixing 0.2 ~ 1.0 weight part of the Rhus verniciflua extract with tomato ketchup or sauce according to the conventional method.

<2-3> Preparation of flour food

0.5 ~ 5.0 weight part of the Rhus verniciflua extract was added to the flour. Health enhancing foods such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method,

<2-4> Preparation of soups and gravies

0.1 ~ 5.0 weight part of the Rhus verniciflua extract was added to soups and gravies. Health enhancing meat products, soups and gravies were prepared with this mixture by the conventional method.

<2-5> Preparation of ground beef

Health enhancing ground beef was prepared by mixing 10 weight part of the Rhus verniciflua extract with ground beef according to the conventional method.

<2-6> Preparation of dairy products

5 ~ 10 weight part of the Rhus verniciflua extract was added to milk. Health enhancing dairy products such as butter and ice cream were prepared with the milk mixture according to the conventional method.

<2-7> Preparation of Sun-Sik

Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders.

Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders. The Rhus verniciflua extract was concentrated under reduced pressure, spray-dried and pulverized to obtain 60- mesh dry powders.

Sun-Sik was prepared by mixing the dry powders of the grains, seeds and the Rhus verniciflua extract according to the below ratio.

Grains (brown rice: 30 weight part, Yulmu: 15 weight part, barley: 20 weight part),

Seeds (wild sesame: 7 weight part, black soybean: 8 weight part, black sesame: 7 weight part), Dry powders of the compound isolated from the Rhus

verniciflua extract (3 weight part),

Ganoderma lucidum (0.5 weight part), Rehmannia glutinosa (0.5 weight part)

Manufacturing Example 3 : Preparation of beverages <3-l> Preparation of health beverages

Rhus verniciflua extract 1000 mg

Citric acid 1000 mg

Oligosaccharide 100 g Maesil (Prunus mume) Extract 2 g

Taurine 1 g

Purified water up to 900 ml

The above constituents were mixed according to the conventional method for preparing health beverages. The mixture was heated at 85 °C for 1 hour with stirring and then filtered. The filtrate was loaded in 2 liter sterilized containers, which were sealed and sterilized again, stored in a refrigerator until they would be used for the preparation of a composition for health beverages. The constituents appropriate for favorite beverages were mixed according to the preferred mixing ratio but the composition ratio can be adjusted according to regional and national preferences, etc.

<3-2> Preparation of vegetable juice

Health enhancing vegetable juice was prepared by adding 5 g of the Rhus verniciflua extract of the present invention to 1,000 ml of tomato or carrot juice according to the conventional method.

<3-3> Preparation of fruit juice

Health enhancing vegetable juice was prepared by adding 1 g of the Rhus verniciflua extract of the present invention to 1,000 ml of apple or grape juice according to the conventional method.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.