KR101074555B1 - The preparing method of extracts of momordica charantia for treating gout and the extracts prepared thereof - Google Patents

Abstract

The present invention relates to a method for preparing gout extracts (momordica carantia, bitter cucumber) and to extracts produced by the method.

In more detail, the method for preparing gout extract for gout treatment of the present invention and the bitum extract prepared by the method have the effect of effectively lowering the concentration of uric acid, which is an indicator of gout, in the blood to treat gout, and also the present invention. Yeoju extract has the effect of preventing inflammation in gout by inhibiting the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).

Litchi extract of the present invention is a concentrated extract of the dried fermented fruit using 70% ethanol, characterized in that extracted in the form that can be administered to the human body.

 Momordica charantia, gout, uric acid

Description

Method of producing gout extracts for gout treatment and extracts produced by the same method {TH PREPARING METHOD OF EXTRACTS OF MOMORDICA CHARANTIA FOR TREATING GOUT AND THE EXTRACTS PREPARED THEREOF}

The present invention relates to a method for preparing gout extracts (momordica carantia, bitter cucumber) and to extracts produced by the method.

Yeoju (Momordika Charantia, Bitter Cucumber) is a perennial vine plant of ferns widely cultivated in Asia and rarely planted in Korea, but in China, fruit has been used for food and medicinal use since ancient times. In addition, as a specialty of tropical countries such as India, South Africa, the Philippines and Vietnam, the Momordica species found in the West are different in form and characteristics from the above tropical species.

Various parts of this plant, especially fruit, have been used in hypoglycemic pharmaceutical compositions. Fruits are long oval, narrow at both ends, hump-like projections, and ripened to reddish red, irregularly split, and covered with red flesh. It is called Yeoju because the fruit is similar to Yeoji. The young fruit and red seedlings are used for food and the seeds are used for medicinal purposes. Recently, it has been reported that it can be used to treat diabetes. (Miura T. et.al., J. Nut. Sci. Vitaminol, 47 (5), 340-344, 2001; Grover JK, et. Al., J. Ethnopharmacol., 76 (3), 233-238, 2001)

Yeast contains 120 milligrams of vitamin C, which is much higher than strawberries (80 mg / 100 g), lemons (90 mg / 100 g) and cabbage (40 mg / 100 g). Especially, vitamin C in Yeoju is not destroyed by applying heat. Yeast also contains minerals such as beta-carotene and iron and potassium, which are converted to vitamin A in the body, but especially important components of yeast are vegetable insulins that help to burn glucose and prevent glucose from resynthesizing in the body. It is charantin, a fat-soluble component that activates the pancreas to secrete insulin. Vegetable insulin acts similar to insulin in the body and is a kind of peptide that is contained in fruits and seeds.

Although Momordica sp . Is a subject that has been studied extensively worldwide, it is mainly being studied and developed as a hypoglycemic agent (Patent Publication No. 10-2007-0004569, Patent Registration No. 10). -0831621, Patent Registration No. 10-0509438, Patent Publication No. 10-2004-0081733, Patent Registration No. 10-0749944.

Gout is a metabolic disease in which the crystals of uric acid, formed by long-lasting high levels of uric acid in the blood, cannot be discharged out of the body and accumulate in various tissues causing various symptoms. An increase in uric acid in the blood may occur due to excessive production of uric acid and abnormal excretion through the kidneys. High levels of uric acid in the blood does not cause gout immediately, but high levels of uric acid make uric acid crystals easily deposited on various tissues for 10 to 20 years, causing gout by triggering factors. This is common among men in their 4's and 50's, but the onset age is getting lower due to dietary changes and environmental changes.Women's renal function is poor after long-term post-menopausal or long-term use of diuretics. Are most likely to develop in.

Looking at the causes of this gout, most of the cells that make up our body has a nucleus, which consists of a nucleic acid containing genetic information. Nucleic acid consists of purine or pyrimidine, which is destroyed at the end of cell life, and breaks down the purine body in the nucleus to make large amounts of uric acid, and crystals of sodium urate deposit on tissues It is known to cause hyperuricemia, gouty arthritis, gout renal disease, gout nephrolithiasis (Yagi K., Chem . Phys . Lipids , 45, p337, 1987).

During metabolism of the purine body, adenine is made of uric acid via hypoxanthine and xanthine, and guanine is directly metabolized to xanthined and decomposed into uric acid, where xanthine oxidase) is known to be involved. When xanthine oxidase converts xanthine to uric acid, it generates a large amount of superoxide radicals, which give oxidative stress to surrounding cells, resulting in gout, hypertension, hyperlipidemia, arteriosclerosis, diabetes It is known to be accompanied by such comorbidities (Storch J et al., Anal. Biochem., 169, p262, 1988). (See picture below)

The pathway in which uric acid is produced can be broadly divided into external uric acid (from the purine body contained in the ingested food) and endogenous uric acid (from the cells destroyed by the body). Most of the uric acid produced is excreted in the urine through a complex process in the kidneys, and problems arise when the balance between production and discharge is broken. In normal men, blood uric acid concentrations are below 7-8 mg / dL, and in normal women, blood uric acid concentrations are less than 6 mg / dL.

Blood uric acid levels are individual differences, but if more than 8 mg / 통증 pain appears, there is a risk of causing vascular disorders of the heart, kidneys, brain, seizures occur more than 10 mg / ㎗. Gout is the most common symptom of arthritis, but it can also cause acute or chronic deterioration of renal function.

     Colchicine (used when the diagnosis is not clear), nonsteroidal anti-inflammatory drug (used when there is no complication and the diagnosis is clear, but there are many potential side effects such as peptic ulcer, renal dysfunction, and edema), steroid ( In single arthritis, it is usually used for intraarticular injection or intramuscular injection.

Modern medicine, however, has not been developed to treat gout fundamentally. However, antihyperacids include uric acid production inhibitor "allopurinol" and uric acid renal excretion promoter "probenecid", but allopurinol has skin rash, gastrointestinal disorders, myelosuppression, itching, nausea and muscle pain. There are serious side effects such as, there is a need for the invention of a material that can prevent and treat gout safe and without side effects.

Gout is a metabolic and chronic disease that cannot be cured with short-term treatment, so it is a regulated disease that requires continuous treatment. As described above, excessive side effects have been derived from existing commercial medicines. And the development of dietary supplements.

In the inflammatory process, nitric oxide (NO) and prostaglandin-2 (prostaglandin E2, PGE2) are known as important pro-inflammatory mediators.

In mammalian cells, nitric oxide synthase (NOS) has three types of isoforms, namely type I, type II and type III type nitric oxide synthases (NOS). Type I (nNOS) and Type III (eNOS) are classified as cNOS because they are constantly present in cells, but type II inducible nitric oxide synthase (iNOS) is a cytokine (cytokine) and lipopolysaccharide (LPS) It is expressed after exposure of certain stimuli. In NOS, iNOS is primarily involved in the inflammatory response.

Cyclooxygenase (COX) converts arachidonic acid to prostagladin (PG). Like NOS, COX exists as two isomers, COX-1 and COX-2. COX-2 is very low in normal cells, but is induced by various stimuli (cytokine, endotoxin, mitogen) in inflammation-related cells and is involved in the production of PGs that are involved in pain or inflammation. In particular, Arachidonic acid is converted to PG by cyclooxgenase, which is later synthesized as prostacycline and thromboxane. Aspirin and acetaminophen, which are used as anti-inflammatory drugs, are well known as COX-2 inhibitors.

Many pharmaceutical compositions have been developed to inhibit the expression of COX-2 and iNOS to prevent inflammation. (US 6,248,745 B1 (AstraZeneca AB), June 19, 2003)

Gout is a metabolic and chronic disease that cannot be cured with short-term treatment, so it is a regulated disease that requires continuous treatment. As described above, excessive side effects have been derived from existing commercial medicines. And the development of dietary supplements.

In addition, the conventional method of refining natural products mainly used organic solvents, but since there is residual or toxic and inverse smell of organic solvents, it was difficult to use them directly for food and medicinal use to extract the form that can be administered to the human body. Is required.

In order to achieve the above object, the present invention is characterized in that the ethanol extract is produced by using ethanol, and the extract of the present invention is an inos (iNOS: Inducible nitric oxide synthase) and cox- 2 (COX-2: Cyclooxigenase-2) inhibits the expression and reduces the amount of uric acid (uric acid) in the blood, it is intended to use for the treatment of gout and inflammation in gout.

Yeoju extract according to the present invention inhibits inos (iNOS: Induciblenitric oxide synthase) and Cox-2 (COX-2: Cyclooxigenase-2) involved in the inflammatory reaction and reduces the amount of uric acid in the blood It is effective in treating gout and preventing inflammation in gout.

( Example )

Hereinafter, the actual experimental example of the present invention will be described in detail.

The following experimental example illustrates the experimental results for the therapeutic effect through the in vivo gout induction experiment using the alcohol extract.

[ Example  1] Preparation of Yeoju extract using ethanol

In order to use Korean Yeoju (Gyeongnam Hamyang) for food and medicine, ethanol and ethanol were used as extraction solvents. The raw material of Yeoju is finely pulverized to 40 mesh or less, dried to 10% or less of water content, and 5 to 10 times 95% medicinal alcohol or alcohol alcohol (stored at -20 ° C, After the first extraction at 4 ° C. for at least 24 hours and centrifugation, the ethanol layer, the supernatant, is discarded, and 5 to 10 times 70% ethanol (-20 times the amount of precipitate not dissolved in ethanol). And ethanol layer obtained by secondary extraction at 4 ° C. for 12 hours or more were evaporated to dryness to obtain a highly viscous material. By the above extraction method was able to obtain about 6 ~ 7 g of the extract extract per 100 g of dried Yeoju dry powder, using the extract was carried out the following experiment.

[ Example  2] Inflammatory Inhibitory Effects of Gout-induced Animal Models on Hepatocytes

In order to confirm the inhibitory effect of the inflammatory protease iNOS and Cox-2 expression of the Yeoju extract of the present invention, after inducing hyperuricemia by administering a potassium oxonate (potassium oxonate) inhibitor, the blood uric acid concentration of Yeoju extract A comparative experiment was performed using a gout disease model administered with allopurinol (Allopurinol, Samil Pharm.), A uric acid synthesis inhibitor, as a positive control for the reducing effect.

2-1. Experimental animal

    The experimental animals were 9-week-old male Sparague-dawley rats, males, 250-280, kept in a SPF chamber maintained at a humidity of 50 ± 5% and a temperature of 24 to 26 ° C. g, Samtaco) was used, and was used in the experiment after being adapted in a laboratory environment for one week with sufficient feed and water.

2-2. Inhibitory Effects of Yeoju Extracts on iNOS and COX-2 Protein Expression

     In this experiment, all groups except the normal control group were treated with potassium oxonate (150 mg / kg), a uric acid-lowering enzyme preparation, intraperitoneally the day before the sample to induce hyperuricemia. To confirm the induction of hyperuricemia after administration of potassium oxonate, the uric acid content before and after administration of potassium oxonate was measured and compared.

Potassium oxonate was suspended in 0.5% sodium cearboxymethylcellulose (CMC-Na, 0.5% CMC with 0.1M sodium acetate (pH 5)). The experimental animals were divided into five groups, and were treated with normal control, hyperuricemia control (Model control), allopurinol administration (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group ( Drug 100) divided into five groups for each group was tested.

      Allopurinol and Yeoju extract administration groups were allopurinol and Yeoju extracts suspended in PBS containing 0.1% tween 80, respectively, orally for 3 days after induction of hyperuricemia, and the hyperuricemia control group was replaced with the same amount of solvent instead of Yeoju extract. . The allopurinol-administered group was orally administered in two concentrations of 50 mg / kg and Yeoju extract, 50 mg / kg and 100 mg / kg, respectively, based on the dosage of the commercially available drug.

After oral administration for 3 days, the animals were fasted one night, anesthetized with dry ice, and then opened, and the liver was extracted, weighed, and then frozen and stored at -80 ° C for freezing. 100 mg of the total liver (liver) was taken to separate proteins using cell lysis buffer, and the proteins were measured using BCA protein assay. The same amount of protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membrane (PVDF). Transfer completed PVDF membrane was reacted with PBST (0.01% polyoxyethylene sorbitan monooleate-0.01M phosphate buffered saline) containing 5% non-fat dry milk for 1 hour to block nonspecific proteins. Subsequently, the primary antibody was reacted overnight, and then reacted with anti-goat IgG and anti-rabbit IgG, which are secondary antibodies to each antibody, for 1 hour. Each reaction was washed 5 times with PBST for 5 minutes. After the final washing, the corresponding protein bands for each antibody were expressed using a supersignal west pico chemiluminescent substrate (Pierce, Rockford, IL, USA).

As a result, as shown in Figure 1, the expression of COX-2 protein in liver tissue than the allopurinol administration group (Positive contorl) in the Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) This concentration-dependent inhibition was found, especially COX-2 protein expression was significantly reduced in Yeoju extract 100 mg / kg administration group (Drug 100), iNOS protein expression was reduced in a similar trend to COX-2 protein expression I could confirm it. In particular, the control drug allopurinol administration group (Positive contorl) rather increased the expression of inflammatory enzymes iNOS and COX-2, which is considered to be part of the side effects of commercial drugs. Therefore, based on the above experimental results, it can be seen that the extract of Yeoju has an effect of suppressing the inflammatory response due to gout. (See Figure 1).

[ Example  3] Blood and in Gout-Induced Animal Models Urinary  Uric Acid Reduction Experiment

      In order to confirm the anti-gout activity of the extract of Yeoju of the present invention, an experiment was performed in which blood and urine uric acid contents were changed using a gout disease model.

     Induced hyperuricemia by administering potassium oxonate, a uric acid-degrading enzyme inhibitor, to experimental rats. The positive control group was administered an allopurinol (tripurinol), an inhibitor of uric acid synthesis, and the anti-gout efficacy was compared by measuring the amount of uric acid in the blood.

3-1. Experimental animal

     Experimental animals used a 9-week-old male Sparague-dawley rat (male, 250-280 g, Samtaco) bred in an SPF chamber maintained at a humidity of 50 ± 5% and a temperature of 24 to 26 ° C. Adapted in a laboratory environment for 1 week while supplying enough feed and water, it was used for the experiment.

3-2. Blood Uric Acid Lowering Effect Test of Yeoju Extract

      In this experiment, all groups except the normal control group were administered uric acid degrading enzyme potassium oxonate (150 mg / kg) intraperitoneally one day before the sample to induce hyperuricemia. To confirm the induction of hyperuricemia after administration of potassium oxonate, the uric acid content before and after administration of potassium oxonate was measured and compared.

Potassium oxonate was suspended in 0.5% sodium carboxymethylcellulose (CMC-Na, 0.5% CMC with 0.1M sodium acetate (pH 5)). The experimental animals were divided into five groups, and were treated with normal control, hyperuricemia control (Model control), allopurinol administration (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group ( Drug 100) divided into five groups for each group was tested.

      Allopurinol and Yeoju extract administration groups were allopurinol and Yeoju extracts suspended in PBS containing 0.1% tween 80, respectively, orally for 3 days after induction of hyperuricemia, and the hyperuricemia control group was replaced with the same amount of solvent instead of Yeoju extract. . The allopurinol-administered group was orally administered in two concentrations of 50 mg / kg and Yeoju extract, 50 mg / kg and 100 mg / kg, respectively, based on the dosage of the commercially available drug.

The urine (urine) of each experimental animal was collected within 2 hours after the last oral administration, and the serum was immediately dissected to measure the concentration of uric acid in the blood and urine to evaluate uric acid concentration, and to evaluate the renal dysfunction. In order to measure the concentration of creatinine and gamma glutamyl transferase (γ-GT) was measured (DRI-CHEM, FUJIFILM, Model No. 3200).

After blood collection, liver tissue was fixed by perfusion and then separated and used as a material for evaluating anti-inflammatory markers at the cellular level. Urinary uric acid concentration was slightly higher in Yeoju extract 100 mg / kg administration group (Drug 100), but this could be seen as an error according to the metabolic rate of the experimental animal and lower than the hyperuricemia control (Model control).

Blood uric acid concentrations in the 50 mg / kg group (Drug 50) and the 100 mg / kg group (Drug 100) were reduced to levels of allopurinol-positive control. Considering the various side effects, it is considered a very useful substance to replace it. (See Figure 2)

Figure 3 is the normal control, hyperuricemia control (Model control), allopurinol administration group (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) It is a graph showing the concentration of creatinine (mg / dl), which shows the result of decreasing the concentration of creatinine in blood in the allopurinol-treated group (Positive contorl) and Yeoju extract 100 mg / kg group (Drug 100) compared to the model of hyperuricemia control (Model control). It was. There is no significant decrease in creatinine in the Yeoju extract 50mg / kg group (Drug 50), but the Yeoju extract 100mg / kg group (Drug 100) shows a significant reduction effect.

Figure 4 is the γ of normal control, hyperuricemia control (Model control), allopurinol administration group (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) It is a graph showing the blood concentration of -GT (U / L), and the blood concentration of γ-GT is normal, model control, allopurinol administration, and Yeoju extract administration. 50, 100) did not show a significant difference.

Therefore, if you look at the blood concentration of creatinine and γ-GT to determine the renal function abnormality, it is determined that the possibility of serious side effects affecting the kidney function of the Yeoju extract of the present invention is all within the normal range. (See Figs. 3 and 4)

1 is a normal control, hyperuricemia control (Model control), allopurinol administration group (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) Figure showing the expression level of the induced enzymes iNOS and COX-2.

Figure 2 is the concentration of uric acid in the blood and urine in allopurinol administered group (Positive contorl), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) compared to the hyperuricemia control group (Model control) Graph showing (mg / dl).

Figure 3 is the normal control, hyperuricemia control (Model control), allopurinol administration group (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) Graph showing concentration of creatinine (mg / dl).

Figure 4 is the γ of normal control, hyperuricemia control (Model control), allopurinol administration group (Positive control), Yeoju extract 50 mg / kg administration group (Drug 50), Yeoju extract 100 mg / kg administration group (Drug 100) -Graph showing blood levels of GT (U / L).

Claims (9)

delete delete delete delete delete Grinding the bitter gourd to 40 mesh or less and drying it to 10% or less of water content; 95% ethanol for primary extraction of 5 to 10 times the amount of dried litchi was added to the dried litchi and left at 4 ° C. for at least 24 hours, followed by centrifugation to remove the ethanol layer, the supernatant, and 95% for primary extraction. Obtaining a precipitate insoluble in% ethanol; And 5 to 10 times the second extraction of 70% ethanol for secondary extraction to the precipitate and left at 4 ℃ for more than 12 hours after the step of evaporating and drying the ethanol of the ethanol layer comprising the step of producing a highly viscous extract Method for preparing gout extract for gout treatment, characterized in that. The method of claim 6, wherein the 95% ethanol for primary extraction and 70% ethanol for secondary extraction are medicinal alcohols stored at -20 ° C. The method of claim 6, wherein the 95% ethanol for the first extraction and 70% ethanol for the second extraction is a ethanol alcohol stored at -20 ° C. A gout extract for treating gout prepared by the method of any one of claims 6 to 8 gout for extract.

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