KR100645391B1 - A isolation method triterpenoid from rumex japonicus and a triterpenoid - Google Patents

Abstract

A method of isolating a triterpenoid compound from Rumex japonicus Houtt. and a triterpenoid compound are provided to restrain the production of the advanced glycation end products(AGEs) and be used for treating and preventing diabetes complications. The triterpenoid compound separated from Rumex japonicus Houtt. is characterized by 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norurc-4(23),12-dien-28-oicacid. The method of isolating a triterpenoid compound comprises the steps of: (a) obtaining crude extract of Rumex japonicus Houtt. by reduced pressure concentration, after extracting the Rumex japonicus Houtt. using 20-80% alcohol; (b) suspending the crude extract in distilled water, and obtaining normal hexane soluble fraction by adding normal hexane and separating normal hexane layer; (c) obtaining ethylacetate soluble fraction by adding ethylacetate in layer remaining after separating normal hexane layer and separating the ethylacetate layer; (d) obtaining 12 number of fractions by performing silica gel chromatography using concentration gradient of chloroform:methanol mixing solvent; (e) obtaining 10 number of fractions by subjecting no.5 fraction among 12 number of fractions to silica gel chromatography using concentration gradient of normal hexane:ethylacetate:methanol mixing solvent; (f) obtaining 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norurc-4(23),12-dien-28-oicacid by subjecting no.3 fraction among fractions of the step(e) to reverse silica gel chromatography using methanol:water mixing solvent.

Description

Isolation method of triterpenoid compounds isolated from Chamsori and A triterpenoid compound thereof {A isolation method triterpenoid from Rumex japonicus and A triterpenoid}

1 is a structural formula of 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid, isolated from Chamsori, according to the present invention,

2 is 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid cosy and HMBC isolated from Chamsori, according to the present invention.

Figure 3 is the NOE of 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid, isolated from Charsoridae according to the present invention,

Figure 4 shows the inhibition rate of the final glycation product production of 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid isolated from the sangryukja according to the present invention Is a graph representing

5 is a graph showing the inhibition rate of the final glycation product production of aminoguanidine.

The present invention relates to a triterpenoid compound that is isolated from Chamsori, and more particularly, 2 alpha, 3 alpha, 19 alpha-tree, which are new triterpenoid compounds isolated from Chamsori. Hydroxy-24-Norrus-4 (23), 12-diene-28-oic acid is used to inhibit the formation of AGEs, a major factor in the development of diabetic complications, and is effective in preventing or treating diabetic complications and preventing or delaying aging. The present invention relates to a triterpenoid compound which is isolated from Chamsori, and a method for separating the same.

Diabetes is one of the most important adult diseases all over the world. In recent years, the prevalence of diabetes mellitus and the prevalence of diabetes have reached 7-8% in Korea, especially since the overall lifespan until complications is prolonged. It became impossible. In general, almost 10 to 20 years after diabetes, almost all organs in the body are damaged, resulting in diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. diabetic neuropathy). Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end stage renal failure, and diabetic cataracts cause blindness and eventually death.

Mechanisms that induce diabetic complications are largely explained by nonenzymatic glycation of proein, polyol pathway and oxidative stress.

Nonenzymatic glycation of protein refers to the condensation reaction of a group of amino acids such as protein lysine residues and reducing sugars by enzymatic reaction, that is, milliard reaction, without enzymatic action. As a result of this reaction, advanced glycation endproducts, AGEs). Non-enzymatic glycosylation of protein (1) an amino acid group such as a lysine residue of a protein and an aldehyde or ketone of a reducing sugar undergo a nucleophilic addition reaction without enzymatic action to form a Schiff base, an early product. When the Schiffbase and the adjacent ketoamine adduct condensate with each other to produce a reversible Amadori type of early glycosylated product, and (2) the hyperglycemic state is continued, and a reversible Amadori type of early glycated product is produced. It can be divided into steps that are rearranged without degradation and cross-linked with the protein to produce an irreversible final glycation product.

Unlike the reversible Amadori type of early glycosylated products, the final glycosylated product is an irreversible reaction product. Once produced, blood sugar does not decompose even when it returns to normal, but accumulates in the tissues during protein survival, causing abnormal changes in tissue structure and function. Causes complications throughout tissues (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur . J. Biochem ., 210: 729-739).

For example, glycated albumin, one of the final glycation products produced by the reaction of glucose and various proteins, is an important factor in causing chronic diabetic nephropathy. Glycosylated albumin enters renal glomeruli more easily than normal albumin without glycosylation, and high concentrations of glucose stimulate mesangium cells to increase extracellular matrix synthesis. Excessly introduced glycated albumin and increased extracellular matrix result in fibrosis of the glomeruli. These mechanisms continue to damage the glomerulus, leading to the stage of extreme treatment methods such as hemodialysis or organ transplantation. In addition, chronic diabetes has been reported to accumulate collagen in the arterial wall and basal membrane protein in the renal glomeruli and to accumulate in tissues (Brownlee, M., et al., 1986, Sciences , 232, 1629-). 1632).

As described above, glycosylation occurs in proteins such as basement membrane, plasma albumin, lens protein, fibrin, and collagen by non-enzymatic protein glycosylation, and the resulting glycosylated product abnormally changes the structure and function of the diabetic diabetic retinopathy. chronic diabetes complications such as retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy.

In addition, it has been reported that lipid metabolism abnormalities occur in the process of producing the final glycated product in hyperglycemic state and oxidative stress is caused by deterioration of defense system function against harmful oxygen free radicals (Yokozawa, T., et. al, 2001, J. of Trad . Med ., 18: 107-112). As such, the mechanism of action of non-enzymatic glycosylation and oxidative stress is related.

The polyol pathway is a process consisting of (1) reduction from aldose or ketose by aldose reductase (AR) to form sorbitol, and (2) sorbitol is oxidized by sorbitol dehydrogenase to produce fructose. . Hyperglycemia activates the polyol pathway, resulting in excessive production of sorbitol and fructose and accumulation in tissues resulting in increased osmotic pressure. Increased osmotic pressure causes water to enter, leading to diabetic retinopathy, diabetic cataract, and diabetic neuropathy (Diabetes, Kim Eung-jin, et al. Medicine, p. 483; Soulis-Liparota, T., et al., 1995, Diabetologia , 38: 357-394).

Final glycation end products have been reported to activate aldose reductase (AR), the main enzyme of the polyol pathway, in human microvascular endothelial cells (Nakamura, N., et al., 2000, Free Radic) Biol . Med ., 29: 17-25). In a steady state, aldose reductase has a very low affinity for glucose, but at high concentrations of glucose, aldose reductase is activated to excessively reduce glucose to sorbitol, and this sorbitol is converted to fructose by sorbitol dehydrogenase. This fructose is about 10 times faster than non-enzymatic glycosylation of protein. Thus, high concentrations of fructose bind to the protein and eventually accelerate the formation of the final glycated product.

As such, non-enzymatic glycosylation, polyol pathways and oxidative stress mechanisms of action are linked to each other to cause diabetic complications. Therefore, it has been found that it is very important to inhibit the formation of final glycation end products in order to delay, prevent or treat the development of diabetic complications (Brownlee, M., et al., 1988, N. Engl. Med . , 318, 1315-1321).

Currently, aminoguanidine, a synthetic glycosylation inhibitor, is a nucleophilic hydrazine, which binds with the amadori product to prevent cross-linking with proteins, thereby inhibiting the formation of the final glycation product and delaying its development into complications. Or prevented (Brownlee, M., et al., 1986, Sciences , 232, 1629-1632; Edelstein, D. et al., 1992, Diabetes , 41, 26-29). Aminoguanidine is the most promising synthetic drug for the prevention and treatment of diabetic complications, but until the phase 3 clinical trial, it was stopped due to the problem of causing toxicity during prolonged administration.In addition, sorbitol was inhibited by inhibiting the action of aldose reductase enzyme. Preventing accumulation and preventing diabetic retinopathy Japan's epilestat, which has excellent therapeutic efficacy, has also been withdrawn from the market due to its post-market toxicity.

Therefore, the development of natural drugs with excellent safety and efficacy is desired.

Therefore, the present inventors have identified a novel triterpenoid compound, 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12, which was isolated from the ethyl acetate layer of the alcoholic extract of Chamsori. The present invention has been completed by discovering that -diene-28-oic acid can be used for the treatment and prevention of diabetic complications by inhibiting the production of the final glycation end products.

In order to solve the above problems, the present applicant has continuously studied the causes, prescriptions, and treatment methods of diabetic complications. Ethyl obtained by systemic separation of triterpenoid compounds, 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruz-4 (23), 12-diene-28-oic acid from Chassori extract The purpose is to provide it separately from the acetate layer.

Another object of the present invention is the prevention of diabetic complications containing 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid as an active ingredient and It provides a therapeutic or anti-aging and delayed pharmaceutical composition.

Another object of the present invention is the prevention of diabetic complications containing 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid as an active ingredient. And to provide a functional or anti-aging and delayed functional food.

The triterpenoid compound to be separated from the chassam in accordance with the present invention for achieving the object of the present invention is extracted by using the alcohol of 20 ~ 80%, then concentrated under reduced pressure to obtain a crude extract of Chamsori Steps; A second step of suspending the crude extract from Chamsori, and then adding normal hexane to separate components dissolved in the normal hexane layer and vacuum drying to obtain a normal hexane soluble fraction; A third step of separating the normal hexane layer in the second step, adding ethyl acetate to the remaining aqueous layer, separating the component dissolved in ethyl acetate, and vacuum drying to obtain an ethyl acetate soluble fraction; A fourth step of subjecting the ethyl acetate soluble fraction to silica gel column chromatography using chloroform: methanol mixed solvent concentration tool (1: 0 → 0: 1) to obtain 12 fractions; The fifth fraction of the 12 fractions of the fourth step was subjected to silica gel column chromatography with a normal hexane: ethyl acetate: methanol mixed solvent concentration gradient (6: 3.5: 0.5 → 2: 2: 1) to obtain 10 fractions. A fifth step of obtaining; The third fraction of the ten fractions of the fifth step was subjected to reverse phase silica gel column chromatography with an eluate of methanol: water 3: 1, followed by 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-nor Urus-4 (23), 12-diene-28- Oye seed is characterized in that the sixth step to obtain.

In addition, the pharmaceutical composition and functional food for preventing and treating diabetic complications according to the present invention are the 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28- It is characterized in that it contains OIC acid as an active ingredient.

In addition, the anti-aging or delayed pharmaceutical composition and functional food according to the present invention is the 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid It is characterized by containing as an active ingredient.

Chamsori used in the present invention is a perennial herb of Polygonaceae which is widely distributed in Korea, and the root of this plant is called Yangje Geun or Rhubarb Rhubarb, which is a tradition of treating phlegm, jaundice, constipation, improvement, and uterine bleeding. It has been used as a medicinal herb (Bae K., "Medicinal Plants of Korea," Kyo-Hak Publishing Co., Seoul, 2000; p 92). In addition, studies on physiological and pharmacological activities include antioxidant activity (Li et al., J. Dermatology , 27 , 761-768, 2000), cytotoxicity (Kim et al., Yakhak) . Hoeji , 42 , 233- 237, 1998), antimicrobial activity (Nishina et al., J. Agric . Food Chem ., 41 , 1772-1775, 1993; Odani et al., Shoyakugaku Zasshi , 31 , 151-154, 1977 A variety of activities have been reported, including studies on flavonoids (Aritomi et al., Chem . Pharm . Bull. , 13 , 1470-1471, 1965), anthraquinone derivatives (Kim et al., Yakhak Hoeji , 42 , 233-237, 1998; Hwang et al., J. Korean Soc . Appl . Biol . Chem., 47 , 274-278, 2004) and naphthalene derivatives (Kim et al. , Yakhak Hoeji, 42, 233-237, 1998; Nishina et al, J. Agric Food Chem, 41, 1772-1775, 1993;....... Aritomi et al, Chem Pharm Bull, 13, 1470-1471 , 1965; Zee et al., Arch. Pharm . Res ., 21 , 485-486, 1998).

However, despite the ongoing study of Chassam, no teachings have been made on the use of Chamsoris as a prophylactic or therapeutic agent for diabetes complications and related chemical components.

Hereinafter, the present invention will be described in detail with reference to Examples.

I. Triterpenoids  Produce

1. The first step: A bullshit ( Rumex japonicus Houtt . ) Crude extract  Produce

Rumex japonicus Houtt. Was collected in the vicinity of Jeonmin-dong, Yuseong-gu, Daejeon. Extraction was performed three times at room temperature three times, and the extract was concentrated under reduced pressure to obtain 185 g of the extract of Chamsori.

2. The second stage: normal hexane Soluble fraction  remove

180 g of the crude extract obtained from the first step was suspended in 1.5 L of distilled water, and then dissolved by adding 1.5 L of normal hexane, which was then dried in vacuo by separating only the components dissolved in the normal hexane layer. This process was repeated three times to obtain 7.6 g of normal hexane soluble fraction.

3. Third step: A bullshit  Ethyl acetate Soluble fraction  Produce

In the second step, the soluble fraction of normal hexane was separated, and 1.5 L of ethyl acetate was added to the remaining aqueous layer, and only the components dissolved in ethyl acetate in the fractional filter were separated and dried in vacuo. This process was repeated three times to obtain 24.0 g of an ethyl acetate soluble fraction.

4. Step 4 ~ Step 6: 2 alpha , 3 alpha , 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid

end. 4th step

21 g of the ethyl acetate soluble fraction was divided into 12 fractions by silica gel column chromatography (silica gel 60, 70-230 mesh, Merch) using a chloroform: methanol mixed solvent concentration gradient (1: 0 → 0: 1). .

I. 5th step

The fifth fraction (1.98 g) separated from the first column of the fourth step was taken and subjected to silica gel column chromatography (Normal hexane: ethyl acetate: methanol mixed solvent gradient (6: 3.5: 0.5 → 2: 2: 1). Silica gel 60, 230-400 mesh, Merch) was carried out and divided into 10 fractions.

All. 6th step

The third fraction separated from the second column of the fifth step was subjected to reverse phase silica gel column chromatography (ODS 12 nm S-150 μm, YMC) using methanol: water 3: 1 as an eluent to obtain the following physical and chemical properties. 12 mg of 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid having a separation were isolated.

※ Physical and chemical properties of 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid separated by the first to sixth steps

-. Condition: White Powder

-. Melting Point (mp): 234 ~ 236 ℃

-. [a] D25 +73.0 ( c = 0.05, MeOH); IR (KBr) cm-1: 3570, 3412, 3075, 2930, 2863, 1689, 1459, 1380, 1238, 1047, 905

-. ¹ H- (pyridine- d 5, 300 MHz) and ¹³ C- NMR (pyridine- d 5, 75 MHz) data, see Table 1

-. EIMS m / z (rel. Int.): 472 [M] + (7), 439 (17), 426 (61), 354 (14), 264 (9), 246 (26), 201 (25), 146 (100)

-. HRESIMS m / z : 495.3081 ([M + Na] < + >, Calcd for C29H44O5Na: 495.3098).

¹ H- and ¹³ C NMR data for Compound 1 (δ ppm) location C H (J, Hz) location C H (J, Hz)  One  44.5 1.87 t (12.0) 2.08 (overlap)  16  26.9 2.05 (overlap) 3.12dt (12.8,3.0) 2 69.9 4.12 brdt (11.4,4.2) 17 48.8 3 77.0 4.66d (2.4) 18 55.2 3.06 s 4 153.7 19 73.1 5 45.8 2.69d (2.4) 20 42.9 1.52 (overlap) 6 21.4 21 27.4 7 32.7 22 38.7 8 41.0 23 109.9 4.77s, 5.14s 9 45.7 2.17 (overlap) 24 - - 10 38.9 25 14.8 0.84 s  11  25.4 2.11 brd (13.2) 2.28 brd (13.2)  26  17.8  1.16 s 12 128.6 5.62brs 27 25.1 1.66 s 13 140.7 28 181.2 14 42.8 29 27.6 1.44 s  15  29.6 1.26 brd (13.2) 2.34dt (13.2,2.4)  30  17.3  1.13d (6.6)

II. Separated 2 alpha , 3 alpha , 19 Alpha-Trihydrock-24-Norrus-4 (23), 12-Diene-28-Oemic Acid

The separated compound is a white powder, and from the molecular ion ([M] +) m / z 495.3080 (C ₂₉ H ₄₄ O ₅ Na) obtained in the HRESIMS experiment, it was found that the molecular formula is C ₂₉ H ₄₄ O ₅ . 1). IR spectra of the compounds showed absorption of carboxyl (1689 cm ⁻¹ ), hydroxyl (hydroxyl, 3432 cm ⁻¹ ) and exocyclic methylene (3075, 1652 cm ⁻¹ ).

¹ H- NMR data of the compound (Table 1) and COSY spectrum analysis result of 4 methyl groups (δ 1.66, 1.44, 1.16 and 0.84, s, 3H), secondary methyl group (δ 1.13, J = 6.6 Hz, d, 3H), trisubstituted olefin double bond (δ 5.62, br s), and exocyclic methylene groups (δ 5.14 and 4.77, s, 1H) were confirmed.

In addition, two oxymethine protons were identified at positions δ 4.66 (d, J = 2.4 Hz, 1H) and 4.12 (br dt, J = 11.4 and 4.2 Hz, 1H), and a single δ 3.06 position was observed. Characteristic double of triplet signal at position δ 3.12 shifted to low field by anisotropic effect of line (H-18) and 19-hydroxy group, J = 12.8 and 3.0 Hz, 1H, H-16) demonstrates that the compound is a type of urs-12-ene derivative with a hydroxyl at the C-19 position (Aquino et al., J. Nat. Prod ., 53 , 559-564, 1990).

¹³ C-NMR spectra of the compounds (Table 1) also show olefin groups at δ 128.6 and 140.7 (C-12 and C-13), exocyclic methylene groups at δ 109.9 and 153.7 (C-4 and C-23), δ The presence of a carboxyl group at 181.2 (C-28) and three hydroxylated carbons at δ 69.9, 73.1 and 77.0 (C-2, C-3 and C-19). The location of all substituents was confirmed via COSY and HMBC NMR techniques (FIG. 2).

The EIMS spectrum is also typical of the retro- Diels-Alder cleavage fragment ions of the Urus-12-ene compounds having a carboxyl group at position 17 and a hydroxyl group at the D or E ring [fragment ions, m / z 264, 246 (264-H2O), 201 (246-COOH)] (Aquino et al., J. Nat. Prod ., 53 , 559-564, 1990). 2 and 3 of myrianthic acid ( J = 2.7 Hz) (Jin et al., Arch. Pharm. Res ., 27 , 376-380, 2004), which are analogous to compound 1 ( J = 2.4 Hz) 2,3-dihydroxy-24-nor-4 (23), which has a similar coupling constant between hydrogens and another similar chemical compound with carbon shifts of 1, 5, 10, 23, and 25, The conformation of the two hydroxyl groups at positions 2 and 3 of the compound is almost identical to 12-oleanadien-28-oic acid (Ballesta-Acosta et al., J. Nat. Prod. , 65 , 1513-1515, 2002) . Both structures were expected as α. This prediction was evidenced by the NOESY NMR spectral data (FIG. 3) of the compounds showing strong NOE correlations between hydrogen 2 and hydrogen 3 and 5.

Thus the structure of this new compound is 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid.

Reagents used in the experiments conducted to investigate the physicochemical properties and structure of the 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid; The device is as follows.

Melting point is IA9100 Melting Point Analyzer (Barnstead International, USA), optical rotation is a P-1020 digital polarimeter (Jasco, Japan), IR spectrum is FTS 165 FT-IR spectrophotometer (Bio -Rad, CA), and LREI and HRESIMS were measured by Autospec (Micromass, UK) and Mariner mass spectrometer (Perspective Biosystem, USA), respectively. NMR experiments were performed with DRX-300 FT-NMR (Bruker, Germany) and TLC analysis with Kieselgel 60 F254 (Merck) plates (silica gel, 0.25 mm layer thickness). Color development was heated at 110 ° C. for 5-10 minutes after spraying 10% sulfuric acid solution (Aldrich). Silica gels (Merck 60A, 70-230 or 230-400 mesh), Sephadex LH-20 (Amersham Pharmacia Biotech), and reversed phase silica gel (YMC Co., ODS-A 12 nm S-150 m) were used.

III. 2 alpha , 3 alpha , 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28- Oicked   In vitro Final Glycation Product  Production Inhibition Effect Analysis

Bovine serum albumin (hereinafter referred to as BSA: Sigma, USA) was selected as a protein source. BSA was prepared by adding 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / mL. As a sugar source, a mixture of 0.2 M fructose and 0.2 M glucose was used. A fructose and glucose mixture was added to the prepared BSA solution. After dissolving the sesame extract and each fraction layer of the concentration between 5 ~ 100 ㎍ / ㎖ in 15% tween 80 as a sample group, it was added to the mixture of BSA and sugar and incubated at 37 ℃ for 7 days.

At this time, 0.02% sodium azide and antimycotics were added as antibacterial and antifungal agents. The control group was cultured with a mixture of BSA and sugar, and the blank group of the test group and the control group was prepared and not cultured. On the other hand, aminoguanidine was used as a positive control which is an index that can compare the superiority of efficacy. All cultures were prepared in four pieces to avoid errors. After 7 days the content of the final glycation product produced in the culture was analyzed and the result was shown. The final glycosylated product is fluorescence, brown, not only has a physicochemical property that can cross-link, but also has a ligand that can be recognized by cell membrane receptors. The amount of the final glycated product having these characteristics was measured by a microplate reader (Excitation: 350 nm, Emission: 450 nm) and analyzed for the degree of inhibition of production (Vinson, JA et al., J. Nutr . Biochem . , 7: 659- 663, 1996). The results are shown in Table 2 and FIG.

The production inhibition rate is calculated by the following equation.

Production Inhibition Rate (%) = 100- (Fluorescence Intensity of Sample-Fluorescence Intensity of Sample Blank) / (Fluorescence Intensity of Control-Fluorescent Intensity of Control Blank) x 100

Comparative Example 1. Efficacy of Inhibiting the Final Glycation Product Production

Aminoguanidine was dissolved in distilled water, and prepared at concentrations of 18.5 μg / ml, 37 μg / ml, and 55.5 μg / ml, followed by incubation at 37 ° C. for 7 days. After 7 days, the amount of the final glycosylated product produced in the culture was measured by a microplate reader (Excitation: 350 nm, Emission: 450 nm), and the results are shown in Table 2 and FIG. 5.

 division Final glycation product production inhibition rate (%) IC ₅₀ value (μg / ml) Concentration Suppression rate 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norurus-4 (23), 12-diene-28-oic acid 5 15.27 ± 1.56  22.81 (50.1㎛) 10 33.98 ± 1.80 25 52.27 ± 1.67  Aminoguanidine 18.5 25.29 ± 3.22  37.11 (500㎛) 37 54.63 ± 0.88 55.5 69.69 ± 0.88

As shown in Table 2, the final glycation product production inhibitory IC ₅₀ value of the compound was 22.81 μg / ml (50.1 μm). This was 100 times better than the positive control aminoguanidine (37.11 μg / ml; (500 μm).

As described above, the triterpenoid compounds isolated from the Chamsori, according to the present invention effectively inhibit the production of the final glycation product which is one of the causes of diabetic complications, the final at low concentrations compared to the positive control aminoguanidine Since it inhibits the production of glycated products, it can be used as a preventive and therapeutic agent for diabetic complications. In addition, the inhibition rate of oxidative stress is reduced when inhibiting the production of the end glycated product, it can be applied as a pharmaceutical composition and functional food for preventing and delaying aging caused by oxidative stress.

Claims (6)

A first step of extracting Chamsori using 20-80% alcohol and then concentrating under reduced pressure to obtain a crude extract of Chamsori; A second step of suspending the crude extract from Chamsori, and then adding normal hexane to separate components dissolved in the normal hexane layer and vacuum drying to obtain a normal hexane soluble fraction; A third step of separating the normal hexane layer in the second step, adding ethyl acetate to the remaining aqueous layer, separating the component dissolved in ethyl acetate, and vacuum drying to obtain an ethyl acetate soluble fraction; A fourth step of subjecting the ethyl acetate soluble fraction to silica gel column chromatography using a chloroform: methanol mixed solvent concentration gradient (1: 0 → 0: 1) to obtain 12 fractions; The fifth fraction of the 12 fractions of the fourth step was subjected to silica gel column chromatography with a normal hexane: ethyl acetate: methanol mixed solvent concentration gradient (6: 3.5: 0.5 → 2: 2: 1) to obtain 10 fractions. A fifth step of obtaining; The third fraction of the ten fractions of the fifth step was subjected to reverse phase silica gel column chromatography with an eluate of methanol: water 3: 1, followed by 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-nor Separation method of triterpenoid compounds separated from Chamsori, comprising a sixth step of obtaining Urus-4 (23), 12-diene-28-oic acid. Isolated from Chamsori, characterized by 2 alpha, 3 alpha, 19 alpha-trihydrox-24-norruth-4 (23), 12-diene-28-oic acid, separated from the chariot by claim 1 Triterpenoid compounds. A pharmaceutical composition for the prevention and treatment of diabetic complications of claim 1 comprising 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid. Functional foods for the prevention and treatment of diabetic complications of claim 1, wherein the 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid as an active ingredient. The anti-aging or delaying pharmaceutical composition according to claim 1, wherein the 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid is an active ingredient. The anti-aging or delayed functional food comprising 2 alpha, 3 alpha, 19 alpha-trihydroxy-24-norruth-4 (23), 12-diene-28-oic acid of claim 1 as an active ingredient.

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