KR20010068159A - Manufacturing of low molecular weight angiotensin converting converting enzyme inhibitor from hot water extracts of the roots of Ixeris dentat Nakai - Google Patents

Abstract

PURPOSE: A manufacturing process for a low molecular weight of angiotensin converting enzyme(ACE) inhibitor from hot water extract of lettuce root is provided, which is an efficient and economical process. CONSTITUTION: The manufacturing process for a low molecular weight of ACE inhibitor from lettuce root comprises the steps of: removing an impurities by washing lettuce, separating its root, adding distilled water to the root, and extracting by making reflux for 2hrs at 100deg.C, 60deg.C; filtering, centrifuging for 30min. at 4deg.C, 8000rpm, and collecting the upper solution; ultra-filtering the upper solution using ultra-filter membrane(limit of M.W. is 1000) to prepare the extract; filtering through membrane filter paper, loading on high performance liquid chromatography, and separating a fraction, F4 with highest activity; and then gathering the F4, freeze-drying, and purifying using reversed phase HPLC.

Description

Manufacturing of low molecular weight angiotensin converting converting enzyme inhibitor from hot water extracts of the roots of Ixeris dentat Nakai}

Hypertension is a chronic degenerative disease of the circulatory system and is explained by the physiochemical mechanism of the renin-angiotensin system, which plays an important role in the regulation of blood pressure and electrolyte balance. In particular, angiotensin converting enzyme (ACE) is an enzyme that converts dipeptide (His-Leu) from decapeptide (decapeptide) to engiotensin II having vasoconstrictive action. to be. An increase in engiotensin II causes hypertension by promoting strong blood pressure synergism and the secretion of the antidiuretic hormone aldosterone and inhibiting the excretion of water and sodium, increasing the amount of circulating blood. In addition, ACE decomposes and inactivates bradykinin, which acts as a vasorelaxant, and as a result, increases blood pressure. Therefore, ACE inhibitor is a drug that provides a breakthrough in the treatment of hypertension because it can suppress blood vessel constriction by inhibiting the action of ACE. As a representative ACE inhibitor, captopril, a chemical synthesis agent, has been developed and used as an antihypertensive agent, but there are many side effects such as dry cough, headache, edible weakness, taste disorder, rash, and white blood cell reduction. Is focusing on developing ACE inhibitors in Substances exhibiting ACE inhibitory activity are broadly classified into peptide and polyphenolic substances. Since peptide inhibitors have been reported from snake venom for the first time, it has been reported that tuna, sardine, mackerel, horse mackerel, bonito, shrimp, scallops, etc. Enzyme hydrolysates, milk protein casein, egg albumin (geal), gelatin (gelatin), and peptides isolated from animal plasma have been reported. [G. Oshima, et al., Biochimica et Biophysica Acta, 566, 128, 1979; S. Maruyama, et al., Agric. Biol. Chem., 46 (5), 1393, 1982; S. Maruyama, et al., Agric. Biol. Chem., 51 (6), 1581, 1987; M. Kohmura, et al., Agric. Biol. Chem., 54 (4), 1101, 1990; Yasuo Ariyoshi, Trends in Food Sci. Tech., 4, 139, 1993; Song, K. B., et al., Biotech. Tech., 10 (7), 479, 1996; Song, K. B., et al., Agric. Chem. Biotech., 40 (1), 39, 1997] Examples of polyphenolic substances include extracts from persimmon leaves and mandarin, tannins from green tea, aloe acetylmannin, chitosan oligosaccharides, onion seasonings, and tannins from mandarin skins. It has been found to have an effect. Matsubara, Y., et al., Agric. Biol. Chem., 49 (4), 909, 1985; Cho, Y., et al., Korean J. Food Sci. Tech., 25 (3), 238, 1993; Ryu, I. W., et al., Korean J. Food Sci. Tech., 29 (1), 1269, 1997; Hong, S. P., et al., Korean J. Food Sci. Tech., 30 (6), 1476, 1998; Park, E. J., et al., Food Sci. Biotech., 9 (3), 163, 2000] In Korea, there are studies on ACE inhibitory peptides derived from doenjang, rice, squid, slaughter blood plasma, seaweeds and mushrooms [Yang, H. C., et al., Agric. Chem. Biotech., 37 (6), 441, 1994; Shin, J. I., et al., J. Food Sci. Tech., 27 (2), 230, 1995; Ko, Y. S., et al., J. Korean Fish. Soc., 32 (4), 427, 1999; Song, K. B., et al., Korean Patent No. 10-215090; Song, K. B., et al., Korean Patent No. 10-215091; Suh, H. J., et al., Food Res. Int., 34, 177, 2001] There is no study on the preparation of substances with ACE inhibitory effects from crust.

It is a perennial herb belonging to the Asteraceae family, its length is 25-50cm, flowers bloom in May-July, usually yellow or white flowers, and is distributed in Korea, Japan, China, and Manchuria. Traditionally, all parts including roots are used for edible or medicinal purposes such as herbs in spring, and have been used for indigestion and appetite as sedatives, health and anti-inflammatory agents. It has also been used for. Therefore, the present invention is to produce an ACE inhibitor for the purpose of developing a new functional food material containing a bioregulatory substance from the moth.

Until now, research on ACE inhibitors has mainly produced functional peptides having an ACE inhibitory effect from hydrolysates obtained by hydrolyzing food proteins with proteases such as pepsin and trypsin, but functional products prepared from hydrolysates of food proteins. Peptides have problems in terms of structural and functional and physiological interpretations and economics. Therefore, the present invention has the technical utility in terms of producing the ACE inhibitor using an efficient and economical material to manufacture a naturally-occurring ACE inhibitor from the edible moth.

FIG. 1 shows a high performance liquid chromatogram of hot water extracts of the nirvana root, with major fractions indicated.

FIG. 2 shows the chromatogram of reloading fraction F4 containing angiotensin converting enzyme inhibitory active substance in FIG. 1 into reverse phase high performance liquid chromatography.

The present invention is to prepare an ACE inhibitor exhibiting an antihypertensive effect from crust. The present invention relates to a method for preparing an ACE inhibitor using hot water extraction, ultrafiltration, high-speed liquid chromatography, etc., which is suitable for use as a food material, such as a functional beverage, without using a protease, which is a conventional method for preparing an ACE inhibitor. .

The present invention will be described through examples.

Example 1

After washing the moth and removing foreign substances, the root part was separated, distilled water was added, and the mixture was refluxed at 100 ° C. and 60 ° C. for 2 hours. The extract was filtered using a filter paper and centrifuged at 8000 rpm for 30 minutes at 4 ° C to obtain only the supernatant. The supernatant was ultrafiltered using an ultrafiltration membrane (molecular weight limit 1000) to prepare a crude extract.

The crude extract was used as a sample to measure angiotensin converting enzyme inhibitory activity, which was carried out according to the method of Cushman and Cheung (Cushman, DW and Cheung, HS. Biochem. Pharmacol., 20, 1637. 197) ACE inhibition degree Hypuryl-L-histidyl-L-leucine (HHL) was used as a substrate, and HHL was dissolved in 50 mM sodium borate buffer solution containing 300 mM NaCl (pH 8.3) to dissolve 5 mM HHL. made. Enzyme was used as enzyme for ACE enzyme (ACE) isolated from rabbit lung. The sample was placed in a substrate and the reaction solution to which the enzyme was added was reacted at 37 ° C. for 30 minutes, and then the reaction was stopped by adding 1N hydrochloric acid. Ethyl acetate was added to the reaction mixture, mixed for 30 seconds, centrifuged at 3000 rpm for 15 minutes, and only the supernatant ethyl acetate layer was taken and dried at 100 ° C. for 1 hour. After drying, distilled water was completely added to the hyplic acid remaining in the test zone, and then absorbance was measured at 228 nm to calculate the degree of ACE inhibition compared with the control. For the control, 50 μl of sodium borate buffer was added instead of the sample solution.

As a result of measuring the inhibitory activity of angiotensin I-converting enzyme of the extract by the same method as described above, as shown in Table 1, the inhibition rate of the hot water extract of ciscarrot root was 46.93%.

Example 2

The moth root hot water extract prepared from Example 1 was filtered through a 0.2 μm membrane filter and loaded on high performance liquid chromatography. In high performance liquid chromatography, the column was Resource RPC, distilled water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% TFA as solvent. 1.0 mL of solvent was supplied. The ACE inhibitor was isolated by performing a linear gradient over 40 minutes from 0% to 45% at a flow rate of / min. As shown in FIG. 1, the fractions were divided into six main peaks, and the inhibitory activity of ACE converting enzyme was measured for each fraction. As a result, as shown in Table 1, the fraction having the highest activity was fraction F4, which showed an inhibition rate of 65.82%.

Example 3

In Example 2, F4 was collected, lyophilized, and purified using reversed phase high performance liquid chromatography. The column was C ₁₈ and the solvent was distilled water containing 0.1% TFA and acetonitrile containing 0.1% TFA. To supply solvent. As shown in FIG. 2, the inhibitory active substance showing an ACE inhibition rate of 81.5% was eluted with a single peak in 33% of acetonitrile.

[Table 1]

Target Sample % Inhibition Example 1 46.9% Example 2 F4 65.8% Example 3 81.5%

It is effective to develop an efficient and economical process for producing ACE inhibitor by extracting hot water from moth root, ultrafiltration with molecular weight limit of 1,000 Daltons, high performance liquid chromatography and reverse phase high performance liquid chromatography.

Claims (3)

Method for preparing an angiotensin converting enzyme inhibitor using ultrafiltration and high-speed liquid chromatography The method according to claim 1, wherein in the case of ultrafiltration, the molecular weight limit is in the range of 500 to 3000. The method of claim 1, wherein the production of food, such as beverages containing the produced material