JPS63223096A - Purified quaiac oil and its production - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to purified guaiac butter suitable as a color indicator for diagnostic agents aimed at detecting glucose and occult blood in body fluids, and a method for producing the same.

[Conventional technology]

Diagnostic agents aimed at detecting glucose and occult blood in body fluids use guaiac oil as a color indicator. Glucose or occult blood in body fluids can be detected semi-quantitatively by utilizing the fact that guaiac oil undergoes oxidation and turns blue when a diagnostic agent comes into contact with body fluids containing glucose or occult blood. It is possible. The commonly used guaiac fat is natural guaiac fat, which is extracted from the heartwood of the tropical tree guaya, and therefore contains α-guaiaconic acid, β-guaiaconic acid, guaiaretic acid, and guaiacic acid. It contains multiple impurities in addition to the active ingredients, such as rezene, gum buds, and essential oil, and the composition of the active ingredients and impurities is not constant. Diagnosis aimed at detecting glucose and occult blood in body fluids using natural guaiac oil as a color indicator [! Li drugs have problems in that the coloration during diagnosis is unclear due to impurities contained in natural guaiac oil, and the storage stability of the diagnostic agent is poor.

Problem to be Solved by the Invention 1 The present invention provides a material containing substantially only active ingredients with reduced impurities and suitable as a color indicator for diagnostic agents aimed at detecting glucose in body fluids and occult blood. The purpose of the present invention is to provide purified guaiac fat and a method for producing the same. Further, the present invention aims to reduce production costs by making the column reusable. [Means for Solving the Problems] The present invention is produced by separating natural guaiac butter using a porous hydroxyl group-containing polymeric gel using a column chromagraph treatment using an organic solvent, and peroxidase and hydrogen peroxide. The specific extinction coefficient E)2 at 600n11 measured after the reaction of is 200 or more, and the RF value is 0.45.
(Toluene/dioxane/glacial acetic acid = 90/25/10
(mixture) of purified guaiac fat. The guaiac butter of the present invention is prepared by dissolving natural guaiac fat in acetone, adding toluene to remove the generated precipitate, evaporating the filtrate to dryness, and dissolving the residue in an organic solvent. This solution was separated by column chromatography using an organic solvent through a column filled with a porous hydroxyl group-containing polymer gel, and RFM 0.45 (toluene/dioxane/glacial acetic acid = 90/25/10 mixture) was separated. The purified guaiac fat is separated and recovered from the collected solution. As the porous hydroxyl group-containing polymer material gel used in the present invention, porous dextran gel (trade name: 5ephadex
Lll, manufactured by Pharmacia ■), porous hydrophilic polyvinyl gel (trade name: Toyovar, manufactured by Toyo Soda Kogyo @)
etc. Further, examples of organic solvents used in the present invention include lower alcohols and acetone. Highly polar organic solvents such as acetate are preferred. The purified guaiac oil obtained by the present invention is suitable as a coloring indicator for test strips for the purpose of detecting glucose and occult blood in body fluids. Body fluid test specimens are manufactured by various known methods, but
For example, in addition to color indicators, sugar oxidase, peroxidase, wetting agents, etc. Fi1 degree adjustment stabilizer. An ink composition for glucose detection is prepared by dissolving or dispersing a pH 1iii agent, a binder, a water-absorbing powder, etc. in a non-aqueous solvent.
It can be obtained by making a liquid and coating it on a substrate. Glucose in body fluids reacts with oxygen in the air through the action of glucose oxidases such as glucose oxidase, and is finally oxidized to gluconic acid and hydrogen peroxide. The generated hydrogen peroxide produces nascent oxygen through the action of peroxidase, which immediately reacts with guaiac oil to cause the indicator to develop color. Based on the degree of color development, the presence or absence of glucose in the body fluid and its amount are determined semi-quantitatively. [Examples] The present invention will be explained below using examples. Dissolve 100g of purified natural guaiac butter in 150% acetone,
Add 1.5 grams of toluene dropwise with stirring to remove the precipitated material (approximately 20 g
9) was suction filtered. Concentrate the filtrate in vacuo. Add 100% ethanol to the dried and removed residue (about 70g)
- was added and dissolved by ultrasonication. This solution was added to a column (diameter 8 (1'J1. height 70σ) of hydrophobic dextran gel (trade name 5ephadex LH-20, manufactured by Pharmacia) that had been equilibrated with ethanol in advance, and separated and eluted with ethanol. .-Aliquot in 100d batches,
After testing each fraction by thin layer chromatography, the desired fractions were collected and concentrated to 100 d. About 11 g of purified guaiac fat was obtained by adding this solution to n-hexane at 2° C. and recrystallizing it. Purification Confirmation of guaiac fat The obtained purified guaiac fat was developed on a silicic acid gel preparation plate with a mixture of toluene/dioxane/glacial acetic acid (90/25/10), and by spraying an aqueous solution of peroxidase-202, a blue color was obtained. Tokoro, RFI
O, 45 w. 10% H20□ aqueous solution 2-9, ethanol 4-9 50 my of horseradish peroxysidase was prepared to 9.9 d with distilled water, and the obtained purified guaiac fat 2 q was mixed with ethanol 20-
Add 0.1 d of solution dissolved in . When the transmittance of the rapidly mixed solution was measured after 30 seconds at 60 Or+1 in a 14 J cuvette, a specific extinction coefficient of 1% [250] was obtained. Cm Buto Manufacturing method and performance evaluation of test specimen for bag detection After finely dispersing the ink composition for glucose detection with the following composition in a homomixer, a rectangular shape with a side of 51 m was printed on a white polystyrene sheet with a thickness of 300 μ by screen printing. I printed it like this. The screen plate used had 80 meshes, and the total thickness of the resist and screen gauze was 130 μm. Ink composition for glucose detection Glucose oxidase (manufactured by Toyobo: Grade II)
3.6ffif 1 part peroxidase (manufactured by Toyobo; Grade l[) 2
．． 4 parts by weight refined guaiac fat 4.8
Parts by weight Sorbitan monolaurate (Nispan 20 manufactured by Chemical Soap Co., Ltd.) 7.2 parts by weight Ascorbyl palmitate 0.48 parts by weight Citric acid 2.8 parts by weight Sodium citrate 11.0 parts by weight Methyl vinyl ether/non-fomaleic acid copolymer Amyl alcohol ester of combination (manufactured by G, ^, Company F: Gauntlets ^N-169) 7.0 parts by weight Cellulose fine powder (manufactured by Seikasei: Avicel SF> 171 parts by weight n-amyl alcohol 228 parts by weight Butyl cellosolve acetate 33 0.5 parts by weight of the obtained product marked 91 was dried at 60°C for 40 minutes and cut into sticks to obtain test specimens for glucose detection at t7.
dl, 100■/dl, 2501Mg/dl, 5
The sticks were dissolved to concentrations of 00I#g/dl and 2000#g/dl and used as specimens, and the sticks were taken out immediately after immersion, left to stand for 1 minute, and the color tone of the test reagent portion was observed. The coloration of the test reagent portion was uniform and clear, and no change in color tone was observed even when the colored test specimen was left standing at room temperature for 5 minutes. Further, the color density increased stepwise as the glucose concentration in the sample increased, and it was possible to clearly distinguish the glucose concentration in the sample within the above range. The obtained specimen was sealed in a glass container and heated at 40°C for 12
After storage for several months, a test was conducted in the same manner as above, and the color of the test reagent was uniform, clear, and stable as before storage, and the grape Ill concentration in the sample was also the same as before storage. It was clearly distinguishable. [Comparative example] Production of test specimen for sugar test 1. A test specimen for glucose detection was prepared in the same manner as in the example except that natural guaiac oil was used instead of the purified guaiac oil shown in the evaluation example. β-D-glucose in normal urine and normal urine 50/q/old, 100η/old, 250■/old, 500IRg/d
The stick was dissolved to a concentration of 2000 M/dl and 2000 M/dl as a specimen, and the stick was immediately taken out after immersion, left to stand for 1 minute, and the color tone of the test reagent portion was observed. The coloration of the test reagent portion was unclear and non-uniform compared to the example. The Koshin concentration increased stepwise as the glucose concentration in the sample increased, and it was possible to distinguish glucose in the sample to some extent within the above range. In addition, after sealing the test specimen in a glass container and storing it at 40°C for 6 months,
When a test was conducted in the same manner as above, the coloration of the test reagent part became unrestricted and unclear compared to before storage, and it was difficult to determine the grape vJ concentration in the sample. K Effects of the invention is obtained. Furthermore, the present invention has extremely good separation ability and is economical because the column can be reused. As described in the Examples, a diagnostic agent for the purpose of detecting glucose in body fluids and occult blood using purified guaiac fat produced by the production method of the present invention as a coloring indicator does not contain substantially impurities. In addition, the color appearance at the time of diagnosis is salmon bright, and the storage stability of the diagnostic agent is good.

Claims (1)

[Scope of Claims] 1. Obtained by separating natural guaiac butter using a porous hydroxyl group-containing polymer gel using column chromatography using an organic solvent, and measured at 600 nm after reaction with peroxidase and hydrogen peroxide. Specific extinction coefficient E^
1^%_1_c_m is 200 or more, and R_F value is 0.45 (toluene/dioxane/glacial acetic acid = 90/25
/10 mixture). 2. The purified guaiac fat according to claim 1, wherein the porous hydroxyl group-containing polymer substance gel is a porous dextran gel. 3. The purified guaiac butter according to claim 1, wherein the organic solvent is a lower alcohol, acetone, acetic ester, or a mixture thereof. 4. Dissolve natural guaiac butter in acetone, remove the precipitate formed by adding toluene, evaporate the filtrate to dryness, dissolve the residue in an organic solvent, and use the organic solvent to form a porous hydroxyl group-containing It was passed through a column filled with molecular substance gel and separated by column chromatography, with an R_F value of 0.45 (toluene/dioxane/glacial acetic acid = 90/25).
1. A method for producing purified guaiac fat, which comprises collecting a fraction having a mixture of 1. 5. The method for producing purified guaiac fat according to claim 4, wherein the porous hydroxyl group-containing polymer substance gel is a porous dextran gel. 6. The method for producing purified guaiac butter according to claim 4, wherein the organic solvent is a lower alcohol, acetone, acetic ester, or a mixture thereof.