JPH05227993A - Determination of antioxidation activity - Google Patents

Abstract

PURPOSE:To accurately determine the antioxidation activity of a specimen with simple operation by using a reaction liquid containing bacterial cells, an alkyl peroxide, an iron complex and a specimen and determining the alkylperoxyl radical elimination activity of the specimen by measuring the number of survived bacterial cells. CONSTITUTION:The antioxidation activity of a specimen to eliminate alkylperoxyl radical which is a main cause of e.g. cytotoxicity and cell degeneration is determined by using a reaction liquid containing three essential elements consisting of (A) a bacterial strain such as Staphylococcus aureus (ATCC 25923), (B) a solution containing t-butyl hydroperoxide as the alkyl peroxide component of an alkylperoxy radical forming system and a hemin as an iron complex compound and (C) the specimen to be determined, incubating the reaction liquid at 37 deg.C for 30min, adding a part of the reaction liquid to an agar plate medium, culturing for a night and counting the number of formed colonies of Staphylococcus aureus, thereby determining the number of living bacterial cells in the reaction liquid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple method for measuring an antioxidant activity, among many antioxidant activities, which eliminates an alkylperoxyl radical, which is a main cause of cytotoxicity, cell degeneration and the like. .

[0002]

2. Description of the Related Art Among various radical molecular species, it is well known that an alkylperoxyl radical is a reaction intermediate of oxidative deterioration of fats and oils and polymer materials in foods. Therefore, an antioxidant that scavenges alkylperoxyl radicals can serve as an antioxidant for these industrial products.

Further, it is known that the peroxidation of lipids constituting the human body is enhanced in various diseases such as inflammation and arteriosclerosis, and plays an important role in the development of these diseases. The alkylperoxyl radical derived from lipids is known. Is an intermediate of lipid peroxidation reaction (edited by Kunio Yagi and Minoru Nakano, edited by Takeo Futaki and Hiroyuki Shimazaki, "Reactive Oxygen-Chemistry, Biology and Medicine", Ito Dental Publishing Co., Ltd., 1987). Furthermore, it has been reported that alkylperoxyl radicals have strong cytotoxicity (Sumiko Ijiri et al., Biochemistry, 1990, Vol. 62, No. 7 8
Page 05). It is also known to have the activity of promoting the carcinogenic process (Takato Kogi et al., 1991, 50th Annual Meeting of the Japanese Cancer Society, p. 16). Therefore, the alkylperoxyl radical scavenger may be a drug such as an anti-inflammatory drug, an arteriosclerosis preventive drug, and a carcinogen preventive drug, and it is very important to screen a substance having an alkylperoxyl radical scavenging activity. There is a demand for a simple method for measuring the antioxidant activity of.

The methods for measuring lipid peroxidation or antioxidant activity have hitherto been the thiobarbituric acid method, iodometry, chemiluminescence method, conjugated diene amount measuring method, oxygen absorption measuring method, electron spin resonance ( ESR) spectrometry is known. Of these methods, only ESR spectrometry can specifically measure only the alkylperoxyl radical and its scavenging activity.

[0005]

ESR spectrometry currently requires extremely expensive measurement equipment. Therefore, there are not many facilities that can use this method. In addition, it is difficult to operate and analyze unless the person has a high degree of specialized knowledge. Furthermore, the alkylperoxyl radical must be measured as the spin adduct formed by reaction with the radical scavenger. Therefore,
In this method, when the sample is a substance that decomposes this spin adduct, it is erroneously evaluated as having the activity even if the sample does not have the alkylperoxyl radical scavenging activity. Further, the radical scavenger is expensive and the supply thereof is not sufficient. In ESR spectrometry, one measurement requires 5 to 10 minutes, and when preparations for standardization are included, it takes several hours to measure various samples with varying concentrations, and the labor is large.

The present invention provides an antioxidant activity measuring method which can economically and conveniently measure the alkylperoxyl radical scavenging activity of a large number of samples at the same time.

[0007]

As a result of research, the present inventors have found that alkylperoxyl radicals have a strong bactericidal action against bacteria, particularly Gram-positive bacteria, and eliminate alkylperoxyl radicals. It is revealed that the substance suppresses its bactericidal action in a concentration-dependent manner,
The present invention has been completed.

That is, the present invention provides a reaction solution containing 1) bacteria, 2) an alkyl peroxide and an iron complex compound, and 3) an analyte to be measured. It is a method for measuring antioxidant activity, characterized in that it is quantified by measuring the survival number of bacteria in it.

Grams-positive bacteria, which are highly sensitive to alkylperoxyl radicals, are excellent as the bacteria used in this measuring method. Gram-positive bacteria include Staphylococcus, Micrococcus, Streptococcus, Bacillus, Clostridium, Lactobacillus, Bifidobacterium, Listeria and the like. Among them, Staphylococcus aureus of the genus Staphylococcus is salt-tolerant, so it can be cultivated in a medium containing a high concentration of salt that can prevent the growth of miscellaneous bacteria, so it is not necessary to operate in a sterile environment. ing.

Alkyl peroxides used in this measuring method include t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, menthane hydroperoxide, dimethylhexane dihydroperoxide, tetramethylbutyl hydroperoxide, linoleic acid and other fatty acids, Alkyl hydroperoxides such as hydroperoxides of their esters, methyl ethyl ketone peroxides, cyclohexanone peroxides, trimethyl cyclohexanone peroxides, methyl cyclohexanone peroxides, acetylacetone peroxides and other ketone peroxides, especially t-butyl hydroperoxide, cumene hydroperoxide, methyl ethyl ketone peroxide. , Or fatty acids and Hydroperoxides esters thereof are preferred.

As the iron complex compound used in this measuring method, a heme compound is preferable, and in particular, hemoglobin, methemoglobin, myoglobin, hemin, and cytochrome C.
Is preferred. There are no particular restrictions on the type and concentration of the buffer used in the preparation of the buffer used in this measurement method, but the pH of the buffer is preferably 4 to 10, and particularly preferably 6 to 8. .

Next, the procedure of this measuring method will be described. To the buffer was added various concentrations of analyte, 10 ⁵ cells / ml to 10 ⁷ cells / ml of bacteria, 0.5 to 30 μmol / ml of alkyl peroxide, and 2 to 50 nmol / ml of iron equivalent iron complex compound. The reaction solution is prepared and incubated at 4 to 40 ° C for 10 to 60 minutes. If the substance that eliminates the alkylperoxyl radical is not present in the reaction solution, the bacteria in the reaction solution will be completely killed under this condition. The suppression of bacterial killing by the sample under these conditions is evaluated by measuring the viable cell count.

The viable cell count can be measured by 1) adding a part of the reaction solution to an agar plate medium and culturing, and then measuring the number of bacterial colonies formed, 2) adding a part of the reaction solution to the medium After culturing by culture, there is a method of measuring the change in pH of the medium. Instead of measuring the pH, 3) a method of measuring the turbidity of the culture can be used. In order to make method 2) simpler, it is desirable to use the following method. That is, a liquid medium containing a pH indicator is added to each well of the multiwell plate, and the reaction solution after incubation is subjected to limiting dilution of viable bacteria by serial dilution on this multiwell plate to culture. The pH of the medium in the wells in which live cells are present decreases with the growth of bacteria and is easily observed as a change in color of the pH indicator. Therefore, the stronger the alkylperoxyl radical scavenging activity of the sample is, the more bacterial growth is observed in the well where the reaction solution is diluted.

As described above, when the substance which eliminates the alkylperoxyl radical is not present in the reaction solution, the bacteria are completely killed, so that no change in pH is observed in all wells. If there is an antioxidant activity that eliminates alkylperoxyl radicals, bacterial survival is observed depending on the strength of the activity. The minimum effective concentration of analyte required for observing bacterial survival can be evaluated for its antioxidant activity in eliminating alkylperoxyl radicals.

When Staphylococcus aureus is used as the Gram-positive bacterium, a mannitol salt medium can be used as a medium for culturing the reaction solution.

[0016]

EFFECT OF THE INVENTION The method for measuring the antioxidant activity for scavenging an alkylperoxyl radical of the present invention does not require a special analytical instrument or a special reagent, and therefore can be easily performed in most facilities. Further, since the operation is simple and the incubation is sufficient for a short time, it is possible to measure many samples at once. Further, unlike ESR spectroscopy using a radical scavenger, there is no erroneous measurement due to a reaction between a spin adduct of an alkylperoxyl radical and a radical scavenger and a sample, and thus the reliability is high.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 1.0 mM as a bacterium in 10 mM sodium phosphate buffer (pH 7.0)
A solution containing 10 μmol / ml t-butyl hydroperoxide as an alkyl peroxide and 6 μM hemin as an iron complex compound was used for an alkylperoxyl radical producing system with S. aureus (ATCC 25923) of 10 ⁶ cells / ml. Prepared. As a sample having alkylperoxyl radical scavenging activity, various concentrations of α-tocopherol, ascorbic acid, superoxide dismutase for scavenging superoxide, or dimethyl sulfoxide for scavenging hydroxy radicals were added to bring the total amount to 1 ml, After incubating at 30 ° C. for 30 minutes, a part of the reaction solution was added to an agar plate medium, and after overnight culture, the number of Staphylococcus aureus colonies formed was measured to determine the number of viable cells in the reaction solution. Table 1 shows the viable cell counts in the presence of various concentrations of the specimen. As a result, the minimum effective concentrations of α-tocopherol and ascorbic acid were 11.1 mM and 1%, respectively.
Whereas 11 mM, superoxide dismutase and dimethyl sulfoxide did not scavenge the t-butylperoxyl radical.

Example 2 1.0 × 10 ⁶ Staphylococcus aureus (ATCC 25923) in 10 mM sodium phosphate buffer (pH 7.0), 10 μmol / ml t-as an alkylperoxyl radical producing system Butyl hydroperoxide and 100 μg / ml methemoglobin, and various concentrations of α-tocopherol, glutathione,
After incubating 1 ml of reaction solution containing ascorbic acid, uric acid, β-carotene, dimethyl sulfoxide, or superoxide dismutase as a sample for 30 minutes at 37 ° C, phenol red added mannitol salt medium (in 1 liter of water Beef extract 3
g, 10 g of peptone, 5 g of salt, 10 g of D-mannitol and 0.0035 g of phenol red), and serially diluted 3-fold on a 96-well multiplate. this
After culturing overnight at 37 ° C, the color change of the medium in each well was observed.

When the reaction solution without the addition of the sample was cultured as described above, the medium remained red and no yellowing of the medium due to bacterial growth, that is, decrease in pH was observed.
On the other hand, as a sample having an alkylperoxyl radical scavenging activity, when α-tocopherol, glutathione, ascorbic acid, or uric acid was added,
Yellowing of the medium was observed depending on the concentration. That is, it can be said that these specimens suppressed the bactericidal action of the t-butylperoxyl radical.

In Table 2, the results of the inhibitory effect of each sample on sterilization by t-butylperoxyl radical are shown as the minimum effective concentration. It is indicated that the lower this minimum effective concentration is, the stronger the antioxidant activity for eliminating the t-butylperoxyl radical is.

Example 3 The same measurement as in Example 2 was carried out using 5 μg / ml cumene hydroperoxide as the alkyl peroxide, and the results are shown in Table 3.

Example 4 The same measurement as in Example 2 was carried out using 0.5 μmol / ml of methyl ethyl ketone peroxide as the alkyl peroxide, and the results are shown in Table 4. Example 5 The same measurement as in Example 2 was carried out using 1 μmol / ml of linoleic acid methyl hydroperoxide as the alkyl peroxide. The results are shown in Table 5.

[0023]

[Table 1]

[0024]

[Table 2]

[0025]

[Table 3]

[0026]

[Table 4]

[0027]

[Table 5]

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Keizo Sato No. 24202, Heimoji-cho, Kumamoto-shi, Kumamoto 24202 No. 202 (72) Inventor Yuichiro Kojima 4-8-13-105 Higashi-machi, Kumamoto-shi, Kumamoto

Claims (2)

[Claims] 1. A method for measuring the antioxidant activity of a sample, which comprises a reaction solution containing 1) bacteria, 2) an alkylperoxide and an iron complex compound, and 3) a sample to be measured, which is alkylperoxy of the sample. A method for measuring antioxidant activity, characterized in that the radical scavenging activity is quantified by measuring the number of surviving bacteria in the reaction solution. 2. The reaction solution is transferred to a medium containing a pH indicator,
The method for measuring antioxidant activity according to claim 1, wherein the number of surviving bacteria is measured by the change in pH after culturing.