KR101134532B1 - Analytic method of ?-amino butyric acid from germinated rice - Google Patents

Abstract

In the present invention, a primary extractant composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform is added to 1 part by weight of water to germination containing GABA (γ-amino butyric acid), followed by centrifugation. (A) separating the primary supernatant and the precipitate by separating; (B) obtaining a secondary supernatant by mixing the precipitate obtained from step (a) by adding a secondary extraction solvent composed of 0.3 to 0.9 parts by weight of chloroform with respect to 1 part by weight of water, followed by centrifugation; (C) obtaining a tertiary supernatant by mixing the first supernatant separated from step (a) and the second supernatant separated from step (b), followed by centrifugation; (D) volatilizing and lyophilizing the organic solvent contained in the tertiary supernatant obtained from step (c); (E) dissolving the lyophilisate obtained from step (d) in water and filtering; (F) derivatizing GABA (γ-amino butyric acid) contained in the filtrate obtained from step (e) in the form of 6-aminoquinoline; And a filtrate comprising GABA derivatized in the form of 6-aminoquinoline, and GABA of the reference material derivatized in the form of 6-aminoquinoline through the step (f) by HPLC. Checking the GABA amount by detecting the amount of 6-aminoquinoline contained (g); relates to a GABA content analysis method of germination, can be easily analyzed GABA content of germination have.

GABA (γ-amino butyric acid), germination, analysis, purification

Description

Analytical method of γ-amino butyric acid from germinated rice}

The present invention relates to a method for analyzing the GABA content of germination.

As life expectancy extends, infectious diseases seen in the past have sharply decreased, while adult diseases of cancer, dementia, hypertension and diabetes are becoming social problems. Recently, the food and drug industry is actively researching the development of bioactive materials for the prevention and treatment of adult diseases caused by aging society and eating habits, and the market size is rapidly increasing.

GABA (γ-amino butyric acid) is a global functional food material that has been used since the mid-1980s and began to form a market in earnest around 2001.

GABA mainly plays a role as an excitatory inhibitory neurotransmitter, and is involved in physiological mechanisms such as improving brain blood flow, increasing oxygen supply, and promoting brain cell metabolism, and thus reducing stress, improving memory, suppressing blood pressure rise, and blood Increases cholesterol and triglycerides, relieves depression insomnia, prevents paralytic dementia, prevents obesity, prevents menopausal disorders, improves vision, anti-anxiety, and anticonvulsions (Krogagaard- Larsen, 1989; Mody et al., 1994) . Recently, it has been found to have an effect of inhibiting the metastasis or proliferation of stroke, colon cancer and colon cancer cells, and has attracted much attention as a pharmacological food material (Oh SH, Oh CH, 2003; 2004).

On the other hand, rice germ and germinated brown rice products with increased GABA content have been found to be effective in preventing stroke and dementia, enhancing memory, and insomnia by increasing cerebral blood flow, increasing oxygen supply, and activating brain cell metabolism. It has been found to be effective in improving circulatory disease due to blood pressure lowering action, and also has a function of removing hangover due to liver function and alcohol metabolism promoting function.

Therefore, GABA has recently been in the spotlight as a material having excellent physiological activity, and is evaluated as a functional material that can secure infinite marketability depending on its application.

On the other hand, the research on the technology that can analyze the above GABA (γ-amino butyric acid) from germination is still insignificant, and has not been specifically established.

Accordingly, an object of the present invention is to establish and provide a GABA (γ-amino butyric acid) content analysis method of germination.

In order to achieve the above object, the present invention provides a primary extraction solvent composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform, based on 1 part by weight of water in the germination containing ABA (γ-amino butyric acid). (A) separating the primary supernatant from the precipitate by addition and mixing, followed by centrifugation; (B) obtaining a secondary supernatant by mixing the precipitate obtained from step (a) by adding a secondary extraction solvent composed of 0.3 to 0.9 parts by weight of chloroform with respect to 1 part by weight of water, followed by centrifugation; (C) obtaining a tertiary supernatant by mixing the first supernatant separated from step (a) and the second supernatant separated from step (b), followed by centrifugation; (D) volatilizing and lyophilizing the organic solvent contained in the tertiary supernatant obtained from step (c); (E) dissolving the lyophilisate obtained from step (d) in water and filtering; (F) derivatizing GABA (γ-amino butyric acid) contained in the filtrate obtained from step (e) in the form of 6-aminoquinoline; And a filtrate comprising GABA derivatized in the form of 6-aminoquinoline, and GABA of the reference material derivatized in the form of 6-aminoquinoline through the step (f) by HPLC. It provides a method for analyzing the GABA content of germination, comprising the step (g) of determining the amount of GABA by detecting the amount of 6-aminoquinoline contained.

Hereinafter, the problem solving means of the present invention will be described in detail.

Step (a); The primary supernatant and the precipitate were mixed by adding GABA-containing germination to the 1 part by weight of water and adding a primary extraction solvent composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform, followed by centrifugation. Separating steps

This step (a) is carried out by adding a primary extraction solvent composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform to 1 part by weight of water to the germination containing GABA, and then centrifuged This is the step of separating the supernatant and the precipitate.

In order to purify GABA from germination, the present invention adds and mixes a primary extracting solvent composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform to 1 part by weight of water containing GABA. Centrifuge.

The chemical formula of GABA is as follows.

The present invention adds a primary extraction solvent composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform to 1 part by weight of water to easily elute GABA from germination.

Step (b); To obtain a secondary supernatant by adding a precipitate obtained from the step (a), by mixing with a secondary extraction solvent composed of 0.3 to 0.9 parts by weight of chloroform per 1 part by weight of water, followed by centrifugation.

This step (b) is added to the precipitate obtained from the step (a), by mixing with a second extraction solvent composed of 0.3 ~ 0.9 parts by weight of chloroform per 1 part by weight of water, followed by centrifugation to separate the secondary supernatant It is a step of obtaining.

In order to separate excess GABA contained in the precipitate obtained from step (a), the present invention uses a secondary extractant composed of 0.3 to 0.9 parts by weight of chloroform with respect to 1 part by weight of water in the precipitate obtained from step (a). After addition and mixing, the secondary supernatant is obtained by centrifugation.

Step (c); Primary separated from step (a) Supernatant  Secondary separated from step (b) Supernatant  After mixing, the third step by centrifugation Supernatant  Obtaining Step

This step (c) is a step of obtaining a tertiary supernatant by mixing the first supernatant separated from step (a) and the second supernatant separated from step (b), followed by centrifugation.

The present invention efficiently concentrates GABA from germination, and after mixing the primary supernatant separated from step (a) and the secondary supernatant separated from step (b) to remove impurities except GABA. The third supernatant is obtained by centrifugation.

Step (d); From step (c) The obtained  3rd In supernatant  Volatilizing and freeze-drying the organic solvent

This step (d) is a step of volatilizing and lyophilizing the organic solvent contained in the tertiary supernatant obtained from the step (c).

The tertiary supernatant obtained from the step (c) is a state containing an organic solvent by using methanol and chloroform to elute GABA from germination, in this step (d) the organic solvent contained in the tertiary supernatant Volatilization and the residue was lyophilized to give a GABA-containing lyophilisate.

Step (e); From step (d) The obtained  Freeze-dried in water and filtered

This step (e) is a step of dissolving the lyophilisate obtained from step (d) in water and filtering.

Step (f); From step (e) The obtained  Contained in the filtrate GABA 6- Aminoquinoline  In the form Derivatization  Steps to

This step (f) is a step of derivatizing GABA contained in the filtrate obtained from step (e) in the form of 6-aminoquinoline. Since GABA has a structurally linear molecular structure, it cannot be identified using HPLC. Therefore, it is necessary to derivatize it in the form of 6-aminoquinoline, which is a form having a benzene ring, and identify it indirectly.

Step (g); remind Step (f)  6- with rough Aminoquinoline  In the form Derivatized  Filtrate containing GABA, 6- Aminoquinoline  In the form Derivatized  Standard GABA Was analyzed by HPLC and 6- Aminoquinoline  By detecting the amount GABA  Step to confirm the quantity

This step (g) is the step (f), the filtrate containing the derivatized GABA in the form of 6-aminoquinoline and the standard GABA derivatized in the form of 6-aminoquinoline using HPLC It's a step.

The GABA to be analyzed in the present invention is derivatized in the form of 6-aminoquinoline, and standards can also be analyzed by HPLC using GABA derivatized in the form of 6-aminoquinoline.

As described above, the GABA (γ-amino butyric acid) content analysis method of germination of the present invention can easily analyze the GABA content of germination.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirit.

Example  One: GABA (γ- amino butyric acid A) sample preparation

GABA of the standard used in this example was purchased from 'Sigma-Aldrich Korea (St, Louis, MO, USA)', water, chloroform, methanol, acetonitrile was' JT Baker Co. (Phillipsburg, NJ, USA) ), And the 'Accq-Fluor ^TM , a reagent needed to derivatize GABA, Reagent Kit and the mobile phase required for analysis were purchased from Waters (Milford, MA, USA).

Meanwhile, germinated brown rice was used as a sample (FIG. 1). First, the sample was pulverized finely using a grinder, and then weighed by 1.0 g in three 'Conical centrifuge tubes'. For primary extraction of GABA contained in the sample, 12 mL of methanol, 5 mL of chloroform, and 3 mL of water were added to the sample, followed by shaking for 30 minutes by adjusting the stirrer (R S-1, JEIO TECH, Korea) to 290 rpm. After 30 minutes, the extract was centrifuged for 15 minutes using a centrifugal separator (Combi-514R, Hanil, Korea) set at a relative centrifugal force (RCF) of 12,000 × g and 4 ° C. to separate the aqueous solution and the precipitate.

The separated aqueous solution was taken in another 'Conical centrifuge tube', and the separated precipitate was added with 3 mL of chloroform and 5 mL of water for secondary extraction of GABA. After centrifugation under the same conditions as the first centrifugation, the supernatant was taken from the aqueous solution separated into two layers and combined with the aqueous solution obtained at the first stage.

Thereafter, the aqueous solution obtained in the first and second centrifuged once again to obtain only the supernatant. The organic solvent was evaporated by distillation under reduced pressure in a 30 ° C. constant temperature water bath, and a lyophilizer (PVTFD 10R, Ilshin lab) was used. By lyophilization.

Thereafter, 10 mL of water was added to the lyophilisate, dissolved, filtered through a 0.45 μm PVDF (Millipore Corporation Bedford, MA 01730, USA) filter, and the GABA contained in the lyophilizate was transformed into 6-aminoquinoline. To do this, the following procedure was carried out.

The heating block was preheated to 55 ° C. for transformation to 6-aminoquinoline, and then 10 μL of the filtrate obtained above was placed in a 2 mL tube. 70 μL of 'Accq-Fluor Borate Buffer' was added to the tube containing the filtrate and mixed, and then 20 μL of 'Accq-Fluor Reagent' was added and mixed again. Thereafter, the mixture was left at room temperature for 3 minutes, transferred to 150 μL 'clear glass conical insert' using a glass pipette, and then put into a vial and heated for 10 minutes with a preheated heating block to prepare a sample to be used in the following HPLC.

On the other hand, the preparation of a standard solution to be used for the preparation of a calibration curve for HPLC detection was performed by the following method. First, 20 mg of GABA was weighed and dissolved in 10 ml of water to make a stock solution. Thereafter, the stock solution was diluted with water to prepare standard solutions of 1, 2, 4, 8, and 100 µg / mL, followed by derivatization of GABA according to the above method, followed by injection into HPLC. Thereafter, a calibration curve was prepared by the concentration-area ratio of the chromatogram obtained by injection into HPLC. The calibration curve is shown in FIG. 2, and the HPLC results for the standard solution are shown in FIG. 3.

Meanwhile, according to the established analytical conditions, the sample prepared above was injected into HPLC to analyze the amount of GABA contained in germinated brown rice.

HPLC was used for GABA analysis, and the analysis conditions were as shown in Table 1 below. Column and mobile phase A were purchased from Waters for amino acid analysis and used in the experiment.

The GABA content in each sample was analyzed by ANOVA SAS 9.1 and significance test ( p <0.05) was performed by Duncan's multiple range test.

device Waters 1525 Binary HPLC pump
Waters 474 Scanning fluorescence column Accq-Tag Amino Acid Analysis Column 3.9mmＸ150mm Mobile phase Gradient mode A: Water Accq-Tag Eluent A 100 mL + water 1L
B: Acetonitrile-Water (60:40, v / v) Minutes Mobile phase A% 0 100 0.5 98 15 93 19 90 32 67 33 67 34 0 37 0 wavelength Excitation: 250 nm
Emission: 390 nm Flow rate 1.0 mL / min Injection volume 10

It was confirmed from FIG. 4 that the sample pretreated from the germinated brown rice contained GABA, and the content of GABA contained in the sample was 257.5 ± 6.8 (µg / g).

Example  2: From germination GABA (γ- amino butyric acid Of how to analyze

First, the linearity of the analysis method was confirmed by the calibration curve of GABA. The calibration curve was prepared as the area ratio of the peak to the concentration of the analyte, and the slope of the calibration curve and the correlation coefficient (R ² ) were shown. (Table 2).

On the other hand, the following experiment was conducted to determine the intraday precision and accuracy and the daily precision and accuracy for the method. The experiment was repeated five times. Germinated brown rice was selected as a sample. The experiment was performed by adding 1000 μg of GABA to the sample, followed by pretreatment, derivatization, and HPLC analysis in the same manner as in Example 1 above. After the experiment, the recovery was calculated by substituting the detected concentration of GABA into the calibration curve. The precision was expressed by the coefficient of variation (CV), and the accuracy was expressed by the percent recovery (%). (Table 2).

On the other hand, the limit of detection (LOD) and limit of quantification (limit of quantification, LOQ) was prepared by the same method as the standard solution 1㎛ / mL was analyzed by HPLC. The experiment was repeated seven times in total and calculated according to the method of Harris (Harris, Exploring Chemical Analysis, 2004) (Table 2).

Values Linearity

Range, μg g ^-1 1-100 Equation y = 205678x + 1156818 R ² 0.9808 Sensitivity
Limit of detection, LOD, μg g ^-1 0.67 Limit of quantification (LOQ, μg g ^-1 ) 2.25 Intraday
(n = 5)
Accuracy Percent recovery,%
(mean ± SD) 111.6 ± 15.0 Precision CV,% 13.5 Interday
(n = 5)
Accuracy Percent recovery,%
(mean ± SD) 146.9 ± 5.8 Precision CV,% 4.0

As a result of verifying the method of analyzing GABA from germination as described above (Table 2), the linearity showed good linearity with an R ² value of 0.9808 in the concentration range of 1-100 µg / g.

LOD was 0.67 µg / g and LOQ was 2.25 µg / g.

In addition, the intraday accuracy was 111.6%, and the precision was 13.5%, which is 20% or less, indicating that it has sufficient precision. The daily accuracy was 146.9%, and the precision was 4.0%, which is less than 20%, indicating that there was sufficient precision.

1 is a photograph of germinated brown rice used in the experiment.

Figure 2 is a diagram showing the standard curve of GABA (γ-amino butyric acid).

Figure 3 is a diagram showing the HPLC results of the GABA standard solution.

Figure 4 is a diagram showing the HPLC results of GABA contained in the sample (germinated brown rice).

Claims (1)

To the germination containing GABA (γ-amino butyric acid), a primary extractant composed of 2.4 to 5.6 parts by weight of methanol and 0.8 to 2.6 parts by weight of chloroform was added to 1 part by weight of water, followed by centrifugation. (A) separating the primary supernatant from the precipitate; (B) obtaining a secondary supernatant by mixing the precipitate obtained from step (a) by adding a secondary extraction solvent composed of 0.3 to 0.9 parts by weight of chloroform with respect to 1 part by weight of water, followed by centrifugation; (C) obtaining a tertiary supernatant by mixing the first supernatant separated from step (a) and the second supernatant separated from step (b), followed by centrifugation; (D) volatilizing and lyophilizing the organic solvent contained in the tertiary supernatant obtained from step (c); (E) dissolving the lyophilisate obtained from step (d) in water and filtering; (F) derivatizing GABA (γ-amino butyric acid) contained in the filtrate obtained from step (e) in the form of 6-aminoquinoline; And, The filtrate containing GABA derivatized in the form of 6-aminoquinoline and GABA of the reference material derivatized in the form of 6-aminoquinoline through step (f) were analyzed in HPLC and contained in the filtrate. Determining the amount of GABA by detecting the amount of 6-aminoquinoline present (g); GABA content analysis method of germination

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