GABA - IARSCS - University of Sindh

76 downloads 30688 Views 518KB Size Report
Jun 28, 2014 - Extraction and analysis of GABA and lysine in rice varieties ... data, ANOVA was done using Analysis ToolPak add in of Excel 2010. 3. Results ...
Journal of Cereal Science 60 (2014) 356e360

Contents lists available at ScienceDirect

Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs

Simultaneous HPLC determination of gamma amino butyric acid (GABA) and lysine in selected Pakistani rice varieties by pre-column derivatization with 2-Hydroxynaphthaldehyde Amir Hayat a, *, Taj Muhammad Jahangir a, Muhammad Yar Khuhawar a, Malik Alamgir a, Amna Jabbar Siddiqui b, Syed Ghulam Musharraf b a b

Institute of Advance Research Studies in Chemical Sciences, University of Sindh, Jamshoro 76062, Pakistan H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, 75270, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 September 2013 Received in revised form 13 May 2014 Accepted 21 May 2014 Available online 28 June 2014

A selective and sensitive HPLC method for the simultaneous determination of gamma amino butyric acid (GABA) and lysine in Pakistani rice varieties was developed. Both analytes were detected in rice varieties as Schiff base derivatives with 2-hydroxynaphthaldehyde. The derivatives were analyzed on a reverse phase C-18 column with Diode Array Detector (DAD) at 254 nm. The calibration curves were found linear over a concentration range of 3.83e34.58 mg/mL for GABA and 5.16e48.68 mg/mL for lysine with a correlation coefficient of 0.998 for both standards. GABA and lysine contents were found higher in brown rice varieties (4.1e6.58 mg/100 g for GABA and 15.1e27.6 mg/100 g for lysine) than the polished varieties (0.32e0.47 mg/100 g GABA and 13.1e19.8 mg/100 g lysine). This method could be used for separation and quantification of GABA and lysine simultaneously in food samples, especially in cereal seeds. © 2014 Elsevier Ltd. All rights reserved.

Keywords: GABA Lysine HPLC Rice

1. Introduction Rice is the major staple food in the world and one of the important crops cultivated in Pakistan along with wheat, maize and cotton. According to the economic survey of Pakistan, total land used for rice production during 2011e12 was 2571 thousand Hectares and the production of rice during 2011e12 was 6160 thousand tons. Gamma amino butyric acid (GABA), a four-carbon non protein amino acid is an important component of the free amino acid pool (Yu-Haey et al., 2003). It is known as the “brain's natural calming agent” and by inhibiting over-stimulation of the brain, GABA may help promote relaxation and ease nervous tension (Zhang et al., 2006). The inhibitory neurotransmitter, GABA is widely distributed throughout the brain and is reported to be found in 30%e40% of all synapses (Oh, 2003). It is located predominantly in the cortex, basal ganglia, hippocampus, hypothalamus, amygdale, cerebellum, medulla and spinal cord. There is currently great interest in several physiological functions of GABA such as antihypertensive and

* Corresponding author. E-mail addresses: [email protected], (A. Hayat). http://dx.doi.org/10.1016/j.jcs.2014.05.011 0733-5210/© 2014 Elsevier Ltd. All rights reserved.

[email protected]

diuretic effects (Sakai et al., 2005). Taken at bedtime, supplemental GABA may assist some people in the initiation of sleep and produce a deeper and more beneficial sleep (Christensen et al., 1994). Several reports suggested that plant extracts containing high GABA level were effective for lowering the blood pressure of experimental animals and human, and recovery of alcohol-related symptoms (Van der et al., 2008; Polanuer et al., 1992). Due to its biological importance, GABA content has been increased in different plants such as GABA-enriched green tea by anaerobic or cyclic treatments of tea leaves or shoots (Rowley et al., 1995), GABA-enriched rice germ by soaking in water and GABAenriched brown rice by high-pressure treatment and germination, tempeh-like fermented soybeans (Smolders et al., 1995). Moreover, it also found in vegetables such as spinach, potatoes, cabbage, asparagus, and tomatoes and in fruits, such as apples and grapes and in cereals, such as barley and maize (Bianchi et al., 1999; Yang et al., 1999; Piepponen and Skujins, 2001). Lysine is an essential amino acid; it means that the human body cannot synthesize it. Although rice is lower in protein as compared to other cereal grains, its protein quality is good because it contains relatively high levels of lysine and, during the polishing of rice, its protein content decreases followed by a remarkable decrease in lysine content (Abbas et al., 2011).

A. Hayat et al. / Journal of Cereal Science 60 (2014) 356e360

GABA and other amino acids such as lysine have been determined by LC followed by UV, fluorescence, or colorimetric detection (Clark et al., 2007). Many methods have utilized the pre-column derivatization with orthophthalaldehyde (OPA) (Kehr, 1998; Saller and Czupryna, 1989; Yamamoto et al., 1985; Nasholm et al., 1987; Sawai et al., 2001), phthaldehyde-2-mercaptoethanol (Lee et al., 2010), dansyle chloride (Mazur et al., 2011), 2,4,6-trinitro benzene sulphonic acid (Clark et al., 2007) and 9-fluorenyl-methyl chloroformate (Roohinejad et al., 2009) for the detection of GABA. In this study, 2-hydroxynaphthaldehyde is used as a derivatizing reagent. This derivatizing reagent is reported in GABA determination in biological samples (Khuhawar and Rajper, 2003). The GABA content has been analyzed from Malaysian rice (Yingguo et al., 2010), from Thai rice, (Jannoey et al., 2010a,b; Komatsuzakia et al., 2007) Chinese rice (Jannoey et al., 2010a,b), Slovakian rice (Rowley et al. 1995) and Japanese rice (Varanyanond et al., 2005; Maisont and Narkrugsa, 2010). The present study examines the GABA and lysine contents in Pakistani rice varieties. 2. Experimental 2.1. Reagents GABA and lysine were purchased from SigmaeAldrich (USA). All chemicals and solvents used were supplied by Merck (Darmstadt, Germany) and were of analytical and HPLC grade, respectively. Deionized water was used from a Milli-Q water purification system (Millipore MA, USA) throughout the study. 2.2. Rice samples Five rice varieties, namely basmati super, basmati-385, basmati2000 varieties in brown and polished forms were obtained from National Agricultural Research Centre, Islamabad, Pakistan, whereas Irri-6 and Irri-9 varieties in brown and polished conditions were gifted by Rice Research Institute, Dokri, Sindh, Pakistan. 2.3. Equipments All pH measurements were made with an Orion (420 Aþ) pH meter (Orion Pvt. Ltd., Boston, USA) with combined glass electrode and internal reference electrode. A vortex mixer was used for sample extraction and centrifugation was carried out on Centrifuge-5804 R (Eppendorf, USA). Rotavpor R-210 with vacuum pump and heating bath (from Buchi, Switzerland) was used for the evaporation of solvent. Spectrophotometric studies were carried out with a double beam Lamda-35 Spectrophotometer (Perkin Elmer Ltd., USA) with duel 1 cm quartz cuvettes. HPLC analysis was carried out on an Agilent 1200 HPLC system (Agilent Technology Inc., USA) equipped with an auto sampler, a degasser, a binary pump and photodiode array detection (DAD) system. Chemstation software was used to run the HPLC. 2.4. Preparation of standard solutions and derivatization Stock standard solutions were prepared in water as 500 mg/mL of each standard by dissolving 25 mg of GABA (99% pure) and hydrochloric salt of lysine (98% pure) in 50 mL volumetric flasks. The stock solutions were kept in the dark at 4  C and then working dilutions of each standard were prepared for spectrophotometric and HPLC studies after derivatization. Five working solutions of each GABA and lysine were prepared through dilution with water from the stock solution and were subjected to derivatization. Each working solution (1 mL) was treated with 1 mL of 2-hydroxynaphthaldehyde (1.5% in methanol)

357

followed by the addition of 0.5 mL borate-HCl buffer (pH 8) in a 5 mL volumetric flask. The resulting mixture was heated at 80  C for 10 min in a water bath. The solution was allowed to cool at room temperature and then the volume was adjusted to 5 mL with methanol. The absorption spectra were recorded on a spectrophotometer from 500 to 200 nm against a reagent blank. The reagent blank was prepared following the same procedure, without the addition of analytes (standards). 2.5. Chromatographic conditions The HPLC analysis was performed on an Agilent 1200 HPLC System. The solution (5 mL) was injected on a reverse phase SB-C18 column (2.1  50 mm) with a methanol (B) and water (A) gradient system (at 0 min, solvent B 50%; at 2 min, solvent B was 60%; at 5 min, B was 70%; at 8 min, B was 80%; at 10 min, B was 90%; and at 12 min, B was 50%) with a flow rate of 0.8 mL/min and UV detection by photodiode array was at 254 nm. The identification of each peak was based on the comparison of retention time of the standard and by an external addition method. The method sensitivity was estimated with respect to the limit of detection (LOD), limit of quantification (LOQ) and correlation coefficient. In order to evaluate LOD and LOQ, calibration curves were used and were evaluated by using the following equation: LOD ¼ 3.3 d/S, LOQ ¼ 10 d/S where, S ¼ the slope of the calibration curve, d ¼ the residual standard deviation of the regression line or the Y-intercept of the regression line. The LOD and LOQ were estimated as 3 and 10 times the noise level, correspondingly. The Intra- and inter-day variation for the estimation of GABA and lysine were evaluated for method precision. Repeated analyses were carried out on the same day for intra-day analysis while the same practice was repeated next day for inter-day analysis. Intra- and inter-day analyses were performed to check the repeatability and reproducibility of the method, respectively and results were statistically evaluated in terms of % R.S.D. Moreover, to check the stability of GABA and lysine derivatives, the derivatized samples were analyzed every week in a month time period. The accuracy of the method was assessed by performing a recovery study of pre-analyzed samples, spiked with extra amounts of GABA and lysine standards followed by derivatization. The analysis of spiked samples was repeated three times. 2.6. Extraction and analysis of GABA and lysine in rice varieties The sample extraction form rice was carried out according to the method reported by (Jannoey et al., 2010a,b) with slight modifications. Rice powder (5 g) was placed in a 15 mL Falcon tube followed by the addition of 5 mL 80% (v/v) ethanol and shaken on a vortex mixer for 5 min. The sample was centrifuged (5000 rpm) for 10 min at 4  C and the upper portion was filtered through a 0.4 mm Millipore filter paper and the filtrate was collected in a vial. Similar extraction steps were repeated twice and extracts were collected in the same vial. The combined extract was allowed to evaporate with the help of a rotary evaporator. Dried extract was redissolved in 5 mL water for further analysis. The resultant solution (1 mL) was then subjected to derivatization according to the procedure as mentioned in the previous section. For statistical evaluation of the data, ANOVA was done using Analysis ToolPak add in of Excel 2010. 3. Results and discussion In this study, GABA and lysine contents were determined through a post-column derivatization with 2hydroxynaphthaldehyde followed by HPLC analysis. In total, five rice varieties in brown and polished forms were analyzed by the developed method.

358

A. Hayat et al. / Journal of Cereal Science 60 (2014) 356e360

CHO OH

O H2N

O

OH

N

pH 8

+

OH OH

Gamma amino butyric acid 2-Hydroxynaphthaldehyde O CHO

O H2N

+ OH NH2 lysine

N

OH

OH pH 8

OH

NH2

2-Hydroxynaphthaldehyde

Fig. 1. Schiff base derivatives of GABA and lysine with 2-Hydroxynaphthaldehyde.

3.1. Optimization of derivatization conditions and spectrophotometric analysis The derivatizing reagent, 2-hydroxynaphthaldehyde was used which reacts with amino groups of GABA and lysine to form imine derivatives as shown in Fig. 1. The reaction was initially monitored by the spectrophotometer for each compound to optimize the reaction conditions for the maximum formation of the derivatives. The reaction conditions were optimized with respect to concentration of reagent, concentration of derivatizing reagent, effect of pH and heating time of the reaction. The optimization conditions were obtained after a number of analyses. The absorbance was measured against reagent blank prepared under the same conditions except the addition of analyte. The effect of pH was examined from pH 1e10 with the addition of unit interval and reagent 2hydroxynaphthaldehyde in methanol was added 1 mL of 0.5%e 3.0% w/v with an interval of 0.5%. The reaction mixture was heated in a water bath at 80  C for 5e20 min with an interval of 5 min. The spectrophotometric study has shown that the best reaction conditions were observed in basic medium within pH 6e8 and the maximum was observed at pH 8. Amount of derivatizing reagent was selected as 1.5% (w/v) which was enough for the complete consumption of standards during the reaction and a similar response was observed after the addition of 1 mL and higher amounts of the reagent solution. Optimized heating time was 10 min at 80  C as shown by maximum conversion. The derivatives once formed were highly stable and did not show any change in absorbance up to 48 h. Both the derivatives obeyed Beers law at l 254 nm within the range of determination.

was found within the range of 3.83e34.58 mg/mL with a correlation coefficient of 0.998. Similarly, a linear regression of the calibration curve was found for the lysine standard within the range of 5.16e48.68 mg/mL with a correlation coefficient of 0.998. Inter

3.2. HPLC method optimization HPLC analyses were carried out by setting different wavelengths at the detector within the UV-VIS range simultaneously (i.e. 254 nm, 360 nm, 420 nm). Better results were observed at 254 nm and this was set for further analysis throughout the study. GABA and lysine derivatives showed good separation on the reverse phase C-18 column under a gradient elution system of methanol and water at Rt 7.925 and 9.214 respectively. However, the excessive derivatizing reagent was eluted before the derivatives at Rt 6.423 (Fig. 2). In order to validate the developed analytical procedure for the determination of GABA and lysine in the cereals (rice), linearity, repeatability, limit of detection (LoD), limit of quantization (LoQ) and accuracy were verified. A linear relationship was observed between peak height/peak area and analyte concentration for each standard. A straight line calibration curve for the standard GABA

Fig. 2. HPLC spectrum of GABA and Lysine Derivatives with 2-Hydroxynaphthaldehyde for (A) Standard GABA and Lysine (B) brown rice extract (C) Polished Rice Extract. Peaks Identification (1) Excessive Derivatizing Reagent (2) GABA (3) Lysine.

A. Hayat et al. / Journal of Cereal Science 60 (2014) 356e360

359

Table 1 GABA and lysine contents in polished and brown rice varieties. Rice form

Polished Brown

Basmati supper

Basmati 385

GABA

Lysine

GABA

Lysine

Basmati 2000 GABA

Lysine

Irri-6 GABA

Lysine

Irri-9 GABA

Lysine

0.74 ± 0.02 6.58 ± 0.25

13.11 ± 0.95 15.06 ± 0.55

0.46 ± 0.02 5.24 ± 0.25

15.24 ± 0.85 21.77 ± 1.12

0.42 ± 0.03 5.42 ± 0.28

16.02 ± 1.11 23.29 ± 2.58

0.39 ± 0.03 4.95 ± 0.18

23.34 ± 1.24 29.11 ± 3.12

0.32 ± 0.03 4.16 ± 0.15

19.87 ± 1.22 27.66 ± 2.33

Results shown as mean of three replicates mg/100 g rice ± standard deviation.

Table 2 Recovery percentage analysis of GABA and lysine. Standard name

Content of standard (mg/ml)

Added amount (mg)

Determined amount (mg/ml)

Recovery rate (%)

GABA

1.486 2.486 5.415 1.256 3.415 4.145

1.056 2.732 1.086 0.854 1.654 2.135

2.486 5.325 6.455 2.085 5.128 6.134

97.79 102.05 99.29 98.81 101.16 97.67

Lysine

Results shown as means of five replicates.

(n ¼ 5) and intra (n ¼ 7) day variation was observed with R.S.D. within 0.1% and 1.05% in retention times and 0.12% and 1.25% peak area, respectively. LOD and LOQ for this method are set as a peak to noise ratio of 3:1 and 10:1, respectively. The detection limits were observed 1.15 and 1.55 mg/mL whereas limits of quantization was found 3.83 and 5.16 mg/mL for GABA and lysine, respectively. The average percentage recovery of GABA and lysine in rice varieties were found 98 ± 3 with C.V. 2.25% (n ¼ 3). GABA and lysine derivatives were also analyzed every week in a month time period and found to be consistent in their retention time and peak area. 3.3. GABA and lysine analysis in rice varieties and statistical evaluation The results of GABA and lysine contents in brown and polished rice varieties are summarized in Tables 1 and 2. Both GABA and lysine were found higher in brown rice varieties compared to the

polished rice varieties which means that during the polishing process, GABA and lysine contents decreased. The GABA content in rice samples was found 0.32e0.47 mg/ 100 g in polished form and 4.1e6.58 mg/100 g in brown form at controlled condition whereas, lysine content found 13.11e19.8 mg/ 100 g in polished rice varieties and 15.1e27.6 mg/100 g in brown rice varieties. The highest GABA content was found in Basmati supper variety (0.74 mg/100 g in polished form and 6.58 mg/100 g in brown form) followed by basmati-385 variety and basmati-2000 variety, whereas the lysine content was found higher in Irri-6 variety (13.1 mg/100 g in polished form and 15.06 mg/100 g in brown form) followed by Irri-9 variety both in polished and brown conditions. The recovery experiment for both GABA and lysine in rice samples was carried out and the method showed high recovery rate, 99 ± 3% for both (Table 3). Moreover, the concentrations in mg/ 100 g with standard deviations were plotted in Fig. 3. The results of ANOVA shows that the calculated value lay in the critical region (Fcal ¼ 2.025358 < Ftab ¼ 6.3882330.05(4,4)), so it is suggested that the two varieties of rice have no significant different content of GABA, while for lysine, the calculated value lay outside the critical region (Fcal ¼ 16.64046 > 6.3882330.05(4,4)), so it is suggested that the two varieties of rice have significantly different content of lysine. 4. Conclusion In this study, 2-hydroxynaphthaldehyde has been examined as a useful pre-column derivatizing reagent for the analysis of GABA and lysine in food samples, especially cereal seeds such as rice seeds. The reaction of standards with derivatizing reagent is quantitative and reproducible. The derivative seems to be quite stable and provided high sensitivity in the UV region and separated easily with a simple gradient elution system of methanol and water. The selectivity and reproducibility of the method can allow determination and separation of GABA and lysine from the plant seeds in less than 10 min. The GABA content was found to be higher in basmati super rice variety followed by Basmati-385 and Basmati-2000 varieties whereas, the lysine content was found higher in Irri-6 variety followed by Irri-9 rice variety. Both analytes showed high concentration in brown rice in comparison with the polished form. Overall, analyzed Pakistani rice varieties have shown a healthy amount of GABA. Brown rice, especially, is a good source of GABA. References

Fig. 3. Graphical presentation of GABA and lysine content in polished and brown rice varieties.

Abbas, A., Murtaza, M., Aslam, F., Khawar, A., Rafique, S., Naheed, S., 2011. Effect of processing on nutritional value of rice (Oryza sativa). World J. Med. Sci. 6, 68e73. Bianchi, L., Della, C.L., Tipton, K.F., 1999. Simultaneous determination of basal and evoked output levels of aspartate, glutamate, taurine and 4-aminobutyric acid during microdialysis and from super fused brain slices. J. Chromatogr. B 723, 47e59. Christensen, H.N., Greene, A.A., Kakuda, D.K., MacLeod, C.L., 1994. Special transport and neurological significance of two amino acids in a configuration conventionally designated as D. J. Exp. Biol. 196, 297e305.

360

A. Hayat et al. / Journal of Cereal Science 60 (2014) 356e360

Clark, G., O’Mahony, S., Malone, G., Dinan, T.G., 2007. An isocratic high performance liquid chromatography method for the determination of GABA and glutamate in discrete regions of the rodent brain. J. Neurosci. Methods 160, 223e230. Jannoey, P., Niamsup, H., Lumyong, S., Suzuki, T., Katayama, T., Chairote, G., 2010a. Compression of gamma-aminobutyric acid production in Thai rice grains. World J. Microbiol. Biotechnol. 26, 257e263. Jannoey, P., Niamsup, H., Lumyong, S., Tajima, S., Nomura, M., Chairote, G., 2010b. gAminobutyric acid (GABA) accumulation in Rice during germination. Chiang Mai J. Sci. 37, 124e133. Kehr, J., 1998. Determination of glutamate and aspartate in microdialysis samples by reversed-phase column liquid chromatography with fluorescence and electrochemical detection. J. Chromatogr. B Biomed. Sci. Appl. 708, 27e38. Khuhawar, M.Y., Rajper, A.D., 2003. Liquid Chromatographic determination of gamma e aminobutyric acid in cerebrospinal fluid using 2hydroxynaphthaldehyde as derivatizing reagent. J. Chromatography B 788, 413e418. Komatsuzaki, N., Tsukaharab, K., Toyoshimac, H., Suzukic, T., Shimizua, N., Kimuraa, T., 2007. Effect of soaking and gaseous treatment on GABA content in germinated brown rice. J. Food Eng. 78, 556e560. Lee, B.J., Kim, J.S., Mi, K.Y., Lim, J.H., Kim, M.Y., Lee, S.M., Jeong, M.H., Ahn, C.B., Je, J.Y., 2010. Antioxidant activity and g-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods. J. Food Chem. 122, 271e276. Maisont, S., Narkrugsa, W., 2010. The effect of germination on GABA content, chemical composition, Tolal Phenolics content and antioxidant capacity of thai Waxy Paddy Rice. Kasetsart J. Nat. Sci. 4, 912e923. Mazur, R., Kovalovska, K., Hudecin, J., 2011. Change in selectivity of gammaaminobutyric acid formation effected by fermentation condition and microorganisms. J. Microbiol. Biotechnol. Food Sci. 1, 164e171. Nasholm, T., Sandberg, G., Ericsson, A., 1987. Quantitative analysis of amino acids in conifer tissues by high-performance liquid chromatography and fluorescence detection of their 9-fluorenylmethyl chloroformate derivatives. J. Chromatogr. A 369, 225e236. Oh, S.H., 2003. Stimulation of g-aminobutyric acid synthesis activity in brown Rice by a Chitosan/Glutamic acid germination solution and Calcium/Calmodulin. J. Biochem. Mol. Biol. 36, 319e325. Piepponen, T.P., Skujins, A., 2001. Rapid and sensitive step gradient assays of glutamate, glycine, taurine and Gamma-aminobutyric acid by highperformance liquid chromatography-fluorescence detection with o-phthalaldehyde mercapto ethanol derivatization with an emphasis on microdialysis samples. J. Chromatogr. B 757, 277e283. Polanuer, B., Sholin, A., Demina, N., Rumiantseva, N., 1992. Determination of glutamine, glutamic acid and pyroglutamic acids using high-performance liquid chromatography on dynamically modified silica. J. Chromatogr. A 594, 173e178.

Roohinejad, S., Mirhosseini, H., Saari, N., Mustafa, S., Alias, I., Hussin, A.S.M., Hamid, A., Manap, M.Y., 2009. Evaluation of GABA, crude protein and amino acid composition from different varieties of Malaysian's brown rice. Aust. J. Crop Sci. 3, 184e190. Rowley, H.L., Martin, K.F., Marsden, C.A., 1995. Determination of in vivo amino acid neurotransmitters by high-performance liquid chromatography with ophthalaldehyde- sulphite derivatisation. J. Neurosci. Methods 57, 93e99. Sakai, T., Okada, H., Kise, M., Komatsu, T., Yamamoto, S., 2005. g- Aminobutyric acid (GABA) suppresses antigen-specific immune responses in ovalbumin g (OVA)immunized BALB/c mice. Am. J. Immunol. 1, 101e105. Saller, C.F., Czupryna, M.J., 1989. Gamma-Aminobutyric acid, glutamate, glycine and taurine analysis using reversed-phase high-performance liquid chromatography and ultraviolet detection of dansyl chloride derivatives. J. Chromatogr. B 487, 167e172. Sawai, Y., Yamaguchi, Y., Miyama, D., Yoshitomi, H., 2001. Cyclic Treatment of anaerobic incubation increases the content of g-aminobutyric acid in tea shoots. J. Amino Acids 20, 331e334. Smolders, I., Sarre, S., Michotte, Y., Ebinger, G., 1995. The analysis of excitatory, inhibitory and other amino acids in rat brain microdialysates using microbore liquid chromatography. J. Neurosci. Methods 57, 47e53. Van der, Z.M., Oldenziel, W.H., Rea, K., Cremers, T.I., Westerink, B.H., 2008. Microdialysis of GABA and glutamate: analysis, interpretation and comparison with micro sensors. Pharmacol. Biochem. Behav. 90, 135e147. Varanyanond, W., Tungtrakul, P., Surojanametakul, V., Watanasiritham, L., Luxiang, W., 2005. Effect of water soaking on Gamma-Aminobutyric Acid (GABA) in germ of different Thai Rice varieties. Kasetsart J. Nat. Sci. 39, 411e415. Yamamoto, T., Nanjoh, C., Kuruma, I., 1985. Neurochem Determination of endogenous GABA released from the cerebral cortex slices of the rat by highperformance liquid chromatography with a series-dual electrochemical detector. J. Neurochem. Int. 7, 77e82. Yang, C.S., Tsai, P.J., Chen, W.Y., Tsai, W.J., Kuo, J.S., 1999. On-line derivatization for continuous and automatic monitoring of brain extracellular glutamate levels in anesthetized rats: a microdialysis study. J. Chromatogr. B 734, 1e6. Yingguo, L., Zhang, H., Meng, X., Wang, L., Guo, X., 2010. A validated HPLC method for determination of GABA by pre-column derivatization with 2, 4-dinitrofluorodinitrobenzene and its application to plant gad activity study. Anal. Lett. 43, 2663e2671. Yu-Haey, K., Fumio, I., Fernand, L., Roger, V.P., 2003. Neuroactive and other free amino acids in seed and young plants of Panax ginseng. J. Phytochem. 62, 1087e1091. Zhang, H., Yao, H.Y., Chen, F., 2006. Accumulation of gamma-aminobutyric acid in Rice germ using Protease. J. Biosci. Biotechnol. Biochem. 70, 1160e1165.